Eur J Biochem 271, 4335–4360 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04380.x REVIEW ARTICLE Models and mechanisms of O-O bond activation by cytochrome P450 A critical assessment of the potential role of multiple active intermediates in oxidative catalysis Peter Hlavica Walther-Straub-Institut fuăr Pharmakologie und Toxikologie der LMU, Muănchen, Germany Cytochrome P450 enzymes promote a number of oxidative biotransformations including the hydroxylation of unactivated hydrocarbons Whereas the long-standing consensus view of the P450 mechanism implicates a high-valent ironoxene species as the predominant oxidant in the radicalar hydrogen abstraction/oxygen rebound pathway, more recent studies on isotope partitioning, product rearrangements with Ôradical clocksÕ, and the impact of threonine mutagenesis in P450s on hydroxylation rates support the notion of the nucleophilic and/or electrophilic (hydro) peroxo-iron intermediate(s) to be operative in P450 catalysis in addition to the electrophilic oxenoid-iron entity; this may contribute to the remarkable versatility of P450s in substrate modification Precedent to this mechanistic concept is given Introduction Cytochrome P450 (P450 or CYP) enzymes (EC 1.14.14.1), a superfamily of b-type hemoproteins found in organisms from all domains of life [1], are major catalysts in the oxidative biotransformation of a structural diversity of endogenous and exogenous compounds [2] While the general chemistry of substrate hydroxylation has been assessed on a broad basis, the specific problem of dioxygen activation during P450 cycling is still the most important and intriguing one in the area of P450 research Here, the need for an active oxidant capable of insertion into Correspondence to P Hlavica, Walther-Straub-Institut fur Pharmakă ologie und Toxikologie, Goethestr 33, D-80336 Munchen, Germany ă Fax: +49 89 218075701, Tel.: +49 89 218075706, E-mail: hlavica@lrz.uni-muenchen.de Abbreviations: TSR, two-state reactivity; KIE, kinetic isotope effects; Hb, haemoglobin; Mb, myoglobin; HO, heme oxygenase; PDO, phthalate dioxygenase; TDO, toluene dioxygenase; NDO, naphthalene 1,2-dioxygenase; PMO, putidamonooxin; BLM, bleomycin; NOS, nitric oxide synthases Enzymes: Cytochrome P450 (EC 1.14.14.1); NADPH-cytochrome P450 oxidoreductase (EC 1.6.2.4); heme oxygenase (EC 1.14.99.3); phthalate oxygenase reductase (EC 1.18.1); phthalate dioxygenase (EC 1.14.12.7); toluene dioxygenase (EC 1.14.12.11); naphthalene 1,2-dioxygenase (EC 1.14.12.12); putidamonooxin (EC 1.14.99.15); nitric oxide synthases (EC 1.14.13.39) (Received 29 July 2004, revised 27 September 2004, accepted 28 September 2004) by studies with natural and synthetic P450 biomimics While the concept of an alternative electrophilic oxidant necessitates C-H hydroxylation to be brought about by a cationic insertion process, recent calculations employing density functional theory favour a Ôtwo-state reactivityÕ scenario, implicating the usual ferryl-dependent oxygen rebound pathway to proceed via two spin states (doublet and quartet); state crossing is thought to be associated with either an insertion or a radicalar mechanism Hence, challenge to future strategies should be to fold the disparate and sometimes contradictory data into a harmonized overall picture Keywords: (hydro)peroxo-iron; iron-oxene; O2-activation; P450 biomimics; P450 unactivated C-H bonds in hydrocarbons and related compounds has extensively captured the imagination of biochemists owing to the unfavourable thermodynamics of the dissociation event [3] Early views of such a mechanism focused on an oxygen insertion pathway promoted by an electrophilic, high-valent iron-oxo species (compound I) [4] This hypothesis was soon supplanted by the Ôhydrogen abstraction/oxygen reboundÕ concept implicating the existence of radical intermediates, as developed on the basis of the well-known chemical properties of peroxidases and porphyrin model systems [5,6] The mechanistic details of oxygen transfer have been addressed elsewhere [7,8] Mounting evidence provided during the past decade suggests that hydroxylation reactions are more complex than previously anticipated, and are not compatible with the idea of a single reaction pathway The picture began to cloud when the application of ultrafast Ôradical clocksÕ to time the oxygen-rebound step disclosed the amounts of rearranged products not to correlate with the radical rearrangement rate constants [9] Moreover, the use of a probe that could distinguish between radical and cationic species hinted at the interference of cationic rearrangements, predicting the hydroxylation to occur via an insertion reaction in place of abstraction and recombination [9] The former process thus necessitated the insertion into a C-H bond of the elements of OH+, implying that the ultimate electrophilic oxidant was either hydroperoxo-iron or ironcomplexed hydrogen peroxide [10] In addition, examination of the oxidative deformylation of cyclic aldehydes as a model for the demethylation reaction mediated by steroidogenic P450s strongly favoured nucleophilic attack on the Ó FEBS 2004 4336 P Hlavica (Eur J Biochem 271) substrates by an iron-peroxo intermediate [11] The sum of these findings points at the involvement of more than one active oxidant in the diverse types of P450-catalyzed substrate processing [12–15] The goal of the present perspective is to provide a critical update of several aspects of the current state of biochemistry relating to the apparently complex machinery of dioxygen activation, which is considered to possibly implicate multiple oxygenating species in P450 catalysis Emphasis will be put on the evaluation of comparative studies with non-P450 hemoproteins, nonheme metalloenzymes as well as biomimetic model systems to discuss the Ômultiple oxidantÕ vs the Ôtwo-state reactivityÕ theory Iron-oxene acting as an electrophilic oxidant in P450-catalyzed hydroxylations The consensus mechanism for hydrocarbon hydroxylation by P450 enzymes involves hydrogen atom abstraction from the hydrocarbon by a high-valent iron-oxo species, best described as an O ¼ Fe(IV) porphyrin p-cation radical, followed by homolytic substitution of the alkyl radical thus formed in the so-called Ôoxygen reboundÕ step [5–8] (Scheme 1) Using CYP2B isoforms as the catalysts, radical collaps was demonstrated to occur at highly variable rates exceeding those of the gross molecular motions of many enzyme-bound substrates and depending on the stereochemical specificities of the compounds to be acted upon [16,17] Reduction of ferric P450 to the ferrous state sets the stage for dioxygen binding, the event that commits the Scheme Rebound mechanism for P450-catalyzed hydroxylations Reproduced from [6] with permission hemoprotein to the step-by-step production of the active oxidant (Scheme 2) Association of dioxygen with ferrous microsomal CYP1A2 [18], certain CYP2B isoforms [19–21], and CYP2C3 [18] to yield hexacoordinate low-spin complexes has been shown to be characterized by absorption bands around 420 and 557 nm in the absolute spectra and broad maxima at about 440 and 590 nm in the difference spectra Similar optical perturbations were also observed upon O2 binding to so-called class I P450s, comprising mitochondrial and bacterial isozymes such as CYP11A1 [22–24] and CYP101 [25,26], respectively The rapid initial step in molecular oxygen activation by both class I and class II P450s, as measured at varying temperatures, usually exhibits monophasic kinetic behaviour, with the secondorder rate constants ranging from 0.58 to 8.41 · 106 M)1Ỉs)1 [18,20,24,25] Interestingly, the presence of certain substrates such as aromatic amines appears to favour homotropic cooperativity in dioxygen binding to P450s: using liver microsomal samples from untreated rabbits, the O2 saturation kinetics for acetanilide 4-hydroxylation have been reported to bear sigmoidal character corresponding to a Hill interaction coefficient, n, of 2.2 [27] Similar experiments with N-alkyl arylamines gave concave upward doublereciprocal plots of velocity vs O2 concentration, from which n could be calculated to have a value of 2.0–2.1 [28,29] Apparent cooperativity in dioxygen association was found to be highly sensitive to changes in hydrogen ion concentration and was most pronounced at physiological pH, whereas CO, acting as a positive effector, abolished autoactivation at all pH values examined (Fig 1) [30] In view of the well-known microheterogeneity of several rabbit liver P450s [31], the amine-induced cooperativity in O2 complexation has been argued to involve the equilibrium between multiple, kinetically distinct protein conformations [32] Alternatively, the oligomeric nature of P450 [33] might offer the possibility of substrate–specific subunit interactions, as has been proposed for the fractional saturation of hemoglobin by dioxygen [34] Results from resonance Raman spectroscopy [35] and Mossbauer studies [36] with microbial CYP101 indicate that ă O O e Fe(III) O2 Fe(II) O Fe(III) O O H O O + H+ + H+ H2O Fe(IV) Fe(III) Fe(III) peroxo-iron nucleophilic oxidant oxo-ion, low spin (inserts O) hydroperoxy-iron (inserts OH+?) spin inversion + H+ HO OH Fe(III) e OH HO Fe(III) iron-complexed hydrogen peroxide + (inserts OH ?) O Fe(IV) oxo-ion, high spin + (abstracts H ) Scheme The putative iron-oxygen intermediates in P450 and their possible roles as oxidants Data collated from [10,15] with permission Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur J Biochem 271) 4337 Fig Effect of hydrogen ion concentration on the Hill interaction coefficient n for oxygen binding Rabbit liver microsomal N-oxide formation from N,N-dimethylaniline was measured in the absence (d) and presence (s) of 490 lM CO Reproduced from [30] with permission the ÔoxyÕ intermediate of P450 most likely exists in the lowspin ferric-superoxide form, with the sixth 3d electron largely transferred to O2 in an autoxidative process (Scheme 2) Spontaneous autodecomposition of oxy-cytochrome 2B4 to release ferric pigment and superoxide [37] has been shown to occur in a biphasic [21,38] or even triphasic [39] fashion, while monophasic first-order kinetics were observed for autoxidation of substrate-bound adrenocortical CYP11A1 [23,24] and bacterial CYPs 101 and 102 [25,26,40], as measured above °C or at subzero temperatures Abortive decay of oxygenated P450 is retarded in the presence of hydroxylatable substrate [23,26,38], preserving the complex for arrival of the second electron, and is inversely proportional to the coupling efficiency of the system [41] Moreover, the steady-state level of oxyferrous P450 has been recognized to be governed by the hydrogen ion concentration and ionic strength of the reaction medium [21,24,25] In view of the strategic importance of the oxyferro intermediate in the process of dioxygen activation, the influence of the physiological redox partner, cytochrome b5, on its autoxidative breakdown has been examined in detail: though increasing the rate of regeneration of ferric enzyme from oxygenated CYP2B4 by a factor of about 8, reduced donor protein added to the assay mixtures failed to undergo substantial reoxidation, suggesting the electron carrier to act as an allosteric effector in this reaction [38,42] In accord with this, both apocytochrome b5 and aporubredoxin reportedly stimulate autoxidative transformation of oxy CYP101 to the ferric state [43] Superoxide departing from regenerated P450 has been found to serve as a source of hydrogen peroxide usually generated during NADPH/O2 consumption [44] Addition of an electron to oxyferrous P450 (Scheme 2) results in the formation of an optimized species, 37 kcalỈmol)1 higher in energy, with elongated Fe-O distance but unchanged O-O bond characteristics [45] Significant O-O bond weakening occurs upon protonation, the calculated proton affinity being )442.1 kcalỈmol)1 [45] The proton-delivering machinery has been recognized to involve a highly conserved active-site threonine residue [46,47] working in tandem with an essential aspartate [48– 50] The residue pair has been ascribed a critical role in orchestrating the dynamic organization of active-site water molecules [46], forming a hydrogen-bonded network capable of pumping protons to the reduced FeO unit [51] to generate the hydroperoxo-iron derivative (compound O; Scheme 2) Intermediacy of the end-on Fe(III)-OOH species has been unequivocally proven by electronic absorption, EPR and ENDOR spectroscopic techniques upon cryoradiolytic reduction of oxy CYP101 [52–55] and CYP119 [56] at 77 K The same intermediate was also obtained by reacting ferrous CYP101 with KO2 [57] or bioreduction of oxyferrous CYP101 with putidaredoxin [58] Unless the protonated peroxide complex decays in a nonproductive mode to liberate ferric enzyme and H2O2 [18], conversion to the actual oxidant proceeds with a significant energy release of 50 kcalỈmol)1 [59] While acylation of the distal oxygen to make it a better leaving group prior to Fenton-type homolytic O-O bond rupture has been vitiated owing to discrepancies between theory and measured data [60], the most favoured activation pathway is heterolytic O-O bond scission to formally produce a [FeO]3+ species (Scheme 2) [6,8], having a midpoint potential of 1.5–2.0 V [61] The so-called ÔpushÕ effect of the thiolate ligand in P450s has been shown to promote heterolytic cleavage of heme-bonded dioxygen by increasing electron density at the iron atom [62–66] The electrondonating properties of the active-site thiolate of CYP101 have been demonstrated to be enhanced by putidaredoxininduced alterations in enzyme conformation [50–67] Attempts were made to characterize the P450 reactive oxygen intermediate Thus, iodosylbenzene, a single-oxygen donor [68], as well as peroxides and peracids, acting as versatile O2 surrogates in oxidative reactions [69–71], have been revealed to elecit spectral perturbations with P450s closely resembling those of the green, high-valent FeOPor + species (compound I) of peroxidases, including the thiolateligated, P450-like chloroperoxidase enzyme [72–75] These findings lent credit to the notion, that an analogous key oxidant might be operative in P450-catalyzed monooxygenations, too, albeit there is a significant difference between P450 and peroxidase models regarding the displacement of the iron atom from the porphyrin plane, resulting in longer Fe-O bond in the P450 active intermediate [76] Density functional studies demonstrate that both enzyme systems, though looking very similar, behave like chemical chameleons, in which small alterations in the environment can cause drastic changes in the reactivity of the active species [76] Further support in favour of the idea of the involvement of a high-valent iron-oxene in P450 catalysis came from experiments with metalloporphyrin models [5,6,77] Of particular importance, a green oxo-ferrylporphyrin p-cation radical intermediate could be isolated and spectrophotometrically and chemically characterized, that was capable of • 4338 P Hlavica (Eur J Biochem 271) oxygen transfer reactions [78] Nevertheless, identification of the [FeO]3+ adduct by UV-visible spectroscopic analysis of CYP119 [79] or transient X-ray crystallography using CYP101 [80] appears to be quite tentative The proportion of the putative iron-oxene species not used for monooxygenations undergoes uncoupling to generate ferric P450 and water [81] in a 4-electron reductive process [82], uncoupling being controlled by substrate accessibility [83] In fact, the presence of substrate has been shown to stabilize the active oxy complex produced with CYP2B4 and organic hydroperoxide, and the protective effect is intensified by cytochrome b5 binding [84,85] Active oxidant thus preserved is thought to promote hydrogen transfer from substrate to initiate monooxygenation (Scheme 1); this step, which proceeds with a remarkable low free–energy barrier, has been suggested to be governed by peripheral heme substituents in the P450 molecule [86] Firm evidence for the nonconcerted hydrogen abstraction/ oxygen rebound chemistry presented in Scheme is provided by a plethora of experimental observations such as (a) the stereochemical scrambling in norbornane [87] and camphor [88] hydroxylation (b) the allylic rearrangements found in the hydroxylation of unsaturated hydrocarbons [89] (c) the correlation of susceptibility toward oxidative attack with C-H bond strength [90] (d) the large kinetic isotope effects (KIE; kH/kD % 11) for C-H activation using norbornane [87], diphenylpropane [91] or difluorocamphor [92] as the hydroxylatable substrates and (e) the results from investigations with Ôradical clockÕ probes such as bicyclopentane, having highly strained carbocyclic structures to permit the unmasking of radical intermediates that rearrange at a rate faster than that of the recombination step [16,93] Despite the apparent predominance of the hydrogen transfer mechanism as the initial step in substrate hydroxylation, electron transfer to generate a carbocation, followed by capture of a hydroxyl anion has been discussed as an alternative oxygenating principle [94,95] The net outcome would be oxidation of an otherwise unactivated C-C single bond Although cations may be the logic precursor for certain substrates with low oxidation potentials, such a pathway cannot be reconciled with the large KIE and stereochemical scrambling detailed above To quantitatively assess the significance of electron transfer in the transition states of hydroxylation reactions, studies on the regioselectivity of nitroacenaphthene oxygenation were conducted with various oxometalloporphyrins; hydrogen abstraction was shown to be the preferred route for all models examined [96] Hydroperoxo-iron acting as an alternative electrophilic oxidant in P450-catalyzed hydroxylations Evidence from kinetic analysis of P450 function Studies on the oxidative transformation of 1-methyl-2phenylcyclopropane and its mono-, di-, and trideuteriomethyl congeners by microsomal CYP2B1 and CYP2E1 suggested that, judging from the large magnitudes of the combined