Characterization of the Key Step for Light-driven Hydrogen Evolution in Green Algae
2009; Elsevier BV; Volume: 284; Issue: 52 Linguagem: Inglês
10.1074/jbc.m109.053496
ISSN1083-351X
AutoresMartin Winkler, Sebastian Kuhlgert, Michael Hippler, Thomas Happe,
Tópico(s)Electrocatalysts for Energy Conversion
ResumoUnder anaerobic conditions, several species of green algae perform a light-dependent hydrogen production catalyzed by a special group of [FeFe] hydrogenases termed HydA. Although highly interesting for biotechnological applications, the direct connection between photosynthetic electron transport and hydrogenase activity is still a matter of speculation. By establishing an in vitro reconstitution system, we demonstrate that the photosynthetic ferredoxin (PetF) is essential for efficient electron transfer between photosystem I and HydA1. To investigate the electrostatic interaction process and electron transfer between PetF and HydA1, we performed site-directed mutagenesis. Kinetic analyses with several site-directed mutagenesis variants of HydA1 and PetF enabled us to localize the respective contact sites. These experiments in combination with in silico docking analyses indicate that electrostatic interactions between the conserved HydA1 residue Lys396 and the C terminus of PetF as well as between the PetF residue Glu122 and the N-terminal amino group of HydA1 play a major role in complex formation and electron transfer. Mapping of relevant HydA1 and PetF residues constitutes an important basis for manipulating the physiological photosynthetic electron flow in favor of light-driven H2 production. Under anaerobic conditions, several species of green algae perform a light-dependent hydrogen production catalyzed by a special group of [FeFe] hydrogenases termed HydA. Although highly interesting for biotechnological applications, the direct connection between photosynthetic electron transport and hydrogenase activity is still a matter of speculation. By establishing an in vitro reconstitution system, we demonstrate that the photosynthetic ferredoxin (PetF) is essential for efficient electron transfer between photosystem I and HydA1. To investigate the electrostatic interaction process and electron transfer between PetF and HydA1, we performed site-directed mutagenesis. Kinetic analyses with several site-directed mutagenesis variants of HydA1 and PetF enabled us to localize the respective contact sites. These experiments in combination with in silico docking analyses indicate that electrostatic interactions between the conserved HydA1 residue Lys396 and the C terminus of PetF as well as between the PetF residue Glu122 and the N-terminal amino group of HydA1 play a major role in complex formation and electron transfer. Mapping of relevant HydA1 and PetF residues constitutes an important basis for manipulating the physiological photosynthetic electron flow in favor of light-driven H2 production. IntroductionAmong all photosynthetic organisms, only green algae can couple light-driven electron transport originating from water splitting with hydrogen production (1.Melis A. Happe T. Photosynth. Res. 2004; 80: 401-409Crossref PubMed Scopus (81) Google Scholar). Hydrogen evolution in the unicellular green alga Chlamydomonas reinhardtii is naturally induced upon nutrient deprivation (2.Melis A. Zhang L. Forestier M. Ghirardi M.L. Seibert M. Plant Physiol. 2000; 122: 127-136Crossref PubMed Scopus (869) Google Scholar). Especially in the absence of sulfur, the photosynthetic oxygen evolution rate drops below the respiratory rate leading to intracellular anaerobiosis. Under anaerobic conditions, the oxygen-sensitive (2.Melis A. Zhang L. Forestier M. Ghirardi M.L. Seibert M. Plant Physiol. 2000; 122: 127-136Crossref PubMed Scopus (869) Google Scholar, 3.Wykoff D.D. Davies J.P. Melis A. Grossman A.R. Plant Physiol. 