MINIREVIEW Reaction mechanisms of thiamin diphosphate enzymes: redox reactions Kai Tittmann Albrecht-von-Haller-Institut fu ¨ r Pflanzenwissenschaften und Go ¨ ttinger Zentrum fu ¨ r Molekulare Biowissenschaften, Georg-August-Universita ¨ t Go ¨ ttingen, Germany Introduction The oxidative decarboxylation of 2-keto acids, such as pyruvate, branched-chain keto acids and ketoglu- tarate, is a key reaction of intermediary metabolism in virtually all organisms and is catalyzed by thiamin diphosphate (ThDP)-dependent enzymes [1]. In view of the central metabolic role of pyruvate, the various biochemical reactions involving pyruvate are the most intensely studied and are well understood. Thus, they serve as prototypical reactions for the enzymic oxidative conversion of 2-keto acids. Hence, the present review mainly focuses on the reaction mechanisms of ThDP enzymes that directly oxidize pyruvate. Special emphasis is devoted to the nature and reactivity of transient intermediates, the coupling of oxidation–reduction and acyl group transfer and electron transfer between cofactors. The review includes a discussion of general aspects of enzyme catalyzed pyruvate oxidation, in addition to individ- ual sections on the different ThDP enzymes that act on pyruvate. Pathways of pyruvate oxidation by ThDP enzymes Generally, there are at least four major different path- ways of ThDP enzyme catalyzed pyruvate oxidation. Keywords electron transfer; flavin; intermediate; iron-sulfur cluster; lipoamide; oxidative decarboxylation; phosphorolysis; pyruvate; radical; X-ray crystallography Correspondence K. Tittmann, Albrecht-von-Haller-Institut fu ¨ r Pflanzenwissenschaften und Go ¨ ttinger Zentrum fu ¨ r Molekulare Biowissenschaften, Georg-August-Universita ¨ tGo ¨ ttingen, Ernst- Caspari-Haus, Justus-von-Liebig-Weg 11, D-37077 Go ¨ ttingen, Germany Fax: +49 551 39 5749 Tel: +49 551 39 14430 E-mail: ktittma@gwdg.de (Received 7 November 2008, revised 3 February 2009, accepted 13 February 2009) doi:10.1111/j.1742-4658.2009.06966.x Amongst a wide variety of different biochemical reactions in cellular car- bon metabolism, thiamin diphosphate-dependent enzymes catalyze the oxi- dative decarboxylation of 2-keto acids. This type of reaction typically involves redox coupled acyl transfer to CoA or phosphate and is mediated by additional cofactors, such as flavins, iron-sulfur clusters or lipoamide swinging arms, which transmit the reducing equivalents that arise during keto acid oxidation to a final electron acceptor. EPR spectroscopic and kinetic studies have implicated the intermediacy of radical cofactor intermediates in pyruvate:ferredoxin oxidoreductase and an acetyl phos- phate-producing pyruvate oxidase, whereas the occurrence of transient on-pathway radicals in other enzymes is less clear. The structures of pyru- vate:ferredoxin oxidoreductase and pyruvate oxidase with different enzymic reaction intermediates along the pathway including a radical intermediate were determined by cryo-crystallography and used to infer electron tunnel- ing pathways and the potential roles of CoA and phosphate for an intimate coupling of electron and acyl group transfer. Viable mechanisms of reduc- tive acetylation in pyruvate dehydrogenase multi-enzyme complex, and of electron transfer in the peripheral membrane enzyme pyruvate oxidase from Escherichia coli, are also discussed. Abbreviations AcThDP, 2-acetyl-ThDP; HEThDP, 2-(1-hydroxyethyl)-ThDP; PDHc, pyruvate dehydrogenase multi-enzyme complex; PFOR, pyruvate:ferredoxin oxidoreductase; POX, pyruvate oxidase; Q 8, ubiquinone 8; ThDP, thiamin diphosphate. 2454 FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS Pyruvate dehydrogenase multi-enzyme complex In mitochondria and respiratory eubacteria, the pyru- vate dehydrogenase multi-enzyme complex (PDHc) catalyzes the essentially irreversible conversion of pyru- vate, CoA and NAD + into CO 2 , NADH and acetyl- CoA (Eqn 1) [2]. The latter is utilized as a precursor for the Krebs cycle and the biosynthesis of fatty acids and steroids, whereas NADH feeds the reducing equiv- alents into the respiratory chain for oxidative phos- phorylation (i.e. ATP synthesis). PDHc:pyruvate þ CoA + NAD þ ! acetyl-CoA+CO 2 þ NADH ð1Þ PDHc is the largest molecular machine known (M r of $ 10 6 –10 7 ) and is composed of multiple copies of three enzyme components: a ThDP-dependent pyru- vate dehydrogenase (termed the E1 component), a dihydrolipoamide transacetylase (E2 component), which carries lipoyl groups covalently attached to lysine residues [N 6 -(lipoyl)lysine, lipoamide], and lipoa- mide dehydrogenase (E3 component) with a nonco- valently yet tightly bound FAD cofactor [3]. In mammals, PDHc contains an additional E3 binding protein and specific kinases and phosphatases, which control the activity of the complex by reversible phos- phorylation ⁄ dephosphorylation of serine side chains in E1 [4]. Initially, E1 catalyzes the irreversible decarbox- ylation of pyruvate and the subsequent reductive acet- ylation of an N 6 -(lipoyl)lysine in E2. E2 itself catalyzes acyl group transfer from the reduced S-acety- ldihydrolipoyl-lysine to CoA. Finally, E3 regenerates the oxidized form of lipoamide and transfers the two reducing equivalents to NAD + . Pyruvate:ferredoxin oxidoreductase In anaerobic organisms, acetyl-CoA is synthesized by the enzyme pyruvate:ferredoxin oxidoreductase (PFOR), which may contain one or multiple [Fe 4 S 4 ] 2+ clusters in addition to ThDP [5]. The two electrons, which arise during oxidation of pyruvate at the ThDP site, are sequentially transferred via the iron- sulfur cluster(s) to final electron acceptors ferredoxin (Fd) or flavodoxin (Eqn 2) [6]. Unlike