primary and secondary KIEs for hydrogen abstraction, rotation in the enzyme pocket was faster than Ó FEBS 2004 its relatively slow reaction (< 106Ỉs)1) with the putative iron-oxene species [97], while the lifetimes of carbon-centred radicals derived from a diverse set of substrates are on the order of about 10)10 s [98] Moreover, the randomness of the apparent intramolecular KIEs for unrearranged and rearranged alcohol products generated from enantiomeric dideuteriomethyl substrate forms implicated that more than one reaction channel existed [99] This concept was reinforced when the KIEs for NADPH-and cumene hydroperoxide-driven N-demethylation of amitryptiline by CYP2D6 were found to be severely discrepant [100] Examination of the competitive intermolecular KIE for sulfoxidation/N-dealkylation reactions mediated by bacterial CYP102 hinted at the involvement of two distinct electrophilic oxidizing species [101], as was also concluded from the intermolecular noncompetitive KIEs for a- and b-hydroxylation of fatty acids by CYP152 peroxygenase isozymes [102] Probing of the metabolism of norcarane by CYP2B4 revealed the formation of a cation-derived rearrangement product not compatible with the hydrogen abstraction mechanism [103] The latter was also challenged by the finding that evaluation of the metabolic transformation of a series of cyclopropane derivatives by CYP2B4 gave unreasonably high rate constants for oxygen rebound (kOH) ranging from 1.5 to · 1012 s)1; this disparate result was rationalized by possible steric effects in the enzyme’s active site causing overestimation of the kOH values [17] However, experiments on the CYP2B1-catalyzed hydroxylation of a new constrained substrate, that would be less likely to be subject to steric constraint, also yielded an incredibly high apparent kOH value of 1.4 · 1013 s)1 [104] Moreover, the plot of the ratio of rearranged to unrearranged alcohol products vs the rate constant for rearrangement of the putative radical intermediate (kr) revealed a lack of correlation between these parameters [104] In addition, hypersensitive radical probe studies with four P450 isozymes gave consistently small amounts of rearranged products, hampered radical ring opening on steric grounds being unlikely [105] The sum of these findings thus suggested that there was either an error in the kinetic scale for fast radical reactions or the mechanistic paradigm of P450-mediated hydroxylations was incomplete To solve this problem, further hypersensitive radical probe substrates were introduced, that could distinguish between radical and carbocation intermediates on the basis of the identity of the rearranged products [9,106,107] Oxidation of these probes with several members of the CYP2 family gave cationderived rearrangement products, disproving the assumption that such rearrangements arose exclusively from radical species Variable partitioning between the radical and carbocation mechanisms thus was concluded to explain the wide range of kOH values described above [106] From the small amounts of radical rearrangement products generated from the hypersensitive probes, the radical lifetimes in the P450-catalyzed reactions could be calculated to range from 70 to 200 fs [106,107], which are too short for true radical intermediates, but rather correspond to vibrational lifetimes or the lifetimes of transition states Hence, the cationic intermediates observed could be ruled out to originate from oxidation of such transient radicals, so that their occurrence necessitated another mechanistic enigma Ó FEBS 2004 OH O-O Bond activation by cytochrome P450 (Eur J Biochem 271) 4339 H O O O H OH O H H OH O Fe 3+ SCys A Fe 3+ Fe 3+ SCys Fe 3+ SCys SCys B C to be obligatory when peroxide substituted for reduced cofactor and dioxygen While N-(4-chlorophenyl) hydroxylamine was found to be the major metabolic product under mixed-function conditions, a marked change to the preponderant formation of 1-chloro-4-nitrobenzene was observed when organic hydroperoxide served as the oxygen donor [116] Involvement in the N-oxidative process of CmO (Cm2 ) radicals could be safely ruled out owing to insensitivity of the reaction toward radical scavengers, whereas blockage of turnover by cyanide hinted at an iron-based mechanism [116] The sum of these findings raised serious questions as to the commonness of the oxygenating species operative in the NADPH- and hydroperoxide-sustained hydroxylations In fact, evidence has been provided for the existence of fairly stable Fe(III)-OOR intermediates generated by reacting organic hydroperoxides with mononuclear iron catalysts [118–120] or intact CYP2C11 [121], and their ability to transfer oxygen to substrates prior to heterolytic cleavage at low temperatures has been ascertained [122–124] As N-hydroxylation of 4-chloroaniline by the putative Fe(III)-OOR species must compete not only with conversion of the intermediate to [FeO]3+, but also with self-destructive oxidation of the heme moiety of P450 [125], it seems worth mentioning that the rate of cumene hydroperoxide-induced loss of CO-reactive CYP2B4 [85] could be demonstrated to be far below that of release of N-oxy product from the ternary complex [116] There is also reason to envisage iron-bound hydroperoxide as a potential oxidant in NADPH-promoted N-oxygenation of N,N-dimethylaniline by CYP2B4: the presence of superoxide dismutase inhibits the reaction by 75%, whereas catalase or mannitol leave N-oxide formation unaffected, dismissing free H2O2 or OH radicals to act as catalysts [126] Notably, investigations with a superoxidegenerating system ruled out O2 ) itself to function as the active intermediate, so that superoxide was invoked to serve as a source for production of the ultimate oxygenating species, presumably Fe(III)-OOH, catalyzing attack on the electron-rich nitrogen centre of the tertiary arylamine [126–128] The active oxidant thus was anticipated to arise from interaction, in the presence of protons, of newly generated O2 ) with either ferrous or oxyferrous [Fe(III)O2 )] P450, as given in Eqns and [129–132] That Fe(III)OOH generated in this way would only serve as a precursor in the transformation to: • Scheme Potential Ôsecond oxidantÕ species in P450 catalysis Data adapted from [108] with permission In this regard, the most plausible premise is insertion of OH+ into a C-H bond to generate protonated alcohol species that can undergo solvolysis-type reactions to yield cationic rearrangement products [9,107] This route requires heterolytic O-O bond fission of the hydroperoxo-iron state of P450 (Scheme 3A) to release OH+ and [FeO]+ [106,107] However, density functional analysis of mechanisms involved in ethylene epoxidation by a Fe(III)–OOH model disclosed barriers for the various pathways of 37–53 kcalỈ mol)1 [108] This was taken to indicate that hydroperoxoiron, as such, could not be the ultimate oxidant, in line with its significant basicity and poor electron-accepting capabilities [108] Moreover, molecular orbital calculations carried out with a similar model system unveiled nonrepulsive potential curves only for peroxo-iron, but not for hydroperoxo-iron as the catalytic intermediate in the turnover of aniline and fluorobenzene [109] Comparative investigations on the NADPH/O2- and iodosylbenzene-dependent metabolism of lauric acid by CYP2B4 favoured the Fe(III)-H2O2 complex (Scheme 3B) as acting as an alternative electrophilic oxidant [110] This postulate is in accord with data from measurements with hypersensitive radical clocks [9,106], albeit there is some objection to this idea: protonation of the proximal oxygen in the reduced ferrous dioxygen unit is usually thought to trigger Fe-O bond weaking followed by uncoupling of monooxygenation reactions [111] On the other hand, stable end-on iron(III)-hydrogen peroxide complexes have been shown to incur in the catalytic cycle of cytochrome c peroxidase [112], horseradish peroxidase [113] and chloroperoxidase [114], but their immediate participation in monooxygenation processes has not been established Finally, molecular dynamics simulations employing the CYP101 crystal structure proposed the diprotonated species displayed in Scheme 3C to be an oxidant far superior to compound I [115] As can be readily seen, the question of the nature of the alternative oxygenating intermediate remains inherently elusive The functional importance of hydroperoxo-iron or ironcoordinated hydrogen peroxide as the putative second oxidant in P450 catalysis is also corroborated by studies on heteroatom oxidation Thus, comparative investigations on the NADPH/O2- and cumene hydroperoxide-driven N-hydroxylation of 4-chloroaniline by CYP2B4 indicated discrepancies in the positions of the Soret maxima in the absolute spectra of the individual oxy complexes [116] Noteworthy, transformation of P450 to the denatured P420 form through treatment with either p-chloromercuribenzoate or deoxycholate rendered the hemoprotein a more powerful peroxygenase [116], but disrupted NADPH-linked monooxygenase activity [117] Hence, resonanace stabilization via the thiolate Ôpush effectÕ (see above) did not appear ã ã ã ã FeIIị ỵ Oặ ỵ Hỵ ! ẵFeIIIị OOH 1ị ẵFeIIIị Oặ ỵ Oặ ỵ Hỵ ! ẵFeIIIị OOH þ O2 2 ð2Þ iron-oxene as the actual catalyst could be discounted on kinetic grounds As an example, the reaction sequence given in Eqn follows second-order kinetics with a rate constant of · 103 M)1Ỉs)1 [133], while injection into Fe(II)-O2 of the ÔsecondÕ electron to produce compound I during regular catalytic cycling is a diffusion-controlled process characterized by a rate constant of · 1010 M)1Ỉs)1 [134] Comparison of these data no doubt precludes the major portion of ferryl material required for efficient substrate turnover to originate from the dismutation-type bypass reaction As 4340 P Hlavica (Eur J Biochem 271) conversion of the hydroperoxo entity to [FeO]3+ is a second-order event, encompassing interaction of the peroxo intermediate with a proton source to initiate O-O bond cleavage with water release, the half-life of this step is inversely correlated with the initial concentration of Fe(III)-OOH Hence, the much lower rate of production of the latter in the superoxide-supported pathway necessarily results in a depressed level, within the time scale of the measurements, of hydroperoxo-iron component relative to the standard redox situation (Scheme 2) This is likely to cause an increase in both the half-life of the scission process and the lifetime of Fe(III)-OOH, possibly fostering direct oxygen insertion into substrate Evidence from experiments with genetically engineered P450 enzymes Inferential evidence of two electrophilic oxidants acting as catalysts in substrate hydroxylations came from studies with mutated P450s The crystal structures of bacterial CYPs 101, 102 and 108 contain a highly conserved active-site threonine within H-bonding distance to the peroxo-iron unit [135] Of particular interest, attenuated camphor and laurate hydroxylation was observed, when T252/268 in the CYP101 and CYP102 polypeptide, respectively, were replaced with alanine [136,137] Nevertheless, the T252A variant was found to accept electrons from NADH and reduce dioxygen to H2O2 [137] via the intermediacy of hydroperoxo-iron [53] Mutation was considered to disrupt a key step in H+ delivery, presumably introduction of the second proton to hamper OO bond dissociation [53] Therefore, P450 mutants devoid of the active-site threonine were regarded ideal means for testing the direct involvement of hydroperoxo-iron in epoxidations Indeed, a drastic increase in the ratio of epoxide to hydroxy products derived from various camphor analogues during catalysis by the T252A congener of CYP101 could be demonstrated in comparison to the wildtype parent [138] Similar findings were made with truncated CYPs 2B4 and 2E1 lacking the active-site threonine: the mutants mediated alkene metabolism at an increased ratio of epoxidation to allylic hydroxylation [139] Using the same wild-type and engineered P450 pairs, the potential involvement of Fe(III)-OOH in hydroxylation reactions was inferred from mutant-induced changes in regioselectivity during the oxidation of probes designed to give different rearrangement products with radical and cationic intermediates [99,105,107,140] Moreover, truncated CYP2E1 with T303 replaced by alanine was shown to exhibit considerably higher activity than the parent enzyme in eliminating p-substituents in phenols to yield hydroquinones [141] The participation of an alternative electrophilic intermediate in heteroatom oxygenation was assessed by employing the T268A mutant of CYP102: the engineered enzyme fostered sulfoxidation of p-(N,N-dimethylamino)thioanisole relative to N-dealkylation of the substituted amine function [101] A mutant of truncated CYP2B4 with exchange of alanine for threonine at position 302 turned out to have decreased ability to catalyze NADPH-dependent N-oxide formation from N,N-dimethylaniline, questioning an obligatory hydroperoxo-iron-promoted mechanism [142] Ó FEBS 2004 However, when the measurements were conducted with iodosylbenzene in place of NADPH/O2 to directly generate the favoured [FeO]3+ entity [68], the enzyme variant still mediated N-oxygenation of the tertiary arylamine at a rate less than half that of the wild-type-catalyzed reaction [142], so that reasonable interpretation of the data seems difficult Evidence from comparative studies with non-P450 hemoproteins and metalloporphyrin models Hemoglobin (Hb) and myoglobin (Mb) When operative in its natural environment, the erythrocyte Hb exerts P450like monooxygenase activity [143] in that the concurrence of multiple reductase systems permits NAD(P)H-supported electron transfer to the pigment [144,145] In fact, isolated Hb reconstituted with NADPH-cytochrome P450 oxidoreductase (EC 1.6.2.4) has been demonstrated to bring about NADPH/O2-promoted alkane hydroxylation [146] as well as N- and O-dealkylation reactions [147], albeit at considerably lower catalytic potency as compared with P450 enzymes Of particular interest, Hb has been found to mediate formation of p-aminophenol from aniline both in intact erythrocytes, supplemented with glucose to allow NADPH production via the pentose phosphate pathway [148], and in a reconstituted system containing P450 reductase, NADPH and atmospheric oxygen [149] In the latter case, catalase inhibited enzyme activity by about 94% in the absence or presence of reductase, suggestive of a hypothetical mechanism for p-hydroxylation of the aromatic amine involving H2O2, formed through dismutation of autoxidatively generated superoxide, to produce the active intermediate, Hb(III)-OOH [149] In line with this, alkaline hemin (ferriprotoporphyrin IX) has been shown to activate O2 to the hydroperoxide anion in the presence of NAD(P)H [150] with the immediate insertion of oxygen into the benzene ring of aniline to yield p-aminophenol [151] Formation of the oxygenating species has been recognized to be facilitated by the binding to HbO2 of aniline and some of its derivatives, causing distortion of the iron-oxygen bond to such an extent as to accelerate autoxidation by alleviating electron transfer from ferrous iron to O2 [147,152] Superoxide displaced from HbO2 has been postulated to contribute to production of the hydroperoxo-methemoglobin entity by reducing hemebound oxygen in the presence of a proton source as given in Eqn [131,153] Indeed, radiolytically reduced samples of oxygenated Hb [154,155] and Mb [156–158] at cryogenic temperatures have been shown by EPR studies to generate the peroxo-bound hemoproteins, with the Fe-O-O unit being stabilized by bonding to the distal histidine proton; the latter was detected to be transferred upon annealing to give the hydroperoxo derivatives Similar results were obtained, when metmyoglobin was reacted with H2O2 at 77 K [159] The ability of Hb to catalyze heteroatom oxygenation is well established Thus, methemoglobin and H2O2 transform thianthrene 5-oxide to both the 5,5-and 5,10-dioxide metabolites [160] The finding that most, if not all, of the sulfoxide oxygen in the 5,5-dioxide product originates from hydrogen peroxide but not from 18O2 has been rationalized by the possible participation in this reaction of a peroxoiron catalyst This view is compatible with the capability of mononuclear peroxo intermediates derived from iron and Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur J Biochem 271) 4341 titanium porphyrin complexes upon treatment with superoxide and H2O2, respectively, to directly promote sulfoxidation reactions [161] Similarly, Hb has been reported to perform N-hydroxylation of 4-chloroaniline both in erythrocyte suspensions [162] and in aerobic systems reconstituted with either NADPH-P450 reductase or the NADH-cytochrome b5 reductase/cytochrome b5 segment of the electron transfer chain in the presence of NAD(P)H [163,164] Under these conditions, addition to the assays of superoxide dismutase or catalase disrupted N-oxygenating activity by about 70%, again posing emphasis on the pivotal role of H2O2 in forming the active oxidant It should be noted that N-oxidative metabolism of 4-chloroaniline is associated with optical changes characterized by a Soret band at 418 nm in the absolute spectrum [163], closely resembling the spectral perturbations arising from reduction of MbO2 by hydrated electrons [134] or reaction of ferrous CYP101 with superoxide [57] to yield Fe(III)-OO(H) Importantly, N-(4-chlorophenyl)hydroxylamine, generated as the primary metabolic product, has been found to be prone to hydroperoxo-methemoglobin-promoted conversion to the 4-chlorophenyl nitroxyl radical [165] The same mechanism appears to also apply to one-electron oxidation of the nitrogen centres in N,N-disubstituted p-phenylenediamines to give Wurster’s blue aminyl radicals: the HbO2-dependent processes have been shown to be decelerated by up to 50% in the presence of catalase, whereas superoxide anion was likely to be of minor importance in formation of the radical cations [166] Collectively, the drastic disruption of