1998; 117: 129-139Crossref PubMed Scopus (392) Google Scholar) [FeFe] hydrogenase HydA is synthesized and catalyzes light-dependent H2 production, thereby dissipating excess redox equivalents under conditions in which the Calvin cycle is down-regulated (4.Hemschemeier A. Fouchard S. Cournac L. Peltier G. Happe T. Planta. 2008; 227: 397-407Crossref PubMed Scopus (171) Google Scholar).The extraordinarily small monomeric [FeFe] hydrogenases of green algae only consist of the catalytic core unit containing the active site (H-cluster), whereas other [FeFe] hydrogenases possess an additional N-terminal F-domain harboring one to four accessory iron-sulfur clusters (5.Peters J.W. Curr. Opin. Struct. Biol. 1999; 9: 670-676Crossref PubMed Scopus (199) Google Scholar, 6.Peters J.W. Lanzilotta W.N. Lemon B.J. Seefeldt L.C. Science. 1998; 282: 1853-1858Crossref PubMed Google Scholar). Because Chlorophyta-type [FeFe] hydrogenases lack any accessory clusters (7.Happe T. Hemschemeier A. Winkler M. Kaminski A. Trends Plant Sci. 2002; 7: 246-250Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 8.Kamp C. Silakov A. Winkler M. Reijerse E.J. Lubitz W. Happe T. Biochim. Biophys. Acta. 2008; 1777: 410-416Crossref PubMed Scopus (101) Google Scholar), a direct electron transfer between the native electron donor and the H-cluster has been assumed (9.Florin L. Tsokoglou A. Happe T. J. Biol. Chem. 2001; 276: 6125-6132Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 10.Winkler M. Heil B. Heil B. Happe T. Biochim. Biophys. Acta. 2002; 1576: 330-334Crossref PubMed Scopus (84) Google Scholar). In C. reinhardtii, HydA1 has been shown to be localized in the chloroplast stroma (11.Happe T. Mosler B. Naber J.D. Eur. J. Biochem. 1994; 222: 769-774Crossref PubMed Scopus (146) Google Scholar), and first kinetic examinations with purified proteins demonstrated that the plastidic ferredoxin PetF can interact with HydA1. These results and the fact that H2 production in C. reinhardtii is photosystem I (PSI) 2The abbreviations used are: PSIphotosystem ISDMsite-directed mutagenesisTricineN-tris(hydroxymethyl)methylglycineMVmethyl viologenHPIhelix of PetF interaction. -dependent (4.Hemschemeier A. Fouchard S. Cournac L. Peltier G. Happe T. Planta. 2008; 227: 397-407Crossref PubMed Scopus (171) Google Scholar) led to the hypothesis that PetF is the native electron donor of the plastidic hydrogenase (12.Happe T. Naber J.D. Eur. J. Biochem. 1993; 214: 475-481Crossref PubMed Scopus (243) Google Scholar). Derived from in silico analyses, two possible PetF-HydA2 electron transfer complex models were recently suggested (13.Chang C.H. King P.W. Ghirardi M.L. Kim K. Biophys. J. 2007; 93: 3034-3045Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). However, in contrast to the well studied interaction of PetF with other redox partners like ferredoxin-NADPH oxidoreductase (14.Morales R. Charon M.H. Kachalova G. Serre L. Medina M. Gómez-Moreno C. Frey M. EMBO Rep. 2000; 1: 271-276Crossref PubMed Scopus (99) Google Scholar, 15.Palma P.N. Lagoutte B. Krippahl L. Moura J.J. Guerlesquin F. FEBS Lett. 2005; 579: 4585-4590Crossref PubMed Scopus (23) Google Scholar), the mechanism of the electron transfer process between PetF and the algal hydrogenase is still an open question (9.Florin L. Tsokoglou A. Happe T. J. Biol. Chem. 2001; 276: 6125-6132Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 10.Winkler M. Heil B. Heil B. Happe T. Biochim. Biophys. Acta. 2002; 1576: 330-334Crossref PubMed Scopus (84) Google Scholar, 16.Happe T. Kaminski A. Eur. J. Biochem. 2002; 269: 1022-1032Crossref PubMed Scopus (211) Google Scholar). Recently, we reported the establishment of an efficient system for the heterologous synthesis of [FeFe] hydrogenases, including HydA1 of C. reinhardtii (17.von Abendroth G. Stripp S. Silakov A. Croux C. Soucaille P. Girbal L. Happe T. Int. J. Hydrogen Energy. 