PDHc, PFOR also carries out the reverse reaction, namely the reduc- tive carboxylation of acetyl-CoA to yield pyruvate. PFOR: pyruvate þ CoA + 2Fd ox ! acetyl - CoA þ CO 2 þ2Fd red ð2Þ The low-potential electrons of Fd red formed in the forward direction (E o of pyruvate oxidation $ )540 mV) are used to drive several low-potential transformations such as CO reduction or hydrogen formation [7]. The reverse synthase reaction (pyruvate formation) is central to CO 2 fixation in acetogenic and green photosynthetic bacteria [8]. Acetyl phosphate-producing pyruvate oxidases In Lactobacillae such as Lactobacillus plantarum or Lactobacillus delbrueckii, which are unable to synthe- size hemes and thus lack a respiratory chain for oxi- dative phosphorylation, ATP is mainly generated by fermentation of carbohydrates with lactic acid as a final product. Under aerobic growth conditions, some Lactobacillae convert carbohydrates to the high- energy metabolite acetyl phosphate, which in turn is used for ATP synthesis. A key reaction of this path- way is the oxidative decarboxylation of pyruvate by the enzyme pyruvate oxidase (POX) that requires ThDP, Mg 2+ and FAD as cofactors [9,10]. After binding and decarboxylation of pyruvate, the reduc- ing equivalents are transferred to the neighboring FAD cofactor. The flavin is then reoxidized by the final electron acceptor dioxygen to yield hydrogen peroxide (Eqn 3). POX : pyruvate þ phosphate + oxygen + H þ ! acetyl phosphate + CO 2 +H 2 O 2 ð3Þ Phosphate-independent pyruvate oxidase from E. coli In E. coli , a related ThDP and FAD-dependent pyru- vate oxidase (EcPOX) feeds electrons from the cytosol directly into the respiratory chain at the membrane [11,12]. EcPOX is considered to be a backup system to PDHc and was shown to be important for aerobic growth of E. coli. Unlike POX from Lactobacillae, EcPOX produces acetate rather than acetyl phosphate and its reduced flavin is unreactive towards oxygen. Two-electron reduction of the flavin has been sug- gested to induce a structural rearrangement of the enzyme that exposes a lipid-binding site at the C-termi- nus. After binding to the membrane, the flavin reduces the membrane-bound mobile electron carrier ubiqui- none 8 (Q 8 ) (Eqn 4). EcPOX : pyruvate + Q 8 +OH À ! acetate + CO 2 +Q 8 H 2 ð4Þ K. Tittmann Redox reactions of thiamin diphosphate enzymes FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS 2455 Reaction intermediates in the ThDP catalyzed oxidation of pyruvate The oxidative decarboxylation of pyruvate in PDHc, PFOR and POX involves a series of covalent ThDP intermediates and analogous elementary reactions (Fig. 1) [13]. In pioneering studies on models, Breslow [14] identified C2 of the ThDP thiazolium as the reactive center that, in its carbanionic form, attacks the substrate carbonyl yielding, in the case of pyru- vate, the tetrahedral pre-decarboxylation intermediate 2-lactyl-ThDP. Decarboxylation of the latter gives the resonant a-carbanion ⁄ enamine forms of 2-(1-hydroxy- ethyl)-ThDP (HEThDP). The enamine is sometimes (and more accurately) referred to as 2-(1-hydroxye- thylidene)-ThDP and formally represents the C2a- deprotonated conjugate base of HEThDP in a resonance stabilized form. Essentially, all steps encompassing binding and decarboxylation of pyru- vate are common to PDHc, PFOR and POX. Reac- tion sequences diverge at the HEThDP carbanion ⁄ enamine intermediate, which is highly reducing and may undergo one-electron or two-elec- tron oxidation by proximal redox cofactors. The [Fe 4 S 4 ] clusters in PFOR are exclusive one-electron acceptors, whereas the flavin in POX may function as a one-electron and two-electron capacitor. Model studies suggest that reduction of the redox active dithiolane moiety of lipoic acid is a two-electron process linked to atom transfer [15]. One-electron oxidation of the HEThDP carban- ion ⁄ enamine results in the formation of a ThDP cat- ion radical (termed the HEThDP radical) with different resonance contributors (the most relevant ones are shown in Fig. 1). In principle, the unpaired spin may be delocalized over the hydroxyethyl and thiazolium moieties but it appears likely that the active sites in enzymes are poised to stabilize just one Fig. 1. Intermediates in the ThDP-catalyzed oxidative decarboxylation of pyruvate. Redox reactions of thiamin diphosphate enzymes K. Tittmann 2456 FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS or few electronic states. If electron transfer is coupled to proton transfer (i.e. deprotonation of the Ca-OH), the neutral 2-acetyl-ThDP (AcThDP)-type radical will be formed with a set of resonant forms similar to those described for the HEThDP radical. Two-elec- tron reduction or stepwise one-electron reduction yields the AcThDP intermediate that exists in three distinct forms: the keto form, the hydrate and the tri- cyclic carbinolamine [16]. The keto form undergoes acyl transfer to nucleophilic acceptors or is hydroly- sed with the geminal diol (hydrate) as a transitory state. The occurrence of 2-lactyl-ThDP, HEThDP and AcThDP as reaction intermediates in ThDP enzymes has been confirmed by 1H NMR spectroscopy after acid quench isolation [17]. EPR spectroscopy was employed to detect radical ThDP intermediates [18]. Thermodynamic aspects of pyruvate oxidation In PDHc, PFOR and POX, the thermodynamically favorable oxidation of pyruvate is coupled to forma- tion of the ‘energy-rich’ metabolites acetyl-CoA and acetyl-phosphate, which serve either as chemically acti- vated building blocks in anabolic pathways, or for ATP synthesis because the group transfer potential of the acetyl-CoA thioester (DG° ¢ = )35.7 kJÆmol )1 ) and the acetyl phosphate