the C- and N-oxidative biotransformation of aniline and its 4-chloro derivative [149,163] by the presence of catalase furnishes unequivocal evidence for a vital role of autoxidatively liberated H2O2 in Hb-dependent catalysis It could be argued that Hb(III)-OOH, generated through the reaction of endogenously released hydrogen peroxide with methemoglobin, might not act itself as the oxidant, but represent a transient intermediate in the production of oxygenating ferryl material However, it seems improbable that the route of ferryl-Hb formation should be preferentially via the Ôperoxide shuntÕ: the sluggish autodecomposition of HbO2 (k % 10)3 M)1Ỉs)1) in the presence of the anilines [166] to finally yield H2O2 together with the relatively low rate of peroxide association with ferric globin (k ẳ 4.8 Ã 102 M)1ặs)1) [159] undoubtedly impose considerable constraints on the overall rate of compound I formation, whereas its direct generation upon electron introduction into the oxyferrous entity is a very rapid process as oulined above [134] The sum of these findings refutes compound I to contribute to significant extent to the total amount of hydrogen peroxide-induced active oxidant, but rather favours Hb(III)–OOH itself or the iron(III)-H2O2 adduct [110] to serve in this function (Scheme 3) This view is endorsed by the fact that imidazole, acting as the proximal axial ligand in electron-rich iron-porphyrin model compounds, appears to prolong the lifetime of the H2O2-derived hydroperoxo-iron species, such as to permit direct oxygen insertion into substrate [167] In accord with this, loss of the proximal H93 ligand in Mb through replacement with cysteine results in enhanced O-O bond scission of oxidant produced with organic hydroperoxide [168] Scheme Proposed mechanism of HO-catalyzed conversion of hemin to a-meso-hydroxy-heme The heme unit is shown in a truncated form Reproduced from [125] with permission Heme oxygenase Although heme oxygenase (HO; EC 1.14.99.3) is distinct from P450s, the reactions catalyzed by this enzyme are, nevertheless, part of the same hemedependent reaction manifold that underlies the catalytic action of all hemoproteins The first metabolic process mediated hy HO is self-hydroxylation of heme to form a-meso-hydroxyheme (Scheme 4), using the histidyl-ligated heme group as both a prosthetic unit and substrate [125,169–171] Whereas transformation by plant [172] and bacterial [173] HO enzymes requires electron supply by an NADPH-ferredoxin reductase/ferredoxin couple analogous to mitochondrial and microbial class I P450s, mammalian heme oxygenases accept reducing equivalents, in the presence of dioxygen, from NADPH-P450 reductase, resembling microsomal class II P450s with respect to their ability to functionalize unactivated C-H bonds [170] There is strong evidence for Fe(III)-OOH to act as the mesohydroxylating species in HO catalysis Thus, H2O2 has been found to be able to replace NADPH/O2 in supporting the first step in heme oxidation, while ferryl-forming acyl hydroperoxides were incompetent [174] Moreover, application of ethyl hydroperoxide as the oxidant could be demonstrated to promote generation of a-meso-ethoxyheme [175] Studies with the four meso-methylmesoheme regioisomers disclosed the electron-donating methyl substitutents to govern the regiochemistry of mesohydroxylation on an electronic rather than steric basis, implicating electrophilic addition of the oxygen to the porphyrin ring [176,177] It should be mentioned, in this context, that the H39V mutant of rat outer mitochondrial membrane cytochrome b5 has been shown to be capable of building a coordinate ferric hydroperoxo intermediate upon reduction of the oxyferrous complex with hydrazine, which adds a hydroxyl group to the porphyrin to produce mesohydroxyheme [178] Direct evidence for the occurrence of Fe(III)-OOH during normal catalytic turnover of HO was furnished by EPR and ENDOR experiments [179] Radiolytic cryoreduction/annealing investigations to directly monitor solvent and secondary KIEs, preventing masking of the latter by interference with other reactions, revealed bond formation between the a-meso-carbon of the porphyrin moiety Ó FEBS 2004 4342 P Hlavica (Eur J Biochem 271) and the terminal oxygen atom of the hydroperoxo entity; this process was activated by delivery of the second proton by a carboxyl donor, presumably D140 [180,181] Finally, optical absorption and EPR measurements permitted the detection of a hydroperoxo intermediate derived from a synthetic Fe(III)-porphyrin complex, with electrophilic addition of the axially ligating OOH– to the porphyrin macrocycle to yield the cationic form of meso-hydroxyporphyrin [182] Por – FeIII – OOH Por – FeIV – O H 2O H+ H3C NH H3C H3C N N F– Microperoxidase-8 (MP8) Microperoxidase-8 is a heme-based mini-enzyme, forming a new generation of biomimics, obtained by two subsequent steps of peptic and tryptic digestion of horse-heart cytochrome c [183] It consists of a residual octapeptide, with histidine covalently attached to the ferric heme iron as the fifth ligand (Fig 2) The mini-catalyst has been depicted as an attractive model for studying P450-type oxygen transfer reactions [184] Thus, addition of ascorbate to MP8/H2O2-containing reaction mixtures to block peroxidase-type radical chemistry and, instead, induce a P450-like oxygenation mechanism has been demonstrated to result in a drastic diminution of polymerization products derived from aniline and some of its p- and N-substituted congeners, while formation of p-hydroxylated and dealkylated metabolites was increased; this was attributed to involvement in catalysis of a (hydro)peroxo-iron intermediate [185,186] In accord with this, NADPH/O2-sustained conversion of aniline to p-aminophenol by heme-peptide reconstituted with NADPH-P450 reductase has been shown to be highly susceptible to the presence of catalase [187] Moreover, 18O-labeling experiments with MP8 in the presence of ascorbate revealed the biocatalyst to p-hydroxylate aniline with full transfer of oxygen from H18 O2, while rapid exchange of the labelled oxygens of H2O2 with unlabelled H2O occurred, pointing at reversibility of formation of the high-valent iron-oxene species to produce a porFe(III)-H2O2 complex [188] Similarly, water has been advocated to play a decisive role in regeneration, through reaction with (R +)MP8Fe(IV) ẳ O, of the active ã Fig Chemical structure of microperoxidase-8 Data taken from [190] F Por – Fe III – OOH F Por – Fe OH III O – OH Scheme Proposed reaction mechanism for a MP-8-catalyzed dehalogenation pathway oxidant operative in the microperoxidase/H2O2-driven hydrocarbon oxygenation in bi- and tricyclic aromatic compounds [189] or oxidative aromate dehalogenation [185,190] (Scheme 5) It should be emphasized that reaction of iodosylbenzene with purified CYP2B1 [191] or nonporphyrin iron(III) chelates in basic media [132,192] has been detected to prompt O-O bond formation at the iron centres Alkoxylating dehalogenation of halophenols, carried out by MP8/H2O2 in alcoholic solvents, has been hypothesized to implicate a mechanism, in which the ironoxene resonance form reacts with alcohol to generate an Fe(III)-OOR intermediate [193] Unequivocal indentification of the MP8-based (hydro)peroxo-iron(III) entity has been achieved by optical absorption [194] and rapidfreeze EPR measurements [195] Synthetic metalloporphyrin models Synthetic metalloporphyrins (Fig 3) were selectively tailored as models of the P450 active site to gain more detailed insight into the mechanistic basis of oxygen transfer reactions Using a set of meso-tetraarylporphyrine derivatives (Fig 3A), cisstilbene was found to be subject to H2O2-sustained conversion to the oxide metabolite in aprotic solvent with trace amounts of allylic oxidation products, ruling out ironoxene or OH radicals to be responsible for olefin epoxidation, while hydroperoxo-iron was likely to be the active oxidant [124,167] Similarly, the lack of 18 O-incorporation, at low temperature, from labeled water during perbenzoic acid-supported epoxidation of cyclooctene by the polyhalogenated TPFPP analogue was interpreted to mean that the electronegatively substituted iron porphyrin generated a relatively stable Fe(III)-OOR species, which directly transferred its oxygen to the olefin [123] This postulate is in line with the observation that there is a strict relationship between the selectivity of norbornylene over a-methylstyrene epoxidation by the TDCPP-porphyrinato-iron complex and the structure of the peracids used [196] The same principle also applies to the cyclooctane vs cyclooctene oxidation catalyzed by a thiolate-ligated meso-tetraarylporphyrin model (Fig 3B) with various p-substituted perbenzoic acids [197] Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur J Biochem 271) 4343 R Me Cl R= Me N N N Fe+++ R Me R TMP F Cl F TDCPP F N N N N Fe+++ N S HN F F R TDFPP F O O F TPFPP A B Et Et Et Et N Cl– N Fe+++ H S Fig Chemical structures of iron(III)porphyrin complexes used as P450 model species Data taken from [167,197,202, 203] Moreover, acylperoxo-iron(III) has been claimed to also serve as the effective catalyst in the peracid-driven cyclohexane hydroxylation depending on the nature of the anionic axial ligands of the Fe(TPFPP) adduct [198] Studies with a second genre of metalloporphyrin systems, containing molecular oxygen and a coreductant such as ascorbate to mimic the natural P450-mediated electron transfer pathway, disclosed manganese meso-tetraphenylporphyrin to substantially differ from the iodosylbenzenepromoted route with respect to regioselectivity of olefin epoxidation and reactivity toward tertiary vs secondary C-H bonds; this was tentatively attributed to the involvement of a Mn(III)-peroxo instead of a ferryl complex in the ascorbate/O2-driven process [199] The reactive (hydro)peroxo-metalloporphyrin species in the above model systems have been characterized by visible spectroscopy, NMR, EPR, and Mossbauer data upon ă combination of the tetraphenyl- or octaethylporphyrinmetal adducts (Fig 3D) with superoxide anion [129, 200,201] or reduction of oxygenated macrocycle by ascorbic acid [202] Using the methylmercaptane porphyrin model depicted in Fig 3C, the optimized geometry of the transient reduced ferrous dioxygen form, calculated by applying nonlocal DFT methods, indicated an asymmetric Ôend-onÕ binding fashion of the dioxygen ligand with pronounced elongation of the Fe-O and Fe-S bonds [203] Evidence from comparative studies with mononuclear nonheme iron enzymes and biomimetic metal chelates Rieske-type dioxygenases Rieske oxygenases catalyze the regio-and stereospecific cis-dihydroxylation of aromatic rings, initiating aerobic degradation of aromatic compounds in soil bacteria, and are targets for bioengineering in bioremediation [204] They are a consortium of two or three protein components involving a Rieske Fe2S2 cluster to channel electrons from NAD(P)H via a flavin-containing reductase to a mononuclear iron centre; the latter is believed to be the site of dioxygen and substrate activation [204] This electron transfer chain thus functions like the heme centres in N H H N Et Et C Et D Et class I P450s acting in unison with their associated ironsulfur redox partners [135] Analogous to the role of putidaredoxin as an effector in CYP101 catalysis [50,67], binding of phthalate oxygenase reductase (EC 1.18.1), a flavo-iron-sulfur polypeptide, to phthalate dioxygenase (PDO; EC 1.14.12.7) has been advocated to tune the enzyme’s structure for oxygenating activity on an allosteric basis [205] Moreover, toluene dioxygenase (TDO; EC 1.14.12.11) and naphthalene 1,2-dioxygenase (NDO; EC 1.14.12.12) have been found to mediate P450-like monooxygenations when provided with appropriate substrates [206,207] Availability of the crystal structure of NDO as well as spectroscopic data provide a rationale for the catalytic mechanism of this class of enzymes Naphthalene 1,2dioxygenase is a heterohexamer composed of an equimolar combination of a- and b-subunits, each a-subunit bearing an Fe2S2 cluster and a mononuclear iron site [208] Two histidines and one bidentate aspartate ligand, the socalled Ô2-His-1-carboxylate facial triadeÕ, encountered with various nonheme iron, oxygen-activating enzymes, occupy one side of the mononuclear iron coordination sphere [209] Substrate binding to produce an open coordination position on Fe(II) has been suggested to be critical in O2-activation, allowing two-elctron transfer from both the mononuclear iron centre and the reduced Rieske cluster to generate an Fe(III)-OO(H) intermediate [210,211]; the latter has been proposed to exert a concerted mode of attack on substrate, explaining the cis-specificity of the dihydroxylation reaction In accord with this concept, hydrogen peroxide has been reported to be able of substituting for NAD(P)H/O2 in NDO-dependent cis-dihydrodiol formation, both oxygen atoms in the product deriving primarily from H2O2 [212] Moreover, benzene has been demonstrated to act as both a substrate and an uncoupler of NDO, causing the release of H2O2 during the reaction [213] More recently, the role of a putative end-on (hydro)peroxo-iron catalyst in NDO turnover has been ascertained by the detection, in the enzyme’s crystal structure, of an indole-oxygen adduct bound to the mononuclear iron [214] Circumstantial EPR Ó FEBS 2004 4344 P Hlavica (Eur J Biochem 271) –OOC COO– –OOC –OOC COO– COO– O O Fe++ Fe++ Rred + O2 Fe + Rred Rred efrom reductase –OOC COO– H H –OOC COO– H O HO –OOC COO– O COO– products OH H+ 3 O O Fe + Rox O– (H+) 2– O Fe + Rox –OOC H Fe + Rox spectrometric and solvent isotope effect studies with 4methoxybenzoate O-demethylase, a two-component system comprised of a flavo-iron-sulfur reductase and putidamonooxin (PMO; EC 1.14.99.15) as the terminal oxygenase, lent further support to the idea of peroxo-iron-sustained oxygenation chemistry [215,216] Apart from O-demethylation, PMO can functionalize aliphatic and aromatic C-H bonds, with H2O2 being liberated in the presence of uncoupling compounds [217,218] By a substrate-modulated reaction, PMO has been demonstrated to also act as a peroxotransferase: using vinylbenzoate as the substrate, the enzyme was found to form 4-(1,2-dihydroxyethyl)benzoate with both oxygen atoms being incorporated into the product from atmospheric 18O2 [218] This metabolic pattern might reflect ring opening of an epoxide intermediate at either of its two C-O bonds [219,220] The sum of these findings strongly invokes the notion of oxygen activation during redox cycling of the diverse dioxygenases to proceed along a common track, with Fe(III)-OO(H) serving as the preponderant oxidant Clearly, some contribution to catalysis by high-valent iron-oxene cannot be dismissed [211,221] Scheme outlines the putative pathway of PDO-dependent cis-dihydroxylation as proposed previously [222,223] Credibility of the mechanistic scheme is enhanced by results obtained with iron-based functional models for Rieske dioxygenases Introduction into the ligand frameworks depicted in Fig of more than one 6-methyl substituent to modulate the electronic and steric properties of the ligand environments has been recognized to afford high-spin hydroperoxo-iron species in combination with H2O2, exhibiting strong predilection for cis-dihydroxylation of olefins at the expense of epoxidation [224,225] A side-on Fe(III)-OOH entity, generated by isomerization of its end-on congener, or the cis-iron(V)-oxo(hydroxo) valence tautomer have been implicated as alternative catalysts in the dominant cis-diol formation from alkenes However, evidence for participation in these reactions of the high-valent oxidant seems equivocal in view of the nearly insignificant amount of 18O-incorporation from water into the diol products [225] or Fe + Rox Scheme Possible molecular mechanism for a PDO-promoted cis-dihydroxylation reaction Rred and Rox denote reduced and oxidized Rieske centre, respectively Reproduced from [223] with permission Bleomycin and related metal-based model complexes The bleomycins (BLMs) constitute a family of natural glycopeptide antibiotics produced by the fungus Streptomyces verticillus, which are used as antineoplastic agents owing to their ability to degrade DNA upon bioactivation in the presence of appropriate metal ions and a source of dioxygen [226] Although iron appears to be the most effective BLM cofactor, other metals also bind strongly to the antibiotic [227] A key to the unique reactivity of the nonheme iron(II) site of BLM (Fig 5) toward O2 seems to reside in one of its equatorial ligands, the pyrimidine moiety [228] The mechanism of oxygen activation is strongly reminiscent of P450 cycling [226], the ferric intermediate being reduced either chemically by dithiothreitol and ascorbate [229] or enzymatically by NADPH-P450 reductase [230] Oxygen surrogates such as iodosylbenzene [229], H2O2 or alkylhydroperoxides [231] have been shown to be apt to bypass reductive Fig Iron-bound ligand frameworks used as models of dioxygenases Data taken from [224] 4346 P Hlavica (Eur J Biochem 271) Ó FEBS 2004 Electrochemical investigations support this concept, revealing autoxidation of diphenylhydrazine, when exposed to O2, to liberate hydrogen peroxide, which collapses with iron(II) to give an Fe(II)-H2O2 complex directly leading to metabolic turnover [261] process [263,264] It has been hypothesized that 17ahydroxy-progesterone binds to unprotonated CYP17 such as to interrupt a proton shuttle to Fe(III)-OO–, facilitating C-17 side-chain dissociation through peroxide chemistry [265] Employing recombinant CYP17 and labeled hydroxyandrostene-17b-carbaldehyde, a pregnenolone analogue, as the substrate in combination with 18O2, isotope-partitioning experiments suggested androgen genesis to be closely linked to formation of an iron-peroxy adduct prone to fragmentation [266] A similar paradigm also appears to apply to the final step in aromatase (CYP19)-catalyzed biotransformation of androgens to estrogens [267] As illustrated in Scheme 7, this event entails aromatization of the