Просмотр кода страницы Английская Википедия:Hydrogenase

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A '''hydrogenase''' is an [[enzyme]] that [[Catalysis|catalyses]] the reversible [[Redox|oxidation]] of molecular [[hydrogen]] (H<sub>2</sub>), as shown below:

{{NumBlk|:|H<sub>2</sub> + [[electron acceptor|A]]<sub>ox</sub> → 2H<sup>+</sup> + A<sub>red</sub>|{{EquationRef|1}}}}
{{NumBlk|:|2H<sup>+</sup> + [[electron donor|D]]<sub>red</sub> → H<sub>2</sub> + D<sub>ox</sub>|{{EquationRef|2}}}}

Hydrogen uptake ({{EquationNote|1}}) is coupled to the reduction of [[electron acceptors]] such as [[oxygen]], [[nitrate]], [[sulfate]], [[carbon dioxide]] ({{CO2}}), and [[fumarate]]. On the other hand, proton reduction ({{EquationNote|2}}) is coupled to the oxidation of electron donors such as [[ferredoxin]] (FNR), and serves to dispose excess electrons in cells (essential in [[pyruvate]] fermentation). Both low-molecular weight compounds and proteins such as FNRs, [[cytochrome]] ''c''<sub>3</sub>, and cytochrome ''c''<sub>6</sub> can act as physiological electron donors or acceptors for hydrogenases.<ref>{{cite journal | vauthors = Vignais PM, Billoud B, Meyer J | title = Classification and phylogeny of hydrogenases | journal = FEMS Microbiology Reviews | volume = 25 | issue = 4 | pages = 455–501 | date = August 2001 | pmid = 11524134 | doi = 10.1111/j.1574-6976.2001.tb00587.x | doi-access = free }}</ref>

== Structural classification ==
It has been estimated that 99% of all organisms utilize [[hydrogen]], H<sub>2</sub>. Most of these species are microbes and their ability to use H<sub>2</sub> as a metabolite arises from the expression of [[metalloenzyme]]s known as hydrogenases.<ref>
{{cite journal | vauthors = Lubitz W, Ogata H, Rüdiger O, Reijerse E | title = Hydrogenases | journal = Chemical Reviews | volume = 114 | issue = 8 | pages = 4081–4148 | date = April 2014 | pmid = 24655035 | doi = 10.1021/cr4005814 | authorlink1 = Wolfgang Lubitz }}
</ref> Hydrogenases are sub-classified into three different types based on the active site metal content: iron-iron hydrogenase, nickel-iron hydrogenase, and iron hydrogenase. 
[[File:ActiveSitesCorrected.png|thumb|center|x160px|The structures of the active sites of the three types of hydrogenase enzymes.]]

Hydrogenases catalyze, sometimes reversibly, H<sub>2</sub> uptake. The [FeFe] and [NiFe] hydrogenases are true redox catalysts, driving H<sub>2</sub> oxidation and proton (H<sup>+</sup>) reduction (equation {{EquationNote|3}}), the [Fe] hydrogenases catalyze the reversible heterolytic cleavage of H<sub>2</sub> shown by reaction ({{EquationNote|4}}).
{{NumBlk|:|H<sub>2</sub> {{eqm}} 2 H<sup>+</sup> + 2 e<sup>−</sup>|{{EquationRef|3}}}}
{{NumBlk|:|H<sub>2</sub> {{eqm}} H<sup>+</sup> + H<sup>−</sup>|{{EquationRef|4}}}}

Although originally believed to be "metal-free", the [Fe]-only hydrogenases contain Fe at the active site and no iron-sulfur clusters. [NiFe] and [FeFe] hydrogenases have some common features in their structures: Each enzyme has an active site and a few Fe-S clusters that are buried in protein. The active site, which is believed to be the place where catalysis takes place, is also a metallocluster, and each iron is coordinated by [[carbon monoxide]] (CO) and [[cyanide]] (CN<sup>−</sup>) ligands.<ref>{{cite journal | vauthors = Fontecilla-Camps JC, Volbeda A, Cavazza C, Nicolet Y | title = Structure/function relationships of [NiFe]- and [FeFe]-hydrogenases | journal = Chemical Reviews | volume = 107 | issue = 10 | pages = 4273–4303 | date = October 2007 | pmid = 17850165 | doi = 10.1021/cr050195z }}</ref>