acid anhydride (DG° ¢ = )44.8 kJÆmol )1 ) exceeds that of ATP (DG° ¢ = 31.8 kJÆmol )1 ) [19]. Electrochemical studies on thiazolium models revealed very low subsequent one-electron oxidation potentials of the presumed pyruvate-derived enamine (E° ox(1) = )0.96 V and E° ox(2) = )0.73 V versus satu- rated calomel electrode; E° ox(1) = )0.72 V and E° ox(2) = )0.49 V versus standard hydrogen electrode) [20]. Thus, the high reducing power of the enamine intermediate is by far sufficient to initiate downhill transfer of the reducing equivalents to the final elec- tron acceptors ferredoxin (E° ¢ [Fe ++ ⁄ Fe +++ ] $ )0.4 V versus standard hydrogen electrode at pH 7), NAD + (E° ¢ [NADH, H + ⁄ NAD + ,2H + ]= )0.32 V), ubiquinone (E° ¢ [dihydroquinone ⁄ quinone, 2H + ] = +0.10 V) or oxygen (E° ¢ = +0.29 V for the O 2 ⁄ H 2 O 2 couple). The redox potentials of additional cofactors directing electron transfer from the ThDP enamine onto final electron acceptors may be modulated to some degree by the protein environ- ment but are suspected to lie in between. Redox potentials of [Fe 4 S 4 ] clusters in PFOR and FAD in POX will be discussed in the sections on the different enzymes. Reaction mechanism of pyruvate: ferredoxin oxidoreductase Evidence for a free radical mechanism In the early 1980s, Oesterhelt et al. discovered that mixing PFOR from Halobacterium halobium with its substrate pyruvate led to the formation of an organic free radical that gives rise to a continuous wave X-band EPR signal centered at g = 2.006 [21]. The radical was reported to be stable even at room temper- ature, but was readily depleted upon addition of the second substrate CoA. Quantitative analysis of sub- strate turnover revealed that two moles of final one- electron acceptor ferredoxin are reduced per mole of pyruvate in the presence of CoA. There are several lines of evidence indicating that the organic radical is a HEThDP radical resulting from one-electron oxidation of the HEThDP carbanion ⁄ enamine intermediate by the neighboring FeS cluster (i.e. this PFOR contains a single [Fe 4 S 4 ] cluster). At first, additional experiments with selectively 14 C-labeled pyruvate revealed that radioactivity remained tightly bound to the enzyme when PFOR was reacted with [3- 14 C]pyruvate, whereas no label could be detected after addition of [1- 14 C]pyruvate, clearly suggesting the radical to be formed after decarboxylation [18]. Second, the hyper- fine splitting of the radical EPR signal was shown to be dependent on the chemical nature of the substrate methyl substituent (i.e. the number of nuclear spins at C3 of pyruvate) [5]. When the EPR spectra were recorded at temperatures below 20 K, spin coupling between the ThDP-derived radical and the reduced [Fe 4 S 4 ] 1+ cluster was observable, indicating that the two paramagnetic centers are located at a distance of approximately 1 nm or less [18]. Subsequently, kinetic and spectroscopic studies on PFORs from different organisms including Desulfovib- rio africanus and Clostridium thermoaceticum suggested a common reaction mechanism with an obligate tran- sient ThDP-based radical, the lifetime of which criti- cally depends on the presence of CoA [22]. Unlike the archetypical PFOR from H. halobium, these PFORs contain three [Fe 4 S 4 ] clusters with slightly different midpoint potentials (E 1 = )540 mV, E 2 = )515 mV, E 3 = )390 mV) [23]. Thermodynamics suggest an elec- tron transfer chain from the thiamin radical to the final electron acceptor ferredoxin via the three iron- sulfur clusters involving the initial reduction of the lowest-potential iron-sulfur cluster (suspected to be located in close proximity to the ThDP cofactor), fol- lowed by sequential reduction of the other two clusters leading towards the surface of the protein, where K. Tittmann Redox reactions of thiamin diphosphate enzymes FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS 2457 ferredoxin will be reduced. Remarkably, in the absence of CoA, addition of pyruvate to D. africanus PFOR eventually resulted in the reduction of only the highest potential [Fe 4 S 4 ] cluster (E 3 = )390 mV). No mag- netic interaction between this cluster and the ThDP radical was detectable, and it was concluded that the reduced cluster is distant from the thiamin binding site [23]. In the presence of pyruvate and CoA, all three clusters become reduced. This exciting discovery on different PFORs pinpointed a crucial role of CoA for facilitating transfer of the second electron from the ThDP radical to the iron-sulfur clusters. Structural studies on PFOR and its ThDP radical intermediate In 1999, Fontecilla-Camps et al. solved the X-ray crys- tallographic structure of the homodimeric PFOR from D. africanus in the resting state at 2.3 A ˚ resolution [24]. The ThDP cofactor is deeply buried within the protein, and its reactive center, the thiazolium nucleus of ThDP, is located approximately 10 A ˚ from the most proximal [Fe 4 S 4 ] cluster (referred to as cluster A, prox- imal) (Fig. 2). Clusters A, B (medial) and C (distal) of each subunit are separated by approximately 10–12 A ˚ , thus allowing for facile electron transfer in a suitably organized redox chain (Dutton’s empirical analysis [25] predicts electron tunneling to take place when donor ⁄ acceptor pairs are within 14 A ˚ edge-to-edge distance). Cluster C is located close to the enzyme surface, where the final electron acceptor ferredoxin is suspected to bind. Electron tunneling is likely to proceed dominantly in a through-bond mechanism involving the backbone and coordinating cysteinyl ligands of the iron-sulfur clusters. Clusters B and C are covalently linked to each other via the protein, and only few gaps