A-ring of androstenedione via oxidative decarbonylation of the 19-aldehyde intermediate to release formic acid with the concomitant production of estrone [268] When human placental microsomes fortified with deuteriated androgen precursors in the presence of 18O2 were used to explore the mechanistic course of aromatization, a transient ferri peroxy-hemiacetal-like complex (Scheme 7) turned out to be a strong contender to explain the step of C-C bond scission [269,270] This type of nucleophilic attack on a carbonyl group by peroxo-iron has also been evidenced in the final C-C bond cleaving process during sterol biosynthesis in the lanosterol 14a-demethylase (CYP51) reaction cascade [271,272] Of particular interest, the NADPH/O2-dependent conversion of cyclohexane carboxyaldehyde to cyclohexene by reconstituted CYP2B4 has been evaluated as a potential model for deformylation reactions brought about by steroidogenic P450s: mass-spectral analysis unveiled formate to be formed in about an equimolar amount with respect to olefinic product [11] Similarly, a series of other xenobiotic C-5 aldehydes have been shown to be deformylated to variable extent by highly purified rabbit liver P450s [273] Moreover, externally added H2O2 in place of the usual O2-reducing system has been reported to be active with CYP2B4 in supporting deformylation of aldehydes [11,274] Employing 3-phenylpropionaldehyde as the substrate, an adduct was detected with a mass corresponding to that of native heme modified by a phenylethyl group, presumably arising from the reaction of a peroxo-iron entity with the aldehyde to give a peroxy-hemiacetal [274] Peroxo-iron acting as a nucleophilic oxidant in P450-catalyzed hydroxylations Evidence from experiments with genetically engineered P450 enzymes and molecular modelling entity is relatively inert toward organic substrates such as alkanes and alkenes, protonation to increase electron affinity has been recognized to be a means of generating a highly reactive species [250,251] However, in most cases available data are insufficient to unravel the intricate mechanism of oxygen transfer Judging from the limited number of reports based on isotope labeling and kinetic studies, hydroperoxo-iron or its alkylperoxo analogue can act as direct oxidants in hydrocarbon hydroxylation [252], alkene epoxidation [253,254], and alcohol oxidation [122,255] promoted by iron-bound members of the pyridine/amine ligand family presented in Fig 6C–F Similarly, Cr-OOH, produced with a macrocyclic chromium-based system (Fig 6C), exhibits H+-assisted oxidative reactivity toward triarylphosphines [256] Generally, the peroxidedriven route appears to be under stereochemical control exerted by a-substituents [252] on the pyridyl moiety (Figs 6D,E) or topological constraints [253] imposed by the isomeric nature of the ligands involved (Fig 6F) Finally, combination, in the presence of atmospheric dioxygen, of a relatively labile Fe(II) complex such as bis (2,2¢-bipyridine)iron(II), bis(picolinate) iron(II), or bis(dipicolinate)iron(II) with a reductant such as diphenylhydrazine to constitute a so-called Mimoun system [257] has been shown to provide a useful tool to assess the molecular basis of alkane [258,259] and phenol [260] hydroxylation The mechanistic scheme for such reactions embodies the occurrence of a ternary catalyst/reductant/O2 adduct [258] analogous to P450 chemistry, so that more concerted redox steps can be envisioned Substrate transformation posits the participation of structurally similar hydroperoxo-iron catalysts with different formal oxidation states [258,260], the process being driven to exothermicity via water formation (Eqn 3) RH þ PhNHNHPh þ O2 ! ROH þ PhN ¼ NPh þ H2 O ð3Þ Evidence from kinetic analysis of P450 function Steroidogenic P450s belong to the category of isozymes promoting multifunctional biosynthesis of endogenous compounds Thus, 17a-hydroxylase-17,20-lyase (CYP17) sustains conversion of pregnenolone/progersterone to androstenediol/androstenedione via primary attack on the 17a-position of the pregnene nucleus, followed by oxidative acyl-carbon cleavage of the 17a-hydroxy intermediate(s) formed to eject acetate [262] Incubation of microsomal fractions prepared from pig testes with deuteriated pregnenolone under an atmosphere of 18O2 permitted analysis of the pattern of isotope incorporation into the reaction products, best rationalized by invoking the participation of a nucleophilic peroxo-iron species in the C-C bond fission Taking advantage of the debilitating effect on proton delivery to Fe(III)-OO– exerted by mutagenesis of the highly conserved active-site threonine in P450s (see above), experiments conducted with the T306A mutant of CYP17 disclosed an about sevenfold increase in the proportion of acyl-carbon cleavage vs hydroxylation activity during androgen biosynthesis as compared with the wild-type enzyme [275] This was taken to substantiate the assumption of juxtapositioning of the nucleophilic ferric peroxide anion and the carbonyl group of the substrate to be a compulsory prerequisite for directing the enzymatic flux toward C-C bond rupture Similarly, replacement of glutamate at position 302 in the CYP19 polypeptide with residues such as alanine or valine proved to be deleterious to conversion of androgens to estrogens [276] Based on an active site Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur J Biochem 271) 4347 Scheme Postulated final oxidation step in aromatization catalyzed by CYP19 Data adapted from [268] with permission model constructed by alignment of the CYP19 sequence with the known crystal structures of bacterial P450s, E302 was postulated to be essential to activation of the 19-oxo group of the substrate for attack by the peroxo-iron species [268,277], with D309 playing an important role in the aromatization process in concert with a histidine residue through facilitating abstraction of the 2b-hydrogen in the A-ring of the C-19 substrate and donation of a proton to the 3-keto entity, respectively, to permit enolization [277,278] (Scheme 7) A Ôthreonine switchÕ, conferring regulatory function on the conserved threonine-310 during peroxoiron-mediated aromatization has been proposed, though experimental results obtained with the T310S variant of CYP19 were ambiguous [277] In fact, switching from ironoxene to peroxo-iron chemistry through threonine-302 to alanine mutagenesis of truncated CYP2B4 could be demonstrated by studies comparing the catalytic specificity of deformylation of cyclohexane carboxaldehyde with that of hydroxylation of other compounds [279] Moreover, investigations on the mechanism-based destruction of CYP2B4 by aldehydes revealed augmented inactivating potency with the T302A congener, emphasizing the notion of a kinship to aldehyde deformylation via a peroxyhemiacetal intermediate [280] Evidence from comparative studies with non-P450 hemoproteins and metalloporphyrin models Nitric oxide synthase Nitric oxide synthases (NOS; EC 1.14.13.39) comprise a family of thiolate-ligated constitutive or inducible hemoprotein isoforms [281], exhibiting insignificant sequence identity with P450s in the heme-binding region [282], but bearing a C-terminal flavoprotein fragment in the single polypeptide chain structurally resembling NADPH-P450 reductase; the latter is separated from the heme domain by a calmodulin consensus binding sequence [283] Importantly, NOS enzymes are dimeric proteins, in which flavin-to-heme transfer of electrons provided by NADPH proceeds exclusively between adjacent subunits in the heterodimer, implying domain swapping for proper alignment of the reductase and oxygenase entities [284] Tetrahydrobiopterin (BH4), located close to the heme unit [285], has been shown to contribute to stabilization of the NOS dimers [286] Moreover, the modifier binds cooperatively to the substratebinding region [287] and facilitates electron flow to oxyferrous NOS [288] The physiological role of NOS pertains to the production of NO , an important signaling molecule, and citrulline through oxidative degradation of NG-hydroxy-L-arginine, generated via primary N-hydroxylation of one of the two equivalent guanidino nitrogens of arginine [281] While L-arginine and the homo-L-arginine derivative have been originally thought to be the only true NOS substrates, more recent studies unveiled a series of N-aryl-N¢-hydroxyguanidines to serve as NO donors after oxidative activation [289] Circumstantial analysis of the stoichiometry of the NO-forming reaction disclosed a three-electron process, with decomposition of the N-hydroxyarginine intermediate consuming only 0.5 equivalents of NADPH per mol of O2 during nitroxyl radical ejection [290] Comparative studies with microsomal P450s [291,292] and biopterin-free as well as BH4-containing NOS [293], exhibiting product selectivity with respect to the almost exclusive, superoxide dismutase-insensitive generation of equimolar amounts of urea and NO from arginine and some non-a-amino-acid • • • Ĩ FEBS 2004 4348 P Hlavica (Eur J Biochem 271) Scheme Proposed mechanism for the second step in NOS-dependent conversion of N-hydroxyarginine to citrulline and NO Reproduced from [297] with permission N-hydroxyguanidines, were designed to unravel the puzzling stoichiometric behaviour The results from these investigations support a unifying concept, predicting the conversion of N-hydroxyarginine to citrulline and NO to be mediated by a peroxo-iron catalyst, generated via the donation of one electron by the N-hydroxy intermediate to oxyferrous NOS, formally [Fe(III)-O2 ) ] The Fe(III)-OO– complex derived from this reaction would undergo nucleophilic addition to the C ¼ N bond of the substrate cation radical to initiate decomposition of the adduct with the elimination of NO [289,294,295] A modified version of this mechanism (Scheme 8), developed on thermodynamic reasons, envisages deprotonation of N-hydroxyarginine to yield hydroperoxo-iron as the oxidant and an iminoxyl radical Nucleophilic attack of the Fe(III)-OOH species on the substrate radical triggers fragmentation of the complex to release NO [295–297] There is evidence for a regulatory function of BH4 in NO formation from N-hydroxyarginine [293,298], however, the precise mechanism of this action remains obscure On the whole, the second step in NOS catalysis closely resembles aromatase chemistry as detailed above • • • • • Chemically modified myoglobin (rMb) Although native Mb has been demonstrated to exhibit P450-like monooxygenase activity when combined with an appropriate electron-transport system [143], efforts were undertaken to engineer the catalytic properties of Mb through functionalization of the pigment by chemical modification of its prosthetic heme unit [299] Thus, studies on the NADH-driven deformylation of 2-phenylpropionaldehyde by rMb, reconstituted with an electronaccepting isoalloxazine (flavin) moiety covalently attached to one heme propionate, revealed acetophenone to be the unique product [300] A similar metabolic pattern was observed when the carbonyl group of the secondary aldehyde was subject to nucleophilic attack by biomimetic peroxo-iron(III) porphyrin complexes [301] (see below) This parallelism was thought to be indicative of the participation in the rMb-dependent deformylation pathway of a nucleophilic Fe(III)-OO– catalyst [300] While 1-phenylethanol has been detected to be a further metabolite derived from 2-phenylpropionaldehyde during oxidative deformylation by CYP2B4 [280], the lack of alcohol product in the rMb-promoted reaction was reasoned to arise from competition between oxygen rebound and PhCỈ HCH3 quenching by dissolved O2, as formation of the radical intermediate is believed to occur near the surface of the flavohemoprotein conjugate [300] Synthetic metalloporphyrin models Peroxo-iron(III) derivatives, coordinated in a side-on (g2) geometry, were created by reacting synthetic porphyrin complexes with potassium superoxide in aprotic solvents, yielding oxygenating model systems for comparison with P450 chemistry Such studies have identified certain ferric peroxo porphyrins as remarkably strong nucleophiles capable of oxidizing a variety of electron-deficient molecules [302] In this way, [Fe(III)(TMP)(O2)]– (Fig 3A) and [Fe(III)(PPIXDME)(O2)]–, the peroxo adduct of the dimethyl ester of protoporphyrin IX, were found to epoxidize electron-poor olefins such as 2-cyclohexen-1-one or 2-methyl-1,4-naphthaquinone (menadione) via direct oxygen insertion into an olefinic bond (Scheme 9A), wereas no reactivity was observed toward electron-rich organic substrates such as tetramethylethylene or triphenylphosphine [303,304] Moreover, the particulatly electron-deficient, perfluorinated peroxo species [Fe(III) (F20TPP)(O2)]– (Figure 3A) did not show interaction with menadione when metabolic turnover was assessed in acetonitrile solution [303,304] Surprisingly, epoxidation of the olefin was switched on, when the reaction was allowed to proceed in neat dimethyl sulfoxide, a solvent capable of axial ligation [305] Generally, comparative investigations with iron(III)-, manganese(III)- and titanium(IV)based metalloporphyrin peroxo complexes in the presence of electron-poor olefinic substrates revealed the ferric peroxo congener to be by far the most nucleophilic oxidant [306] Ferric peroxo porphyrins were also employed as surrogates to decipher the molecular events in the final step of estrogen biosynthesis by CYP19 Initial studies demonstrated [Fe(III)(TMP)(O2)]– and structurally related peroxo complexes to be mediators of C-C bond scission in aliphatic aldehydes [301], mimicking the first part of the aromatization process The above peroxo species was also found to be able of rapidly reacting with an enolized analogue of the A and B rings of androstenedione [307] to generate aromatized product with the ejection of formate (Scheme 9B) This observation is in accord with the capacity of H2O2 to slowly Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur J Biochem 271) 4349 Scheme Epoxidation of menadione (A) and deformylation of an enolized mimic of the A/B ring moiety of androstenedione (B) by [Fe(III)(TMP)(O2)]– Data reproduced from [302] with permission react with the enolized version of the natural substrate to yield the corresponding estrogen derivative [308] Conclusions and future prospects While the consensus view of the P450 mechanism, implicating a high-valent iron-oxene species as the predominant catalyst in a host of crucial biological oxidations, has a great deal of weight, accumulating evidence points at the participation in substrate turnover of alternative active oxidants such as (hydro)peroxo-iron intermediate(s) This notion is partly endorsed by isotope partition experiments [99–102], though it must not be disregarded that, with a branched reaction sequence yielding multiple products from the same P450-substrate complex, isotopically sensitive Ômetabolic switchingÕ may perturb the product distribution pattern without a change in the basic oxygenating mechanism [309] Moreover, the involvement of cationic intermediates in some aspects of P450-catalyzed hydroxylations needs unambiguous interpretation Whereas this scenario was regarded to be compatible with OH+ insertion by hydroperoxo-iron into a C-H bond [9,107], carbocation formation trough radical oxidation was envisaged as an alternative possibility [310,311]; the latter route would require that oxidation reactions proceed at higher rates than usual radical rearrangements [98,312] On the other hand, the timing of radical rearrangement (radical clocks) may depend critically on the tightness of the radical cage and the ensemble of steric and electronic forces experienced by the incipient radical within the variable cage [313] For substrates with a very strong C-H bond and a small steric size, both effects would push the reaction coordinate toward a tighter radical cage with drastic shortening of the apparent radical lifetime This might persuade one into questioning the existence of a radical pathway [106,107] A step forward in the analysis of Fe(III)-OO(H) as a potential catalyst was thought to be offered by the use of mutated P450s, bearing alanine or some other amino acid in place of the highly conserved active-site threonine; the latter residue is believed to be the direct proton donor to the ironlinked oxygen However, generalizations as to this hypothesis should be avoided Thus, replacement of T252 in the CPY101 polypeptide with O-methylthreonine gave a variant that was identical to the wild-type enzyme in its catalytic properties [137] Moreover, with certain P450s such as CYP107 [314] or CYP152 [102] there is lack of a conserved threonine near the putative O2-binding site This opens the possibility that threonine mutagenesis rather induces a compensatory change in the organized, H-bonded network of water molecules to facilitate proton delivery [14,51] In fact, the T252A mutant of CYP101 displays distortion of the geometry of the immediate heme vicinity: the I-helical ÔkinkÕ seen in the wild-type enzyme is still apparent, but the centre of this feature is shifted one residue toward the N-terminus in the engineered hemoprotein [315] Similar observations were made with the CYP102 congener [47] In accord with these findings, threonine mutation elicits a marked change in the apparent Kd values for type II interaction of nitrogenous ligands such as metyrapone or phenylimidazole with CYP 1A2 [316] or type I spectral binding of pregnenolone to CYP17 [275] Noteworthy, some experiments were conducted with T302A-mutated CYP2B4 bearing additional deletion of the NH2-terminal signal anchor sequence [139,142,279,280] Truncation of CYP2B4, as such, has been demonstrated to significantly distort the structural integrity of the polypeptide, such as to cause tyrosine(s) and the invariant C-helical W121 residue, aligning with W96 in CYP102, to become buried [317] In agreement with the well-known function of these amino acids in productive contacts with redox partners and iron spin-state modulation [318], respectively, shortened CYP2B4 displayed