The structure and reactivity of the HoxEFU complex from the cyanobacterium Synechocystis sp. PCC 6803

Abstract

Cyanobacterial Hox is a [NiFe] hydrogenase that consists of the hydrogen (H2)-activating subunits HoxYH, which form a complex with the HoxEFU assembly to mediate reactions with soluble electron carriers like NAD(P)H and ferredoxin (Fdx), thereby coupling photosynthetic electron transfer to energy-transforming catalytic reactions. Researchers studying the HoxEFUYH complex have observed that HoxEFU can be isolated independently of HoxYH, leading to the hypothesis that HoxEFU is a distinct functional subcomplex rather than an artifact of Hox complex isolation. Moreover, outstanding questions about the reactivity of Hox with natural substrates and the site(s) of substrate interactions and coupling of H2, NAD(P)H, and Fdx remain to be resolved. To address these questions, here we analyzed recombinantly produced HoxEFU by electron paramagnetic resonance spectroscopy and kinetic assays with natural substrates. The purified HoxEFU subcomplex catalyzed electron transfer reactions among NAD(P)H, flavodoxin, and several ferredoxins, thus functioning in vitro as a shuttle among different cyanobacterial pools of reducing equivalents. Both Fdx1-dependent reductions of NAD+ and NADP+ were cooperative. HoxEFU also catalyzed the flavodoxin-dependent reduction of NAD(P)+, Fdx2-dependent oxidation of NADH and Fdx4- and Fdx11-dependent reduction of NAD+. MS-based mapping identified an Fdx1-binding site at the junction of HoxE and HoxF, adjacent to iron-sulfur (FeS) clusters in both subunits. Overall, the reactivity of HoxEFU observed here suggests that it functions in managing peripheral electron flow from photosynthetic electron transfer, findings that reveal detailed insights into how ubiquitous cellular components may be used to allocate energy flow into specific bioenergetic products. Cyanobacterial Hox is a [NiFe] hydrogenase that consists of the hydrogen (H2)-activating subunits HoxYH, which form a complex with the HoxEFU assembly to mediate reactions with soluble electron carriers like NAD(P)H and ferredoxin (Fdx), thereby coupling photosynthetic electron transfer to energy-transforming catalytic reactions. Researchers studying the HoxEFUYH complex have observed that HoxEFU can be isolated independently of HoxYH, leading to the hypothesis that HoxEFU is a distinct functional subcomplex rather than an artifact of Hox complex isolation. Moreover, outstanding questions about the reactivity of Hox with natural substrates and the site(s) of substrate interactions and coupling of H2, NAD(P)H, and Fdx remain to be resolved. To address these questions, here we analyzed recombinantly produced HoxEFU by electron paramagnetic resonance spectroscopy and kinetic assays with natural substrates. The purified HoxEFU subcomplex catalyzed electron transfer reactions among NAD(P)H, flavodoxin, and several ferredoxins, thus functioning in vitro as a shuttle among different cyanobacterial pools of reducing equivalents. Both Fdx1-dependent reductions of NAD+ and NADP+ were cooperative. HoxEFU also catalyzed the flavodoxin-dependent reduction of NAD(P)+, Fdx2-dependent oxidation of NADH and Fdx4- and Fdx11-dependent reduction of NAD+. MS-based mapping identified an Fdx1-binding site at the junction of HoxE and HoxF, adjacent to iron-sulfur (FeS) clusters in both subunits. Overall, the reactivity of HoxEFU observed here suggests that it functions in managing peripheral electron flow from photosynthetic electron transfer, findings that reveal detailed insights into how ubiquitous cellular components may be used to allocate energy flow into specific bioenergetic products. Synechocystis sp. PCC 6803 has long served as a model photosynthetic organism to address questions on the biochemistry and mechanisms of photosynthetic electron transfer, including the function of peripheral redox enzymes that maintain redox homeostasis (1Foyer C.H. Ruban A.V. Nixon P.J. Photosynthesis solutions to enhance productivity.