MINIREVIEW Structural and mechanistic aspects of flavoproteins: photosynthetic electron transfer from photosystem I to NADP+ Milagros Medina ´ ´ Departamento de Bioquımica y Biologıa Molecular y Celular and BFIF, Universidad de Zaragoza, Spain Keywords electron transfer; ferredoxin; ferredoxin– NADP+ reductase; flavodoxin; hydride transfer; NAD(P)+ ⁄ H; photosystem I; protein–flavin complexes; protein–protein and protein–ligand interaction; redox potential regulation Correspondence ´ M Medina, Departamento de Bioquımica y ´ Biologıa Molecular y Celular, Facultad de Ciencias, Pedro Cerbuna 12, Universidad de Zaragoza, 50009-Zaragoza, Spain Fax: +34 976 762123 Tel: +34 976 762476 E-mail: mmedina@unizar.es (Received 28 January 2009, revised 22 April 2009, accepted May 2009) doi:10.1111/j.1742-4658.2009.07122.x This minireview covers the research carried out in recent years into different aspects of the function of the flavoproteins involved in cyanobacterial photosynthetic electron transfer from photosystem I to NADP+, flavodoxin and ferredoxin–NADP+ reductase Interactions that stabilize protein– flavin complexes and tailor the midpoint potentials in these proteins, as well as many details of the binding and electron transfer to protein and ligand partners, have been revealed In addition to their role in photosynthesis, flavodoxin and ferredoxin–NADP+ reductase are ubiquitous flavoenzymes that deliver NAD(P)H or low midpoint potential one-electron donors to redox-based metabolisms in plastids, mitochondria and bacteria They are also the basic prototypes for a large family of diflavin electron transferases with common functional and structural properties Understanding their mechanisms should enable greater comprehension of the many physiological roles played by flavodoxin and ferredoxin–NADP+ reductase, either free or as modules in multidomain proteins Many aspects of their biochemistry have been extensively characterized using a combination of site-directed mutagenesis, steady-state and transient kinetics, spectroscopy and X-ray crystallography Despite these considerable advances, various key features of the structural–function relationship are yet to be explained in molecular terms Better knowledge of these systems and their particular properties may allow us to envisage several interesting applications of these proteins beyond their physiological functions Introduction Many electron-transfer reactions in biological systems depend on redox chains that involve flavoproteins [1] In these chains, questions remain regarding not only the mechanisms of electron transfer and hydride transfer, but also the role that flavins might play in these events The primary function of photosystem I (PSI) is to reduce NADP+ to NADPH, which is then used in the assimilation of CO2 [2,3] In plants, this occurs via reduction of the soluble [2Fe–2S] ferredoxin (Fd) by PSI Subsequent reduction of NADP+ by Fdrd is catalysed by FAD-containing ferredoxin– NADP+ reductase (FNR) [4] In most cyanobacteria, and some algae under low iron conditions, flavodoxin (Fld) (an FMN flavoprotein), in particular Abbreviations 2¢P-AMP, 2¢-phospho-AMP portion of NADP+ ⁄ H; CTC, charge-transfer complex; Fd, ferredoxin; Fdox, oxidized ferredoxin; Fdrd, reduced ferredoxin; Fld, flavodoxin; Fldhq, hydroquinone flavodoxin; Fldox, oxidized flavodoxin; Fldsq, semiquinone flavodoxin; FNR, ferredoxin–NADP+ reductase; FNRox, oxidized ferredoxin–NADP+ reductase; FNRsq, semiquinone ferredoxin–NADP+ reductase; NMN, nicotinamide mononucleotide portion of NAD(P)+ ⁄ H; PSI, photosystem I 3942 FEBS Journal 276 (2009) 3942–3958 ª 2009 The Author Journal compilation ª 2009 FEBS M Medina Flavoproteins in photosynthetic electron transfer Fldsq ⁄ Fldhq, substitutes for the Fdox ⁄ Fdrd pair in this reaction [5,6] PSIrd ỵ Fldsq ! PSI + Fldhq NADPỵ ỵ 2Fldhq Ă NADPH + 2Fldsq FNR Two Fldsq molecules transfer two electrons from two PSI molecules to one FNR FNR becomes fully reduced through formation of the intermediate, FNRsq, and later transfers both electrons simultaneously to NADP+ [7,8] During this process, the Fld molecule must move between its docking site in PSI and the docking site in FNR, and the formation of short-life transient complexes is required Mutational and structural studies that characterize such interactions are the subject of this minireview Studies on proteins from the cyanobacterium Anabaena are preferentially considered because it is the most thoroughly investigated system containing both Fld and FNR (hereafter, AnFld and AnFNR) [5,8] A Cyanobacterial PSI exists as either a monomer or trimer embedded in the thylacoid membrane It contains 12 subunits, 96 chlorophylls, 22 carotenoids, phylloquinones, lipids and [4Fe–4S] clusters per monomer [9,10] Protein subunits with more relevant functions are conserved between plant and cyanobacteria [11] When light strikes one of the antenna chlorophylls and the exciton is transferred to the pair of chlorophylls in the PSI reaction centre, charge separation occurs Low-potential electrons are transferred across the membrane by a chain that ends in three [4Fe–4S] clusters, FX, FA and FB FX is coordinated by cysteines located in both of the large PSI subunits, PsaA and PsaB, via a loop that also plays a role in the attachment of PsaC [12] PsaC, PsaD and PsaE are located at the cytosolic site (Fig 1A) [2,7,13–16] PsaC carries the terminal FA and FB clusters After binding of the protein carrier to this PSI site, the electron is B K106 (PsaD) K34 (PsaC) PSI architecture FB E301 R16 FA L78 L76 K75 R39 (PsaE) R264 K72 Y303 C D144, E145 D150 Y94 I92 D96, N97 I59 Y94 D90 I59 T12 N58 D146 E20 