with edge-to-edge distances close to van- der-Waals distance (< 4 A ˚ ) exist between ThDP and cluster B, such that through-space tunneling will be scarcely required. Fontecilla-Camps et al. then reported the high-reso- lution X-ray structure of the free AcThDP radical trapped at the active center of PFOR from D. afric- anus [26]. The electron density maps suggested the thiazolium moiety of the cofactor intermediate to be markedly puckered, a structural feature that indicates reduction or even loss of aromaticity. Therefore, the thiazolium ring was proposed to adopt an unprece- dented tautomeric form, in which a proton from the 4-methyl group has undergone transfer to C5 (Fig. 3A). Also, the C2-C2a bond that connects C2 of ThDP with the substrate C2 was reported to be excep- tionally long (1.86 A ˚ ) prompting the authors to suggest a r ⁄ n-type AcThDP radical in which the unpaired spin is mostly confined to the acetyl moiety and, to a lesser degree, to C2 of the cofactor [26]. Fig. 2. Stereo drawing of PFOR structure (Protein Data Bank code: 1kek) in transpar- ent surface representation. The ThDP radical and the three [Fe 4 S 4 ] clusters are shown as sticks. Edge-to-edge distances between all cofactors are indicated. Redox reactions of thiamin diphosphate enzymes K. Tittmann 2458 FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS After fragmentation of the r ⁄ n-type cation radical and formation of an acetyl radical, radical recombination with a CoA thiyl radical was proposed to occur. By contrast to p-type radicals with extensive delocalization of the unpaired spin over aromatic systems, the pro- posed r ⁄ n-type AcThDP radical must be regarded, especially in view of the tenuously bonded acetyl moi- ety, as an unstable high-energy intermediate and, thus, its long lifetime, as demonstrated experimentally both in the crystalline phase and in solution, is seemingly counterintuitive. EPR studies on the free radical in PFOR and role of CoA for electron transfer Ragsdale and Reed [27] thoroughly examined the HEThDP radical in PFOR from Moorella thermoacetica by X-band and D-band EPR spectroscopy. EPR spec- tra of PFOR were recorded for different combinations of native and isotopically labeled cofactor ([2- 13 C], [3- 15 N]) and substrate ([3- 2 H 3 ], [2- 13 C], [3- 13 C]), and further analyzed by spectral simulations. The obtained g-values and 14 N ⁄ 15 N hyperfine-splittings are in good agreement with a planar p-type radical and extensive delocalization of the unpaired spin over the thiazolium ring. The EPR spectra and associated simulations on simplified thiazolium models are not consistent with a r ⁄ n-type AcThDP radical proposed on the basis of pure structural data [26]. Although which protonation state pertains to the radical intermediate cannot be clarified unambiguously, the observed 1 H- and 13 C-hyperfine splittings of the C2ß protons and C2 and C2a carbons would best correspond to an interme- diate state between C2a O-protonated (HEThDP radi- cal) and O-deprotonated (AcThDP radical) forms. The close proximity of the cofactor’s exocyclic 4¢-amino group demonstrated in the X-ray structure favors a hydrogen-bonding interaction between C2a-O and N4¢ (Fig. 3B). As noted above, addition of pyruvate to PFOR gen- erates a stable ThDP radical and one reduced [Fe 4 S 4 ] cluster, which was more recently demonstrated to be the medial cluster [28]. Rapid depletion of the thiamin radical and reduction of all clusters is only achieved after addition of CoA. What is the special role of CoA for propagation of the second electron and the associ- ated 10 5 -fold rate enhancement of electron transfer? In pursuit of this question, Ragsdale proposed several viable mechanisms [7,29]. At first, he considered that CoA itself could comprise part of the electron transfer chain by wiring the HEThDP radical and one iron-sulfur cluster, followed by nucleo- philic attack of the AcThDP formed in that process and eventual release of acetyl-CoA. The observation that the CoA analogue desulfo-CoA induces no rate enhancement of electron transfer, despite only marginally compro- mised binding energy compared to CoA, renders the above-considered mechanism unlikely. In line with that argument, if indeed CoA were to lend its orbitals for bridging the pathway and effective through-bond tunnel- ing, why then does transfer of the first electron proceed so facilely from the HEThDP enamine to cluster B via cluster A in the absence of CoA? Second, a biradical mechanism was proposed that involves one-electron reduction of one iron-sulfur cluster by CoA and subsequent recombination of the Fig. 3. Chemical structures of the HEThDP ⁄ AcThDP radical in PFOR proposed on the basis of structural data (A) [26] or EPR spectroscopic analysis (B) [27]. K. Tittmann Redox reactions of thiamin diphosphate enzymes FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS 2459 resultant CoA thiyl radical and the HEThDP radical to form acetyl-CoA. Support for this comes from the observation that CoA reduced one [Fe 4 S 4 ] cluster in PFOR from C. thermoaceticum, even in the absence of pyruvate [22]. However, such behavior has not been reported for all PFORs and there was no EPR spectro- scopic evidence for the putative CoA sulfur-based thiyl radical. An additional intricacy of this mechanism is the necessity of a structural rearrangement of CoA in the course of catalysis: initially, the reactive thiol group of CoA must be positioned proximal to an iron- sulfur cluster and distant to ThDP but, after oxida- tion–reduction, it would have to swing closer to ThDP. Although a simple bond rotation could account for such conformational transition, diffusion of the CoA radical