=== [NiFe] hydrogenase ===
[[File:NiFe Hydrogenase.png|thumb|left|Crystal structure of [NiFe] hydrogenase]]
The [NiFe] hydrogenases are heterodimeric proteins consisting of small (S) and large (L) subunits. The small subunit contains three [[iron-sulfur cluster]]s while the large subunit contains the active site, a nickel-iron centre which is connected to the solvent by a molecular tunnel.<ref name="Jugder 8142">{{Cite journal| vauthors = Jugder BE, Welch J, Aguey-Zinsou KF, Marquis CP |date=2013-05-14|title=Fundamentals and electrochemical applications of [Ni–Fe]-uptake hydrogenases|journal=RSC Advances|language=en|volume=3|issue=22|page=8142|doi=10.1039/c3ra22668a|bibcode=2013RSCAd...3.8142J |issn=2046-2069}}</ref><ref name="pmid19966788" /> In some [NiFe] hydrogenases, one of the Ni-bound cysteine residues is replaced by [[selenocysteine]]. On the basis of sequence similarity, however, the [NiFe] and [NiFeSe] hydrogenases should be considered a single superfamily.
To date, periplasmic, cytoplasmic, and cytoplasmic membrane-bound hydrogenases have been found. The [NiFe] hydrogenases, when isolated, are found to catalyse both H<sub>2</sub> evolution and uptake, with low-potential multihaem cytochromes such as cytochrome ''c''<sub>3</sub> acting as either electron donors or acceptors, depending on their oxidation state.<ref name="Jugder 8142"/> Generally speaking, however, [NiFe] hydrogenases are more active in oxidizing H<sub>2</sub>. A wide spectrum of H<sub>2</sub> affinities have also been observed in H<sub>2</sub>-oxidizing hydrogenases.<ref>{{cite journal | vauthors = Greening C, Berney M, Hards K, Cook GM, Conrad R | title = A soil actinobacterium scavenges atmospheric H2 using two membrane-associated, oxygen-dependent [NiFe] hydrogenases | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 111 | issue = 11 | pages = 4257–4261 | date = March 2014 | pmid = 24591586 | pmc = 3964045 | doi = 10.1073/pnas.1320586111 | doi-access = free | bibcode = 2014PNAS..111.4257G }}</ref>

Like [FeFe] hydrogenases, [NiFe] hydrogenases are known to be usually deactivated by molecular oxygen (O<sub>2</sub>). Hydrogenase from ''[[Ralstonia eutropha]]'', and several other so-called Knallgas-bacteria, were found to be oxygen-tolerant.<ref name="Jugder 8142"/><ref name="Burgdorf">{{cite journal | vauthors = Burgdorf T, Lenz O, Buhrke T, van der Linden E, Jones AK, Albracht SP, Friedrich B | title = [NiFe]-hydrogenases of Ralstonia eutropha H16: modular enzymes for oxygen-tolerant biological hydrogen oxidation | journal = Journal of Molecular Microbiology and Biotechnology | volume = 10 | issue = 2–4 | pages = 181–196 | year = 2005 | pmid = 16645314 | doi = 10.1159/000091564 | s2cid = 8030367 }}</ref> The soluble [NiFe] hydrogenase from ''Ralstonia eutropha'' H16 can be conveniently produced on [[heterotroph]]ic growth media.<ref name="Jugder 42">{{cite journal | vauthors = Jugder BE, Chen Z, Ping DT, Lebhar H, Welch J, Marquis CP | title = An analysis of the changes in soluble hydrogenase and global gene expression in Cupriavidus necator (Ralstonia eutropha) H16 grown in heterotrophic diauxic batch culture | language = En | journal = Microbial Cell Factories | volume = 14 | issue = 1 | pages = 42 | date = March 2015 | pmid = 25880663 | pmc = 4377017 | doi = 10.1186/s12934-015-0226-4 | doi-access = free }}</ref><ref name="Jugder 242–250">{{cite journal | vauthors = Jugder BE, Lebhar H, Aguey-Zinsou KF, Marquis CP | title = Production and purification of a soluble hydrogenase from Ralstonia eutropha H16 for potential hydrogen fuel cell applications | journal = MethodsX | volume = 3 | pages = 242–250 | date = 2016-01-01 | pmid = 27077052 | pmc = 4816682 | doi = 10.1016/j.mex.2016.03.005 }}</ref> This finding increased hope that hydrogenases can be used in photosynthetic production of molecular hydrogen via splitting water.
Another [NiFe], called Huc or Hyd1 or cyanobacterial-type uptake hydrogenase,<ref>{{cite journal | vauthors = Cordero PR, Grinter R, Hards K, Cryle MJ, Warr CG, Cook GM, Greening C | title = Two uptake hydrogenases differentially interact with the aerobic respiratory chain during mycobacterial growth and persistence | journal = The Journal of Biological Chemistry | volume = 294 | issue = 50 | pages = 18980–18991 | date = December 2019 | pmid = 31624148 | pmc = 6916507 | doi = 10.1074/jbc.RA119.011076 | doi-access = free }}</ref> has been found to be oxygen insensitive while having a very high affinity for hydrogen. Hydrogen is able to penetrate narrow channels in the enzyme that oxygen molecules cannot enter. This allows 
bacteria such as ''[[Mycobacterium smegmatis]]'' to utilize the small amount of hydrogen in the atmosphere as a source of energy when other sources are lacking.<ref>{{cite journal | vauthors = Grinter R, Kropp A, Venugopal H, Senger M, Badley J, Cabotaje PR, Jia R, Duan Z, Huang P, Stripp ST, Barlow CK, Belousoff M, Shafaat HS, Cook GM, Schittenhelm RB, Vincent KA, Khalid S, Berggren G, Greening C | title = Structural basis for bacterial energy extraction from atmospheric hydrogen | journal = Nature | volume = 615 | issue = 7952 | pages = 541–547 | date = March 2023 | pmid = 36890228 | doi = 10.1038/s41586-023-05781-7 | doi-access = free | pmc = 10017518 | bibcode = 2023Natur.615..541G }}</ref><ref>{{cite journal | vauthors = Wilkins A |title=Soil bacteria enzyme generates electricity from hydrogen in the air |journal=New Scientist |date=Mar 8, 2023 |volume=257 |issue=3430 |page=13 |doi=10.1016/S0262-4079(23)00459-1 |bibcode=2023NewSc.257...13W |s2cid=257625443 |url=https://www.newscientist.com/article/2363552-soil-bacteria-enzyme-generates-electricity-from-hydrogen-in-the-air/}}</ref>