2008; 33: 6076-6081Crossref Scopus (67) Google Scholar). Using this system, we generated several variants of HydA1 and PetF that were specifically designed on the basis of predicted electrostatic surface distribution and preceding in silico docking analyses. The characterization of the kinetics of electron transfer processes between these protein variants of HydA1 and PetF allowed us to specify the residues that are essential for a proper interaction of the two proteins. To examine the in vivo relevance of these results, we established an in vitro system by reconstituting a part of the photosynthetic electron transport chain consisting of plastocyanin, PSI, PetF, and [FeFe] hydrogenase. This assay verifies the model of PSI-dependent H2 production, and it also allows mechanistic insights into complex formation and electron transfer between HydA1 and PetF. The experimental data demonstrate that especially Lys396 of HydA1, which is particularly conserved among green algal hydrogenases, is crucial for a successful binding and electron transfer between PetF and HydA1.DISCUSSIONThe objective of this work was to characterize the interaction of photosynthetic electron transport and hydrogen production, which is uniquely found in several green algal species (7.Happe T. Hemschemeier A. Winkler M. Kaminski A. Trends Plant Sci. 2002; 7: 246-250Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 8.Kamp C. Silakov A. Winkler M. Reijerse E.J. Lubitz W. Happe T. Biochim. Biophys. Acta. 2008; 1777: 410-416Crossref PubMed Scopus (101) Google Scholar, 9.Florin L. Tsokoglou A. Happe T. J. Biol. Chem. 2001; 276: 6125-6132Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 10.Winkler M. Heil B. Heil B. Happe T. Biochim. Biophys. Acta. 2002; 1576: 330-334Crossref PubMed Scopus (84) Google Scholar, 16.Happe T. Kaminski A. Eur. J. Biochem. 2002; 269: 1022-1032Crossref PubMed Scopus (211) Google Scholar). Earlier physiological studies have demonstrated that a significant H2 production in green algae is dependent on photosystem I activity (31.Melis A. Happe T. Plant Physiol. 2001; 127: 740-748Crossref PubMed Scopus (502) Google Scholar). It was further shown that the C. reinhardtii [FeFe] hydrogenase HydA1 can accept electrons from artificially reduced ferredoxin PetF in vitro (12.Happe T. Naber J.D. Eur. J. Biochem. 1993; 214: 475-481Crossref PubMed Scopus (243) Google Scholar). However, so far neither the molecular mechanism of the HydA1-PetF interaction nor the electron transport chain from reduced PSI to HydA1 has been analyzed.Reconstitution of Light-driven Hydrogen ProductionIn this study, the in vivo situation was successfully reconstituted by combining isolated elements of the photosynthetic electron transport chain beginning with artificially reduced plastocyanin. The light-dependent assay confirmed that PetF works as an efficient electron donor for HydA1 and demonstrated that the interaction between both proteins could indeed constitute a functional interface between photosynthetic electron transport and hydrogen production in living C. reinhardtii cells. A direct electron transfer between HydA1 and plastocyanin or PSI can be excluded. Interestingly, increasing the concentration of HydA1 above 10 nm had no influence on H2 production in this system, demonstrating that the amount of HydA1 is not the rate-limiting factor. However, H2 production could be enhanced by increasing the PSI concentration, indicating that the electron supply rather than HydA1 catalytic activity is the rate-limiting factor of in vivo H2 production in C. reinhardtii.HydA1 Residues That Are Important for the Reaction with PetFThe mechanism of the HydA1-PetF interaction was further analyzed on the molecular level performing site-directed mutagenesis. Alterations of conserved lysine residues to noncharged amino acids led to a specific decrease of PetF-dependent hydrogenase activity, although the MV-dependent H2 production rate was left unaffected. These residues therefore seem to be specifically important for the electron transfer between HydA1 and PetF. Because their exchange exhibited only a moderate decrease in PetF-dependent activity, it can be assumed that Lys262, Lys179, Lys397, and Lys433 at least take part in the guidance of the two electron transfer partners