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2017; 372 (28808094): 2016037410.1098/rstb.2016.0374Crossref PubMed Scopus (49) Google Scholar, 2Peters J.W. Schut G.J. Boyd E.S. Mulder D.W. Shepard E.M. Broderick J.B. King P.W. Adams M.W. [FeFe]- and [NiFe]-hydrogenase diversity, mechanism, and maturation.Biochim. Biophys. Acta. 2015; 1853 (25461840): 1350-136910.1016/j.bbamcr.2014.11.021Crossref PubMed Scopus (305) Google Scholar). In Synechocystis 6803, one example is the bidirectional hydrogenase HoxEFUYH, which, depending on the metabolic context, either functions to dispense with excess reducing power through the generation of hydrogen or couples the oxidation of hydrogen to the reduction of electron carrier pools (3Carrieri D. Wawrousek K. Eckert C. Yu J. Maness P.C. The role of the bidirectional hydrogenase in cyanobacteria.Bioresour. Technol. 2011; 102 (21514820): 8368-837710.1016/j.biortech.2011.03.103Crossref PubMed Scopus (71) Google Scholar, 4Gutekunst K. Chen X. Schreiber K. Kaspar U. Makam S. Appel J. The bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 is reduced by flavodoxin and ferredoxin and is essential under mixotrophic, nitrate-limiting conditions.J. Biol. Chem. 2014; 289 (24311779): 1930-193710.1074/jbc.M113.526376Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). HoxEFUYH has long been of interest due to the possibility of engineering the enzyme and metabolic pathways to enable photobiological hydrogen production routes (5Tamagnini P. Axelsson R. Lindberg P. Oxelfelt F. Wünschiers R. Lindblad P. Hydrogenases and hydrogen metabolism of cyanobacteria.Microbiol. Mol. Biol. Rev. 2002; 66: 1-2010.1128/MMBR.66.1.1-20.2002Crossref PubMed Scopus (402) Google Scholar). Despite numerous investigations, the underlying features of the biochemistry, structure, and protein-protein interactions of HoxEFU have yet to be fully detailed. A more complete understanding of these properties could be used to improve approaches for engineering photobiohydrogen production, artificial photosynthesis, or catalyst design. HoxEFUYH consists of the small and large [NiFe] hydrogenase subunits, HoxY and HoxH, respectively, and an additional set of subunits that compose the heterotrimeric diaphorase HoxEFU (6McIntosh C.L. Germer F. Schulz R. Appel J. Jones A.K. The [NiFe]-hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 works bidirectionally with a bias to H2 production.J.Am. Chem. Soc. 2011; 133: 11308-1131910.1021/ja203376yCrossref PubMed Scopus (75) Google Scholar) (Fig. 1). Biochemical reactions with ferredoxins 1 and 4 (Fdx1 and Fdx4; ssl0020 and slr0150, respectively), flavodoxin (Fld), and NAD(P)H demonstrate that each of these of catalytic H2 production by Hox in K. Chen X. Schreiber K. Kaspar U. Makam S. Appel J. The bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 is reduced by flavodoxin and ferredoxin and is essential under mixotrophic, nitrate-limiting conditions.J. Biol. Chem. 2014; 289 (24311779): 1930-193710.1074/jbc.M113.526376Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, P. of HoxE in Synechocystis J. 2011; PubMed Scopus Google Scholar, of hydrogen in a of Synechocystis sp. PCC 6803 in the PubMed Scopus Google Scholar) and purified enzyme K. specific for the bidirectional hydrogenase complex of 2002; PubMed Scopus Google Scholar) with the HoxEFUYH have is a for NADH K. Chen X. Schreiber K. Kaspar U. Makam S. Appel J. The bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 is reduced by flavodoxin and ferredoxin and is essential under mixotrophic, nitrate-limiting conditions.J. Biol. Chem. 2014; 289 (24311779): 1930-193710.1074/jbc.M113.526376Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, P. of HoxE in Synechocystis J. 2011; PubMed Scopus Google Scholar, K. specific for the bidirectional hydrogenase complex of 2002; PubMed Scopus Google Scholar, of catalytic properties of hydrogenase isolated from the cyanobacterium PubMed Scopus Google Scholar, hydrogenase in from the cyanobacterium Scholar, J.W. The soluble [NiFe]-hydrogenase from consists of subunits and can be by PubMed Scopus Google Scholar). 