=== [FeFe] hydrogenase ===
[[File:FeFe Hydrogenase.png|thumb|right|Crystal structure of [FeFe] hydrogenase]]
The hydrogenases containing a di-iron center with a bridging [[azadithiolate cofactor|dithiolate cofactor]] are called [FeFe] hydrogenases.<ref>{{cite journal | vauthors = Berggren G, Adamska A, Lambertz C, Simmons TR, Esselborn J, Atta M, Gambarelli S, Mouesca JM, Reijerse E, Lubitz W, Happe T, Artero V, Fontecave M | title = Biomimetic assembly and activation of [FeFe]-hydrogenases | journal = Nature | volume = 499 | issue = 7456 | pages = 66–69 | date = July 2013 | pmid = 23803769 | pmc = 3793303 | doi = 10.1038/nature12239 | bibcode = 2013Natur.499...66B }}</ref> Three families of [FeFe] hydrogenases are recognized:
* cytoplasmic, soluble, monomeric hydrogenases, found in strict anaerobes such as ''Clostridium pasteurianum'' and ''Megasphaera elsdenii''. They catalyse both H<sub>2</sub> evolution and uptake.
* periplasmic, heterodimeric hydrogenases from ''Desulfovibrio'' spp., which can be purified aerobically.
* soluble, monomeric hydrogenases, found in chloroplasts of green alga ''Scenedesmus obliquus'', catalyses H<sub>2</sub> evolution. The [Fe<sub>2</sub>S<sub>2</sub>] ferredoxin functions as natural electron donor linking the enzyme to the photosynthetic [[electron transport chain]].

In contrast to [NiFe] hydrogenases, [FeFe] hydrogenases are generally more active in production of molecular hydrogen. Turnover frequency (TOF) in the order of 10,000 s<sup>−1</sup> have been reported in literature for [FeFe] hydrogenases from ''Clostridium pasteurianum''.<ref>{{cite journal | vauthors = Madden C, Vaughn MD, Díez-Pérez I, Brown KA, King PW, Gust D, Moore AL, Moore TA | title = Catalytic turnover of [FeFe]-hydrogenase based on single-molecule imaging | journal = Journal of the American Chemical Society | volume = 134 | issue = 3 | pages = 1577–1582 | date = January 2012 | pmid = 21916466 | doi = 10.1021/ja207461t }}</ref> This has led to intense research focusing on use of [FeFe] hydrogenase for sustainable production of H<sub>2</sub>.<ref>{{cite journal | vauthors = Smith PR, Bingham AS, Swartz JR | title = Generation of hydrogen from NADPH using an [FeFe] hydrogenase | journal=[[International Journal of Hydrogen Energy]] | year=2012 | volume=37 | issue = 3 | pages=2977–2983 | doi=10.1016/j.ijhydene.2011.03.172}}</ref>

The active site of the diiron hydrogenase is known as the H-cluster. The H-cluster consists of a [4Fe4S] cubane-shaped structure, coupled to the low valent diiron co-factor by a cysteine derived thiol. The diiron co-factor includes two iron atoms, connected by a bridging aza-dithiolate ligand (-SCH<sub>2</sub>-NH-CH<sub>2</sub>S-, adt), the iron atoms are coordinated by carbonyl and cyanide ligands.<ref>{{cite journal | vauthors = Németh B, Esmieu C, Redman HJ, Berggren G | title = Monitoring H-cluster assembly using a semi-synthetic HydF protein | journal = Dalton Transactions | volume = 48 | issue = 18 | pages = 5978–5986 | date = May 2019 | pmid = 30632592 | pmc = 6509880 | doi = 10.1039/C8DT04294B | doi-access = free }}</ref>