E61, D65 E67 W57 E16 K2, K3 T56, W57, N58 Fig (A) Molecular surface with the electrostatic potential of the putative Fd ⁄ Fld-binding site of Synechococcus elongatus PSI (PDB code 1jb0) [10] The surface is transparent to show the internal position of the FA and FB centres (represented as spheres) in the PsaC subunit of PSI (S elongatus numbering is used) (B) Molecular surface with the electrostatic potential of Anabaena FNR at the Fld-docking site (PDB code 1que) [62] The FAD group is drawn in CPK with carbons shown in orange PSI and FNR positions for the interaction with the protein carrier are indicated (C) Molecular surface with electrostatic potential of Anabaena Fld (PDB code 1flv) [28] Detail of residues in the close FMN environment in the oxidized Fld O-down conformation is shown on the right FMN is drawn in CPK (balls or sticks), with carbons shown in orange Figure 1(A,B) is reproduced from the supplementary material in Goni et al [48] ˜ FEBS Journal 276 (2009) 3942–3958 ª 2009 The Author Journal compilation ª 2009 FEBS 3943 Flavoproteins in photosynthetic electron transfer M Medina transferred from FB to Fld (or Fd), which subsequently leaves the PSI site bringing the electron to FNR Flavins: key cofactors in protein electron transfer reactions Free flavins stabilize very little of their one-electron reduced form, the semiquinone, because the midpoint potential for reduction of the oxidized state to the semiquinone, Eox ⁄ sq, is more negative than that for reduction of the semiquinone to the hydroquinone, Esq ⁄ hq [17] Binding of FAD or FMN to the apoprotein usually displaces Eox ⁄ sq to a less negative value, whereas Esq ⁄ hq shifts to a more negative value, stabilizing the semiquinone [1,18,19] This allows flavoproteins to function as key intermediates at the interface between one- and two-electron transfers [20,21] and allows flavoproteins to participate in many biological processes [19,21,22] Flavodoxin The redox activity of Fld derives from its FMN cofactor The Fld semiquinone is exceptionally stable and its midpoint potentials are quite negative This is a direct consequence of the differing stability of the oxidized, semiquinone and reduced ApoFld:FMN complexes [23–25] In AnFld, the values are Eox ⁄ sq = )266 mV and Esq ⁄ hq = )439 mV at pH 8.0 and 25 °C, with a maximum stabilization of the semiquinone of  96% [25,26] Therefore, Fld is proposed to replace Fd (Eox ⁄ rd = )384 mV) by exchanging electrons between its semiquinone and hydroquinone states [5,8] Oxidized Flds fold into a five-stranded parallel b sheet sandwiched between five a helices [27–31], with the FMN group located at the edge of the globular protein and its two isoalloxazine methyls solvent accessible (Fig 1C) The polypeptide chain in the loops surrounding amino acid residue 50 and amino acid residue 90 (50’s and 90’s loops) make close contact with the isoalloxazine and modulate its reduction properties [24–26,32–37] The H-bond network observed in the AnFld FMN environment is conserved in Flds across species [38], but specific interactions around the flavin vary [39–41] The 90’s loop usually provides a Tyr stacked against the FMN si-face (Y94 in AnFld) which makes a large contribution to the midpoint potential [23–25] The residue from the 50’s loop that stacks at the re-inner face is commonly a Trp (W57 in AnFld) [20,28,29,39,42,43], but nonaromatic residues (L, H, M or A) have also been found [29,40,44,45] Both residues ensure that the flavin is in 3944 an electronegative environment which allows tight FMNhq binding while making formation of its anion thermodynamically unfavourable [24,25] In several Flds, rearrangement of the peptide bond equivalent to 58–59 in AnFld allows a main chain carbonyl to flip from an ‘O-down’ conformation to an ‘O-up’ conformation In the ‘O-up’ conformation, an H-bond occurs between this carbonyl and N(5)H from the neutral semiquinone [46,47] In Anacystis nidulans Fld, the flip involves breaking a weak H-bond present in the oxidized state between the FMN N(5) and the NH of V59, in favour of a stronger H-bond between the carbonyl of N58 (‘O-up’ conformation) and FMN N(5)H [41] The semiquinone states of A nidulans and Anabaena Flds are less stable than those from other species because the semiquinone H-bond with the CO of Asn is weaker than the bond formed with the smaller Gly, and because of the presence in the oxidized state of a N(5)–HN59 H-bond that is absent in other Flds Replacements at T56, W57, N58, I59 and E61 in AnFld regulate the ability of the N58–I59 peptide to H-bond with the N(5) or N(5)H, and modulate the energy of its conformational change [25,26,48] Therefore, the backbone rearrangements of N58–I59 provide a versatile device for modulating the strength of FMN binding and Eox ⁄ sq and Esq ⁄ hq in AnFld [25,26,48] Fld has a large excess of acidic residues which produce a strong dipole that orients its negative end towards the FMN isoalloxazine The importance of electrostatic repulsion in the control of Eox ⁄ sq, and particularly of Esq ⁄ hq, has also been demonstrated with several Flds [25,37,41,48–53] Electron nuclear double resonance and 1D and 2D electron spin echo envelope modulation spectroscopies applied to AnFldsq also led to assignment of the interaction parameters of N(1), N(3), H(5), H(6), CH3(8) and N(10) with the electron spin [54,55] Analysis of mutants indicated that the stacking of a bulky residue at the re-face of the flavin decreases the electron-spin density in the benzene ring, whereas an aromatic residue at the si-face increases the spin density at N(5) and C(6) [56] Ferredoxin–NADP+ reductase The first structure obtained for