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The hydrogenase subcomplex of the [NiFe] hydrogenase from insights into and redox Chem. 2011; Scopus (49) Google and in Hox with or the diaphorase from which can have different and The HoxEFU subcomplex a complex of redox the [NiFe] catalytic site with one additional HoxEFU an in HoxF, as as clusters clusters and of hydrogen in a of Synechocystis sp. PCC 6803 in the PubMed Scopus Google Scholar). and have been for of Synechocystis 6803 HoxEFUYH and have with [NiFe] F. F. Schulz R. Appel J. and of the bidirectional [NiFe] hydrogenase from Synechocystis sp. PCC Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). HoxEFUYH is to to form HoxEFU from the hydrogenase subcomplex C. D. Yu J. Nixon P.J. Maness P.C. of the Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 in and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, properties of the isolated diaphorase of the [NiFe]-hydrogenase from 2011; PubMed Scopus Google Scholar, J. D. subunits of the in J. PubMed Scopus Google Scholar, Lindblad P. Cyanobacterial and hydrogen metabolism and J. Mol. Sci. 2015; PubMed Scopus Google Scholar). has the of the HoxEFU subcomplex has a distinct role in the C. D. Yu J. Nixon P.J. Maness P.C. of the Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 in and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Eckert C. E.M. Yu J. Nixon P.J. Appel J. production specific of the Sci. 2014; PubMed Google Scholar). To HoxEFU has been observed in C. D. Yu J. Nixon P.J. Maness P.C. of the Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 in and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) as as in vitro of the by the that in of is into the HoxEFU subcomplex rather than the complete HoxEFUYH complex Eckert C. E.M. Yu J. Nixon P.J. Appel J. production specific of the Sci. 2014; PubMed Google Scholar). The of the biochemistry of Synechocystis 6803 substrate and the role of HoxE in reactions with soluble electron HoxE in Synechocystis 6803 is to be for H2 production by Hox to both NADH and P. of HoxE in Synechocystis J. 2011; PubMed Scopus Google Scholar) and has been in and electron transfer with and it is also to of Hox with the Eckert C. E.M. Yu J. Nixon P.J. Appel J. production specific of the Sci. 2014; PubMed Google Scholar). with have the role of HoxE in electron transfer D. hydrogenase in the photosynthetic PubMed Scopus Google Scholar). the HoxE is for several functions of photosynthetic Hox specific features that to diaphorase The Hox from in the oxidation of NADH in the of it the an J.W. The soluble [NiFe]-hydrogenase from consists of subunits and can be by PubMed Scopus Google Scholar, properties of the isolated diaphorase of the [NiFe]-hydrogenase from 2011; PubMed Scopus Google Scholar) and is to a role in electron The Hox complex from H2 oxidation to NAD+ reduction and has a HoxE a S. S. of the redox in the soluble 2017; PubMed Scopus Google Scholar). how to functions Hox and the role of subunits remain to be resolved. To address questions about the of and properties of Synechocystis 6803 Hox to the Synechocystis 6803 HoxEFU isolated as an and the and were The demonstrate that HoxEFU couples NAD(P)H oxidation and reduction to the of with redox including Fdx and in the of the hydrogenase The and features identified here demonstrate that HoxEFU has the essential properties to redox reactions with photosynthetic electron transfer components in a more and than of HoxEFU in on the for HoxEFUYH in Synechocystis 6803 F. F. Schulz R. Appel J. and of the bidirectional [NiFe] hydrogenase from Synechocystis sp. PCC Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The a on the of and from the in Synechocystis under of HoxEFU of and used to (Fig. and through with a of To HoxEFU can with NAD(P)H and mediate electron transfer reactions the the of NAD(P)H on the of the HoxEFU by The HoxEFU (Fig. due