severely compromised electron acceptance from NADPH-P450 reductase and cytochrome b5, ostensibly arising from disruption of events involved in second-electron transfer to oxyferrous P450, while stability of the active oxy-complex, once formed, remained unaffected; overall enzyme activity was shown to be substantially harassed [317] The sum of these findings suggests extreme caution in interpreting data obtained with the genetically engineered P450s, because changes in the pattern of product distribution, as observed with various diagnostic probes, cannot be savely ruled out to be the direct result of subtle alterations in active-site conformation Extensive approaches to a better understanding of the diversification of the oxygenating pathways focus on comparative studies with natural and bioinspired P450 mimics uniformly bearing nitrogen-chelated iron in their active sites While oxygen binding and activation in these congeners appears to obey identical rules, the specific steric and electronic features of the iron coordination environments, no doubt, are decisive in steering the equilibrium between metal-oxo-/metal-peroxo-driven oxygenating activity, thus modulating the pattern of product distribution [64,168,224,253,305] Despite this diversity, all the systems examined carry out P450-like oxidations, in the presence of both dioxygen and peroxides, obviously using (hydro)peroxo-iron as the common catalyst As P450s are part of this manifold of oxygenating metalloenzymes, it seems permissive to disclose parallels to their biomimics, reinforcing the contention that Fe(III)-OO(H) might be operative as an alternative oxidant in P450 cycling, too Novel concepts have been advanced to harmonize the partly conflicting data on the role of multiple oxidants in P450 chemistry Based on the ability of H2 and methane to inhibit hydrocarbon oxygenations by P450 models, an agostic r-complex between probe and the iron-oxene centre was proposed to rearrange in competition with consumma- Ó FEBS 2004 4350 P Hlavica (Eur J Biochem 271) tion of the hydroxylation reaction [319]; however, investigations with a set of alkylcyclopropanes and CYP2B1 provide little support to this idea [320] Using density functional theory, a viable Ôtwo-state reactivityÕ (TSR) paradigm has been delineated applying to the intermediate iron-oxo species [321–324] The model entails competition between a spin-paired low-spin ensemble and a nonspinpaired high-spin manifold, with state crossing being associated with either an insertion or a radicalar pathway, permitting the formation of cationic derivatives through radical oxidation Spin inversion should be probe dependent The proposed mechanistic scheme has a certain intellectual attraction in satisfactorily explaining controversial P450 data [311,325], especially those pertaining to the unusually high rates of oxygen rebound met with hypersensitive radical clocks [103,104,320] The theory has also been used to characterize alkene epoxidation [326,327] Scheme 10 depicts the potential energy surface for ethene epoxidation This reaction can be brought about in a synchronous or asynchronous mode, the latter being energetically more favourable The activation energies for the low-spin (2TS1) and high-spin (4TS1) states in the asynchronous route are 14.9 and 13.9 kcalỈmol)1, respect- ively While collaps of the quartet high-spin transition states (42-III and 42-IV) to the corresponding high-spin, ironcoordinated epoxy products exhibits an energetic barrier of 7.2 and 2.3 kcalỈmol)1, a tiny barrier of < 0.3 kcalỈmol)1 permits conversion of the doublet low-spin intermediates (22-III and 22-IV) to the epoxide complexes The low energy required for transformation of the transition states in the asynchronous pathways, making them stepwise but apparently concerted, has been advocated to be responsible for preservation of the stereochemistry during alkene epoxidation Albeit there is agreement with respect to the occurrence of cationic rearrangement products in P450-catalyzed reactions, the mechanism of their formation still remains a matter of considerable debate While investigations conducted with enantiomerically enriched isotopomers of 1-alkyl-2-arylcyclopropane and CYP2 members claimed to have disclosed discrepancies between the experimental data and values computed according to the TSR concept [99], giving support to the OH+ insertion theory in carbocation generation, more recent DFT studies applying cyclopropane derivatives have shown a coherent match between the TSR paradigm and experimental results, excluding the partici- 1.396 (1.371) {1.392} 1.897 (2.039) {1.898} 1.743 (1.716) {1.750} 1.463 {1.485} 1.462 {1.499} 2.412 (2.501) {2.413} 1.944 {1.882} 4TS1-IV (2TS1-IV) {4TS1-III} 2.465 {2.523} ∠OCC: 90.5 {85.0} 4TS2-IV {4TS2-III} TS1-III 17.4 14.9 13.9 4TS1 42 (z2) cat 4TS1-IV 42 (xy) cat 22 cat 4TS2-III 10.4 42-III 0.9 0.6 0.0 4,21 + C2H4 22-IV 4TS2-IV 3.2 42-IV 1.486 (1.482) {1.488} [1.465] 1.810 (1.808) {1.821} [1.978] 2.9 1.464 (1.473) {1.469} [1.395] 2.381 (2.421) {2.447} [2.362] ∠OCC: 107.0 (108.2) {107.7} [119.5] ∠FeOCC: -178.8 (-179.2) {-171.0} [53.7] 42-IV rad ( 2-IVrad) { 2-IIIrad} [ 2cat] 43 23 -25.5 -27.9 Scheme 10 Potential energy surface for ethene epoxidation The energies for the low- and high-spin states (2,4TS1), the corresponding transition intermediates (2,42) and epoxide products (2,43) are expressed in kcalỈmol)1 Data taken from [327] with permission Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur J Biochem 271) 4351 pation of free carbocation during C-H hydroxylation of the alkanes [328] Hence, cationic intermediates that appear as intimate ion pair species [HSFe(III)porOH–/Alk+] have been reasoned to arise either from minor dissociation of the high-spin ion pairs or electron transfer from the corresponding radical complexes [HSFe(IV)porOH/Alk ] These puzzling inconsistencies may be accomodated by assuming that the Ôtwo-oxidantÕ and Ôtwo-stateÕ hypotheses are not mutually exclusive (Scheme 2) While there is little dissent regarding the role of peroxoiron as an oxidant in P450-dependent nucleophilic reactions, the interesting concept of Fe(III)-OOH or [Fe(III)-H2O2] acting as alternative electrophilic oxygenating intermediates ` needs more direct evidence vis-a-vis the TSR theory On the other hand, intractability of high-energy intermediates such as ferryl precludes facile experimental proof of the ÔtwostateÕ proposal owing to lack of appropriate technical means Hence, new perspectives for increasing the size of the computational models, such as the combined quantum mechanical/molecular mechanical (QM/MM) methods [329], may afford truly innovative solutions for differentiating the complex pathways of P450-catalyzed oxygen activation on a theoretical basis 12 • References Nelson, D.R., Koymans, L., Kamataki, T., Stegeman, J.J., Feyereisen, R., Waxman, D.J., Waterman, M.R., Gotoh, O., Coon, M.J., Estabrook, R.W., Gunsalus, I.C & Nebert, D.W (1996) P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature Pharmacognetics 6, 1–42 Porter, T.D & Coon, M.J (1991) Cytochrome P450 Multiplicity of isoforms, substrates, and catalytic and regulatory mechanisms J Biol Chem 266, 13469–13472 Berkowitz, J., Ellison, G.B & Gutman, D (1994) Three methods to measure RH bond energies J Phys Chem 98, 2744–2765 Ullrich, V & Staudinger, H.J (1968) Aktivierung von Sauerstoff in Modellsystemen In Biochemie des Sauerstoffs (Hess, B Staudinger, H.J., eds), pp 229–248 Springer, Berlin McMurry, T.J & Groves, J.T (1986) Metalloporphyrin models for cytochrome P-450 In Cytochrome P-450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P.R., ed.), pp 1–28 Plenum Press, New York Groves, J.T & Han, Y (1995) Models and mechanisms of cytochrome P450 action In Cytochrome P450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P.R., ed.), 2nd edn, pp 3–48 Plenum Press, New York Ortiz de Montellano, P.R (1986) Oxygen activation and transfer In Cytochrome P-450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P.R., ed.), pp 217–271 Plenum Press, New York Ortiz de Montellano, P.R (1995) Oxygen activation and reactivity In Cytochrome P450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P.R., ed.), 2nd edn, pp 245– 303 Plenum Press, New York Newcomb, M & Toy, P.H (2000) Hypersensitive radical probes and the mechanisms of cytochrome P450-catalyzed hydroxylation reactions Acc Chem Res 33, 449–455 10 Newcomb, M., Hollenberg, P.F & Coon, M.J (2003) Multiple mechanisms and multiple oxidants in P450-catalyzed hydroxylations Arch Biochem Biophys 409, 72–79 11 Vaz, A.D.N., Roberts, E.S & Coon, M.J (1991) Olefin formation in the oxidative deformylation of aldehydes by 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 cytochrome P450 Mechanistic implications for catalysis by oxygen-derived peroxide J Am Chem Soc 113, 5886–5887 Coon, M.J., Vaz, A.D.N., McGinnity, D.F & Peng, H.M (1998) Multiple activated oxygen species in P450 catalysis Contributions to specificity in drug metabolism Drug Metab Dispos 26, 1190–1193 Vaz, A.D.N (2001) Multiple oxidants in cytochrome P450catalyzed reactions: implications for drug metabolism Curr Drug Metab 2, 1–16 Watanabe, Y (2001) Alternatives to the oxoferryl porphyrin cation radical as the proposed reactive intermediate of cytochrome P450: two-electron oxidized Fe(III) porphyrin derivatives J Biol Inorg Chem 6, 846–856 Coon, M.J (2003) Multiple oxidants and multiple mechanisms in cytochrome P450 catalysis Biochem Biophys Res Commun 312, 163–168 Ortiz de Montellano, P.R & Stearns, R.A (1987) Timing of the radical recombination step in cytochrome P450 catalysis with ring-stained probes J Am Chem Soc 109, 3415–3420 Atkinson, J.K & Ingold, K.U (1993) Cytochrome P450 hydroxylation of hydrocarbons: variation in the rate of oxygen rebound using cyclopropyl radical clocks including two new ultrafast probes Biochemistry 32, 9209–9214 Oprian, D.D., Gorsky, L.D & Coon, M.J (1983) Properties of the oxygenated form of liver microsomal cytochrome P-450 J Biol Chem 258, 8684–8691 Estabrook, R.W., Hildebrandt, A.G., Baron, J., Netter, K.J & Leibman, K (1971) A new spectral intermediate associated with cytochrome P-450 function in liver microsomes Biochem Biophys Res Commun 42, 132–139 Rosen, P & Stier, A (1973) Kinetics of CO aund O2 complexes ă of rabbit liver microsomal cytochrome P450 Biochem Biophys Res Commun 51, 603–611 Bonfils, C., Debey, P & Maurel, P (1979) Highly purified microsomal P-450: the oxyferro intermediate stabilized at low temperature Biochem Biophys Res Commun 88, 1301– 1307 Larroque, C & van Lier, J.E (1980) The subzero temperature stabilized oxyferro complex of purified cytochrome P450scc FEBS Lett 115, 175–177 Tuckey, R.C & Kamin, H (1982) The oxyferro complex of adrenal cytochrome P-450scc Effect of cholesterol and intermediates on its stability and optical characteristics J Biol Chem 257, 9309–9314 Kashem, M.A & Dunford, H.B (1987) The formation and decay of the oxyferrous complex of beef adrenocortical cytochrome P-450scc Rapid-scan and stopped-flow studies Biochem Cell Biol 65, 486–492 Peterson, J.A., Ishimura, Y & Griffin, B.W (1972) Pseudomonas putida cytochrome P-450: characterization of an oxygenated form of the hemoprotein Arch Biochem Biophys 149, 197–208 Eisenstein, L., Debey, P & Douzou, P (1977) P450cam: oxygenated complexes stabilized at low temperature Biochem Biophys Res Commun 77, 1377–1383 Kerekjarto, B & Staudinger, H (1966) Die Sauerstoffabhangigkeit der Hydroxylierung von Acetanilid zu ¨ 4-Acetaminophenol durch Kaninchenlebermikrosomen HoppeSeyler Z Physiol Chem 347, 7–17 Hlavica, P (1971) Hepatic mixed function amine oxidase An allosteric system Xenobiotica 1, 537–538 Hlavica, P (1972) Interaction of oxygen and aromatic amines with hepatic microsomal mixed-function oxidase Biochim Biophys Acta 273, 318–327 Hlavica, P & Kehl, M (1974) Studies on the mechanism of hepatic microsomal N-oxide formation I Effect of carbon 4352 P Hlavica (Eur J Biochem 271) 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 monoxide on the N-oxidation of N,N-dimethylaniline HoppeSeyler Z Physiol Chem 355, 1508–1514 Gasser, R., Negishi, M & Philpot, R.M (1988) Primary structures of multiple forms of cytochrome P-450 isozyme derived from rabbit pulmonary and hepatic cDNAs Mol Pharmacol 32, 22–30 Hlavica, P & Lewis, D.F.V (2001) Allosteric phenomena in cytochrome P450-catalyzed monooxygenations Eur J Biochem 268, 4817–4832 Golly, I., Hlavica, P & Schartau, W (1988) The functional role of cytochrome b5 reincorporated into hepatic microsomal fractions Arch Biochem Biophys 260, 232–240 Monod, J., Wyman, J & Changeux, J.P (1965) On the nature of allosteric transitions: a plausible model J Mol Biol 12, 88–118 Bangcharoenpaurpong, O., Rizos, A.K & Champion, P.M (1986) Resonance Raman detection of bound dioxygen in cytochrome P-450cam J Biol Chem 261, 8089–8092 Sharrock, M., Munck, E., Debrunner, P.G., Marshall, V., ă Lipscomb, J.D & Gunsalus, I.C (1973) Mossbauer studies of ă cytochrome P-450cam Biochemistry 12, 258265 Ingelman-Sundberg, M & Johansson, I (1984) Mechanism of hydroxyl radical formation and ethanol oxidation by ethanolinducible and other forms of rabbit liver microsomal cytochromes P-450 J Biol Chem 259, 6447–6458 Guengerich, F.P., Ballou, D.P & Coon, M.J (1976) Spectral intermediates in the reaction of oxygen with purified liver microsomal cytochrome P-450 Biochem Biophys Res Commun 70, 951–956 Bonfils, C., Balny, C & Maurel, P (1981) Direct evidence for electron transfer from ferrous cytochrome b5 to the oxyferrous intermediate of liver microsomal cytochrome P-450LM2 J Biol Chem 256, 9457–9465 Ost, T.W.B., Clark, J., Mowat, C.G., Miles, C.S., Walkinshaw, M.D., Reid, G.A., Chapman, S.K & Daff, S (2003) Oxygen activation and electron transfer in flavocytochrome P450 BM3 J Am Chem Soc 125, 15010–15020 Schulze, J., Tschop, K., Lehnerer, M & Hlavica, P (2000) ă Residue 285 in cytochrome P450 2B4 lacking the NH2-terminal hydrophobic sequence has a role in the functional association of NADPH-cytochrome P450 reductase Biochem Biophys Res Commun 270, 777–781 Bonfils, C., Balny, C., Douzou, P & Maurel, P (1980) Studies on the reactivity of the oxyferro intermediate of highly purified microsomal P-450 In Biochemistry, Biophysics and Regulation of Cytochrome P-450 (Gustafsson, J.A., Carlstedt-Duke, J., Mode, A & Rafter, J., eds), pp 559–564 Elsevier, Amsterdam Lipscomb, J.D., Sligar, S.G., Namtvedt, M.J & Gunsalus, I.C (1976) Autooxidation and hydroxylation reactions of oxygenated cytochrome P-450cam J Biol Chem 251, 1116– 1124 Kuthan, H., Tsuji, H., Graf, H., Ullrich, V., Werringloer, J & Estabrook, R.W (1978) Generation of superoxide anion as a source of hydrogen peroxide in a reconstituted monooxygenase system FEBS Lett 91, 343–345 Loew, G.H & Harris, D.L (2000) Role of the heme active site and protein environment in structure, spectra, and function of the cytochrome P450s Chem Rev 100, 407–419 Aikens, J & Sligar, S.G (1994) Kinetic solvent isotope effects during oxygen activation by cytochrome P-450cam J Am Chem Soc 116, 1143–1144 Yeom, H & Sligar, S.G (1995) The role of Thr268 in oxygen activation by cytochrome P450BM-3 Biochemistry 34, 14733– 14740 Gerber, N.C & Sligar, S.G (1994) A role of Asp-251 in cytochrome P450cam oxygen activation J Biol Chem 269, 4260– 4266 Ó FEBS 2004 49 Vidakovic, M., Sligar, S.G., Li, H & Poulos, T.L (1998) Understanding the role of the essential Asp251 in cytochrome P450cam using site-directed mutagenesis, crystallography, and kinetic solvent isotope effects Biochemistry 37, 9211–9219 50 Sjodin, T., Christian, J.F., Macdonald, I.D.G., Davydov, R., Unno, M., Sligar, S.G., Hoffmann, B.M & Champion, P.M (2001) Resonance Raman and EPR investigations of the D251N oxycytochrome P450cam/putidaredoxin complex Biochemistry 40, 6852–6859 51 Harris, D.L (2002) Oxidation and electronic state dependence of proton transfer in the enzymatic cycle of cytochrome P450eryF J Inorg Biochem 91, 568–585 52 Davydov, R., Kappl, R., Huttermann, J & Peterson, J.A (1991) ă EPR-spectroscopy of reduced oxyferrous-P450cam FEBS Lett 295, 113–115 53 Davydov, R., Makris, T.M., Kofman, V., Werst, D.E., Sligar, S.G & Hoffman, B.M (2001) Hydroxylation of camphor by reduced oxy-cytochrome P450cam: mechanistic implications of EPR and ENDOR studies of catalytic intermediates in native and mutant enzymes J Am Chem Soc 123, 1403–1415 54 Denisov, I.G., Makris, T.M & Sligar, S.G (2001) Cryotrapped reaction intermediates of cytochrome P450 studied by radiolytic reduction with phosphorus-32 J Biol Chem 276, 11648–11652 55 Makris, T.M., Davydov, R., Denisov, I.G., Hoffman, B.M & Sligar, S.G (2002) Mechanistic enzymology of oxygen activation by the cytochromes P450 Drug Metab Rev 34, 691–708 56 Denisov, I.G., Hung, S.C., Weiss, K.E., McLean, M.A., Shiro, Y., Park, S.Y., Champion, P.M & Sligar, S.G (2001) Characterization of the oxygenated intermediate of the thermophilic cytochrome P450 CYP119 J Inorg Biochem 87, 215–226 57 Kobayashi, K., Iwamoto, T & Honda, K (1994) Spectral intermediate in the reaction of ferrous cytochrome P450cam with superoxide anion Biochem Biophys Res Commun 201, 1348– 1355 58 Benson, D.E., Suslick, K.S & Sligar, S.G (1997) Reduced oxy intermediate observed in D251N cytochrome P450cam Biochemistry 36, 5104–5107 59 Kamachi, T & Yoshizawa, K (2003) A theoretical study on the mechanism of camphor hydroxylation by compound I of cytochrome P450 J Am Chem Soc 125, 4652–4661 60 White, R.E & Coon, M.J (1980) Oxygen activation by cytochrome P-450 Annu Rev Biochem 49, 315–356 61 Macdonald, T.L., Gutheim, W.G., Martin, R.B & Guengerich, F.P (1989) Oxidation of substituted N,N-dimethylanilines by cytochrome P-450: estimation of the effective oxidation-reduction potential of cytochrome P-450 Biochemistry 28, 2071–2077 62 Sono, M., Andersson, L.A & Dawson, J.H (1982) Sulfur donor ligand binding to ferric cytochrome P450cam and myoglobin Ultraviolet-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectrosocopic investigation of the complexes J Biol Chem 257, 8308–8320 63 Sono, M., Roach, M.P., Coulter, E.D & Dawson, J.H (1996) Heme-containing oxygenases Chem Rev 96, 2841–2887 64 Ogliaro, F., de Visser, S.P & Shaik, S (2002) The ÔpushÕ effect of the thiolate ligand in cytochrome P450: a theoretical gauging J Inorg Biochem 91, 554–567 65 Vatsis, K.P., Peng, H.M & Coon, M.J (2002) Replacement of active-site cysteine-436 by serine converts cytochrome P450 2B4 into an NADPH oxidase with negligible monooxygenase activity J Inorg Biochem 91, 542–553 66 Yamaguchi, K., Watanabe, Y & Morishima, I (1992) Push effect on the heterolyic O-O bond cleavage of peroxoiron(III) porphyrin adducts Inorg Chem 31, 156–157 67 Tosha, T., Yoshioka, S., Takahashi, S., Ishimori, K., Shimada, H & Morishima, I (2003) NMR study on the structural changes Ó FEBS 2004 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 O-O Bond activation by cytochrome P450 (Eur J Biochem 271) 4353 of cytochrome P450cam upon the complex formation with putidaredoxin J Biol Chem 278, 39809–39821 Lichtenberger, F., Nastainczyk, W & Ullrich, V (1976) Cytochrome P450 as an oxene transferase Biochem Biophys Res Commun 70, 939–946 Gustafsson, J.A., Rondahl, L & Bergman, J (1979) Iodosylbenzene derivatives as oxygen donors in cytochrome P-450 catalyzed steroid hydroxylations Biochemistry 18, 865–870 Nordblom, G.D., White, R.E & Coon, M.J (1976) Studies on hydroperoxide-dependent substrate hydroxylation by purified liver microsomal cytochrome P-450 Arch Biochem Biophys 175, 524–533 Estabrook, R.W., Martin-Wixtrom, C., Saeki, Y., Renneberg, R., Hildebrandt, A & Werringloer, J (1984) The peroxidatic function of liver microsomal cytochrome P-450: comparison of hydrogen peroxide and NADPH-catalysed N-demethylation reactions Xenobiotica 14, 87–104 Blake, R.C & Coon, M.J (1989) On the mechanism of action of cytochrome P-450: spectral intermediates in the reaction with iodosobenzene and its derivatives J Biol Chem 264, 3694– 3701 Rahimtula, A.D., O’Brien, P.J., Hrycay, E.G., Peterson, J.A & Estabrook, R.W (1974) Possible higher valence states of cytochrome P-450 during oxidative reactions Biochem Biophys Res Commun 60, 695–702 Gustafsson, J.A., Hrycay, E.G & Ernster, L (1976) Sodium periodate, sodium chlorite, and organic hydroperoxides as hydroxylating agents in steroid hydroxylation reactions catalyzed by adrenocortical microsomal and mitochondrial cytochrome P450 Arch Biochem Biophys 174, 440–453 Egawa, T., Shimada, H & Ishimura, Y (1994) Evidence for compound I formation in the reaction of cytochrome P450cam with m-chloroperbenzoic acid Biochem Biophys Res Commun 201, 1464–1469 de Visser, S.P., Shaik, S., Sharma, P.K., Kumar, D & Thiel, W (2003) Active species of horseradish peroxidase (HRP) and cytochrome P450: two electronic chameleons J Am Chem Soc 125, 15779–15788 Mansuy, D., Battioni, P & Battioni, J.P (1989) Chemical model systems for drug-metabolizing cytochrome-P-450-dependent monooxygenases Eur J Biochem 184, 267–285 Groves, J.T., Haushalter, R.C., Nakamura, M., Nemo, T.E & Evans, B.J (1981) High-valent iron-porphyrin complexes related to peroxidase and cytochrome P-450 J Am Chem Soc 103, 2884–2886 Kellner, D.G., Hung, S.C., Weiss, K.E & Sligar, S.G (2002) Kinetic characterization of compound I formation in the thermostable cytochrome P450 CYP119 J Biol Chem 277, 9641– 9644 Schlichting, I., Berendzen, J., Chu, K., Stock, A.M., Maves, S.A., Benson, D.E., Sweet, R.M., Ringe, D., Petsko, G.A & Sligar, S.G (2000) The catalytic pathway of cytochrome P450cam at atomic resolution Science 287, 1615–1622 Staudt, H., Lichtenberger, F & Ullrich, V (1974) The role of NADH in uncoupled microsomal monooxygenations Eur J Biochem 46, 99–106 Gorsky, L.D., Koop, D.R & Coon, M.J (1984) On the stoichiometry of the oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P-450: products of oxygen reduction J Biol Chem 259, 6812–6817 Loida, P.J & Sligar, S.G (1993) Molecular recognition in cytochrome P-450: mechanism for the control of uncoupling reactions Biochemistry 32, 11530–11538 Hlavica, P (1984) On the function of cytochrome b5 in the cytochrome P-450-dependent oxygenase system Arch Biochem Biophys 228, 600–608 85 Golly, I & Hlavica, P (1987) Regulative mechanisms in NADHand NADPH-supported N-oxidation of 4-chloroaniline catalyzed by cytochrome b5-enriched rabbit liver microsomal fractions Biochim Biophys Acta 913, 219–227 86 Guallar, V., Baik, M.H., Lippard, S.J & Friesner, R.A (2003) Peripheral heme substituents control the hydrogen-atom abstraction chemistry in cytochromes P450 Proc Natl Acad Sci USA 100, 6998–7002 87 Groves, J.T., McClusky, G.A., White, R.E & Coon, M.J (1978) Aliphatic hydroxylation by highly purified liver microsomal cytochrome P-450 Evidence for a carbon radical intermediate Biochem Biophys Res Commun 81, 154–160 88 Gelb, M.H., Heimbrook, D.C., Malkonen, P & Sligar, S.G ă ă (1982) Stereochemistry and deuterium isotope effects in camphor hydroxylation by the cytochrome P450cam monooxygenase system Biochemistry 21, 370–377 89 Groves, J.T & Subramanian, D.V (1984) Hydroxylation by cytochrome P-450 and metalloporphyrin models Evidence for allylic rearrangements J Am Chem Soc 106, 2177–2181 90 Frommer, U., Ullrich, V & Staudinger, H.J (1970) Hydroxylation of aliphatic compounds by liver microsomes, I The distribution pattern of isomeric alcohols Hoppe-Seyler Z Physiol Chem 351, 903–912 91 Hjelmeland, L.M., Aronow, L & Trudell, J.R (1977) Intramolecular determination of primary kinetic isotope effects in hydroxylations catalyzed by cytochrome P-450 Biochem Biophys Res Commun 76, 541–549 92 Kadkhodayan, S., Coulter, E.D., Maryniak, D.M., Bryson, T.A & Dawson, J.H (1995) Uncoupling oxygen transfer and electron transfer in the oxygenation of camphor analogues by cytochrome P450cam Direct observation of an intermolecular isotope effect for substrate C-H activation J Biol Chem 270, 28042–28048 93 Bowry, V.W & Ingold, K.U (1991) A radical clock investigation of microsomal cytochrome P-450 hydroxylation of hydrocarbons Rate of oxygen rebound J Am Chem Soc 113, 5699–5707 94 Stearns, R.A & Ortiz de Montellano, P.R (1985) Cytochrome P450-catalyzed oxidation of quadricyclane Evidence for a radical cation intermediate J Am Chem Soc 107, 4081–4082 95 Ullrich, V (2003) Thoughts on thiolate tethering Tribute and thanks to a teacher Arch Biochem Biophys 409, 45–51 96 Khanna, R.K., Sutherlin, J.S & Lindsey, D (1990) Mechanisms in a biomimetic hydroxylation of a chemical probe: 5-nitroacenaphthene J Org Chem 55, 6233–6234 97 Atkinson, J.A., Hollenberg, P.F., Ingold, K.U., Johnson, C.C., Le Tadic, M.H., Newcomb, M & Putt, D.A (1994) Cytochrome P450-catalyzed hydroxylation of hydrocarbons: kinetic deuterium isotope effects for the hydroxylation of an ultrafast radical clock Biochemistry 33, 10630–10637 98 Griller, D & Ingold, K.U (1980) Free-radical clocks Acc Chem Res 13, 317–323 99 Newcomb, M., Aebisher, D., Shen, R., Chandrasena, E.P., Hollenberg, P.F & Coon, M.J (2003) Kinetic isotope effects implicate two electrophilic oxidants in cytochrome P450-catalyzed hydroxylations J Am Chem Soc 125, 6064–6065 100 Hutzler, J.M., Powers, F.P., Wynalda, M.A & Wienkers, L.C (2003) Effect of carbonate anion on cytochrome P450 2D6mediated metabolism in vitro: the potential role of multiple oxygenating species Arch Biochem Biophys 417, 165–175 101 Volz, T.J., Rock, D.A & Jones, J.P (2002) Evidence for two different active oxygen species in cytochrome P450BM3 mediated sulfoxidation and N-dealkylation reactions J Am Chem Soc 124, 9724–9725 102 Matsunaga, I., Yamada, A., Lee, D.S., Obayashi, E., Fujiwara, N., Kobayashi, K., Ogura, H & Shiro, Y (2002) Enzymatic 4354 P Hlavica (Eur J Biochem 271) 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 reaction of hydrogen peroxide-dependent peroxygenase cytochrome P450s: kinetic deuterium isotope effects and analyses by resonance Raman spectroscopy Biochemistry 41, 1886– 1892 Newcomb, M., Shen, R., Lu, Y., Coon, M.J., Hollenberg, P.F., Kopp, D.A & Lippard, S.J (2002) Evaluation of norcarane as a probe for radicals in cytochrome P450- and soluble methane monooxygenase-catalyzed hydroxylation reactions J Am Chem Soc 124, 6879–6886 Newcomb, M., Le Tadic, M.H., Putt, D.A & Hollenberg, P.F (1995) An incredibly fast apparent oxygen rebound rate constant for hydrocarbon hydroxylation by cytochrome P-450 enzymes J Am Chem Soc 117, 3312–3313 Toy, P.H., Newcomb, M., Coon, M.J & Vaz, A.D.N (1998) Two distinct electrophilic oxidants effect hydroxylation in cytochrome P-450-catalyzed reactions J Am Chem Soc 120, 9718–9719 Newcomb, M., Le Tadic-Biadatti, M.H., Chestney, D.L., Roberts, E.S & Hollenberg, P.F (1995) A nonsynchronous concerted mechanism for cytochrome P-450 catalyzed hydroxylations J Am Chem Soc 117, 12085–12091 Newcomb, M., Shen, R., Choi, S.Y., Toy, P.H., Hollenberg, P.F., Vaz, A.D.N & Coon, M.J (2000) Cytochrome P450-catalyzed hydroxylation of mechanistic probes that distinguish between radicals and cations Evidence for cationic but not for radical intermediates J Am Chem Soc 122, 2677–2686 Ogliaro, F., de Visser, S.P., Cohen, S., Sharma, P.K & Shaik, S (2002) Searching for the second oxidant in the catalytic cycle of cytochrome P450: a theoretical investigation on the iron(III)hydroperoxo species and its epoxidation pathways J Am Chem Soc 124, 2806–2817 Zakharieva, O., Trautwein, A.X & Veeger, C (2000) PorphyrinFe(III)-hydroperoxide and porphyrin-Fe(III)-peroxide anion as catalytic intermediates in cytochrome P450 catalyzed hydroxylation reactions: a molecular orbital study Biophys Chem 88, 11–34 Pratt, J.M., Ridd, T.I & King, L.J (1995) Activation of H2O2 by P450: evidence that the hydroxylating intermediate is iron(III)-coordinated H2O2 and not the ferryl FeO3+ complex J Chem Soc., Chem Commun 2297–2298 Harris, D.L & Loew, G.H (1998) Theoretical investigation of the proton assisted pathway to formation of cytochrome P450 compound I J Am Chem Soc 120, 8941–8948 Collins, J.R., P & Loew, G.H (1992) Molecular dynamics simulations of the resting and hydrogen peroxide-bound states of cytochrome c peroxidase Biochemistry 31, 11166–11174 Harris, D.L & Loew, G.H (1996) Identification of putative peroxide intermediates of peroxidases by electronic structure and spectra calculations J Am Chem Soc 118, 10588–10594 Dawson, J.H (1988) Probing structure-function relationships in heme-containing oxygenases and peroxidases Science 240, 433– 439 Hata, M., Hoshino, T & Tsuda, M (2000) An ultimate species in the substrate oxidation process by cytochrome P-450 Chem Commun 2037–2038 Hlavica, P., Golly, I & Mietaschk, J (1983) Comparative studies on the cumene hydroperoxide- and NADPH-supported N-oxidation of 4-chloroaniline by cytochrome P-450 Biochem J 212, 539–547 Hlavica, P (1982) Biological oxidation of nitrogen in organic compounds and disposition of N-oxidized products CRC Crit Rev Biochem 12, 39–101 Zang, Y., Elgren, T.E., Dong, Y.L & Que, Y (1993) A highpotential ferrous complex and its conversion to an alkylperoxoiron(III) intermediate A lipoxygenase model J Am Chem Soc 115, 811–813 Ó FEBS 2004 119 Wada, A., Ogo, S., Watanabe, Y., Mukai, M., Kitagawa, T., Jitsukawa, K., Masuda, H & Einaga, H (1999) Synthesis and characterization of novel alkylperoxo mononuclear iron(III) complexes with a tripodal pyridylamine ligand: a model for peroxo intermediates in reactions catalyzed by non-heme iron enzymes Inorg Chem 38, 3592–3593 120 Balch, A.L (1992) The reactivity of spectroscopically detected peroxy complexes of iron porphyrins Inorg Chim Acta 198–200, 297–307 121 Tajima, K., Edo, T., Ishizu, K., Imaoka, S., Funae, Y., Oka, S & Sakurai, H (1993) Cytochrome P-450-butyl peroxide complex detected by ESR Biochem Biophys Res Commun 191, 157–164 122 Kim, J., Harrison, R.G., Kim, C & Que, L (1996) Fe (TPA) catalyzed alkane hydroxylation Metal-based oxidation vs radical chain autoxidation J Am Chem Soc 118, 4373–4379 123 Lee, K.A & Nam, W (1997) Determination of reactive intermediates in iron porphyrin complex-catalyzed oxygenations of hydrocarbons using isotopically labeled water: mechanistic insights J Am Chem Soc 119, 1916–1922 124 Nam, W., Lim, M.H., Lee, H.J & Kim, C (2000) Evidence for the participation of two distinct reactive intermediates in iron(III) prophyrin complex-catalyzed epoxidation reactions J Am Chem Soc 122, 6641–6647 125 Yoshida, T & Migita, C.T (2000) Mechanism of heme degradation by heme oxygenase J Inorg Biochem 82, 33–41 126 Hlavica, P & Kunzel-Mulas, U (1993) Metabolic N-oxide foră mation by rabbit-liver microsomal cytochrome P-450 2B4: involvement of superoxide in the NADPH-dependent N-oxygenation of N,N-dimethylaniline Biochim Biophys Acta 1158, 83–90 127 Hlavica, P (2002) N-Oxidative transformation of free and N-substituted amine functions by cytochrome P450 as means of bioactivation and detoxication Drug Metab Rev 34, 451– 477 128 Lewis, D.F.V (1998) The P450 catalytic cycle and oxygenation mechanism Drug Metab Rev 30, 739–786 129 McCandlish, E., Miksztal, A.R., Nappa, M., Sprenger, A.Q., Valentine, J.S., Stong, J.D & Spiro, T.G (1980) Reactions of superoxide with iron porphyrins in aprotic solvents A high spin ferric porphyrin peroxo complex J Am Chem Soc 102, 4268– 4271 130 Welborn, C.H., Dolphin, D & James, B.R (1981) One-electron electrochemical reduction of a ferrous porphyrin dioxygen complex J Am Chem Soc 103, 2869–2871 131 Fridovich, I (1986) Biological effects of the superoxide radical Arch Biochem Biophys 247, 1–11 132 Sauer-Masarwa, A., Herron, N., Fendrick, C.M & Busch, D.H (1993) Kinetics and intermediates in the autoxidation of synthetic, non-porphyrin iron(II) dioxygen carriers Inorg Chem 32, 1086–1094 133 Sutton, H.C., Roberts, P.B & Winterbourn, C.C (1976) The rate of reaction of superoxide radical ion with oxyhaemoglobin and methaemoglobin Biochem J 155, 503–510 134 Kobayashi, K & Hayashi, K (1981) One-electron reduction in oxyform of hemoproteins J Biol Chem 256, 12350–12354 135 Peterson, J.A & Graham-Lorence, S.E (1995) Bacterial P450s Structural similarities and functional differences In Cytochrome P450: Structure, Mechanism, and Biochemistry (Ortiz de Montallano, P.R., ed.), 2nd edn, pp 151–180 Plenum Press, New York 136 Martinis, S.A., Atkins, W.M., Stayton, P.S & Sligar, S.G (1989) A conserved residue of cytochrome P-450 is involved in hemeoxygen stability and activation J Am Chem Soc 111, 9252– 9253 Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur J Biochem 271) 4355 137 Kimata, Y., Shimada, H., Hirose, T & Ishimura, Y (1995) Role of Thr-252 in cytochrome P450cam: a study with unnatural amino acid mutagenesis Biochem Biophys Res Commun 208, 96–102 138 Jin, S., Makris, T.M., Bryson, T.A., Sligar, S.G & Dawson, J.H (2003) Epoxidation of olefins by hydroperoxo-ferric cytochrome P450 J Am Chem Soc 125, 3406–3407 139 Vaz, A.D.N., McGinnity, D.F & Coon, M.J (1998) Epoxidation of olefins by cytochrome P450: evidence from site-specific mutagenesis for hydroperoxo-iron as an electrophilic oxidant Proc Natl Acad Sci USA 95, 3555–3560 140 Chandrasena, R.E.P., Vatsis, K.P., Coon, M.J., Hollenberg, P.F & Newcomb, M (2004) Hydroxylation by the hydroperoxy-iron species in cytochrome P450 enzymes J Am Chem Soc 126, 115–126 141 Vatsis, K.P & Coon, M.J (2002) Ipso-Substitution by cytochrome P450 with conversion of p-hydroxybenzene derivatives to hydroquinone: evidence for hydroperoxo-iron as the active oxygen species Arch Biochem Biophys 397, 119–129 142 Guengerich, F.P., Vaz, A.D.N., Raner, G.N., Pernecky, S.J & Coon, M.J (1997) Evidence for a role of a perferryl-oxygen complex, FeO3+, in the N-oxygenation of amines by cytochrome P450 enzymes Mol Pharmacol 51, 147–151 143 Mieyal, J.J (1985) Monooxygenase activity of hemoglobin and myoglobin In Reviews in Biochemical Toxicology (Hodgson, E., Bend, J & Philpot, R.M., eds), pp 1–66 Elsevier, New York 144 Huennekens, F.M., Caffrey, R.W., Basford, R.E & Gabrio, B.W (1957) Erythrocyte metabolism IV Isolation and properties of methemoglobin reductase J Biol Chem 227, 261–272 145 Passon, P.G & Hultquist, D.E (1972) Soluble cytochrome b5 reductase from human erythrocytes Biochim Biophys Acta 275, 62–73 146 Starke, D.W & Mieyal, J.J (1989) Hemoglobin catalysis of a monooxygenase-like aliphatic hydroxylation reaction Biochem Pharmacol 38, 201–204 147 Starke, K.S., Blisard, D.W & Mieyal, J.J (1984) Substrate specificity of the monooxygenase activity of hemoglobin Mol Pharmacol 25, 467–475 148 Blisard, K.S & Mieyal, J.J (1979) Characterization of the aniline hydroxylase activity of erythrocytes J Biol Chem 254, 5104– 5110 149 Mieyal, J.J., Ackerman, R.S., Blumer, J.L & Freeman, L.S (1976) Characterization of enzyme-like activity of human hemoglobin Properties of the hemoglobin-P-450 reductase– coupled aniline hydroxylase system J Biol Chem 251, 3436– 3441 150 Brown, W.D & Snyder, H.E (1969) Nonenzymatic reduction and oxidation of myoglobin and hemoglobin by nicotinamide adenine dinucleotides and flavins J Biol Chem 244, 6702–6706 151 Adams, P.A & Berman, M.C (1982) Hemin-mediated para hydroxylation of aniline: a potential model for oxygen activation and insertion reactions of mixed function oxidases J Inorg Biochem 17, 1–14 152 Mieyal, J.J & Blumer, J.L (1976) Acceleration of the autooxidation of human oxyhemoglobin by aniline and its relation to hemoglobin-catalyzed aniline hyroxylation J Biol Chem 251, 3442–3446 153 Lynch, R.E., Lee, G.R & Cartwright, G.E (1976) Inhibition by superoxide dismutase of methemoglobin formation from oxyhemoglobin J Biol Chem 251, 1015–1019 154 Kappl, R., Hohn-Berlage, M., Huttermann, J., Bartlett, N & ă ă Symons, M.C.R (1985) Electron spin and electron nuclear double resonance of the [FeO2]– centre from irradiated oxyhemoand oxymyoglobin Biochim Biophys Acta 827, 327–343 155 Davydov, R., Satterlee, J.D., Fujii, H., Sauer-Masarwa, A., Busch, D.H & Hoffman, B.M (2003) A superoxo-ferrous state 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 in a reduced oxy-ferrous hemoprotein and model compounds J Am Chem Soc 125, 16340–16346 Leibl, W., Nitschke, W & Huttermann, J (1986) Spină density distribution in the [FeO2] complex Electron spin resonance of myoglobin single crystals Biochim Biophys Acta 870, 