a photosynthetic FNR was from spinach (spFNR) spFNR folds in two domains, one of which presents a noncovalently bound FAD molecule and the other binds NADP+ [57,58] Structures from other species have also been reported [59–62] The FAD-binding domain in AnFNR includes residues 1–138 and is made up of six antiparallel b strands arranged in two perpendicu- FEBS Journal 276 (2009) 3942–3958 ª 2009 The Author Journal compilation ª 2009 FEBS M Medina lar b sheets, with a short a helix at the bottom and another a helix and a long loop that is maintained by a small two-stranded antiparallel b sheet at the top (Fig 1B) The NADP+-binding domain includes residues 139–303 and consists of a core of five parallel b strands surrounded by seven a helices [62] FAD is bound outside the antiparallel b barrel, and its isoalloxazine lies between two tyrosines, Y79 and the Cterminal Y303 in AnFNR The two one-electron midpoint potentials of the flavin in FNRs are close to each other, therefore these proteins stabilize only 10–20% of the maximal amount of semiquinone [63,64] An Eox ⁄ hq value of )325 mV has been reported for recombinant AnFNR at pH 8.0 and 10 °C [63,65] Despite differences in buffer, temperature and pH, the reported values for AnFNR are in good agreement, however, slightly more negative midpoint potentials are reported for other species [64,66,67] Replacements for Y79 have been reported for the pea and spinach enzymes, Y89 and Y95, respectively, suggesting that the aromaticity of this residue is essential for FAD binding and that H-bonding through the Tyr-OH is involved in the correct positioning of the NADP+ substrate for efficient catalysis [61,68] Replacement of Y303 with Ser and of E301 (situated at the active site) with Ala shift the flavin midpoint potential to considerably less negative values, although semiquinone stabilization is severely hampered, introducing constraints into one-electron transfer processes [26,63] In addition, L76, L78 and particularly K75 at the FAD-domain modulate Eox ⁄ hq [63,65] FNR:Fd complexation correlates with the AnFd midpoint potential becoming 15 mV more negative and that of AnFNR becoming 27–40 mV less negative [69] As seen with the spinach proteins, complexation makes electron transfer thermodynamically more favourable [70] Similarly, the midpoint potential for reduction of NADP+ in complex with FNR is 40 mV less negative than that of the free NADP+ ⁄ NADPH pair [66,71] Assignment of hyperfine couplings to nuclei of the isoalloxazine semiquinone have also been reported for AnFNRsq and pFNRsq [72] These studies indicated that the net effect of the C-terminal Tyr is withdrawal of electron density from the benzene ring towards the pyrazine ring, placing the accepted electron nearer to a site where it can best be neutralized by protonation – the N5 position Electron transfer from PSI to flavodoxin Fd and Fld differ in size and in the chemical nature of their redox cofactors There is no sequence homology Flavoproteins in photosynthetic electron transfer between them, but structural alignment based on their surface electrostatic potentials shows cofactor superposition in the region where both proteins accumulate the negative end of their molecular dipole moments [73] Their biding site on PSI was analysed by studying the kinetic behaviour of site-directed mutants and by electron microscopy on cross-linked complexes [12,15,16,74,75] The cytosolic subunits of Synechococcus elongatus (Sy) PSI, PsaC, PsaD and PsaE, and the extrinsic loop of PsaA, present a positively charged surface potential (Fig 1A) and are proposed to participate in electrostatic docking of the negatively charged Fd or Fld (Fig 1C) [13,16] The PsaC subunit cannot be deleted without loss of PSI activity because it carries the FB donor [14] PsaD contributes to the electrostatic steering of Fd toward its binding site [76,77], whereas several roles are proposed for PsaE [78,79] K35 from the PsaC subunit of Chlamydomonas reinhardtii is critical for the interaction and, therefore, efficient electron transfer [15,80] The residues of PSI and Fd facing each other have not yet been identified, with the exception of K106 in SyPsaD, which interacts with E93 in SyFd (E95 in AnFd) [77,81] The scarce data about the interaction of Fld and PSI suggest similar functions for PsaC, PsaD and PsaE, but there are insufficient data to propose a Fld docking site [13,16,82,83] Analysis of different AnFd and SyFd mutants revealed that E31, R42, T48, D67, D68, D69, E94, and particularly D59, D62 and E95 (AnFd numbering) influence electron transfer and are involved in either the binding process or electron transfer itself [84–86] Although the Fldsq ⁄ Fldhq pair is involved in shuttling electrons between PSI and FNR, a physiological role for the Fldox ⁄ Fldsq pair cannot be precluded [7,14] Reduction of AnFldox to the semiquinone state by PSI has been a useful model with which to analyse the interaction forces and electron transfer parameters involved in the physiological reaction [48] Wild-type AnFld forms a transient PSI:Fldox complex prior to electron transfer [87] Site-directed mutagenesis has been used to find the role of specific AnFld side chains in the interaction and electron transfer with PSI [53,84,88–90] Many of these Fld mutants (T12V, E16Q, T56G, W57 replaced by K, R, F, L, A and Y, I59 replaced by A and K, Y94 replaced by A and F, N97K, I59A ⁄ I92A and I59E ⁄ I92E) accept electrons from PSI following transient complex formation [53,87,90,91] For some (T12V, W57Y and Y94F), ket was lowered considerably, suggesting that the complex is not optimal for electron transfer Changes in the midpoint