to the of as NAD(P)H or the To the of the reduced HoxEFU the reduced with redox hydrogen The redox of and from reactions with and hydrogen J. PubMed Scopus Google in a with an that for (Fig. with the of for of of HoxEFU by the NAD(P)H to that of the with complete reduction (Fig. and and the of the to with The with the from one of the fully reduced by NAD(P)H, by a at a of with an in with and in reduction of HoxEFU by NAD(P)H more in reducing the clusters than HoxEFUYH at F. F. Schulz R. Appel J. and of the bidirectional [NiFe] hydrogenase from Synechocystis sp. PCC Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is due to HoxEFUYH under for H2 production which reduction of which could the of fully reduced clusters under reduced the of the and features at the at The coupling the reduced with function in electron transfer for HoxEFU F. F. Schulz R. Appel J. and of the bidirectional [NiFe] hydrogenase from Synechocystis sp. PCC Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, and of spectroscopy in the Soc. Rev. PubMed Google Scholar). features at the and of the to the for the HoxEFUYH complex F. F. Schulz R. Appel J. and of the bidirectional [NiFe] hydrogenase from Synechocystis sp. PCC Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and the could be on and power (Fig. at of and and 4 at of and which with and To these were on the HoxEFU (Fig. the on HoxEFUYH F. F. Schulz R. Appel J. and of the bidirectional [NiFe] hydrogenase from Synechocystis sp. PCC Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the were the of a to for the The and and and to clusters on the which were in with the in the and one and that for the in the (Fig. The can be to a on the the of in the that it may have from additional the may be a of a different as power at The to the components of the is due to the of the also be that the is observed under NAD(P)H or it may be in the fully reduced and form or by in the The HoxEFUYH complex couples the oxidation of reduced electron carriers to the production of HoxEFU is for the reactivity with electron the site interactions and To the and of HoxEFU for different electron we the reactivity in reactions with redox The oxidation of NAD(P)H by HoxEFU is a of the catalyzed by HoxEFUYH, with of and for NADH and The of HoxEFU for NADH of is the of for the HoxEFUYH complex of hydrogen in a of Synechocystis sp. PCC 6803 in the PubMed Scopus Google Scholar, of catalytic properties of hydrogenase isolated from the cyanobacterium PubMed Scopus Google the HoxEFU for of 1 is than for H2 production by Synechocystis 6803 of hydrogen in a of Synechocystis sp. PCC 6803 in the PubMed Scopus Google Scholar). may in or in the and reactivity of HoxEFU for in the of on Hox H2 or on K. Chen X. Schreiber K. Kaspar U. Makam S. Appel J. The bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 is reduced by flavodoxin and ferredoxin and is essential under mixotrophic, nitrate-limiting conditions.J. Biol. Chem. 2014; 289 (24311779): 1930-193710.1074/jbc.M113.526376Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, P. of HoxE in Synechocystis J. 2011; PubMed Scopus Google Scholar, of catalytic properties of hydrogenase isolated from the cyanobacterium PubMed Scopus Google Scholar, hydrogenase in from the cyanobacterium Scholar) or used with purified enzymes the of enzyme (6McIntosh C.L. Germer F. Schulz R. Appel J. Jones A.K. The [NiFe]-hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 works bidirectionally with a bias to H2 production.J.Am. Chem. Soc. 2011; 133: 11308-1131910.1021/ja203376yCrossref PubMed Scopus (75) Google Scholar, J. P. The hydrogenase subcomplex of the [NiFe] hydrogenase from insights into and redox Chem. 2011; Scopus (49) Google Scholar). it is to specific here on purified HoxEFU with of Synechocystis 6803 and oxidation to for oxidation to for oxidation to for for in a The NAD(P)H reactions the redox rather than the natural substrates To of a HoxEFU for reduction to and flavodoxin from the cyanobacterium Synechocystis PCC