20–30 Gasyna, Z (1979) Intermediate spin-states in one-electron reduction of oxygen-hemoprotein complexes at low temperature FEBS Lett 106, 213–218 Ibrahim, M., Denisov, I.G., Makris, T.M., Kincaid, J.R & Sligar, S.G (2003) Resonance Raman spectroscopic studies of hydroperoxo-myoglobin at cryogenic temperatures J Am Chem Soc 125, 13714–13718 Egawa, T., Yoshioka, S., Takahashi, S., Hori, H., Nagano, S., Shimada, H., Ishimori, K., Morishima, I., Suematsu, M & Ishimura, Y (2003) Kinetic and spectroscopic characterization of a hydroperoxy compound in the reaction of native myoglobin with hydrogen peroxide J Biol Chem 278, 41597–41606 Alvarez, J.C & Ortiz de Montellano, P.R (1992) Thianthrene 5-oxide as probe of the electrophilicity of hemoprotein oxidizing species Biochemistry 31, 8315–8322 Miksztal, A.R & Valentine, J.S (1984) Reactivity of the peroxo ligand in metalloporphyrin complexes Reaction of sulfur dioxide with iron and titanium porphyrin peroxo complexes to give sulfato complexes or sulfate Inorg Chem 23, 3548–3552 Lenk, W & Sterzl, H (1984) Peroxidase activity of oxyhemoglobin in vitro Xenobiotica 14, 3548–3552 Golly, I & Hlavica, P (1983) The role of hemoglobin in the N-oxidation of 4-chloroaniline Biochim Biophys Acta 760, 69– 76 Golly, I & Hlavica, P (1983) Mechanisms of extrahepatic bioactivation of aromatic amines: the role of hemoglobin in the N-oxidation of 4-chloraniline In Extrahepatic Drug Metabolism and Chemical Carcinogenesis (Rydstrom, J., Montelius, J & ă Bengtsson, M., eds), pp 235–236 Elsevier, Amsterdam Lenk, W., Riedl, M & Andersson, L.O (1991) Relevance of primary and secondary nitroxide radicals in biological oxidations In N-Oxidation of Drugs Biochemistry, Pharmacology, Toxicology (Hlavica, P & Damani, L.A., eds), pp 393–422 Chapman & Hall, London Eyer, P (1991) Activation of aromatic amines by oxyhaemoglobin In N-Oxidation of Drugs Biochemistry, Pharmacology, Toxicology (Hlavica, P & Damani, L.A., eds), pp 371–391 Chapman & Hall, London Nam, W., Lee, H.J., Oh, S.Y., Kim, C & Jang, H.G (2000) First success of catalytic epoxidation of olefins by an electron-rich iron(III) porphyrin complex and H2O2: imidazole effect on the activation of H2O2 by iron porphyrin complexes in aprotic solvent J Inorg Biochem 80, 219–225 Adachi, S., Nagano, S., Ishimori, K., Watanabe, Y., Morishima, I., Egawa, T., Kitagawa, T & Makino, R (1993) Roles of proximal ligand in heme proteins: replacement of proximal histidine of human myoglobin with cysteine and tyrosine by sitedirected mutagenesis as models for P-450, chloroperoxidase, and catalase Biochemistry 32, 241–252 Ortiz de Montellano, P.R (1998) Heme oxygenase mechanism: evidence for an electrophilic, ferric peroxide species Acc Chem Res 31, 543–549 Ortiz de Montellano, P.R & Wilks, A (2001) Heme oxygenase structure and mechanism Adv Inorg Chem 51, 359–407 Lad, L., Schuller, D.J., Shimizu, H., Friedman, J., Li, H., Ortiz de Montellano, P.R & Poulos, T.L (2003) Comparison of the heme-free and -bound crystal structures of human heme oxygenase-1 J Biol Chem 278, 7834–7843 Beale, S.I (1993) Biosynthesis of phycobilins Chem Rev 93, 785–802 4356 P Hlavica (Eur J Biochem 271) 173 Wilks, A & Schmitt, M.P (1998) Expression and characterization of heme oxygenase (Hmu O) from Corynebacterium diphtheriae J Biol Chem 273, 837–841 174 Wilks, A & Ortiz de Montellano, P.R (1993) Rat liver heme oxygenase High level expression of a truncated soluble form and nature of the meso-hydroxylating species J Biol Chem 268, 22357–22362 175 Wilks, A., Torpey, J & Ortiz de Montellano, P.R (1994) Heme oxygenase (HO-1) Evidence for electrophilic oxygen addition to the porphyrin ring in the formation of a-meso-hydroxyheme J Biol Chem 269, 29553–29556 176 Torpey, J & Ortiz de Montellano, P.R (1996) Oxidation of the meso-methylmesoheme regioisomers by heme oxygenase Electronic control of the reaction regiospecificity J Biol Chem 271, 26067–26073 177 Torpey, J & Ortiz de Montellano, P.R (1997) Oxidation of ameso-formylmesoheme by heme oxygenase Electronic control of the reaction regiospecificity J Biol Chem 272, 22008–22014 178 Avila, L., Huang, H., Damaso, C.O., Lu, S., Moenne-Loccoz, P & Rivera, M (2003) Coupled oxidation vs heme oxygenation: insights from axial ligand mutants of mitochondrial cytochrome b5 J Am Chem Soc 125, 4103–4110 179 Davydov, R.M., Yoshida, T., Ikeda-Saito, M & Hoffman, B.M (1999) Hydroperoxy-heme oxygenase generated by cryoreduction catalyzes the formation of a-meso-hydroxyheme as detected by EPR and ENDOR J Am Chem Soc 121, 10656–10657 180 Davydov, R., Kofman, V., Fujii, H., Yoshida, T., Ikeda-Saito, M & Hoffman, B.M (2002) Catalytic mechanism of heme oxygenase through EPR and ENDOR of cryoreduced oxy-heme oxygenase and its Asp140 mutants J Am Chem Soc 124, 1798– 1808 181 Davydov, R., Matsui, T., Fujii, H., Ikeda-Saito, M & Hoffman, B.M (2003) Kinetic isotope effects on the rate-limiting step of heme oxyenase catalysis indicate concerted proton transfer/heme hydroxylation J Am Chem Soc 125, 16208–16209 182 Tajima, K., Tada, K., Shigematsu, M., Kanaori, K., Azuma, N & Makino, K (1997) Mechanistic study on meso-hydroxyoctaethylporphyrin formation from an FeIII(oep)-H2O2 complex Chem Commun 1069–1070 183 Aron, J., Baldwin, D.A., Marques, M.M., Pratt, J.M & Adams, P.A (1986) 1: Preparation and analysis of the heme-containing octapeptide (microperoxidase-8) and identification of the monomeric form in aqueous solution J Inorg Biochem 27, 227– 243 184 Osman, A.M., Koerts, J., Boersma, M.G., Boeren, S., Veeger, C & Rietjens, I.M.C.M (1996) Microperoxidase/H2O2-catalyzed aromatic hydroxylation proceeds by a cytochrome-P-450-type oxygen-transfer mechanism Eur J Biochem 240, 232–238 185 Boersma, M.G., Primus, J.L., Koerts, J., Veeger, C & Rietjens, I.M.C.M (2000) Heme-(hydro) peroxide mediated O- and N-dealkylation A study with microperoxidase Eur J Biochem 267, 6673–6678 186 Primus, J.L., Boersma, M.G., Mandon, D., Boeren, S., Veeger, C., Weiss, R & Rietjens, I.M.C.M (1999) The effect of iron to manganese substitution on microperoxidase catalysed peroxidase and cytochrome P450 type of catalysis J Biol Inorg Chem 4, 274–283 187 Rusvai, E., Vegh, M., Kramer, M & Horvath, I (1988) Hydroxylation of aniline mediated by heme-bound oxy-radicals in a heme peptide model system Biochem Pharmacol 37, 4574– 4577 188 van Haandel, M.J.H., Primus, J.L., Teunis, C., Boersma, M.G., Osman, A.M., Veeger, C & Rietjens, I.M.C.M (1998) Reversible formation of high-valent-iron-oxo porphyrin intermediates in heme-based catalysis: revisiting the kinetic model for horseradish peroxidase Inorg Chim Acta 275–276, 98–105 Ó FEBS 2004 189 Dorovska-Taran, V., Posthumus, M.A., Boeren, S., Boersma, M.G., Teunis, C.J., Rietjens, I.M.C.M & Veeger, C (1998) Oxygen exchange with water in heme-oxo intermediates during H2O2-driven oxygen incorporation in aromatic hydrocarbons catalyzed by microperoxidase-8 Eur J Biochem 253, 659–668 190 Veeger, C (2002) Does P450-type catalysis proceed through a peroxo-iron intermeditate? A review of studies with microperoxidase J Inorg Biochem 91, 35–45 191 Macdonald, T.L., Burka, L.T., Wright, S.T & Guengerich, F.P (1982) Mechanisms of hydroxylation by cytochrome P-450: exchange of iron-oxo intermediates with water Biochem Biophys Res Commun 104, 620–625 192 Guajardo, R.J., Hudson, S.E., Brown, S.J & Mascharak, P.K (1993) [Fe(PMA)]n+ (n¼1,2): good models of Fe-bleomycins and examples of mononuclear non-heme iron complexes with significant O2-activation capabilities J Am Chem Soc 115, 7971– 7977 193 Osman, A.M., Boeren, S., Boersma, M.G., Veeger, C & Rietjens, I.M.C.M (1997) Microperoxidase/H2O2-mediated alkoxylating dehalogenation of halophenol derivatives in alcoholic media Proc Natl Acad Sci USA 94, 4295–4299 194 Wang, J.S., Baek, H.K & van Wart, H.E (1991) High-valent intermediates in the reaction of N-acetyl microperoxidase-8 with hydrogen peroxide: models for compounds 0, I and II of horseradish peroxidase Biochem Biophys Res Commun 179, 1320– 1324 195 Primus, J.L., Grunenwald, S., Hagedoorn, P.L., Albrecht-Gray, A.M., Mandon, D & Veeger, C (2002) The nature of the intermediates in the reactions of Fe(III)- and Mn(III)-microperoxidase-8 with H2O2: a rapid kinetics study J Am Chem Soc 124, 1214–1221 196 Machii, K., Watanabe, Y & Morishima, I (1995) Acylperoxoiron(III) porphyrin complexes: a new entry of potent oxidants for the alkene epoxidation J Am Chem Soc 117, 6691–6697 197 Suzuki, N., Higuchi, T & Nagano, T (2002) Multiple active intermediates in oxidation reaction catalyzed by synthetic hemethiolate complex relevant to cytochrome P450 J Am Chem Soc 124, 9622–9628 198 Nam, W., Lim, M.H., Moon, S.K & Kim, C (2000) Participation of two distinct hydroxylating intermediates in iron(III) porphyrin complex-catalyzed hydroxylation of alkanes J Am Chem Soc 122, 10805–10809 199 Fontecave, M & Mansuy, D (1984) Monooxygenase-like oxidations of olefins and alkanes catalyzed by manganese porphyrins: comparison of systems involving either O2 and ascorbate or iodosylbenzene Tetrahedron 40, 4297–4311 200 Shirazi, A & Goff, H.M (1982) Characterization of superoxidemetalloporphyrin reaction products: effective use of deuterium NMR spectroscopy J Am Chem Soc 104, 6318–6322 201 Burstyn, J.N., Roe, J.A., Miksztal, A.R., Shaevitz, B.A., Lang, G & Valentine, J.S (1988) Magnetic and spectroscopic characterization of an iron porphyrin peroxide complex peroxoferrioctaethylporphyrin(1-) J Am Chem Soc 110, 1382– 1388 202 Tajima, K., Shigematsu, M., Jinno, J., Ishizu, K & OhyaNishiguchi, H (1990) Generation of FeIIIOEP-hydrogen peroxide complex (OEP¼octaethylporphyrinato) by reduction of FeIIOEP-O2 with ascorbic acid sodium salt J Chem Soc., Chem Commun 144–145 203 Harris, D., Loew, G & Waskell, L (1998) Structure and spectra of ferrous dioxygen and reduced ferrous dioxygen model cytochrome P450 J Am Chem Soc 120, 4308–4318 204 Bertini, I., Cremonini, M.A., Ferretti, S., Lozzi, I., Luchinat, C & Viezzoli, M.S (1996) Arene hydroxylases: metalloenzymes catalysing dioxygenation of aromatic compounds Coord Chem Rev 151, 145–160 Ó FEBS 2004 O-O Bond activation by cytochrome P450 (Eur J Biochem 271) 4357 205 Batie, C.J., LaHaie, E & Ballou, D.P (1987) Purification and characterization of phthalate oxygenase and phthalate oxygenase reductase from Pseudomonas cepacia J Biol Chem 262, 1510– 1518 206 Wackett, L.P., Kwart, L.D & Gibson, D.T (1988) Benzylic monooxygenation catalyzed by toluene dioxygenase from Pseudomonas putida Biochemistry 27, 1360–1367 207 Resnick, S.M., Lee, K & Gibson, D.T (1996) Diverse reactions catalyzed by naphthalene dioxygenase from Pseudomonas sp strain NCIB 9816 J Ind Microbiol 17, 438–457 208 Kauppi, B., Lee, K., Carredano, E., Parales, R.E., Gibson, D.T., Eklund, H & Ramaswamy, S (1998) Structure of an aromatic ring-hydroxylating dioxygenase-naphthalene 1,2-dioxygenase Structure 6, 571–586 209 Que, L (2000) One motif – many different reactions Nat Struct Biol 7, 182–184 210 Karlsson, A., Parales, J.V., Parales, R.E., Gibson, D.T., Eklund, H & Ramaswamy, S (2003) Crystal structure of naphthalene dioxygenase: side-on binding of dioxygen to iron Science 299, 1039–1042 211 Wolfe, M.D., Parales, J.V., Gibson, D.T & Lipscomb, J.D (2001) Single turnover chemistry and regulation of O2 activation by the oxygenase component of naphthalene 1,2-dioxygenase J Biol Chem 276, 1945–1953 212 Wolfe, M.D & Lipscomb, J.D (2003) Hydrogen peroxide-coupled cis-diol formation catalyzed by naphthalene 1,2-dioxygenase J Biol Chem 278, 829–835 213 Lee, K (1999) Benzene-induced uncoupling of naphthalene dioxygenase activity and enzyme inactivation by production of hydrogen peroxide J Bacteriol 181, 2719–2725 214 Carredano, E., Karlsson, A., Kauppi, B., Choudhury, D., Parales, R.E., Parales, J.V., Lee, K., Gibson, D.T., Eklund, H & Ramaswamy, S (2000) Substrate binding site of naphthalene 1,2dioxygenase: functional implications of indole binding J Mol Biol 296, 701–712 215 Twilfer, H., Bernhardt, F.H & Gersonde, K (1985) Dioxygenactivating iron center in putidamonooxin Electron spin resonance investigation of the nitrosylated putidamonooxin Eur J Biochem 147, 171–176 216 Twilfer, H., Sandfort, G & Bernhardt, F.H (2000) Substrate and solvent isotope effects on the fate of the active oxygen species in substrate-modulated reactions of putidamonooxin Eur J Biochem 267, 5926–5934 217 Bernhardt, F.H & Kuthan, H (1981) Dioxygen activation by putidamonooxin The oxygen species formed and released under uncoupling conditions Eur J Biochem 120, 547–555 218 Wende, P., Bernhardt, F.H & Pfleger, K (1989) Substratemodulated reactions of putidamonooxin The nature of the active oxygen species formed and its reaction mechanism Eur J Biochem 181, 189–197 219 Feig, A.L & Lippard, S.J (1994) Reactions of non-heme iron(II) centers with dioxygen in biology and chemistry Chem Rev 94, 759–805 220 Que, L & Ho, R.Y.N (1996) Dioxygen activation by enzymes with mononuclear non-heme iron active sites Chem Rev 96, 2607–2624 221 Wolfe, M.D., Altier, D.J., Stubna, A., Popescu, C.V., Munck, E ă & Lipscomb, J.D (2002) Benzoate 1,2-dioxygenase from Pseudomonas putida: single turnover kinetics and regulation of a two-component Rieske dioxygenase Biochemistry 41, 9611– 9626 222 Ballou, D & Batie, C (1988) Phthalate oxygenase, a Rieske ironsulfur protein from Pseudomonas cepacia In Oxidases and Related Redox Systems (King, T.E., Mason, H.S & Morrison, M., eds), pp 211–226 Alan R Liss, Inc., New York 223 Solomon, E.I., Brunold, T.C., Davis, M.I., Kemsley, J.N., Lee, S.K., Lehnert, N., Neese, F., Skulan, A.J., Yang, Y.S & Zhou, J (2000) Geometric and electronic structure/function correlations in non-heme iron enzymes Chem Rev 100, 235–349 224 Chen, K., Costas, M., Kim, J., Tipton, A.K & Que, L (2002) Olefin cis-dihydroxylation versus epoxidation by non-heme iron catalysts: two faces of an Fe(III)-OOH coin J Am Chem Soc 124, 3026–3035 225 Chen, K., Costas, M & Que, L (2002) Spin state tuning of nonheme iron-catalyzed hydrocarbon oxidations: participation of Fe(III)-OOH and Fe(V)¼O intermediates J Chem Soc., Dalton Trans 672–679 226 Hecht, S.M (2000) Bleomycin new perspectives on the mechanism of action J Nat Prod 63, 158–168 227 Burger, R.M (1998) Cleavage of nucleic acids by bleomycin Chem Rev 98, 1153–1169 228 Loeb, K.E., Zaleski, J.M., Westre, T.E., Guajardo, R.J., Mascharak, P.K., Hedman, B., Hodgson, K.O & Solomon, E.I (1995) Spectroscopic definition of the geometric and electronic structure of the non-heme iron active site in iron(II) bleomycin: correlation with oxygen reactivity J Am Chem Soc 117, 4545– 4561 229 Murugesan, N & Hecht, S.M (1985) Bleomycin as an oxene transferase: catalytic oxygen transfer to olefins J Am Chem Soc 107, 493–500 230 Scheulen, M.E., Kappus, H., Thyssen, D & Schmidt, C.G (1981) Redox cycling of Fe(III)-bleomycin by NADPH-cytochrome P-450 reductase Biochem Pharmacol 30, 3385–3388 231 Burger, R.M., Peisach, J & Horwitz, S.B (1981) Activated bleomycin A transient complex of drug, iron, and oxygen that degrades DNA J Biol Chem 256, 11636–11644 232 Burger, R.M., Kent, T.A., Horwitz, S.B., Munck, E & Peisach, ă J (1983) Mossbauer study of iron bleomycin and its activation ă intermediates J Biol Chem 258, 15591564 233 Sam, J.W., Tang, X.J & Peisach, J (1994) Electrospray mass spectrometry of iron bleomycin: demonstration that activated bleomycin is a ferric peroxide complex J Am Chem Soc 116, 5250–5256 234 Westre, T.E., Loeb, K.E., Zaleski, J.M., Hedman, B., Hodgson, K.O & Solomon, E.I (1995) Determination of the geometric and electronic structure of acitvated bleomycin using X-ray absorption spectroscopy J Am Chem Soc 117, 1309–1313 235 Veselov, A., Sun, H., Sienkiewicz, A., Taylor, H., Burger, R.M & Scholes, C.P (1995) Iron coordination of activated bleomycin probed by Q- and X-band ENDOR: hyperfine coupling to activated 17O oxygen, 14N, and exchangeable 1H J Am Chem Soc 117, 7508–7512 236 Boger, D.L., Ramsey, T.M., Cai, H., Hoehn, S.T & Stubbe, J.A (1998) A systematic evaluation of the bleomycin A2, 1-threonine side chain: its role in preorganization of a compact conformation implicated in sequence-selective DNA cleavage J Am Chem Soc 120, 9139–9148 237 Ishida, R & Takahashi, T (1975) Increased DNA chain breakage by combined action of bleomycin and superoxide radical Biochem Biophys Res Commun 66, 1432–1438 238 Ciriolo, M.R., Magliozzo, R.S & Peisach, J (1987) Microsomestimulated activation of ferrous bleomycin in the presence of DNA J Biol Chem 262, 6290–6295 239 Stubbe, J.A., Kozarich, J.W., Wu, W & Vanderwall, D.E (1996) Bleomycins: a structural model for specificity, binding, and double strand cleavage Acc Chem Res 29, 322–330 240 Burger, R.M (2000) Nature of activated bleomycin Struct Bond 97, 287–303 241 Neese, F., Zaleski, J.M., Loeb-Zaleski, K & Solomon, E.I (2000) Electronic structure of activated bleomycin: oxygen 4358 P Hlavica (Eur J Biochem 271) 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 intermediates in heme versus non-heme iron J Am Chem Soc 122, 11703–11724 Wu, W., Vanderwall, D.E., Turner, C.J., Kozarich, J.W & Stubbe, J (1996) Solution structure of Co bleomycin A2 green complexed with d(CCAGGCCTGG) J Am Chem Soc 118, 1281–1294 Mialane, P., Nivorojkine, A., Pratviel, G., Azema, L., Slany, M., Godde, F., Simaan, A., Banse, F., Kargar-Grisel, T., Bouchoux, G., Sainton, J., Horner, O., Guilhem, J., Tchertanova, L., Meunier, B & Girerd, J.J (1999) Structures of Fe(II) complexes with N,N,NÕ-tris(2-pyridylmethyl)ethane-1,2-diamine type ligands Bleomycin-like cleavage and enhancement by an alkylammonium substituent on the N’ atom of the ligand Inorg Chem 38, 1085–1092 Simaan, A.J., Dopner, S., Banse, F., Bourcier, S., Bouchoux, ă G., Boussac, A., Hildebrandt, P & Girer, J.J (2000) FeIII-hydroperoxo and peroxo complexes with aminopyridyl ligands and the resonance Raman spectroscopic identification of the Fe-O and O-O stretching modes Eur J Inorg Chem 1627– 1633 Heimbrook, D.C., Carr, S.A., Mentzer, M.A., Long, E.C & Hecht, S.M (1987) Mechanism of oxygenation of cis-stilbene by iron bleomycin Inorg Chem 26, 3835–3836 