[FeFe]-hydrogenases can be separated into four distinct [[Phylogenetics|phylogenetic]] groups A−D.<ref name=":0">{{Cite journal| vauthors = Land H, Senger M, Berggren G, Stripp ST |date=2020-05-28|title=Current State of [FeFe]-Hydrogenase Research: Biodiversity and Spectroscopic Investigations |journal=ACS Catalysis|volume=10|issue=13|pages=7069–7086|doi=10.1021/acscatal.0c01614|s2cid=219749715 |issn=2155-5435}}</ref> Group A consists of prototypical and [[electron bifurcation|bifurcating]] [FeFe]-hydrogenases. In nature, prototypical [FeFe]-hydrogenases perform hydrogen [[turnover number|turnover]] using [[ferredoxin]] as a redox partner while bifurcating types perform the same reaction using both ferredoxin and [[NADH|NAD(H)]] as electron donor or acceptor.<ref>{{cite journal | vauthors = Schuchmann K, Chowdhury NP, Müller V | title = Complex Multimeric [FeFe] Hydrogenases: Biochemistry, Physiology and New Opportunities for the Hydrogen Economy | journal = Frontiers in Microbiology | volume = 9 | pages = 2911 | date = 2018-12-04 | pmid = 30564206 | pmc = 6288185 | doi = 10.3389/fmicb.2018.02911 | doi-access = free }}</ref> In order to conserve energy, anaerobic bacteria use [[electron bifurcation]] where [[Exergonic process|exergonic]] and [[Endergonic reaction|endergonic]] redox reactions are coupled to circumvent [[Thermodynamics|thermodynamic barriers]]. Group A comprises the best characterized and catalytically most active enzymes such as the [FeFe]-hydrogenase from ''[[Chlamydomonas reinhardtii]]'' (''Cr''HydA1),<ref>{{cite journal | vauthors = Happe T, Naber JD | title = Isolation, characterization and N-terminal amino acid sequence of hydrogenase from the green alga Chlamydomonas reinhardtii | journal = European Journal of Biochemistry | volume = 214 | issue = 2 | pages = 475–481 | date = June 1993 | pmid = 8513797 | doi = 10.1111/j.1432-1033.1993.tb17944.x | doi-access = free }}</ref> [[Desulfovibrio desulfuricans]] (''Dd''HydAB or ''Dd''H),<ref>{{cite journal | vauthors = Glick BR, Martin WG, Martin SM | title = Purification and properties of the periplasmic hydrogenase from Desulfovibrio desulfuricans | journal = Canadian Journal of Microbiology | volume = 26 | issue = 10 | pages = 1214–1223 | date = October 1980 | pmid = 7006765 | doi = 10.1139/m80-203 }}</ref> and ''[[Clostridium pasteurianum]]'' and ''[[Clostridium acetobutylicum]]'' (''Cp''HydA1 and ''Ca''HydA1, referred to as ''Cp''I and ''Ca''I).<ref>{{cite journal | vauthors = Nakos G, Mortenson L | title = Purification and properties of hydrogenase, an iron sulfur protein, from Clostridium pasteurianum W5 | journal = Biochimica et Biophysica Acta (BBA) - Enzymology | volume = 227 | issue = 3 | pages = 576–583 | date = March 1971 | pmid = 5569125 | doi = 10.1016/0005-2744(71)90008-8 }}</ref> No representative examples of Group B has been characterized yet but it is phylogenetically distinct even when it shares similar amino acid [[Structural motif|motifs]] around the H-cluster as Group A [FeFe]-hydrogenases. Group C has been classified as "sensory" based on the presence of a [[PAS domain|Per-Arnt-Sim domain]].<ref name="Calusinska 1575–1588">{{cite journal | vauthors = Calusinska M, Happe T, Joris B, Wilmotte A | title = The surprising diversity of clostridial hydrogenases: a comparative genomic perspective | journal = Microbiology | volume = 156 | issue = Pt 6 | pages = 1575–1588 | date = June 2010 | pmid = 20395274 | doi = 10.1099/mic.0.032771-0 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Greening C, Biswas A, Carere CR, Jackson CJ, Taylor MC, Stott MB, Cook GM, Morales SE | title = Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival | journal = The ISME Journal | volume = 10 | issue = 3 | pages = 761–777 | date = March 2016 | pmid = 26405831 | pmc = 4817680 | doi = 10.1038/ismej.2015.153 | bibcode = 2016ISMEJ..10..761G }}</ref> One example of a Group C [FeFe]-hydrogenase is from ''[[Thermotoga maritima]]'' (''Tm''HydS) which shows only modest catalytic rates compared to Group A enzymes and an apparent high sensitivity toward hydrogen (H<sub>2</sub>).<ref>{{cite journal | vauthors = Chongdar N, Birrell JA, Pawlak K, Sommer C, Reijerse EJ, Rüdiger O, Lubitz W, Ogata H | title = Unique Spectroscopic Properties of the H-Cluster in a Putative Sensory [FeFe] Hydrogenase | journal = Journal of the American Chemical Society | volume = 140 | issue = 3 | pages = 1057–1068 | date = January 2018 | pmid = 29251926 | doi = 10.1021/jacs.7b11287 }}</ref> A closely related subclass from Group D has a similar location on the bacterial gene and share similar domain structure to a subclass from Group E but it lacks the PAS domain.<ref name=":0" /><ref name="Calusinska 1575–1588"/> Within Group D, the [FeFe]-hydrogenase from ''Thermoanaerobacter mathranii'' (referred to as ''Tam'' HydS) has been characterized.<ref>{{cite journal | vauthors = Land H, Sekretareva A, Huang P, Redman HJ, Németh B, Polidori N, Mészáros LS, Senger M, Stripp ST, Berggren G | title = Characterization of a putative sensory [FeFe]-hydrogenase provides new insight into the role of the active site architecture | journal = Chemical Science | volume = 11 | issue = 47 | pages = 12789–12801 | date = September 2020 | pmid = 34094474 | pmc = 8163306 | doi = 10.1039/D0SC03319G }}</ref> 

=== [Fe]-only hydrogenase ===
[[File:Fe Hydrogenase.png|thumb|left|Crystal structure of [Fe] hydrogenase]]
[[5,10-methenyltetrahydromethanopterin hydrogenase]] (EC [http://enzyme.expasy.org/EC/1.12.98.2 1.12.98.2]) found in [[methanogen]]ic [[Archaea]] contains neither nickel nor iron-sulfur clusters but an iron-containing cofactor that was recently characterized by X-ray diffraction.<ref>{{cite journal | vauthors = Shima S, Pilak O, Vogt S, Schick M, Stagni MS, Meyer-Klaucke W, Warkentin E, Thauer RK, Ermler U | title = The crystal structure of [Fe]-hydrogenase reveals the geometry of the active site | journal = Science | volume = 321 | issue = 5888 | pages = 572–575 | date = July 2008 | pmid = 18653896 | doi = 10.1126/science.1158978 | s2cid = 206513302 | bibcode = 2008Sci...321..572S }}</ref>