Biophys. Acta. PubMed Scopus Google the a of the with kinetic for NAD+ and of (Fig. The of suggests that HoxEFU into of or NAD+ to one HoxEFU could on of or NAD+ at additional HoxEFU C. and kinetic in to on a of an Sci. PubMed Scopus Google Scholar). In NADP+ with and a of for The for both NAD+ and NADP+ reduction were than for NAD(P)H oxidation 1). The kinetic assays demonstrate a for a with of H2 production by of hydrogen in a of Synechocystis sp. PCC 6803 in the PubMed Scopus Google Scholar, of catalytic properties of hydrogenase isolated from the cyanobacterium PubMed Scopus Google Scholar, hydrogenase in from the cyanobacterium Scholar). as an electron carrier in peripheral pathways and to H2 production by HoxEFUYH in K. Chen X. Schreiber K. Kaspar U. Makam S. Appel J. The bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 is reduced by flavodoxin and ferredoxin and is essential under mixotrophic, nitrate-limiting conditions.J. Biol. Chem. 2014; 289 (24311779): 1930-193710.1074/jbc.M113.526376Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). To HoxEFU is to with as a the reduction of with reduced The redox couples of and for the fully reduced and and flavodoxin from the cyanobacterium Synechocystis PCC Biophys. Acta. PubMed Scopus Google Scholar). on the of the redox couples it that HoxEFU could mediate reduction of as as the HoxEFU oxidation of to the reduction of at a than the and flavodoxin from the cyanobacterium Synechocystis PCC Biophys. Acta. PubMed Scopus Google Scholar). The of the observed with the of In the HoxEFU NADH and reduced to the with a of an In to the reduction of from NAD(P)H oxidation is more The of reduction of NAD+ by oxidation of than the In reduced H2 production than with a than K. Chen X. Schreiber K. Kaspar U. Makam S. Appel J. The bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 is reduced by flavodoxin and ferredoxin and is essential under mixotrophic, nitrate-limiting conditions.J. Biol. Chem. 2014; 289 (24311779): 1930-193710.1074/jbc.M113.526376Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). on kinetic it is that be reactivity HoxEFU and HoxEFUYH that may the substrate in HoxEFU to Fdx The of HoxEFU to suggests that soluble redox carriers with may also as redox To diaphorase under for additional Fdx from Synechocystis 6803 in C. F. and of ferredoxins in the Synechocystis 2014; Google Scholar). assays were of Fdx and the may be to of Synechocystis 6803 HoxEFU with Fdx reduction oxidation and flavodoxin from the cyanobacterium Synechocystis PCC Biophys. Acta. PubMed Scopus Google D. S. R. A ferredoxin as a in the of photosynthetic Sci. PubMed Scopus Google D. S. R. A ferredoxin as a in the of photosynthetic Sci. PubMed Scopus Google C. F. and of ferredoxins in the Synechocystis 2014; Google as P. D. R. C. cyanobacterial ferredoxin with and functional Biophys. PubMed Scopus Google of the from C. F. and of ferredoxins in the Synechocystis 2014; Google identified in as K. Chen X. Schreiber K. Kaspar U. Makam S. Appel J. The bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 is reduced by flavodoxin and ferredoxin and is essential under mixotrophic, nitrate-limiting conditions.J. Biol. Chem. 2014; 289 (24311779): 1930-193710.1074/jbc.M113.526376Abstract Full Text Full Text PDF PubMed Scopus (94) Google of the from P. D. R. C. cyanobacterial ferredoxin with and functional Biophys. PubMed Scopus Google identified in J. K. P. C. K. D. J. R. and a to PubMed Scopus Google Scholar). in a has an of D. S. R. A ferredoxin as a in the of photosynthetic Sci. PubMed Scopus Google to the of the has a role with in homeostasis and and it is in photosynthetic C. F. and of ferredoxins in the Synechocystis 2014; Google Scholar, P. ferredoxins and PubMed Scopus Google Scholar). the of NADH oxidation to reduction of it than that of The of NAD+ reduction by is to have different and C. F. and of ferredoxins in the Synechocystis 2014; Google Scholar). of the Fdx from a of P. D. R. C. cyanobacterial ferredoxin with and