potential are proposed to be responsible for the W57Y Fld behaviour [90], but FEBS Journal 276 (2009) 3942–3958 ª 2009 The Author Journal compilation ª 2009 FEBS 3945 Flavoproteins in photosynthetic electron transfer M Medina changes in the electrostatics within the FMN environment which favour a complex less efficient for electron transfer explain the results for the other mutants [88] By contrast, ket was enhanced considerably for most of the remaining mutants More or less negative values of Eox ⁄ sq for these Flds compared with wildtype Fld might influence this kinetic behaviour by decreasing or increasing, respectively, the driving force of the reaction In addition, analysis of multiple charge reversal Fld mutants has recently indicated that changes in the orientation and magnitude of the molecular dipole moment have a critical effect on electron transfer [48] However, for some Fld mutants (E20K, T56S, W57E, N58C, N58K, I59E, E61A, E61K, I92 replaced by A, E and K and D96N and also some multiple charge reversal mutants), electron transfer from PSI to Fldox takes place via a collisional-type mechanism [48,53,91] Noticeably, some mutants show rates higher than those obtained for the wild-type in all the PSI ⁄ Fld ratios assayed [88] These effects are not related to a change in Fld midpoint potentials They might be interpreted as being caused by a modification in the accessibility of the flavin, but more generally can be explained by a conformational change in the orientation of the interacting PSI and Fld surfaces, leading to a smaller edge-to-edge distance between FB and the flavin ring [48] However, for other mutations, rates at a given concentration are lower than the corresponding rate for wild-type Fld Because, in general, the introduced mutations are not in the direct isoalloxazine coordination, this is unlikely to be because of differences in the structural FMN environment, but rather because the orientation between the protein dipoles is not optimal for electron transfer or because of a change in the electrostatic potential of the protein [48] In conclusion, subtle changes in the isoalloxazine environment influence Fld binding ability and modulate the electron-exchange process by producing different orientations and distances between redox centres Observations indicate that these side chains contribute to the orientation of AnFld on the PSI, producing a wild-type complex that is not the most optimal for electron transfer Mutation of these residues changes Fld surface topology, and the module and orientation of the molecular dipole, contributing to their altered behaviour Mutational studies on I59 and I92 AnFld indicate that their hydrophobicity is far from critical, suggesting that either hydrophobic interactions not play a crucial role or that the hydrophobic surface of Fld must be provided by the solvent-exposed portion of FMN 3946 Electron flow from flavodoxin to NADP+ mediated by FNR Interaction and electron transfer between Fld and FNR Crystal structures of Fd:FNR complexes have been reported for Anabaena [92] and maize leaf [93] Despite the two structures exhibiting a different orientation for Fd [94], the [2Fe–2S] cluster lies close to the FAD of FNR in both Mutants of the two partners have also contributed to the identification of residues essential for complex formation and electron transfer [5,65,95– 102] Both electrostatic and hydrophobic interactions play an important role in the association and dissociation processes in these complexes [5,8,92] K72 (K88 in spFNR), K75, L76, L78 and V136 in AnFNR are key for the interaction with Fd [5,97,100,103] Similarly, residues on the AnFd surface have a moderate effect on complex stability and electron transfer with the reductase, with E94, F65 and S47 being crucial [69,104–106] Despite proposals that FNR interacts with Fld and Fd using the same region [100,101], in general, replacing some FNR residues had more drastic effects in processes involving Fld, suggesting that the individual residues not contribute equally to complex formation with both partners This was the case for R16, K72, and particularly K75 [65,89,100,107] In addition, K138 and R264 in the NADP+-binding domain of AnFNR are more important in establishing interactions with AnFld than with AnFd [100,108] Moreover, although removal of the E139 AnFNR negative charge has a deleterious effect on electron transfer reactions with AnFd, it appears to enhance electron transfer with AnFld [109] Electron transfer with Fld is severely diminished upon the introduction of negatively charged side chains at L76, L78 and V136 in AnFNR [89] Therefore, these nonpolar residues participate in the establishment of interactions with both AnFld and AnFd With this in mind, it was expected that one or more negatively charged or hydrophobic residues on the Fld surface would interact with some of the above specified residues on FNR A number of AnFld variants containing replacements, either at the putative interaction surface with FNR or in the FMN environment, have been analysed None of the E16, E20, T56, I59, E61, D65, I92, Y94, D96 and N97 positions is key, but they contribute cooperatively to the orientation and strengthening of the FNR:Fld complexes [53,88] Simultaneous replacement of I59 and I92 indicated that they are not involved in crucial specific interactions [53,89] T12, W57 and N58 seem to be more important in the inter- FEBS Journal 276 (2009) 3942–3958 ª 2009 The Author Journal compilation ª 2009 FEBS M Medina Flavoproteins in photosynthetic electron transfer action [53,88–90] and, in addition, FMN might be critical [53] It is somehow unclear whether the