into a final stable intermolecular electron transfer complex. The low residual activity of only 18% resulting from exchanging Lys396 of HydA1 by glutamine indicates a strong influence of this lysine residue on forming and stabilizing the electron transfer complex between PetF and HydA1.As most of the examined lysine residues are specifically conserved among hydrogenases of green algae (Fig. 1B), it seems as if these positions have not gained their importance until the differentiation of the chlorophyta type [FeFe] hydrogenases (8.Kamp C. Silakov A. Winkler M. Reijerse E.J. Lubitz W. Happe T. Biochim. Biophys. Acta. 2008; 1777: 410-416Crossref PubMed Scopus (101) Google Scholar, 9.Florin L. Tsokoglou A. Happe T. J. Biol. Chem. 2001; 276: 6125-6132Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 10.Winkler M. Heil B. Heil B. Happe T. Biochim. Biophys. Acta. 2002; 1576: 330-334Crossref PubMed Scopus (84) Google Scholar, 16.Happe T. Kaminski A. Eur. J. Biochem. 2002; 269: 1022-1032Crossref PubMed Scopus (211) Google Scholar), which can be distinguished from other [FeFe] hydrogenases by lacking any accessory FeS clusters (7.Happe T. Hemschemeier A. Winkler M. Kaminski A. Trends Plant Sci. 2002; 7: 246-250Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 8.Kamp C. Silakov A. Winkler M. Reijerse E.J. Lubitz W. Happe T. Biochim. Biophys. Acta. 2008; 1777: 410-416Crossref PubMed Scopus (101) Google Scholar). Therefore, a close interaction between HydA1 and PetF is necessary to allow electron transfer between the active site redox clusters.Residues of PetF Important for Binding and Electron Transfer to HydA1The docking complex between HydA1 and PetF was further examined by analyzing mutant variants of PetF. Although exchanging the amino acids PetF-Glu60 and PetF-Glu123 had no significant influence on PetF-dependent HydA1 activity, charge neutralization of positions Asp90, Asp64, and Asp95 resulted in a decreased affinity of HydA1 for these PetF variants, indicating their importance for intermolecular attraction and orientation in the first stages of complex formation. On the other hand, the exchange of Asp56 to asparagine mainly influenced the Vmax value. Obviously, Asp56 has a more important role in stabilizing the final complex orientation.Residues Glu122 and Phe93 constitute a third group, because neutralizing exchanges of these resulted in strong effects on both the Vmax and the Km values of PetF-dependent hydrogenase activity. Because the in vitro assays were conducted using sodium dithionite in excess to completely reduce the respective PetF variants (32.Mayhew S.G. Petering D. Palmer G. Foust G.P. J. Biol. Chem. 1969; 244: 2830-2834Abstract Full Text PDF PubMed Google Scholar), a dominating influence of a possible change in the PetF redox potential is unlikely (33.Hurley J.K. Weber-Main A.M. Stankovich M.T. Benning M.M. Thoden J.B. Vanhooke J.L. Holden H.M. Chae Y.K. Xia B. Cheng H. Markley J.L. Martinez-Júlvez M. Gómez-Moreno C. Schmeits J.L. Tollin G. Biochemistry. 1997; 36: 11100-11117Crossref PubMed Scopus (96) Google Scholar). Exchanging the residues Glu94 and Phe65 in PetF of Anabaena (corresponding to Glu122 and Phe93 in PetF of C. reinhardtii) had a similar impact on electron transfer between PetF and ferredoxin-NADPH oxidoreductase as described here for the PetF-HydA1 complex (33.Hurley J.K. Weber-Main A.M. Stankovich M.T. Benning M.M. Thoden J.B. Vanhooke J.L. Holden H.M. Chae Y.K. Xia B. Cheng H. Markley J.L. Martinez-Júlvez M. Gómez-Moreno C. Schmeits J.L. Tollin G. Biochemistry. 1997; 36: 11100-11117Crossref PubMed Scopus (96) Google Scholar, 34.Mayoral T. Martínez-Júlvez M. Pérez-Dorado I. Sanz-Aparicio J. Gómez-Moreno C. Medina M. Hermoso J.A. Proteins. 