functional Biophys. PubMed Scopus Google and it has been to H2 production in Synechocystis 6803 HoxEFUYH K. Chen X. Schreiber K. Kaspar U. Makam S. Appel J. The bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 is reduced by flavodoxin and ferredoxin and is essential under mixotrophic, nitrate-limiting conditions.J. Biol. Chem. 2014; 289 (24311779): 1930-193710.1074/jbc.M113.526376Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). it that it NAD+ reduction by HoxEFU to a as and to be the with a of than that of is on the as and thus is a is more to than Fdx and is more to C. F. and of ferredoxins in the Synechocystis 2014; Google Scholar). the we HoxEFU with for either NAD+ reduction or NADH The of may be due to in the that in the in vitro as E.M. of and the in the of ferredoxin in Synechocystis sp. PCC PubMed Scopus Google or it may be a of and electron transfer and additional cyanobacterial which we and as a Fdx of by the D. S. P. C. The in protein-protein 2017; PubMed Scopus Google Scholar) the photosynthetic components and to be to the reduction of NAD+ to NADH by HoxEFU with a of with a that of the a reactivity for HoxEFU which may photosynthetic electron the with the different Fdx demonstrate that HoxEFU has the to with a of electron carriers and that the of the is and that may and The of these to that to the Fdx from Synechocystis 6803 that has a is S. The of and to Mol. Biol. PubMed Scopus Google Scholar). The of Fdx that can with HoxEFU that Hox functions a complex reactivity and suggests the possibility that HoxEFU may with electron as ferredoxins, or The diaphorase of function independently of and hydrogenase to reducing among the carrier pools in in The here demonstrate that the HoxEFU subcomplex has substrate both to and distinct from the HoxEFUYH complex for coupling redox reactions NAD(P)H, and Fdx or The that HoxEFUYH is to or NAD(P)H to H2 that it may a redox redox substrates K. Chen X. Schreiber K. Kaspar U. Makam S. Appel J. The bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 is reduced by flavodoxin and ferredoxin and is essential under mixotrophic, nitrate-limiting conditions.J. Biol. Chem. 2014; 289 (24311779): 1930-193710.1074/jbc.M113.526376Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). reactivity be to the electron that has been observed for from J.W. R. Jones A.K. King P.W. Mulder D.W. S. Schut G.J. for electron Chem. Biol. PubMed Scopus Google Scholar, electron a of energy Rev. PubMed Scopus Google Scholar). the the reactivity of HoxEFU may be specific to the subcomplex to of electron flow among substrate has also been that HoxEFU functions to and thereby the [NiFe] site in HoxYH, a hypothesis that is by (6McIntosh C.L. Germer F. Schulz R. Appel J. Jones A.K. The [NiFe]-hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 works bidirectionally with a bias to H2 production.J.Am. Chem. Soc. 2011; 133: 11308-1131910.1021/ja203376yCrossref PubMed Scopus (75) Google Scholar). The of HoxEFU to mediate an of the NAD(P)H, and pools in of the that cyanobacterial photosynthetic complex from reduced ferredoxin J. P. of photosynthetic complex enable electron PubMed Scopus Google Scholar). In to the observed by the kinetic we on in which the interactions of NADH with HoxEFU by the The that NADH to with a of and a of to the of NADH in the reduction (Fig. also the of NAD+ and NADP+ to Both with of and for NAD+ and respectively, and of and (Fig. B and NADP+ from HoxEFU more than with that a for NAD+ The also that the of of for Synechocystis 6803 in and of Synechocystis sp. PCC 6803 by PubMed Scopus Google Scholar). on the that a of HoxEFU with at with a in to The with the and a function of the HoxEFU subcomplex in catalytic oxidation of NAD(P)H to reduction of soluble rather than the reduction of oxidation of NAD(P)H observed as an artificial electron oxidation observed with is a of of reducing a more Fdx by the more of NAD(P)H (Fig. different is a of to as in of J. P. in the cyanobacterium Synechocystis sp. PCC a for in of