Unlike the other two types, [Fe]-only hydrogenases are found only in some hydrogenotrophic methanogenic archaea. They also feature a fundamentally different enzymatic mechanism in terms of redox partners and how electrons are delivered to the active site. In [NiFe] and [FeFe] hydrogenases, electrons travel through a series of metallorganic clusters that comprise a long distance; the active site structures remain unchanged during the whole process. In [Fe]-only hydrogenases, however, electrons are directly delivered to the active site via a short distance. Methenyl-H4MPT<sup>+</sup>, a cofactor, directly accepts the hydride from H<sub>2</sub> in the process. [Fe]-only hydrogenase is also known as H<sub>2</sub>-forming methylenetetrahydromethanopterin (methylene-H4MPT) dehydrogenase, because its function is the reversible reduction of methenyl-H4MPT<sup>+</sup> to methylene-H4MPT.<ref>{{cite journal | vauthors = Salomone-Stagni M, Stellato F, Whaley CM, Vogt S, Morante S, Shima S, Rauchfuss TB, Meyer-Klaucke W | title = The iron-site structure of [Fe]-hydrogenase and model systems: an X-ray absorption near edge spectroscopy study | journal = Dalton Transactions | volume = 39 | issue = 12 | pages = 3057–3064 | date = March 2010 | pmid = 20221540 | pmc = 3465567 | doi = 10.1039/b922557a }}</ref> The hydrogenation of a methenyl-H4MPT+ occurs instead of H<sub>2</sub> oxidation/production, which is the case for the other two types of hydrogenases. While the exact mechanism of the catalysis is still under study, recent finding suggests that molecular hydrogen is first heterolytically cleaved by Fe(II), followed by transfer of hydride to the carbocation of the acceptor.<ref>{{cite journal | vauthors = Shima S, Vogt S, Göbels A, Bill E | title = Iron-chromophore circular dichroism of [Fe]-hydrogenase: the conformational change required for H2 activation | journal = Angewandte Chemie | volume = 49 | issue = 51 | pages = 9917–9921 | date = December 2010 | pmid = 21105038 | doi = 10.1002/anie.201006255 }}</ref>

== Mechanism ==
The molecular mechanism by which protons are converted into hydrogen molecules within hydrogenases is still under extensive study. One popular approach employs mutagenesis to elucidate roles of [[amino acid]]s and/or [[ligand]]s in different steps of catalysis such as intramolecular transport of substrates. For instance, Cornish et al. conducted mutagenesis studies and found out that four amino acids located along the putative channel connecting the active site and protein surface are critical to enzymatic function of [FeFe] hydrogenase from ''Clostridium pasteurianum'' (CpI).<ref>{{cite journal | vauthors = Cornish AJ, Gärtner K, Yang H, Peters JW, Hegg EL | title = Mechanism of proton transfer in [FeFe]-hydrogenase from Clostridium pasteurianum | journal = The Journal of Biological Chemistry | volume = 286 | issue = 44 | pages = 38341–38347 | date = November 2011 | pmid = 21900241 | pmc = 3207428 | doi = 10.1074/jbc.M111.254664 | doi-access = free }}</ref> On the other hand, one can also rely on computational analysis and simulations. Nilsson Lill and Siegbahn have recently taken this approach in investigating the mechanism by which [NiFe] hydrogenases catalyze H<sub>2</sub> cleavage.<ref>{{cite journal | vauthors = Lill SO, Siegbahn PE | title = An autocatalytic mechanism for NiFe-hydrogenase: reduction to Ni(I) followed by oxidative addition | journal = Biochemistry | volume = 48 | issue = 5 | pages = 1056–1066 | date = February 2009 | pmid = 19138102 | doi = 10.1021/bi801218n }}</ref> The two approaches are complementary and can benefit one another. In fact, Cao and Hall combined both approaches in developing the model that describes how hydrogen molecules are oxidized or produced within the active site of [FeFe] hydrogenases.<ref>{{cite journal | vauthors = Cao Z, Hall MB | title = Modeling the active sites in metalloenzymes. 3. Density functional calculations on models for [Fe]-hydrogenase: structures and vibrational frequencies of the observed redox forms and the reaction mechanism at the Diiron Active Center | journal = Journal of the American Chemical Society | volume = 123 | issue = 16 | pages = 3734–3742 | date = April 2001 | pmid = 11457105 | doi = 10.1021/ja000116v }}</ref> While more research and experimental data are required to complete our understanding of the mechanism, these findings have allowed scientists to apply the knowledge in, e.g., building artificial catalysts mimicking active sites of hydrogenases.<ref>{{cite journal | vauthors = Tard C, Liu X, Ibrahim SK, Bruschi M, De Gioia L, Davies SC, Yang X, Wang LS, Sawers G, Pickett CJ | title = Synthesis of the H-cluster framework of iron-only hydrogenase | journal = Nature | volume = 433 | issue = 7026 | pages = 610–613 | date = February 2005 | pmid = 15703741 | doi = 10.1038/nature03298 | s2cid = 4430994 | bibcode = 2005Natur.433..610T }}</ref>