2005; 59: 592-602Crossref PubMed Scopus (24) Google Scholar). Accordingly, in the crystal structure of the PetF-ferredoxin-NADPH oxidoreductase complex from Anabaena, residue Glu94 has been located as part of an important intermolecular salt bridge, whereas Phe65 turned out to be involved in hydrophobic interactions that stabilize the final complex orientation (14.Morales R. Charon M.H. Kachalova G. Serre L. Medina M. Gómez-Moreno C. Frey M. EMBO Rep. 2000; 1: 271-276Crossref PubMed Scopus (99) Google Scholar).Although a comparison might be difficult as two different isoenzymes are examined, the suggested patterns of electrostatic interactions derived from the published in silico models of the PetF-HydA2 complex do not match the experimental results for the PetF-HydA1 interaction presented here (13.Chang C.H. King P.W. Ghirardi M.L. Kim K. Biophys. J. 2007; 93: 3034-3045Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In the favored PetF-HydA2 complex (solution 16) residues Glu122 and Asp56 of PetF would be of no importance, although Glu60, which has been experimentally demonstrated to have no influence on complex formation with HydA1 in this study, was assumed to participate in electrostatic interaction. In both models that were suggested to be the most likely ones (13.Chang C.H. King P.W. Ghirardi M.L. Kim K. Biophys. J. 2007; 93: 3034-3045Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), position Lys407 of HydA2 has no significant influence on PetF-HydA2 complex stability, whereas residue Lys396 of HydA1, which corresponds to Lys407 of HydA2, was proven in this study to be the most important residue for complex formation between PetF and HydA1.Modeling the Final and Productive Electron Transfer Complex between HydA1 and PetFOn the basis of the results obtained by site-directed mutagenesis, a single complex model was filtered out of 5000 pre-designed in silico docking complexes. With about 670 Å2, the contact interface of the complex lies at the lower range of typical protein contacts (800 ± 200 Å2) and thus corresponds to values of transient interactions (35.Lo Conte L. Chothia C. Janin J. J. Mol. Biol. 1999; 285: 2177-2198Crossref PubMed Scopus (1755) Google Scholar) characteristic for electron transfer complexes.The salt bridge contacts include some of the residues examined in this study. Interestingly, for the respective participants of pairings 2 and 3 (see Fig. 5A), the experimentally determined level of influence on the electron transfer between PetF and HydA1 corresponds to each other. Three of the HydA1 residues (marked with an asterisk in the legend of Fig. 5) originate from a single α-helix (helix of PetF interaction, HPI), which has a high sequence conservation level among green algal [FeFe] hydrogenase enzymes (see Fig. 1B). Furthermore, according to this model complex, Phe93 of PetF would be located close to the hydrophobic N-terminal part of the HPI (from Gly391 to Gly393) and thus might further stabilize the contact of the HPI with PetF on the level of hydrophobic interactions.As indicated by the electron transfer complex model, electrostatic contacts of HydA1 residue Lys396 with the C terminus of PetF (Tyr126) on the one hand and between Glu122 of PetF and the N terminus of HydA1 (Ala57) of the other hand could play a central role in complex formation, which corresponds to the experimental results obtained with the variants HydA1-K396Q and PetF-E122Q, respectively. The interaction of Glu122 with the amino group of the hydrophobic HydA1 N terminus might even open up a "hydrophobic lid" covering the passage to the H-cluster generated by the first five amino acids of the processed HydA1 molecule (A1APAA5) (Fig. 5B).Furthermore, the homologous positions of Glu122 and the C-terminal tyrosine group (Tyr126) in PetF of Anabaena have been described to take part in a hydrogen bond network originating from the loop that surrounds the 2Fe2S cluster (33.Hurley J.K. Weber-Main A.M. Stankovich M.T. Benning M.M. Thoden J.B. Vanhooke J.L. Holden H.M. Chae Y.K. Xia B. Cheng H. Markley J.L. Martinez-Júlvez M. Gómez-Moreno C. Schmeits J.L. Tollin G. Biochemistry. 1997; 36: 11100-11117Crossref PubMed Scopus (96) Google Scholar, 36.Morales R. Charon M.H. Hudry-Clergeon G. Pétillot Y. Norager S. Medina M. Frey M. Biochemistry. 