residues stacking the FMN ring, W57 and Y94, truly affect protein binding, or if the altered electron transfer properties in mutations be explained in terms of altered flavin accessibility and ⁄ or thermodynamic parameters [24,25,90] Therefore, electron transfer processes between FNR and Fld resulted in the modulation (either negatively or positively) of some mutants, but in no case was electron transfer prevented Considering also that the crystallographic structure of the AnFNR:AnFld interaction appears highly elusive, these observations suggest that the interaction of Fld with FNR is less specific than that of Fd Theoretical models of this interaction, one based on the rat NADPH–cytochrome P450 reductase (CPR) structure [95] and the other obtained by docking [110], confirm that a mutual orientation between FNR and Fld, similar to the corresponding binding domains in CPR, is highly probable and places the redox centres closer than observed in the FNR:Fd structures [92,93,95] The docking model fits well with the experimental data, showing that all Fld residues important for the interaction with FNR are in contact with the FAD cofactor Different interface propensities for the same FNR residues, with either Fd or Fld, are consistent with experimental observations indicating that, A although FNR uses the same site for interaction with Fd and Fld, each individual residue does not participate to the same extent in interactions with each of the partners [100] This is in agreement with the fact that, although multiple chemical modifications produced Flds less suitable for electron transfer [111], site-directed mutagenesis has not revealed any residues critical for the interaction with FNR [53,88–90] Replacement of the few Fld positions, T12, W57, N58 and Y94, with a high interface propensity produced opposing effects: some Fld:FNR complexes can be either weaker or stronger and less optimal for electron transfer than those with wild-type Fld, but others can appear more optimal for a particular electron transfer process Docking suggests that wild-type Fld could adopt different orientations on the FNR surface without significantly altering the distance between the methyl groups of FAD and FMN (Fig 2A) This might explain why subtle changes in the Fld still produce functional complexes Moreover, the enhanced or hindered reactivity can also be explained if there is a single orientation of Fld in the complex that is retained and changes either the overall interaction or the electron transfer parameters Recent analysis of multiple charge-reversal mutations on the Fld surface concluded that interactions not rely on a precise complementary surface in the reacting molecules In B E301 S80 Y79 R264 L263 Fig Proposed interactions in the Anabaena FNR active site leading to electron transfer ⁄ hydride transfer (A) Model of a ternary Fld:FNR:NADP+ complex FNR is shown as a grey surface with the atoms of Y303 CPK coloured, with C shown in violet The position of NADP+ on the FNR surface corresponds to the X-ray FNR:NADP+ complex (PDB code 1gjr), in which Y303 prevents stacking of the flavin and nicotinamide rings The figure also shows several positions determined by docking of Fld onto FNR (Fld in green, light orange and pink correspond to docking solutions ranked 1, and respectively) (B) Detail of the proposed FNR active site centre in a model of ternary complexes competent for hydride transfer FNR active site residues are given as sticks and CPK coloured, with C in grey The nicotinamide portion of the coenzyme presents the position derived from the structure in complex with Y303S FNR (PDB code 2bsa) [117] The dotted surface around the nicotinamide indicates the position of Y303 in wild-type FNR For both structures, FAD in FNR, FMN in Fld and NADP+ are show as sticks and are CPK coloured, with carbons in yellow, orange and pink, respectively FEBS Journal 276 (2009) 3942–3958 ª 2009 The Author Journal compilation ª 2009 FEBS 3947 Flavoproteins in photosynthetic electron transfer M Medina fact, analysis indicates that the initial orientation, driven by alignment of the Fld molecule dipole moment with that of FNR, contributes to the formation of several alternative binding modes competent for the efficient electron transfer reaction [48] Similar behaviours have been reported in other electron transfer systems, where dynamic ensembles, as opposed to single conformations, contribute to the electron transfer process [112,113] Electrostatic nonspecific interactions as major determinants of the efficient interaction between Fld and its counterparts Some mutations appear to favour single orientations which improve the association and electron transfer with a particular partner and native Fld complexes are not the most optimal for electron transfer Such observation, in agreement with docking analysis [110], suggests that the flavin atoms might be mainly involved in the interaction and be solely responsible for electron transfer Therefore, subtle changes in the isoalloxazine environment not only influence Fld-binding abilities, but also modulate the electron transfer process by producing different orientations and distances between the redox centres This further confirms that Fld interacts with different structural partners through nonspecific interactions, which in turn decrease the potential efficiency that could be achieved if unique and more favourable orientations were produced with a reduced number of partners During Fld-dependent photosynthetic electron transfer, the Fld molecule must move from its docking site in PSI to that in FNR In vivo, the formation of transient complexes of Fld with PSI and