out of the active site and abortive side reactions of the highly reactive thiyl radical could successfully compete with radical recombination. Furthermore, direct access to the clusters is sterically occluded by different loops, so the structural confine- ments of the active site channel render the proposed double duty of CoA (cluster reduction and radical recombination) unlikely, unless binding of CoA would enforce large structural rearrangements of the protein. Third, it was proposed that the rate enhancement of electron transfer by CoA could result from a chemical and kinetic coupling of oxidation–reduction and acyl group transfer [7]. This mechanism would generate a covalent adduct between the AcThDP-type radical and CoA to form an anion radical, the reducing power of which can be anticipated to be much higher than of a charge-neutral AcThDP radical, thus increasing the driving force of the redox reaction. As noted earlier above, in model studies, it was established that the potential of the thiazolium enamine ⁄ radical couple (E° ox(1) = )0.72 V versus standard hydrogen elec- trode) is more negative than that of the radical ⁄ acetyl couple (E° ox(2) = )0.49 V). It is conceivable that the redox potential of the former is low enough to reduce the lowest potential PFOR cluster (E 1 = )0.54 V), whereas the reducing ability of the latter might be insufficient in that concern. Thus, conclusively, the formation of a low potential anion radical may be thermodynamically mandatory to drive re-reduction of the lowest potential clusters in PFOR. Mechanism of pyruvate oxidation in pyruvate dehydrogenase multi-enzyme complex As noted earlier, oxidation of pyruvate in the E1 com- ponent of PDHc is coupled to reductive acetylation of lipoic acid covalently attached in amide linkage to the e-amino function of a lysine in the E2 component. By contrast to iron-sulfur clusters in PFOR or FAD in POX, the N 6 -(lipoyl)lysine conjugate is structurally flexible, a ‘swinging arm’ that permits active site cou- pling between E1, E2 and E3 components by rotation of the lipoyl moiety itself and by additional movement of the whole protein domain (‘swinging domain’) that carries the lipoyl-lysine, thus providing a ‘super arm’ that is capable to span the gaps between the active centers on the different components [2,30]. Oxidation–reduction chemistry of lipoic acid in models and implications for reductive acetylation in pyruvate dehydrogenase Lipoic acid exists in an oxidized disulfide form with a slightly strained five-membered dithiolane ring (LipS 2 ) and in the two-electron reduced acyclic dithiol form (dihydrolipoic acid, Lip(SH) 2 ). The standard redox potential of the Lip(SH 2 ) ⁄ LipS 2 couple has been determined by polarographic analysis to be approxi- mately )0.32 V (pH 7) and is thus more positive than the two subsequent one-electron oxidation potentials of thiazolium enamine models, making oxidation of the enamine by LipS 2 thermodynamically favorable [31]. However, LipS 2 will be electrochemically reduced only at extremely negative potentials in acetonitril solution ()1.92 V versus saturated calomel electrode) and it even resists reduction in water [15]. Low-poten- tial single-electron reductants such as reduced methyl viologen do not undergo oxidation–reduction with LipS 2 , clearly indicating that sequential one-electron reduction is an unlikely mechanism for two-electron reduction of LipS 2 [15]. A lipoic acid disulfide anion radical can be generated by one-electron reduction using hydrated electrons as reductants, but the reduc- ing power of the disulfide radical is much higher than that of the HEThDP enamine, disfavoring its one-electron oxidation by LipS 2 [32]. Because the complete reduction of LipS 2 was easily achieved by reaction with molecular hydrogen, it was concluded that reduction and concomitant cleavage of the disulfide linkage must be coupled to atom (proton) transfer [15]. In line with these early investigations, Jordan et al. observed that, in chemical models, reductive acetyla- tion of lipoic acid by thiazolium enamine occurs extremely slowly and requires the addition of a mercury trapping reagent [33]. Subsequently, the same laboratory used S-methylated lipoic acid [LipS(SCH 3 ) + ] as a viable chemical model for the S-protonated form of LipS 2 [34]. MS analysis revealed the existence of a tetrahedral adduct with an S-C Redox reactions of thiamin diphosphate enzymes K. Tittmann 2460 FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS linkage formed between lipoic acid and the thiazolium C2a [34]. Very remarkably, LipS(SCH 3 ) + easily oxi- dizes thiazolium enamine models with second-order rate constants that, in view of the effective molarity of the lipoyl-lysine in the multi-enzyme complex, can account for the observed turnover number of PDHc. This intriguing observation suggests that reductive acetylation in PDHc requires an acid ⁄ base catalyst to protonate the dithiolane part of lipoamide. Two dif- ferent mechanisms were envisioned to explain the cat- alytic role of a proton donor. At first, the ThDP enamine might attack at one of the sulfurs to form the tetrahedral adduct, and the free thiolate anion would then be protonated by a proximal proton source. Alternatively, LipS 2 could be protonated in a pre-equilibrium to give a highly reactive thiolanium cation LipS 2 H + , followed by nucleophilic attack by the enamine and concomitant cleavage of the disulfide bond. An important factor concerning the latter mechanism is the low pKa of the thiolanium cation, so that only minor fractions will be present in equili- brium under physiological conditions. Mechanistic analysis of reductive acetylation