1999; 38: 15764-15773Crossref PubMed Scopus (122) Google Scholar). Comparative examinations on the crystal structures of reduced and oxidized PetF indicate that this network stabilizes the redox state of PetF by fixing the position of the peptide bond between Ser75 and Cys74, which has been demonstrated to perform a flipping movement upon the switch of the redox center from the reduced to the oxidized state (36.Morales R. Charon M.H. Hudry-Clergeon G. Pétillot Y. Norager S. Medina M. Frey M. Biochemistry. 1999; 38: 15764-15773Crossref PubMed Scopus (122) Google Scholar). Considering the importance of the Ser75–Cys74 bond in C. reinhardtii PetF, residue Phe93 could exert its effect by influencing this bond, because it seems to be very close to the Ser75–Cys74 pair. Additionally, the interaction of Phe93 and Tyr126 might play an important role for the stabilization of PetF, because it has been discussed that the homologous residues fulfill this function in Anabaena PetF (36.Morales R. Charon M.H. Hudry-Clergeon G. Pétillot Y. Norager S. Medina M. Frey M. Biochemistry. 1999; 38: 15764-15773Crossref PubMed Scopus (122) Google Scholar). Therefore interactions of the C. reinhardtii PetF amino acids Glu122, Tyr126, and Phe93 with residues of HydA1 might even be involved in the change of the redox state that initiates the electron transfer (Fig. 6).Besides PETF, the nuclear genome of C. reinhardtii contains at least five additional genes encoding proteins with significant homology to plant-type ferredoxins (FDX2-6) (37.Merchant S.S. Allen M.D. Kropat J. Moseley J.L. Long J.C. Tottey S. Terauchi A.M. Biochim. Biophys. Acta. 2006; 1763: 578-594Crossref PubMed Scopus (178) Google Scholar, 38.Terauchi A.M. Lu S.F. Zaffagnini M. Tappa S. Hirasawa M. Tripathy J.N. Knaff D.B. Farmer P.J. Lemaire S.D. Hase T. Merchant S.S. J. Biol. Chem. 2009; 284: 25867-25878Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Compared with PetF, Fdx2 exhibits the highest level of sequence homology (65% identity), although for the other ferredoxins the similarity lies between 47% (Fdx5) and 29% (Fdx6) sequence identity. Indeed, several of the PetF residues that contribute to complex formation with HydA1 are conserved in Fdx2, Fdx3, and Fdx5 (supplemental Fig. S4).Recent examinations demonstrated the tendency of Fdx2 for NADPH oxidation via ferredoxin-NADPH oxidoreductase. Furthermore, reduced Fdx2 obviously has a substrate specificity for nitrite reductase. This behavior suggests an equivalent role of Fdx2 and root-type ferredoxin of higher plants, which operates independently from the photosynthetic electron transport (38.Terauchi A.M. Lu S.F. Zaffagnini M. Tappa S. Hirasawa M. Tripathy J.N. Knaff D.B. Farmer P.J. Lemaire S.D. Hase T. Merchant S.S. J. Biol. Chem. 2009; 284: 25867-25878Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar).Due to the fact that Glu122, one of the most important residues for complex formation with HydA1, is substituted by a serine, it further seems unlikely that Fdx3 would effectively interact with HydA1. Even though the transcript of FDX5 strongly accumulates under anaerobic conditions, corresponding to the expression pattern of HYDA1 (16.Happe T. Kaminski A. Eur. J. Biochem. 2002; 269: 1022-1032Crossref PubMed Scopus (211) Google Scholar), recent experimental evidence suggests that Fdx5 is not able to efficiently donate electrons to HydA1 (26.Jacobs J. Pudollek S. Hemschemeier A. Happe T. FEBS Lett. 2009; 583: 325-329Crossref PubMed Scopus (54) Google Scholar).In the end, it would be surprising if one of the other ferredoxins could replace PetF as an efficient electron mediator for HydA1, as Phe93, another important PetF position, is not conserved in any of the other ferredoxins. Even the fact that the C-terminal end of PetF is unique among the ferredoxins of C. reinhardtii could potentially exclude the other ferredoxins as interaction partners (compare Fig. 6 and supplemental Fig. S4).The effects on PetF-dependent hydrogenase activity