FNR is useful, but not critical, during this process to promote electron transfer and avoid the reduction of oxygen by the donor centres [7,8,14,48,53] Thus, electrostatic alignment appears to be one of the major determinants of the orientation of Fld on the partner surface The fact that simultaneous replacement on the Fld surface did not hinder or enhance processes with PSI and FNR also suggests a different interaction mode with each partner [48] Interaction of FNR with the NADP+ coenzyme and the hydride transfer event Once the FAD cofactor of FNR has accepted two electrons, they have to be transferred to NADP+ The FNR protein portion has a dual role in this process by: (a) modulating the FAD midpoint potential to a value that makes the hydride transfer reversible, and (b) providing the environment for an efficient encoun3948 ter between the N5 of the flavin and the C4 of the nicotinamide FNR is highly specific for NADP+ ⁄ H versus NAD+ ⁄ H and different studies have established a role for several FNR residues in determining coenzyme binding, specificity and enzymatic efficiency [114–120] Three FNR regions appear to be mainly responsible for the interaction: 2¢-phospho-AMP (2¢PAMP) and pyrophosphate of the NADP+ ⁄ H binding sites, and the position occupied by the C-terminal residue where the nicotinamide portion of NADP+ (NMN) is proposed to bind for hydride transfer [114– 117,119] S223 and Y235 at the AnFNR 2¢P-AMP site are critical in determining the specificity and efficient coenzyme orientation [99,115,121] The 155–160 and 261–268 loops, which accommodate the coenzyme pyrophosphate portion, also confer specificity and the volume of residues in the latter loop fine-tunes FNR catalytic efficiency [114,116,119,120] R100 (K166 in spFNR), situated at the FAD-binding domain, allows its guanidinium group to H-bond to the NADP+ pyrophosphate, providing the necessary flexibility to address the NMN moiety of NADP+ towards the active site [99,108,114] Finally, the Tyr at the si-face contributes to the correct positioning of the substrate NADP+ [68], whereas the C-terminal Tyr at the re-face is surely critical for modulating NADP+ ⁄ H biding affinity and selectivity [117,118,122–126] Structural studies have allowed us to postulate a stepwise mechanism in which the nucleotide must bind to a bipartite site [59,62,114,117,127] The first stage is recognition of the 2¢P-AMP moiety [62] The intermediate state represents a narrowing of the cavity to match the adenine and the pyrophosphate, whereas the nicotinamide is placed in a pocket near the FAD [114] However, in this arrangement, the C-terminal Tyr prevents interaction of nicotinamide with the isoalloxazine and its energetically unfavourable displacement is then expected if the hydride transfer optimal interaction is to be achieved (Fig 2A) [59,114,118,127] Only FNR variants in which the C-terminal Tyr has been replaced produced structures with a rearrangement between flavin and nicotinamide that was compatible with hydride transfer, for example Y303S in AnFNR, and Y308W or Y308S in pFNR (Fig 2B) [59,117] These FNRs improved the affinity for NADP+ and produced a close interaction between flavin and nicotinamide [117,118] However, because of this strong binding, they show low catalytic efficiency [59,117,118] All the data indicate a fine-tuning of the FNR efficiency produced by minor structural changes in the regions involved in coenzyme binding [117,119] Reduction of NADP+ by FNRhq occurs by a formal hydride transfer from the flavin anionic hydro- FEBS Journal 276 (2009) 3942–3958 ª 2009 The Author Journal compilation ª 2009 FEBS M Medina quinone to the nicotinamide In vitro, the reaction takes place via a two-step mechanism, in which the first observed process is related to formation of the FNRhq–NADP+ charge-transfer complex (CTC-2) via an intermediate Michaelis–Menten complex (MC-2), followed by hydride transfer to produce an equilibrium mixture of the CTC-2 and FNRox–NADPH (CTC-1) CTCs Both CTCs are also detected for the reverse reaction, although the mechanism has some differences Spectroscopic properties for these CTCs and their hydride transfer rates for interconversion have been estimated [66,121] In AnFNR, CTC-2 accumulates during the reaction and at equilibrium, whereas CTC-1 evolves rapidly into other FNR states Formation of these CTCs appears necessary for efficient hydride transfer and the relative conformation and orientation of FNR and NADP+ ⁄ H during the interaction are critical [121] Hydride transfer in systems involving flavins and pyridine nucleotides is highly dependent on the approach and colinear orientation of the N5 of the flavin, the hydride to be transferred, and C4 of the nicotinamide In FNR, displacement of the C-terminal Tyr appears to be required for the interaction to occur [117,118] The data reported to date suggest good agreement between CTC formation and hydride transfer rates [96,121] However, this hypothesis is based on a limited data set and preliminary characterization of the process for Y303S AnFNR suggests that, at least for this mutant, there might not be a direct correlation [128] Therefore, it may be that for wild-type FNR the most favourable orientation between the nicotinamide and the flavin might present considerably less overlap of the rings than in the mutant structure (Fig 2B), and other relative orientations that maintain C4, H and N5 colinearity might account for the efficient hydride transfer Further work is needed to clarify these points Most data indicate a similar interaction in higher plant and cyanobacterial FNRs, but NADP+ disorders their spectra differently [71] The