== Biological function ==
Assuming that the Earth's atmosphere was initially rich in hydrogen, scientists hypothesize that hydrogenases were evolved to generate energy from/as molecular H<sub>2</sub>. Accordingly, hydrogenases can either help microorganisms to proliferate under such conditions, or to set up ecosystems empowered by H<sub>2</sub>.<ref>{{cite journal | vauthors = Vignais PM, Billoud B | title = Occurrence, classification, and biological function of hydrogenases: an overview | journal = Chemical Reviews | volume = 107 | issue = 10 | pages = 4206–4272 | date = October 2007 | pmid = 17927159 | doi = 10.1021/cr050196r }}</ref> Microbial communities driven by molecular hydrogen have, in fact, been found in deep-sea settings where other sources of energy from [[photosynthesis]] are not available. Based on these grounds, the primary role of hydrogenases are believed to be energy generation, and this can be sufficient to sustain an ecosystem.

Recent studies have revealed other biological functions of hydrogenases. To begin with, bidirectional hydrogenases can also act as "valves" to control excess reducing equivalents, especially in photosynthetic microorganisms. Such a role makes hydrogenases play a vital role in [[Glycolysis|anaerobic metabolism]].<ref>{{cite journal | vauthors = Adams MW, Stiefel EI | title = Biological hydrogen production: not so elementary | journal = Science | volume = 282 | issue = 5395 | pages = 1842–1843 | date = December 1998 | pmid = 9874636 | doi = 10.1126/science.282.5395.1842 | s2cid = 38018712 }}</ref><ref>{{cite journal | vauthors = Frey M | title = Hydrogenases: hydrogen-activating enzymes | journal = ChemBioChem | volume = 3 | issue = 2–3 | pages = 153–160 | date = March 2002 | pmid = 11921392 | doi = 10.1002/1439-7633(20020301)3:2/3<153::AID-CBIC153>3.0.CO;2-B | s2cid = 36754174 | doi-access = free }}</ref> Moreover, hydrogenases may also be involved in membrane-linked energy conservation through the generation of a transmembrane protonmotive force.<sup>[15]</sup>There is a possibility that hydrogenases have been responsible for [[bioremediation]] of chlorinated compounds.  Hydrogenases proficient in H<sub>2</sub> uptake can help heavy metal contaminants to be recovered in intoxicated forms. These uptake hydrogenases have been recently discovered in pathogenic bacteria and parasites and are believed to be involved in their virulence.<sup>[15]</sup>

== Applications ==
Hydrogenases were first discovered in the 1930s,<ref name="Thauer_1998">{{cite journal | vauthors = Thauer RK | title = Biochemistry of methanogenesis: a tribute to Marjory Stephenson. 1998 Marjory Stephenson Prize Lecture | journal = Microbiology (Reading, England) | volume = 144 | issue = Pt 9 | pages = 2377–2406 | date = September 1998 | pmid = 9782487 | doi = 10.1099/00221287-144-9-2377 | doi-access = free }}</ref> and they have since attracted interest from many researchers including [[inorganic chemistry|inorganic chemists]] who have synthesized a variety of [[hydrogenase mimic]]s.  The soluble [NiFe] hydrogenase from ''[[Ralstonia eutropha]]'' H16 is a promising candidate enzyme for H<sub>2</sub>-based biofuel application as it favours H<sub>2</sub> oxidation and is relatively oxygen-tolerant. It can be produced on [[heterotroph]]ic growth media<ref name="Jugder 42"/> and purified via [[Anion-exchange chromatography|anion exchange]] and [[Size-exclusion chromatography|size exclusion chromatography]] matrices.<ref name="Jugder 242–250"/> Understanding the catalytic mechanism of hydrogenase might help scientists design clean biological energy sources, such as algae, that produce hydrogen.<ref>{{cite journal | vauthors = Florin L, Tsokoglou A, Happe T | title = A novel type of iron hydrogenase in the green alga Scenedesmus obliquus is linked to the photosynthetic electron transport chain | journal = The Journal of Biological Chemistry | volume = 276 | issue = 9 | pages = 6125–6132 | date = March 2001 | pmid = 11096090 | doi = 10.1074/jbc.M008470200 | doi-access = free }}</ref>

=== Biological hydrogen production ===
Various systems are capable of splitting water into O<sub>2</sub> and H<sup>+</sup> from incident sunlight. Likewise, numerous catalysts, either chemical or biological, can reduce the produced H<sup>+</sup> into H<sub>2</sub>. Different catalysts require unequal overpotential for this reduction reaction to take place. Hydrogenases are attractive since they require a relatively low [[overpotential]]. In fact, its catalytic activity is more effective than platinum, which is the best known catalyst for H<sub>2</sub> evolution reaction.<ref>{{cite journal | vauthors = Hinnemann B, Moses PG, Bonde J, Jørgensen KP, Nielsen JH, Horch S, Chorkendorff I, Nørskov JK | title = Biomimetic hydrogen evolution: MoS2 nanoparticles as catalyst for hydrogen evolution | journal = Journal of the American Chemical Society | volume = 127 | issue = 15 | pages = 5308–5309 | date = April 2005 | pmid = 15826154 | doi = 10.1021/ja0504690 }}</ref> Among three different types of hydrogenases, [FeFe] hydrogenases is considered as a strong candidate for an integral part of the solar H<sub>2</sub> production system since they offer an additional advantage of high TOF (over 9000 s<sup>−1</sup>)<sup>[6]</sup>.