observed in this study indicate that most of the residues that are involved in PetF-ferredoxin-NADPH oxidoreductase interaction (21.Kurisu G. Kusunoki M. Katoh E. Yamazaki T. Teshima K. Onda Y. Kimata-Ariga Y. Hase T. Nat. Struct. Biol. 2001; 8: 117-121Crossref PubMed Scopus (274) Google Scholar, 30.De Pascalis A.R. Jelesarov I. Ackermann F. Koppenol W.H. Hirasawa M. Knaff D.B. Bosshard H.R. Protein Sci. 1993; 2: 1126-1135Crossref PubMed Scopus (90) Google Scholar, 33.Hurley J.K. Weber-Main A.M. Stankovich M.T. Benning M.M. Thoden J.B. Vanhooke J.L. Holden H.M. Chae Y.K. Xia B. Cheng H. Markley J.L. Martinez-Júlvez M. Gómez-Moreno C. Schmeits J.L. Tollin G. Biochemistry. 1997; 36: 11100-11117Crossref PubMed Scopus (96) Google Scholar) also participate in complex formation between PetF and HydA1. Nevertheless, there are some significant exceptions. Mutagenesis at positions Glu59 and Glu60 have only minor or no effect on interaction with HydA1, although their importance for the PetF- ferredoxin-NADPH oxidoreductase complex was demonstrated for different plants such as spinach and maize (21.Kurisu G. Kusunoki M. Katoh E. Yamazaki T. Teshima K. Onda Y. Kimata-Ariga Y. Hase T. Nat. Struct. Biol. 2001; 8: 117-121Crossref PubMed Scopus (274) Google Scholar, 30.De Pascalis A.R. Jelesarov I. Ackermann F. Koppenol W.H. Hirasawa M. Knaff D.B. Bosshard H.R. Protein Sci. 1993; 2: 1126-1135Crossref PubMed Scopus (90) Google Scholar). Under competitive conditions, PetF mainly delivers electrons to the ferredoxin-NADPH oxidoreductase instead of sustaining H2 production (39.Cinco R. Macinnis J. Greenbaum E. Photosynth. Res. 1993; 38: 27-33Crossref PubMed Scopus (40) Google Scholar). Indeed, the availability of electrons has been pointed out to be a critical aspect in light-driven hydrogen production (see above). Mapping of PetF residues relevant for the interaction with each PetF redox partner will allow us to select positions for site-directed mutagenesis to favor and direct photosynthetic electron flow to the hydrogenase and thus for enhancing light-driven H2 production. IntroductionAmong all photosynthetic organisms, only green algae can couple light-driven electron transport originating from water splitting with hydrogen production (1.Melis A. Happe T. Photosynth. Res. 2004; 80: 401-409Crossref PubMed Scopus (81) Google Scholar). Hydrogen evolution in the unicellular green alga Chlamydomonas reinhardtii is naturally induced upon nutrient deprivation (2.Melis A. Zhang L. Forestier M. Ghirardi M.L. Seibert M. Plant Physiol. 2000; 122: 127-136Crossref PubMed Scopus (869) Google Scholar). Especially in the absence of sulfur, the photosynthetic oxygen evolution rate drops below the respiratory rate leading to intracellular anaerobiosis. Under anaerobic conditions, the oxygen-sensitive (2.Melis A. Zhang L. Forestier M. Ghirardi M.L. Seibert M. Plant Physiol. 2000; 122: 127-136Crossref PubMed Scopus (869) Google Scholar, 3.Wykoff D.D. Davies J.P. Melis A. Grossman A.R. Plant Physiol. 1998; 117: 129-139Crossref PubMed Scopus (392) Google Scholar) [FeFe] hydrogenase HydA is synthesized and catalyzes light-dependent H2 production, thereby dissipating excess redox equivalents under conditions in which the Calvin cycle is down-regulated (4.Hemschemeier A. Fouchard S. Cournac L. Peltier G. Happe T. Planta. 2008; 227: 397-407Crossref PubMed Scopus (171) Google Scholar).The extraordinarily small monomeric [FeFe] hydrogenases of green algae only consist of the catalytic core unit containing the active site (H-cluster), whereas other [FeFe] hydrogenases possess an additional N-terminal F-domain harboring one to four accessory iron-sulfur clusters (5.Peters J.W. Curr. Opin. Struct. Biol. 1999; 9: 670-676Crossref PubMed Scopus (199) Google Scholar, 6.Peters J.W. Lanzilotta W.N. Lemon B.J. Seefeldt L.C. Science. 