different spectra of higher plant FNRs show a peak at  510 nm indicative of a stacking interaction between the nicotinamide and the isoalloxazine, which is not seen in cyanobacterial FNRs [71] Spectra obtained upon addition of NADP+ to C-terminal Tyr mutants produce prominent peaks in AnFNR and pFNR, suggesting greater nicotinamide occupancy of the active site [117] Thus, although the AnFNR UV spectra and electron spin density distribution of AnFNRsq are perturbed by NADP+ [55,72], lower nicotinamide occupancy of the active site is expected in AnFNR relative to higher plant FNRs Therefore, differences Flavoproteins in photosynthetic electron transfer in nicotinamide binding to the active sites cannot be discounted FNR catalytic site The structure of the catalytically competent FNR:NADP+ conformation indicates that in AnFNR, S80, C261, E301 and Y303 constitute the FNR catalytic site [59,117] Y303 plays distinct and complementary roles during the catalytic cycle by lowering the affinity for NADP+ ⁄ H to levels compatible with turnover, by stabilizing the flavin semiquinone required for electron splitting and by modulating the electron transfer rates [59,117,118] Moreover, a role in providing adequate orientation between the reacting rings might be envisaged [128] S80 and C261 contribute to the efficient flavin:nicotinamide interaction through the production of CTCs during hydride transfer (in spFNR S96 and C272) [96,129] This Ser also contributes to semiquinone stabilization, and the volume of the Cys residue modulates the enzyme catalytic efficiency [119] E301 has been studied in AnFNR and spFNR (E312) [98,130] Structural and functional differences were found when the same mutants were produced in both species, again suggesting differences in their mechanisms [131] E301 was more critical for stabilization of the semiquinone and midpoint potential in AnFNR [63,98,130] Studies in spFNR concluded that E301 does not act as a proton donor [98], but whether it transfers protons in AnFNR could not be determined [130] In fact, in the AnFd:AnFNR and AnFld:AnFNR dockings, the carboxylic group of E301 is no longer exposed to solvent and it is one of the residues with highest propensity for being at the interface [110] Similar observations have been extracted from the Fd:FNR crystal structure [92] This suggests a possible pathway for proton transfer between the external medium and the AnFNR isoalloxazine N5 via S80 [58,62] In addition, in both enzymes, this residue is critical for proper binding of the nicotinamide to the active centre, CTC stabilization and efficient flavin reduction by NADPH [98,130] FNR catalytic cycle: the ternary complex The ability of FNR, Fd and NADP+ to form a ternary complex is fully accepted, indicating that NADP+ is able to occupy a site on FNR without displacing Fd [70,71,114,127], and similar behaviour also applies for Fld [132] During catalysis, the order in which substrates are added is not important, although Fd and Fld lower the affinity for NADP+ and occupation of the NADP+-binding site weakens the Fd:FNR FEBS Journal 276 (2009) 3942–3958 ª 2009 The Author Journal compilation ª 2009 FEBS 3949 Flavoproteins in photosynthetic electron transfer M Medina and Fld:FNR complexes [70,132] The two binding sites are not completely independent, and the overall reaction is proposed to work in an ordered two-substrate process, with the pyridine nucleotide binding first [70,133] Complex formation between Fdrd and FNR:NADP+ was found to increase the rate of electron transfer by facilitating the rate-limiting step of the process – dissociation of the product (Fdox) [127] Thus, in the system involving Fd, negative cooperativity in the ternary interaction is translated into positive cooperativity at the kinetic level However, some key features of the process remain to be explained in molecular terms because the expected molecular movements are not apparent and, although reversibile, different mechanisms seem to apply in each direction [127] Binding equilibrium and steady-state studies in wild-type and mutant proteins envisage similar mechanisms for Fld [5,90,130,132] Fld, reduced to Fldhq by PSI, will cycle between Fldhq and Fldsq upon passing a single electron to FNR [5] Fast kinetic methods have been used to analyse binding and electron transfer between FNR and Fld, but to date the reactions have been followed at single wavelengths and have not involved NADP+ ⁄ H [90] Therefore, it remains to the future to better evaluate the intermediate and final species of the equilibrium mixture in the electron transfer process in the binary Fld:FNR and ternary Fld:FNR:NADP+ electron transfer systems Flds and FNRs in nonphotosynthetic organisms and as building blocks for more complex proteins Flds are electron transfer proteins involved in a variety of photosynthetic and nonphotosynthetic reactions in bacteria [134] In eukaryotes, only a few Flds have been reported [135–137], but a descendant of the Fld gene helps to build multidomain proteins [134,138] A photosynthetic function was first proposed for FNR, but flavoproteins with FNR activity have been described in chloroplasts, phototropic and heterotrophic bacteria, apicoplasts, and animal and yeast mitochondria [139] Two unrelated families of proteins can be found in these enzymes: the plant type and the glutathione reductase type [126] Based on their structural and functional properties, plant-type FNRs are classified as plastidic type and bacterial type Plastidic FNRs efficiently catalyse electron transfer and hydride transfer between low-potential one-electron carriers and NADP+ ⁄ H, usually participating in the production