Low overpotential and high catalytic activity of [FeFe] hydrogenases are accompanied by high O<sub>2</sub> sensitivity. It is necessary to engineer them O<sub>2</sub>-tolerant for use in solar H<sub>2</sub> production since O<sub>2</sub> is a by-product of [[water splitting]] reaction. Past research efforts by various groups around the world have focused on understanding the mechanisms involved in O<sub>2</sub>-inactivation of hydrogenases.<ref name="pmid19966788">{{cite journal | vauthors = Liebgott PP, Leroux F, Burlat B, Dementin S, Baffert C, Lautier T, Fourmond V, Ceccaldi P, Cavazza C, Meynial-Salles I, Soucaille P, Fontecilla-Camps JC, Guigliarelli B, Bertrand P, Rousset M, Léger C | title = Relating diffusion along the substrate tunnel and oxygen sensitivity in hydrogenase | journal = Nature Chemical Biology | volume = 6 | issue = 1 | pages = 63–70 | date = January 2010 | pmid = 19966788 | doi = 10.1038/nchembio.276 }}</ref><ref>{{cite journal | vauthors = Goris T, Wait AF, Saggu M, Fritsch J, Heidary N, Stein M, Zebger I, Lendzian F, Armstrong FA, Friedrich B, Lenz O | title = A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]-hydrogenase | journal = Nature Chemical Biology | volume = 7 | issue = 5 | pages = 310–318 | date = May 2011 | pmid = 21390036 | doi = 10.1038/nchembio.555 }}</ref> For instance, Stripp et al. relied on protein film electrochemistry and discovered that O<sub>2</sub> first converts into a reactive species at the active site of [FeFe] hydrogenases, and then damages its [4Fe-4S] domain.<ref>{{cite journal | vauthors = Stripp ST, Goldet G, Brandmayr C, Sanganas O, Vincent KA, Haumann M, Armstrong FA, Happe T | title = How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 106 | issue = 41 | pages = 17331–17336 | date = October 2009 | pmid = 19805068 | pmc = 2765078 | doi = 10.1073/pnas.0905343106 | doi-access = free | bibcode = 2009PNAS..10617331S }}</ref> Cohen et al. investigated how oxygen can reach the active site that is buried inside the protein body by molecular dynamics simulation approach; their results indicate that O<sub>2</sub> diffuses through mainly two pathways that are formed by enlargement of and interconnection between cavities during dynamic motion.<ref>{{cite journal | vauthors = Cohen J, Kim K, King P, Seibert M, Schulten K | title = Finding gas diffusion pathways in proteins: application to O2 and H2 transport in CpI [FeFe]-hydrogenase and the role of packing defects | journal = Structure | volume = 13 | issue = 9 | pages = 1321–1329 | date = September 2005 | pmid = 16154089 | doi = 10.1016/j.str.2005.05.013 | doi-access = free }}</ref> These works, in combination with other reports, suggest that inactivation is governed by two phenomena: [[diffusion]] of O<sub>2</sub> to the active site, and destructive modification of the active site.

Despite these findings, research is still under progress for engineering oxygen tolerance in hydrogenases. While researchers have found oxygen-tolerant [NiFe] hydrogenases, they are only efficient in hydrogen uptake and not production<sup>[21]</sup>. Bingham et al.'s recent success in engineering [FeFe] hydrogenase from ''Clostridium pasteurianum'' was also limited to retained activity (during exposure to oxygen) for H<sub>2</sub> consumption, only.<ref>{{cite journal | vauthors = Bingham AS, Smith PR, Swartz JR | title=Evolution of an [FeFe] hydrogenase with decreased oxygen sensitivity | journal=International Journal of Hydrogen Energy | year=2012 | volume=37 | issue=3 | pages=2965–2976 | doi=10.1016/j.ijhydene.2011.02.048}}</ref>

=== Hydrogenase-based biofuel cells ===
Typical [[enzymatic biofuel cell]]s involve the usage of enzymes as [[electrocatalyst]]s at either both cathode and anode or at one electrode. In hydrogenase-based [[biofuel]] cells, hydrogenase enzymes are present at the anode for H<sub>2</sub> oxidation.<ref name="Jugder 242–250"/><ref name="Jugder 8142"/><ref name=Lubitz>{{cite journal | vauthors = Lubitz W, Ogata H, Rüdiger O, Reijerse E | title = Hydrogenases | journal = Chemical Reviews | volume = 114 | issue = 8 | pages = 4081–4148 | date = April 2014 | pmid = 24655035 | doi = 10.1021/cr4005814 }}</ref>

==== Principle ====
The bidirectional or [[reversible reaction]] catalyzed by hydrogenase allows for the capture and storage of renewable energy as fuel with use on demand. This can be demonstrated through the chemical storage of electricity obtained from a renewable source (e.g. solar, wind, [[hydrothermal]]) as H<sub>2</sub> during periods of low energy demands. When energy is desired, H<sub>2</sub> can be oxidized to produce electricity.<ref name=Lubitz />