1998; 282: 1853-1858Crossref PubMed Google Scholar). Because Chlorophyta-type [FeFe] hydrogenases lack any accessory clusters (7.Happe T. Hemschemeier A. Winkler M. Kaminski A. Trends Plant Sci. 2002; 7: 246-250Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 8.Kamp C. Silakov A. Winkler M. Reijerse E.J. Lubitz W. Happe T. Biochim. Biophys. Acta. 2008; 1777: 410-416Crossref PubMed Scopus (101) Google Scholar), a direct electron transfer between the native electron donor and the H-cluster has been assumed (9.Florin L. Tsokoglou A. Happe T. J. Biol. Chem. 2001; 276: 6125-6132Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 10.Winkler M. Heil B. Heil B. Happe T. Biochim. Biophys. Acta. 2002; 1576: 330-334Crossref PubMed Scopus (84) Google Scholar). In C. reinhardtii, HydA1 has been shown to be localized in the chloroplast stroma (11.Happe T. Mosler B. Naber J.D. Eur. J. Biochem. 1994; 222: 769-774Crossref PubMed Scopus (146) Google Scholar), and first kinetic examinations with purified proteins demonstrated that the plastidic ferredoxin PetF can interact with HydA1. These results and the fact that H2 production in C. reinhardtii is photosystem I (PSI) 2The abbreviations used are: PSIphotosystem ISDMsite-directed mutagenesisTricineN-tris(hydroxymethyl)methylglycineMVmethyl viologenHPIhelix of PetF interaction. -dependent (4.Hemschemeier A. Fouchard S. Cournac L. Peltier G. Happe T. Planta. 2008; 227: 397-407Crossref PubMed Scopus (171) Google Scholar) led to the hypothesis that PetF is the native electron donor of the plastidic hydrogenase (12.Happe T. Naber J.D. Eur. J. Biochem. 1993; 214: 475-481Crossref PubMed Scopus (243) Google Scholar). Derived from in silico analyses, two possible PetF-HydA2 electron transfer complex models were recently suggested (13.Chang C.H. King P.W. Ghirardi M.L. Kim K. Biophys. J. 2007; 93: 3034-3045Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). However, in contrast to the well studied interaction of PetF with other redox partners like ferredoxin-NADPH oxidoreductase (14.Morales R. Charon M.H. Kachalova G. Serre L. Medina M. Gómez-Moreno C. Frey M. EMBO Rep. 2000; 1: 271-276Crossref PubMed Scopus (99) Google Scholar, 15.Palma P.N. Lagoutte B. Krippahl L. Moura J.J. Guerlesquin F. FEBS Lett. 2005; 579: 4585-4590Crossref PubMed Scopus (23) Google Scholar), the mechanism of the electron transfer process between PetF and the algal hydrogenase is still an open question (9.Florin L. Tsokoglou A. Happe T. J. Biol. Chem. 2001; 276: 6125-6132Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 10.Winkler M. Heil B. Heil B. Happe T. Biochim. Biophys. Acta. 2002; 1576: 330-334Crossref PubMed Scopus (84) Google Scholar, 16.Happe T. Kaminski A. Eur. J. Biochem. 2002; 269: 1022-1032Crossref PubMed Scopus (211) Google Scholar). Recently, we reported the establishment of an efficient system for the heterologous synthesis of [FeFe] hydrogenases, including HydA1 of C. reinhardtii (17.von Abendroth G. Stripp S. Silakov A. Croux C. Soucaille P. Girbal L. Happe T. Int. J. Hydrogen Energy. 2008; 33: 6076-6081Crossref Scopus (67) Google Scholar). Using this system, we generated several variants of HydA1 and PetF that were specifically designed on the basis of predicted electrostatic surface distribution and preceding in silico docking analyses. The characterization of the kinetics of electron transfer processes between these protein variants of HydA1 and PetF allowed us to specify the residues that are essential for a proper interaction of the two proteins. To examine the in vivo relevance of these results, we established an in vitro system by reconstituting a part of the photosynthetic electron transport chain consisting of plastocyanin, PSI, PetF, and [FeFe] hydrogenase. This assay verifies the model of PSI-dependent H2 production, and it also allows mechanistic insights into complex formation and electron transfer between HydA1 and PetF. The experimental data demonstrate that especially Lys396 of HydA1, which is particularly conserved among green algal hydrogenases, is crucial for a successful binding and electron transfer between PetF and HydA1.
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