of NADPH Bacterial FNRs generally exhibit considerably slower turnover, provide the cell with reduced electron carriers and are examples of novel 3950 methods of FAD and NADP+ ⁄ H binding However, their structures, the particular residues involved in FAD binding and the residues at the catalytic centre are well conserved [91] In addition, all plant-type FNRs may share a similar catalytic mechanism [140] The general fold found in FNR is also present in other enzymes Many of these enzymes are multidomain proteins that, in addition to the FNR-like domain, also contain Fd- or Fld-like domains These proteins contain FAD (or FMN) and a FMN or Fe–S protein, and shuttle electrons from NAD(P)H to the metal centres via their FNR and Fd ⁄ Fld domains [138,141–144] The Fld and FNR domains in diflavin reductases appear to have evolved independently [141,142,144] Despite most charged residues in Anabaena proteins being conserved in these domains, the dipole moment orientations between the FNR and Fld domains are far from colinear [48] Long-range electrostatic forces to attract their interaction surfaces have been decreased However, residues on the Fld- and FNR-domain interaction surfaces may have been conserved to orientate the Fld domain when pivoting between the FNR domain and the electron acceptor [144] The FNR family also contains NAD+ ⁄ H- and NADP+ ⁄ H-dependent members Some NAD+ ⁄ H-dependent members not present the C-terminal aromatic residue stacking the flavin and a cavity appears open at its re-face [138,145,146], but the NMN moiety is usually not observed in the structures of their complexes and, when observed an interaction between the flavin and the nicotinamide compatible with hydride transfer is not present [114,138,145,147] Catalytic differences between NADP+-dependent members are related to the different energies required to produce stacking of the nicotinamide at the re-face to FAD [119,138] These observations are compatible with a mechanism in which the initial interactions between the enzyme and 2¢P-AMP must evolve towards the production of alternative structures for each protein The fine-tuning of the enzyme catalytic efficiency is governed by the distance between and mutual orientation of the N5 of FAD and the nicotinamide C4 Therefore, it is reasonable to suppose that ancestral FNR adapted its NAD(P)+ ⁄ H-binding site, modulating unique orientations to adapt its efficiency to the coenzyme oxidation or reduction rates required in each particular electron transfer chain Applications of current knowledge about Flds and FNRs The NADPH-producing electron transfer chain has been used to explore the possibility of redesigning FEBS Journal 276 (2009) 3942–3958 ª 2009 The Author Journal compilation ª 2009 FEBS M Medina existing electron transfer systems so that they can perform functions other than that for which they were synthesized [148,149] Strategies to engineer stress tolerance in plants based on the typical stress response of photosynthetic micro-organisms are underway [150– 152] The change in the enzyme specificity with respect to its coenzyme is another example of redesign FNR regions involved in coenzyme binding have been modelled to mimic the site in NAD+ ⁄ H-dependent enzymes [115–118,120], but further work is needed to improve their catalytic efficiency Finally, Flds and FNRs are essential for the survival of some human pathogens and so may be important in the field of drug design [126,153–155] Flavoproteins in photosynthetic electron transfer Acknowledgements This work has been supported by Ministerio de Educa´ cion y Ciencia, Spain (Grant BIO2007-65890-C02-01) ´ I would like to thank Drs J Sancho and C GomezMoreno who initially introduced me in the Flavoproteins and Photosynthesis In particular, I appreciate all the support and collaboration received from Dr ´ Gomez-Moreno over more than 20 years I also thank to Drs M L Peleato, M F Fillat and T Bes, as well as all those collaborators that over the years have helped us to obtain X-ray structures and kinetic data: Drs J A Hermoso, G Tollin, J K Hurley, M A ´ Rosa, M Hervas and J A Navarro Finally, I must thank to my current and former PhD students, Dr M ´ Martı´ nez-Julvez, Dr J Tejero, Dr I Nogues, Dr S ´ Frago, J R Peregrina, G Goni, A Serrano, B Her˜ guedas and I Lans for their collaboration and interest to better understand the FNR system and for all they teach me everyday 10 11 12 13 14 References Muller F (1990) Chemistry and Biochemistry of Flavoă enzymes CRC Press, Boca Raton, FL Golbeck JH (2006) Photosystem I The Light-driven Platocyanin:Ferredoxin Oxidoreductase Springer, Dordrecht Vishniac W & Ochoa S (1952) Fixation of carbon dioxide coupled to photochemical reduction of pyridine nucleotides by chloroplast preparations J Biol Chem 195, 75–93 Arakaki AK, Ceccarelli EA & Carrillo N (1997) Planttype ferredoxin–NADP+ reductases: a basal structural framework and a multiplicity of functions FASEB J 11, 133–140 ´ Medina M & Gomez-Moreno C (2004) 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AnFNR isoalloxazine N5 via S80 [58,62] In addition, in both enzymes, this residue is critical for proper binding of the nicotinamide to the active centre, CTC stabilization and efficient flavin reduction... topology, and the module and orientation of the molecular dipole, contributing to their altered behaviour Mutational studies on I5 9 and I9 2 AnFld indicate that their hydrophobicity is far from critical,... position Electron transfer from PSI to flavodoxin Fd and Fld differ in size and in the chemical nature of their redox cofactors There is no sequence homology Flavoproteins in photosynthetic electron