and Biophys. Acta. 2014; PubMed Scopus Google Scholar, C. Schreiber U. of in Synechocystis PCC 6803 and PubMed Scopus Google or under and K. K. the of in Synechocystis sp. PCC 2014; PubMed Scopus Google HoxEFU may reduction of in a to that catalyzed by The that is a function of it a possibility that the of the subcomplex could The that can to HoxEFU the of HoxEFUYH to mediate Fdx1-dependent of H2 K. Chen X. Schreiber K. Kaspar U. Makam S. Appel J. The bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 is reduced by flavodoxin and ferredoxin and is essential under mixotrophic, nitrate-limiting conditions.J. Biol. Chem. 2014; 289 (24311779): 1930-193710.1074/jbc.M113.526376Abstract Full Text Full Text PDF PubMed Scopus (94) Google a for NAD(P)H additional is that HoxEFUYH is of a as electron which is by the of redox the of identified for HoxEFU in the Fdx1-dependent reduction of NADP+ may be an of a in the of To in the of assays that have HoxEFUYH is an that the demonstrate a the suggests the possibility that is that the NADP+ reduction is observed the S. C. of substrate enzyme in and of Biol. Chem. Full Text Full Text PDF PubMed Google Scholar, J. S. of in the catalytic of a of with Biol. Chem. Full Text PDF PubMed Google Scholar). on has that of a substrate the observed from to in a in Biol. PubMed Scopus (75) Google Scholar, A with Google Scholar). to from in as substrate is or of Rev. Biophys. PubMed Scopus Google Scholar). the of the from a in Sci. PubMed Scopus Google Scholar). The site of on Hox has been to the HoxE the for a specific is K. Chen X. Schreiber K. Kaspar U. Makam S. Appel J. The bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 is reduced by flavodoxin and ferredoxin and is essential under mixotrophic, nitrate-limiting conditions.J. Biol. Chem. 2014; 289 (24311779): 1930-193710.1074/jbc.M113.526376Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, Eckert C. E.M. Yu J. Nixon P.J. Appel J. production specific of the Sci. 2014; PubMed Google Scholar). The of with Hox has to the of and the electron flow Hox To address purified and purified with were to of to HoxEFU The identified in a HoxF, and (Fig. that were identified may or be an of or or in the role of in to HoxF, and electron transfer from to the in either HoxE or HoxF, with the of clusters in for electron transfer routes the site and is to that a to the of which one of the clusters of be an electron in the HoxEFU as as in The model to Synechocystis 6803 Hox is from it HoxE and has a with from Synechocystis 6803 the Synechocystis 6803 model here a in understanding the site of of to Synechocystis 6803 on HoxEFUYH from Synechocystis 6803 have that is a electron for H2 production, which may to the site identified here for additional K. Chen X. Schreiber K. Kaspar U. Makam S. Appel J. The bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 is reduced by flavodoxin and ferredoxin and is essential under mixotrophic, nitrate-limiting conditions.J. Biol. Chem. 2014; 289 (24311779): 1930-193710.1074/jbc.M113.526376Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). that HoxEFU is of functioning in independently of HoxYH, to redox reactions NAD(P)H, and (Fig. The in the of different ferredoxins to with NAD(P)H HoxEFU the that the of ferredoxins in Synechocystis a of redox to be the of the diaphorase to shuttle the NAD(P)H, and pools is of in have identified of which for reactivity with electron a model for how with The of may HoxEFU by electron transfer pathways and the of the Hox of reduction by HoxEFU HoxEFUYH that substrate reactivity in the of or of the functions of HoxEFU identified here may be among Hox to as in which HoxE S. S. of the redox in the soluble 2017; PubMed Scopus Google and C. which HoxE with the J.W. The soluble [NiFe]-hydrogenase from consists of subunits and can be by PubMed Scopus Google could both reactivity and enable to the and mechanisms to electron transfer and the role of HoxEFU in photosynthetic

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