in PDHc A key question related to the mechanism of reductive acetylation in PDHc is whether enzyme-bound dihydrolipoamide is acetylated at S6 or S8 by the HEThDP enamine. It has been impossible, thus far, to directly test the two alternatives or to disprove one of them for the reconstituted multi-enzyme complex; however, elaborate studies conducted by Frey et al. on the E2-catalyzed acetylation of free dihydrolipoamide by acetyl-CoA clearly revealed formation of the 8-S isomer, followed by non-enzymatic isomerization and formation of the 6-S isomer [35]. By invoking the prin- ciple of microscopic reversibility, 8-(S)-acetyl- dihydrolipoylamide is the chemically (and kinetically) competent isomer for the physiologically relevant for- ward reaction of E2 (i.e. the formation of acetyl-CoA) and this must be formed in the preceding reductive acetylation at E1. A further compelling question concerns a possible coupling of oxidation–reduction and acyl group trans- fer. In principle, the two elementary reactions of reduc- tive acetylation could occur simultaneously in a tightly-coupled mechanism or, alternatively, in a step- wise manner. Both mechanisms would involve the tetrahedral adduct between reduced lipoamide and AcThDP; however, AcThDP would be a compulsory on-pathway intermediate only in the stepwise mecha- nism. Frey et al. could isolate AcThDP in the steady state of the overall reaction of PDHc by acid quench trapping [36]. This finding is consistent with a stepwise mechanism of oxidation–reduction and acyl group transfer; however, it cannot disprove a coupled mecha- nism because AcThDP could be generated from the tetrahedral thiamin-lipoamide adduct in an equilibrium side reaction. Further support for a stepwise mechanism comes from the observation that E1-bound AcThDP (formed by enzymic conversion of 3-flouropyruvate) is a chemi- cally competent acyl group donor to externally added dihydrolipoamide [37]. In search of putative free radi- cal intermediates that could be transiently formed in the course of sequential one-electron transfer from the enamine to oxidized lipoyl-lysine, a p-type HEThDP radical could be detected by EPR spectroscopic studies on PDHc from different organisms [13,38]. There was, however, no spectroscopic evidence for a correspond- ing lipoamide sulfur-centered thiyl radical, and the for- mation of the ThDP-based radical appeared to result from an oxygenase side reaction of the HEThDP enamine with dioxygen rather than from on-pathway oxidation by lipoamide. X-ray crystallographic studies on E1 from different dehydrogenase complexes have provided the structural framework for our mechanistic understanding of reductive acetylation and active site coupling between E1 and E2 [39,40]. The active center of E1 with the ThDP cofactor is located at the bottom of a long fun- nel-shaped substrate channel, which is suitable orga- nized to accommodate a flexible E2-linked lipoamide swinging arm for chemical coupling and intermediate channeling. As noted earlier above, reductive acetyla- tion is likely to involve acid ⁄ base chemistry from the protein and ⁄ or ThDP cofactor. General acid catalysis is required for protonation of the lipoamide disulfide, and a general base must be involved to deprotonate the a-OH of the HEThDP enamine. Structural and functional data implicate highly-conserved His side chains at E1 to fulfil this function. Some of the His residues such as His407 in E. coli E1 are located in loops that are flexible in the resting state but become ordered upon binding and processing of pyruvate [41,42]. A probable (and partially modeled) structural snapshot of catalysis showing E2-bound lipoamide prior to reaction with the planar HEThDP enamine intermediate (atomic coordinates of HEThDP enamine taken from POX) at the active center of E1 from E. coli is illustrated in Fig. 4. The lipoamide molecule was modeled into the substrate channel of E1 such that (a) formation of 8-(S)-acetyl-dihydrolipoylamide is more likely than of the 6-S isomer and (b) protonation of the lipoamide dithiolane by His407 K. Tittmann Redox reactions of thiamin diphosphate enzymes FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS 2461 may occur (on the basis of structural considerations and previously available functional data [41]). The resultant model invokes active center residue His640 to be important for deprotonation of the a-OH of the HEThDP enamine. Reaction mechanism of acetyl phosphate-producing pyruvate oxidases Chemical considerations of acetyl phosphate formation by pyruvate oxidases In phosphate-dependent pyruvate oxidases, such as that from L. plantarum (LpPOX), thermodynamically favorable oxidation of pyruvate is coupled to forma- tion of the ‘energy-rich’ metabolite acetyl phosphate that carries an acid anhydride linkage [10]. Owing to its high group transfer potential, acetyl phosphate may undergo favorable phosphotransfer to ADP to give ATP, a process that is catalyzed by the enzyme acetate kinase [19]. Besides ThDP and a divalent cat- ion (Mg 2+ ) required for anchoring the former, pyru- vate oxidases contain FAD as an additional cofactor, of which the apparent catalytic role is to oxidize the HEThDP carbanion ⁄ enamine formed after binding and decarboxylation of pyruvate at the thiamin site. Two-electron oxidation of the HEThDP carban- ion ⁄ enamine by FAD gives AcThDP, an intermediate that was initially suspected to be highly unstable but, in chemical models (AcThDP and 2-acetyl-thiazolium salts), was shown to be relatively stable at low hydroxide ion concentrations or in the absence of trapping nucleophiles [16,43]. Water and other less basic nucleophiles, such as the phosphate dianion, were