==== Advantages ====
This is one solution to the challenge in the development of technologies for the capture and storage of [[renewable]] energy as fuel with use on demand. The generation of electricity from H<sub>2</sub> is comparable with the similar functionality of [[Platinum]] catalysts minus the catalyst poisoning, and thus is very efficient. In the case of H<sub>2</sub>/O<sub>2</sub> fuel cells, where the product is water, there is no [[Greenhouse gas emissions|production of greenhouse gases]].<ref name=Lubitz />

== Biochemical classification ==
;[[Enzyme Commission number|EC]] [http://enzyme.expasy.org/EC/1.12.1.2 1.12.1.2] 
[[hydrogen dehydrogenase]] (hydrogen:NAD<sup>+</sup> oxidoreductase)
: H<sub>2</sub> + NAD<sup>+</sup> {{eqm}} H<sup>+</sup> + NADH

;EC [http://enzyme.expasy.org/EC/1.12.1.3 1.12.1.3] 
hydrogen dehydrogenase (NADP) (hydrogen:NADPH<sup>+</sup> oxidoreductase)
: H<sub>2</sub> + NADP<sup>+</sup> {{eqm}} H<sup>+</sup> + NADPH

;EC [http://enzyme.expasy.org/EC/1.12.2.1 1.12.2.1] 
[[Cytochrome-c3 hydrogenase|cytochrome-''c''<sub>3</sub> hydrogenase]] (hydrogen:ferricytochrome-''c''<sub>3</sub> oxidoreductase)
: 2H<sub>2</sub> + ferricytochrome ''c''<sub>3</sub> {{eqm}} 4H<sup>+</sup> + ferrocytochrome ''c''<sub>3</sub>

;EC [http://enzyme.expasy.org/EC/1.12.5.1 1.12.5.1] 
[[hydrogen:quinone oxidoreductase]]
: H<sub>2</sub> + menaquinone {{eqm}} menaquinol

;EC [http://enzyme.expasy.org/EC/1.12.7.2 1.12.7.2] 
[[ferredoxin hydrogenase]] (hydrogen:ferredoxin oxidoreductase)
: H<sub>2</sub> + oxidized ferredoxin {{eqm}} 2H<sup>+</sup> + reduced ferredoxin

;EC [http://enzyme.expasy.org/EC/1.12.98.1 1.12.98.1] 
[[Coenzyme F420 hydrogenase|coenzyme F<sub>420</sub> hydrogenase]] (hydrogen:coenzyme F<sub>420</sub> oxidoreductase)
: H<sub>2</sub> + coenzyme F<sub>420</sub> {{eqm}} reduced coenzyme F<sub>420</sub>

;EC [http://enzyme.expasy.org/EC/1.12.99.6 1.12.99.6] 
hydrogenase (acceptor) (hydrogen:acceptor oxidoreductase)
: H<sub>2</sub> + A {{eqm}} AH<sub>2</sub>

;EC [http://enzyme.expasy.org/EC/1.12.98.2 1.12.98.2] 
5,10-methenyltetrahydromethanopterin hydrogenase (hydrogen:5,10-methenyltetrahydromethanopterin oxidoreductase)
: H<sub>2</sub> + 5,10-methenyltetrahydromethanopterin {{eqm}} H<sup>+</sup> + 5,10-methylenetetrahydromethanopterin

;EC [http://enzyme.expasy.org/EC/1.12.98.3 1.12.98.3] 
''Methanosarcina''-phenazine hydrogenase [hydrogen:2-(2,3-dihydropentaprenyloxy)phenazine oxidoreductase]
: H<sub>2</sub> + 2-(2,3-dihydropentaprenyloxy)phenazine {{eqm}} 2-dihydropentaprenyloxyphenazine

== References ==
{{reflist|2}}

== External links ==
* [http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=2B0J 2B0J] {{Webarchive|url=https://web.archive.org/web/20080124181058/http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=2B0J |date=2008-01-24 }} - [[Protein Data Bank|PDB]] Structure of the Apoenzyme of the Iron-sulphur cluster-free hydrogenase from ''Methanothermococcus jannaschii''
* [http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1HFE 1HFE] {{Webarchive|url=https://web.archive.org/web/20090116051605/http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1HFE |date=2009-01-16 }} - [[Protein Data Bank|PDB]] structure of [FeFe]-hydrogenase from ''Desulfovibrio desulfuricans''
* [http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1C4A 1C4A] {{Webarchive|url=https://web.archive.org/web/20090116042611/http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1C4A |date=2009-01-16 }} - PDB structure of [FeFe]-hydrogenase from ''Clostridium pasteurianum''
* [http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1UBR 1UBR] {{Webarchive|url=https://web.archive.org/web/20080124181051/http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1UBR |date=2008-01-24 }} - PDB structure of [NiFe]-hydrogenase from ''Desulfovibrio vulgaris''
* [http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1CC1 1CC1] {{Webarchive|url=https://web.archive.org/web/20080124181039/http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1CC1 |date=2008-01-24 }} - PDB structure of [NiFeSe]-hydrogenase from  ''Desulfomicrobium baculatum''
* [https://web.archive.org/web/20050422121233/http://www.kcl.ac.uk/ip/richardcammack/H2/animation/movie1.html Animation] - Mechanism of [NiFe]-hydrogenase

{{Hydrogen oxidoreductases}}
{{Enzymes}}
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[[Category:Iron–sulfur proteins]]
[[Category:EC 1.12]]
[[Category:EC 1.2.1]]
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