Heimbrook, D.C & Sligar, S.G (1981) Multiple mechanisms of cytochrome P450-catalyzed substrate hydroxylations Biochem Biophys Res Commun 99, 530–535 Schardt, B.C & Hill, C.L (1983) Preparation of iodobenzene dimethoxide A new synthesis of [18O]iodosylbenzene and a reexamination of its infrared spectrum Inorg Chem 22, 1563– 1565 Bukowski, M.R., Zhu, S., Koehntop, K.D., Brennessel, W.W & Que, L (2004) Characterization of an FeIII-OOH species and its decomposition product in a bleomycin model system J Biol Inorg Chem 9, 39–48 Girerd, J.J., Banse, F & Simaan, A.J (2000) Characterization and properties of non-heme iron peroxo complexes Struct Bond 97, 145–177 Neese, F & Solomon, E.I (1998) Detailed spectroscopic and theoretical studies on [Fe(EDTA)(O2)]3–: electronic structure of the side-on ferric-peroxide bond and its relevance to reactivity J Am Chem Soc 120, 12829–12848 Ho, R.Y.N., Roelfes, G., Hermant, R., Hage, R., Feringa, B.L & Que, L (1999) Resonance Raman evidence for the interconversion between an [FeIII-g1OOH]2+ and [FeIII-g2O2]+ species and mechanistic implications thereof Chem Commun 2161–2162 Chen, K & Que, L (2001) Stereospecific alkane hydroxylation by non-heme catalysts: mechanistic evidence for an FeV¼O active species J Am Chem Soc 123, 6327–6337 Costas, M & Que, L (2002) Ligand topology tuning of ironcatalyzed hydrocarbon oxidations Angew Chem Int Ed 12, 2179–2181 Nam, W., Ho, R & Valentine, J.S (1991) Iron-cyclam complexes as catalysts for the epoxidation of olefins by 30% aqueous hydrogen peroxide in acetonitrile and methanol J Am Chem Soc 113, 7052–7054 Kim, J., Kim, C., Harrison, R.G., Wilkinson, E.C & Que, L (1997) Fe (TPA) -catalyzed alkane hydroxylation can be a metalbased oxidation J Mol Cat A: Chem 117, 83–89 Pestovsky, O & Bakac, A (2003) Direct kinetic studies of atom transfer and electron transfer reactions of hydroperoxo and highvalent oxo complexes of chromium J Am Chem Soc 125, 14714–14715 ` Mimoun, H & Seree de Roch, I (1975) Activation de 1¢oxygene ´ ` moleculaire – nouveaux systemes d’hydroxylation des hydrocarbures Tetrahedron 31, 777–784 Ó FEBS 2004 258 Davis, R., Durrant, J.L.A & Khan, M.A (1988) A study of the mechanism of alkane hydroxylation using the Fepy4PhNHNHPh-O2 system Polyhedron 7, 425–438 259 Sheu, C & Sawyer, D.T (1990) Activation of dioxygen by bis[(2carboxy-6-carboxylato)pyridine]iron(II) for the bromination (via BrCCl3) and monooxygenation (via PhNHNHPh) of saturated hydrocarbons: reaction mimic for the methane monooxygenase proteins J Am Chem Soc 112, 8212–8214 260 Hage, J.P & Sawyer, D.T (1995) Iron(II)/reductant (DH2) induced activation of dioxygen for the hydroxylation of aromatic hydrocarbons and phenols: reaction mimic for tyrosine hydroxylase J Am Chem Soc 117, 5617–5621 261 Sawyer, D.T., Sugimoto, H & Calderwood, T.S (1984) Base (OÁÀ , e–, or OH–) -induced autoxygenation of organic substrates: a model chemical system for cytochrome P-450-catalyzed monooxygenation and dehydrogenation by dioxygen Proc Natl Acad Sci USA 81, 8025–8027 262 Keeney, D.S & Waterman, M.R (1993) Regulation of steroid hydroxylase gene expression: importance to physiology and disease Pharmacol Ther 58, 301–317 263 Miller, S.L., Wright, J.N., Corina, D.L & Akhtar, M (1991) Mechanistic studies on pregnene side-chain cleavage enzyme (17a-hydroxylase-17,20-lyase) using 18O J Chem Soc., Chem Commun 157–159 264 Akhtar, M., Corina, D., Miller, S., Shyadehi, A.Z & Wright, J.N (1994) Mechanism of the acyl-carbon cleavage and related reactions catalyzed by multifunctional P-450s: studies on cytochrome P-45017a Biochemistry 33, 4410–4418 265 Swinney, D.C & Mak, A.Y (1994) Androgen formation by cytochrome P450 CYP17 Solvent isotope effect and pL studies suggest a role for protons in the regulation of oxene versus peroxide chemistry Biochemistry 33, 2185–2190 266 Lee-Robichaud, P., Shyadehi, A.Z., Wright, J.N., Akhtar, M.E & Akhtar, M (1995) Mechanistic kinship between hydroxylation and desaturation reactions: acylcarbon bond cleavage promoted by pig and human CYP17 (P-45017a; 17a-hydroxylase-17,20lyase) Biochemistry 34, 14104–14113 267 Cole, P.A & Robinson, C.H (1990) Mechanism and inhibition of cytochrome P-450 aromatase J Med Chem 33, 2933–2942 268 Oh, S.S & Robinson, C.H (1993) Mechanism of human placental aromatase: a new active site model J Steroid Biochem Mol Biol 44, 389–397 269 Akhtar, M., Calder, M.R., Corina, D.L & Wright, J.N (1982) Mechanistic studies on C-19 demethylation in oestrogen biosynthesis Biochem J 201, 569–580 270 Stevenson, D.E., Wright, J.N & Akhtar, M (1988) Mechanistic consideration of P-450 dependent enzymic reactions: studies on oestriol biosynthesis J Chem Soc Perkin Trans I, 2043–2052 271 Fischer, R.T., Trzaskos, J.M., Magolda, R.L., Ko, S.S., Brosz, C.S & Larsen, B (1991) Lanosterol 14a-methyl demethylase Isolation and characterization of the third metabolically generated oxidative demethylation intermediate J Biol Chem 266, 6124–6132 272 Shyadehi, A.Z., Lamb, D.C., Kelly, S.L., Kelly, D.E., Schunck, W.H., Wright, J.N., Corina, D & Akhtar, M (1996) The mechanism of the acyl-carbon bond cleavage reaction catalyzed by recombinant sterol 14a-demethylase of Candida albicans (other names are: lanosterol 14a-demethylase, P-450 14DM, and CYP51) J Biol Chem 271, 12445–12450 273 Roberts, E.S., Vaz, A.D.N & Coon, M.J (1991) Catalysis by cytochrome P-450 of an oxidative reaction in xenobiotic aldehyde metabolism: deformylation with olefin formation Proc Natl Acad Sci USA 88, 8963–8966 274 Kuo, C.L., Raner, G.M., Vaz, A.D.N & Coon, M.J (1999) Discrete species of activated oxygen yield different cytochrome Ó FEBS 2004 275 276 277 278 279 280 281 282 283 284 285 286 287 288 O-O Bond activation by cytochrome P450 (Eur J Biochem 271) 4359 P450 heme adducts from aldehydes Biochemistry 38, 10511– 10518 Lee-Robichaud, P., Akhtar, M.E & Akhtar, M (1998) An analysis of the role of active site protic residues of cytochrome P-450s: mechanistic and mutational studies on 17a-hydroxylase17,20-lyase (P-45017a also CYP17) Biochem J 330, 967–974 Graham-Lorence, S., Khalil, M.W., Lorence, M.C., Mendelson, C.R & Simpson, E.R (1991) Structure-function relationships of human aromatase cytochrome P-450 using molecular modeling and site-directed mutagenesis J Biol Chem 266, 11939–11946 Kao, Y.C., Korzekwa, K.R., Laughton, C.A & Chen, S (2001) Evaluation of the mechanism of aromatase cytochrome P450 A site-directed mutagenesis study Eur J Biochem 268, 243–251 Graham-Lorence, S., Amarneh, B., White, R.E., Peterson, J.A & Simpson, E.R (1995) A three-dimensional model of aromatase cytochrome P450 Protein Sci 4, 1065–1080 Vaz, A.D.N., Pernecky, S.J., Raner, G.M & Coon, M.J (1996) Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4 Proc Natl Acad Sci USA 93, 4644–4648 Raner, G.M., Chiang, E.W., Vaz, A.D.N & Coon, M.J (1997) Mechanism-based inactivation of cytochrome P450 2B4 by aldehydes: relationship to aldehyde deformylation via a peroxyhemiacetal intermediate Biochemistry 36, 4895–4902 Mansuy, D & Renaud, J.P (1995) Heme-thiolate proteins different from cytochromes P450 catalyzing monooxygenations In Cytochrome P450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P.R., ed.), 2nd edn, pp 537–574 Plenum Press, New York Renaud, J.P., Boucher, J.L., Vadon, S., Delaforge, M & Mansuy, D (1993) Particular ability of liver P450s3A to catalyze the oxidation of Nx-hydroxyarginine to citrulline and nitrogen oxides and occurrence in NO synthases of a sequence very similar to the heme-binding sequence in P450s Biochem Biophys Res Commun 192, 53–60 Bredt, D.S., Hwang, P.M., Glatt, C.E., Lowenstein, C., Reed, R.R & Snyder, S.H (1991) Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase Nature 351, 714–718 Siddhanta, U., Presta, A., Fan, B., Wolan, D., Rousseau, D & Stuehr, D (1998) Domain swapping in inducible nitric-oxide synthase Electron transfer occurs between flavin and heme groups located on adjacent subunits in the dimer J Biol Chem 273, 18950–18958 Fischmann, T.O., Hruza, A., Niu, X.D., Fossetta, J.D., Lunn, C.A., Dolphin, E., Progay, A.J., Reichert, P., Lundell, D.J., Narula, S.K & Weber, P.C (1999) Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation Nat Struct Biol 6, 233–242 Klatt, P., Schmidt, K., Lehner, D., Glatter, O., Bachinger, H.P ă & Mayer, B (1995) Structural analysis of porcine brain nitric oxide synthase reveals a role for tetrahydrobiopterin and 1-arginine in the formation of an SDS-resistant dimer Eur Mol Biol Organ J 14, 3687–3695 Klatt, P., Schmid, M., Leopold, E., Schmidt, K., Werner, E.R & Mayer, B (1994) The pteridine binding site of brain nitric oxide synthase Tetrahydrobiopterin binding kinetics, specificity, and allosteric interaction with the substrate domain J Biol Chem 269, 13861–13866 Presta, A., Siddhanta, U., Wu, C., Sennequier, N., Huang, L., Abu-Soud, H.M., Erzurum, S & Stuehr, D.J (1998) Comparative functioning of dihydro- and tetrahydropterins in supporting electron transfer, catalysis, and subunit dimerization in inducible nitric oxide synthase Biochemistry 37, 298–310 289 Mansuy, D & Boucher, J.L (2002) Oxidation of N-hydroxyguanidines by cytochromes P450 and NO-synthases and formation of nitric oxide Drug Metab Rev 34, 593–606 290 Stuehr, D.J., Kwon, N.S., Nathan, C.F., Griffith, O.W., Feldman, P.L & Wiseman, J (1991) Nx-Hydroxy-L-arginine is an intermediate in the biosynthesis of nitric oxide from 1-arginine J Biol Chem 266, 6259–6263 291 Jousserandot, A., Boucher, J.L., Henry, Y., Niklaus, B., Clement, B & Mansuy, D (1998) Microsomal cytochrome P450 dependent oxidation of N-hydroxyguanidines, amidoximes, and ketoximes: mechanism of the oxidative cleavage of their C¼N (OH) bond with formation of nitrogen oxides Biochemistry 37, 17179–17191 292 Zhang, Z., Li, Y., Stearns, R.A., Ortiz de Montellano, P.R., Baillie, T.A & Tang, W (2002) Cytochrome P450 3A4-mediated oxidative conversion of a cyano to an amide group in the metabolism of pinacidil Biochemistry 41, 2712–2718 293 Moali, C., Boucher, J.L., Renodon-Corniere, A., Stuehr, D.J & Mansuy, D (2001) Oxidation of NG-hydroxyarginine analogues and various N-hydroxyguanidines by NO synthase II: key role of tetrahydrobiopterin in the reaction mechanism and substrate selectivity Chem Res Toxicol 14, 202–210 294 Marletta, M.A (1993) Nitric oxide synthase structure and mechanism J Biol Chem 268, 12231–12234 295 Mansuy, D., Boucher, J.L & Clement, B (1995) On the mechanism of nitric oxide formation upon cleavage of C¼N (OH) bonds by NO-synthases and cytochrome P450 Biochimie 77, 661–667 296 Korth, H.G., Sustmann, R., Thater, C., Butler, A.R & Ingold, K.U (1994) On the mechanism of nitric oxide synthase-catalyzed conversion of N-hydroxy-L-arginine to citrulline and nitric oxide J Biol Chem 269, 17776–17779 297 Nishida, C.R., Knudsen, G., Straub, W & Ortiz de Montellano, P.R (2002) Electron supply and catalytic oxidation of nitrogen by cytochrome P450 and nitric oxide synthase Drug Metab Rev 34, 479–501 298 Adak, S., Wang, Q & Stuehr, D.J (2000) Arginine conversion to nitric oxide by tetra-hydrobiopterin-free neuronal nitric-oxide synthase Implications for mechanism J Biol Chem 275, 33554– 33561 299 Hayashi, T & Hisaeda, Y (2002) New functionalization of myoglobin by chemical modification of heme propionates Acc Chem Res 35, 35–43 300 Matsuo, T., Hayashi, T & Hisaeda, Y (2002) Reductive activation of dioxygen by a myoglobin reconstituted with a flavohemin J Am Chem Soc 124, 11234–11235 301 Goto, Y., Wada, S., Morishima, I & Watanabe, Y (1998) Reactivity of peroxoiron(III) porphyrin complexes: models for deformylation reactions catalyzed by cytochrome P-450 J Inorg Biochem 69, 241–247 302 Wertz, D.L & Valentine, J.S (2000) Nucleophilicity of ironperoxo porphyrin complexes Struct Bond 97, 37–60 303 Selke, M., Sisemore, M.F & Valentine, J.S (1996) The diverse reactivity of peroxy ferric porphyrin complexes of electron-rich and electron-poor porphyrins J Am Chem Soc 118, 2008– 2012 304 Selke, M., Sisemore, M.F., Ho, R.Y.N., Wertz, D.L & Valentine, J.S (1997) Dioxygen activation by iron complexes The search for reactive intermediates J Mol Cat A: Chem 117, 71–82 305 Selke, M & Valentine, J.S (1998) Switching on the nucleophilic reactivity of a ferric porphyrin peroxo complex J Am Chem Soc 120, 2652–2653 306 Sisemore, M.F., Selke, M., Burstyn, J.N & Valentine, J.S (1997) Metalloporphyrin peroxo complexes of iron(III), manganese(III), and titanium(IV) Comparative studies demonstrating 4360 P Hlavica (Eur J Biochem 271) 307 308 309 310 311 312 313 314 315 316 317 318 that the iron(III) complex is extremely nucleophilic Inorg Chem 36, 979–984 Wertz, D.L., Sisemore, M.F., Selke, M., Driscoll, J & Valentine, J.S (1998) Mimicking cytochrome P-450 2B4 and aromatase: aromatization of a substrate analogue by a peroxo Fe(III) porphyrin complex J Am Chem Soc 120, 5331–5332 Cole, P.A & Robinson, C.H (1991) Mechanistic studies on a placental aromatase model reaction J Am Chem Soc 113, 8130–8137 Jones, J.P., Korzekwa, K.R., Rettie, A.E & Trager, W.F (1986) Isotopically sensitive branching and its effect on the observed intramolecular isotope effects in cytochrome P-450 catalyzed reactions: a new method for the estimation of intrinsic isotope effects J Am Chem Soc 108, 7074–7078 Ruzicka, F., Huang, D.S., Donnelly, M.I & Frey, P.A (1990) Methane monooxyenase catalyzed oxygenation of 1,1-dimethylcyclopropane Evidence for radical and carbocationic intermediates Biochemistry 29, 1696–1700 Auclair, K., Hu, Z., Little, D.M., Ortiz de Montellano, P.R & Groves, J.T (2002) Revisiting the mechanism of P450 enzymes with the radical clocks norcarane and spiro[2,5]octane J Am Chem Soc 124, 6020–6027 Rollick, K.L & Kochi, J.K (1982) Oxidation-reduction mechanisms Inner-sphere and outer-sphere electron transfer in the reduction of iron(III), ruthenium(III), and osmium(III) complexes by alkyl radicals J Am Chem Soc 104, 1319–1330 Groves, J.T (2003) The bioinorganic chemistry of iron in oxygenases and supramolecular assemblies Proc Natl Acad Sci USA 100, 3569–3574 Cupp-Vickery, J.R., Han, O., Hutchinson, C.R & Poulos, T.L (1996) Substrate-assisted catalysis in cytochrome P450eryF Nat Struct Biol 3, 632–637 Raag, R., Martinis, S.A., Sligar, S.G & Poulos, T.L (1991) Crystal structure of the cytochrome P-450cam active site mutant Thr252Ala Biochemistry 30, 11420–11429 Shimizu, T., Sadeque, A.J.M., Sadeque, G.N., Hatano, M & Fujii-Kuriyama, Y (1991) Ligand binding studies of engineered cytochrome P450d wild type, proximal mutants, and distal mutants Biochemistry 30, 1490–1496 Lehnerer, M., Schulze, J., Pernecky, S.J., Lewis, D.F.V., Eulitz, M & Hlavica, P (1998) Influence of mutation of the aminoterminal signal anchor sequence of cytochrome P450 2B4 on the enzyme structure and electron transfer processes J Biochem 124, 396–403 Hlavica, P., Schulze, J & Lewis, D.F.V (2003) Functional interaction of cytochrome P450 with its redox partners: a critical assessment and update of the topology of predicted contact regions J Inorg Biochem 96, 279–297 Ó FEBS 2004 319 Collman, J.P., Chien, A.S., Eberspacher, T.A & Brauman, J.I (1998) An agostic alternative to the P-450 rebound mechanism J Am Chem Soc 120, 425–426 320 Toy, P.H., Newcomb, M & Hollenberg, P.F (1998) Hypersensitive mechanistic probe studies of cytochrome P450catalyzed hydroxylation reactions Implications for the cationic pathway J Am Chem Soc 120, 7719–7729 321 Ogliaro, F., Harris, N., Cohen, S., Filatov, M., de Visser, S.P & Shaik, S (2000) A model ÔreboundÕ mechanism of hydroxylation by cytochrome P450: stepwise and effectively concerted pathways, and their reactivity patterns J Am Chem Soc 122, 8977– 8989 322 Shaik, S., de Visser, S.P., Ogliaro, F., Schwarz, H & Schroder, ă D (2002) Two-state reactivity mechanisms of hydroxylation and epoxidation by cytochrome P-450 revealed by theory Curr Opin Chem Biol 6, 556–567 323 Kumar, D., de Visser, S.P & Shaik, S (2003) How does product isotope effect prove the operation of a two-state ÔreboundÕ mechanism in C-H hydroxylation by cytochrome P450? J Am Chem Soc 125, 13024–13025 324 Sharma, P.K., de Visser, S.P & Shaik, S (2003) Can a single oxidant with two spin states masquerade as two different oxidants? A study of the sulfoxidation mechanism by cytochrome P450 J Am Chem Soc 125, 8698–8699 325 Woggon, W.D., Wagenknecht, H.A & Claude, C (2001) Synthetic active site analogues of heme-thiolate proteins Characterization and identification of intermediates of the catalytic cycles of cytochrome P450cam and chloroperoxidase J Inorg Biochem 83, 289–300 326 De Visser, S.P., Ogliaro, F., Harris, N & Shaik, S (2001) Multistate epoxidation of ethene by cytochrome P450: a quantum chemical study J Am Chem Soc 123, 3037–3047 327 De Visser, S.P., Kumar, D & Shaik, S (2004) how aldehyde side products occur during alkene epocidation by cytochrome P450? Theory reveals a state-specific multi-state scenario where the high-spin component leads to all side products J Inorg Biochem 98, 1183–1193 328 Kumar, D., de Visser, S.P., Sharma, P.K., Cohen, S & Skaik, S (2004) Radical clock substrates, their C-H hydroxylation mechanism by cytochrome P450, and other reactivity patterns: what does theory reveal about the clocks’ behavior? J Am Chem Soc 126, 1907–1920 329 Schoneboom, J.C., Cohen, S., Lin, H., Shaik, S & Thiel, W ă (2004) Quantum mechanical/molecular mechanical investigation of the mechanism of C-H hydroxylation of camphor by cytochrome P450cam: theory supports a two-state rebound mechanism J Am Chem Soc 126, 4017–4034 ... Hlavica, P (1983) Mechanisms of extrahepatic bioactivation of aromatic amines: the role of hemoglobin in the N-oxidation of 4-chloraniline In Extrahepatic Drug Metabolism and Chemical Carcinogenesis... [106,107] A step forward in the analysis of Fe(III)-OO(H) as a potential catalyst was thought to be offered by the use of mutated P450s, bearing alanine or some other amino acid in place of the highly... permit the unmasking of radical intermediates that rearrange at a rate faster than that of the recombination step [16,93] Despite the apparent predominance of the hydrogen transfer mechanism as the