demonstrated to add to 2-acetyl-thiazolium salts and result in tetrahedral adducts; however, only the water adduct underwent further decomposition to acetate, whereas reactions with phosphate gave back the starting material rather than acylated phosphates [43]. This is because phosphate is a better leaving group than the thiazolium ylid, and electron donation to C2 in the tetrahedral phospho adduct is not as extensive as in the water adduct to enable expulsion of the thiazolium ylide. In the case of AcThDP, phosphate did not even form a tetrahedral addition compound [16]. Conclusively, these model studies indicate that acetyl phosphate-producing pyruvate oxidase may not utilize simple oxidation–reduction chemistry followed by acyl transfer to phosphate. Besides overcoming the large barrier for expelling acetyl phosphate from the tetrahedral phospho adduct, the enzyme must also suppress hydrolytic cleavage of the presumed AcThDP intermediate to avoid decoupling of oxidation and acid anhydride bond formation. Molecular architecture of phosphate-dependent pyruvate oxidases and implications for electron transfer The X-ray crystal structure of the homotetrameric LpPOX was solved by Muller and Schulz [44] in the early 1990s and serves as the structural prototype for acetyl phosphate-producing POXs. As in all ThDP- dependent enzymes that have been structurally charac- terized to date, the active site is located at the interface of two corresponding subunits constituting the cata- lytic dimer (Fig. 5). The thiazolium of ThDP and the redox active isoalloxazine of FAD are bound at approximately 7 A ˚ edge-to-edge distance, with the dimethylbenzene part of FAD pointing directly towards the thiazolium. The flavin isoalloxazine is markedly bent over the N5–N10 axis ($ 10–15°), which is a structural feature that increases the driving force of oxidation–reduction because the distorted con- formation resembles the reduced state of the flavin, thus increasing its oxidizing power. The widely- accepted theoretical framework for biological electron transfer (Dutton’s ruler) predicts that pure electron (quantum-chemical) tunneling between both cofac- tors in POX would occur extremely rapidly (k theo $ 10 8 s )1 ) when assuming ‘normal’ free and reorganization energies [25]; however, the packing Fig. 4. Putative structure of E2-bound lipoamide attacking the HET- hDP enamine at the active center of E1 from E. coli in stereo view. A lipoamide molecule in the oxidized state was modeled into the substrate channel of PDHc-E1 from E. coli (Protein Data Bank code: 2g25), thus representing the catalytic state prior to reductive acety- lation. To adequately illustrate the confinements of the substrate channel, the protein is shown in surface representation with a sliced active center pocket and substrate funnel. The HEThDP enamine (modeled according to [47]) and selected His residues implicated as participating in general acid ⁄ base catalysis are shown in a stick representation. Redox reactions of thiamin diphosphate enzymes K. Tittmann 2462 FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS density (i.e. a measure of the volume in the inter-reac- tant space occupied by protein atoms) between the thiazolium and the isoalloxazine is very small so that electron transfer would mostly occur as through-space tunneling. Because of this structural observation, it has been alternatively suggested that electron tunneling might involve the side chains of two Phe residues (Phe479 and Phe121) contributed by different mono- mers as way stations for electron transfer in a com- bined through-space ⁄ through-bond mechanism [44]. In support of this proposal, an arginine side chain sitting atop Phe479 could partially offset the transiently formed negative charge at the phenyl ring. These dif- ferent possible modes notwithstanding, theoretical treatment of oxidation–reduction between the HET- hDP enamine and FAD might not be as straightfor- ward as in other systems because electron transfer in POX is definitely coupled to proton transfer (i.e. deprotonation of C2a-OH of HEThDP and proton- ation of FAD at N5 and N1). The tight and rigid binding of both cofactors excludes a direct carbanion mechanism with covalent linkage between C2a of the HEThDP enamine and C4a of FAD, as suggested for FAD-catalyzed oxidation of other organic substrates (e.g. amino acids). Besides the different hydrophobic active center resi- dues considered above as being involved in electron transfer, there are a few polar side chains (Glu, Gln) in close vicinity to the ThDP cofactor, which are likely to play important roles for catalysis and binding of phosphate. This initially premature functional assign- ment has been corroborated by kinetic and structural analysis of different LpPOX variants (G. Wille and K. Tittmann, unpublished results). Kinetic and spectroscopic analysis of oxidation–reduction in pyruvate oxidase As considered above, the structural confinements of the active site and the long distance between the two cofactors ($ 11 A ˚ between ThDP-C2 and FAD-N5 or FAD-C4a) relegate a direct carbanion mechanism with covalent HEThDP-FAD linkage or an alternative hydride transfer mechanism to minor probability. Fur- ther experimental evidence arguing against a hydride transfer mechanism comes from the finding that no oxidation–reduction can be detected in LpPOX recon- stituted with 5-deaza-5-carba-FAD, which is a FAD analogue that functions as a good hydride acceptor but does not catalyze single electron transfer [45]. Fur- thermore, FAD reduction kinetics exhibits no kinetic solvent isotope effect. Conclusively, two-electron reduction of the flavin by the HEThDP carban- ion ⁄ enamine should take place in two sequential one- electron transfer steps coupled to proton transfer. Stopped-flow kinetics and spectroscopic analysis of the reductive half-reaction (i.e. single turnover reduction of the flavin under anaerobic conditions) could not demonstrate transient formation of flavin radicals [45,46]. Two-electron reduction of the flavin occurred with a k obs of approximately 10 2 s )1 at saturating pyruvate concentrations. The inability to observe radi- cal intermediates cannot, however, rule out a two-step sequential electron transfer mechanism because a kinetic stabilization of radical intermediates requires the transfer of the second electron (k red 2 ) to proceed at a comparable rate or slower than that which occurs for the first electron (k red 1 ). No transient radicals will be kinetically stabilized when k red 2 » k red 1 . Initial Fig. 5. Stereo drawing of the active site of pyruvate oxidase from L. plantarum (Protein Data Bank code: 1pox) showing the cofac- tors ThDP and FAD and selected proximal amino acid residues. The amino acid resi- dues contributed by the corresponding subunits are colored individually (green or pink). The two Phe residues suggested to be involved in electron transfer are indicated. K. Tittmann Redox reactions of thiamin diphosphate enzymes FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS 2463 [...]... 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS 2467 Redox reactions of thiamin diphosphate enzymes K Tittmann mechanism for the thiamin diphosphate- and FADdependent pyruvate oxidase from Lactobacillus plantarum Kinetic coupling of intercofactor electron transfer with phosphate transfer to acetyl -thiamin diphosphate via a transient FAD semiquinone ⁄ hydroxyethyl-ThDP radical pair.. .Redox reactions of thiamin diphosphate enzymes K Tittmann kinetic investigations of the reductive half -reaction of LpPOX were conducted in phosphate buffer [45] Although phosphate is a substrate for the overall reaction, it was not suspected a priori to play a catalytic role for FAD reduction under the chosen single turnover conditions (i.e it was added as a stabilizing agent to the reaction. .. Electrontransfer properties of active aldehydes of thiamin coenzyme models, and mechanism of formation of the reactive intermediates Chemistry Eur J 5, 2810–2818 21 Cammack R, Kerscher L & Oesterhelt D (1980) A stable free-radical intermediate in the reaction of 2-oxoacid – ferredoxin oxidoreductases of Halobacterium halobium FEBS Lett 118, 271–273 22 Menon S & Ragsdale SW (1997) Mechanism of the Clostridium... reductive acyl trans- Redox reactions of thiamin diphosphate enzymes 35 36 37 38 39 40 41 42 43 44 45 46 fers catalyzed by the 2-oxoacid dehydrogenase multienzyme complexes Biochemistry 37, 1357–1364 Yang YS & Frey PA (1986) Dihydrolipoyl transacetylase of Escherichia coli – formation of 8-S-acetyldihydrolipoamide Biochemistry 25, 8173–8178 Gruys KJ, Datta A & Frey PA (1989) 2-Acetylthiamin pyrophosphate... Superposition of the active sites of EcPOX in the resting state (green; Protein Data Bank code: 3ey9) and after proteolytic activation (yellow; Protein Data Bank code: 3eya) in stereo view The position of residue Phe465 suspected to be involved in oxidation–reduction is highlighted FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS 2465 Redox reactions of thiamin diphosphate. .. enamine to FAD in the oxidized state A coupling mechanism of oxidation–reduction and acyl transfer? What could be the catalytic role of phosphate as a mediator for electron transfer between HEThDP and FAD? By theory, an enhanced rate of electron transfer could result from a larger driving force of the reaction (i.e a change of the redox potentials of the donor and ⁄ or acceptor pair), a shorter tunneling... enzymes catalyze an amazing variety of different chemical reactions, among which the oxidative decarboxylation of 2-keto acids is a fundamental process of intermediary metabolism in all organisms ThDP enzyme-catalyzed oxidation of keto acids is coupled to the formation of energy-rich metabolites such as acyl-CoA conjugates or acyl phosphate and the concomitant generation of reducing equivalents The underlying... the packing density between the two cofactors It was also established that phosphate has no impact on the midpoint redox potential of enzymebound FAD (Em $ )0.06 V) and the thermodynamic stabilization of FAD radicals by LpPOX [46] Conclusively, many of the mechanisms considered above are highly unlikely to explain the role of phosphate for facilitating transfer of the second reducing equivalent A mechanism... phosphate Reaction mechanism of pyruvate oxidase from Escherichia coli Pyruvate oxidase from E coli (EcPOX) catalyzes a similar intramolecular redox reaction as LpPOX, also involving two-electron oxidation of the HEThDP enamine by a neighboring FAD cofactor Unlike LpPOX, EcPOX does not produce the energy-rich product acetyl phosphate, but rather acetate [11] Analysis of the reductive half -reaction by... reactions of thiamin diphosphate enzymes Fig 6 Alternative reaction mechanisms of phosphorolysis in LpPOX taking into account the addition of phosphate to a neutral AcThDP radical (A) or HEThDP cation radical (B) It was noted earlier above that EcPOX is a peripheral membrane protein that locates to the cytosol in the resting state and becomes recruited to the biological membrane only after reduction of the . MINIREVIEW Reaction mechanisms of thiamin diphosphate enzymes: redox reactions Kai Tittmann Albrecht-von-Haller-Institut. the S-protonated form of LipS 2 [34]. MS analysis revealed the existence of a tetrahedral adduct with an S-C Redox reactions of thiamin diphosphate enzymes