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A hydrogenase is an enzyme that Catalysis the reversible Redox of molecular hydrogen (H2), as shown below:
Hydrogen oxidation () is coupled to the reduction of electron acceptors such as oxygen, nitrate, Ferric, sulfate, carbon dioxide (), and fumarate. On the other hand, proton reduction () 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 c3, and cytochrome c6 can act as physiological electron donors or acceptors for hydrogenases.
Hydrogenases are sub-classified into three different types based on the active site metal content: nickel-iron hydrogenase, iron-iron hydrogenase, and iron hydrogenase.
Hydrogenases catalyze, sometimes reversibly, H2 uptake. The FeFe and NiFe hydrogenases are true redox catalysts, driving H2 oxidation and proton (H+) reduction (equation ), the Fe hydrogenases catalyze the reversible heterolytic cleavage of H2 shown by reaction ().
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−) ligands.
Like FeFe hydrogenases, NiFe hydrogenases are known to be usually deactivated by molecular oxygen (O2). Hydrogenase from Ralstonia eutropha, and several other so-called Knallgas-bacteria, were found to be oxygen-tolerant. The soluble NiFe hydrogenase from Ralstonia eutropha H16 can be conveniently produced on growth media. 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, 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.
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−1 have been reported in literature for FeFe hydrogenases from Clostridium pasteurianum. This has led to intense research focusing on use of FeFe hydrogenase for sustainable production of H2.
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 (-SCH2-NH-CH2S-, adt), the iron atoms are coordinated by carbonyl and cyanide ligands.
FeFe-hydrogenases can be separated into four distinct Phylogenetics groups A−D. Group A consists of prototypical and bifurcating FeFe-hydrogenases. In nature, prototypical FeFe-hydrogenases perform hydrogen turnover number using ferredoxin as a redox partner while bifurcating types perform the same reaction using both ferredoxin and NADH as electron donor or acceptor. In order to conserve energy, anaerobic bacteria use electron bifurcation where exergonic and endergonic redox reactions are coupled to circumvent Thermodynamics. Group A comprises the best characterized and catalytically most active enzymes such as the FeFe-hydrogenase from Chlamydomonas reinhardtii ( CrHydA1), Desulfovibrio desulfuricans ( DdHydAB or DdH), and Clostridium pasteurianum and Clostridium acetobutylicum ( CpHydA1 and CaHydA1, referred to as CpI and CaI). No representative examples of Group B has been characterized yet but it is phylogenetically distinct even when it shares similar amino acid Structural motif around the H-cluster as Group A FeFe-hydrogenases. Group C has been classified as "sensory" based on the presence of a PAS domain. One example of a Group C FeFe-hydrogenase is from Thermotoga maritima ( TmHydS) which shows only modest catalytic rates compared to Group A enzymes and an apparent high sensitivity toward hydrogen (H2). 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. Within Group D, the FeFe-hydrogenase from Thermoanaerobacter mathranii (referred to as Tam HydS) has been characterized.
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+, a cofactor, directly accepts the hydride from H2 in the process. Fe-only hydrogenase is also known as H2-forming methylenetetrahydromethanopterin (methylene-H4MPT) dehydrogenase, because its function is the reversible reduction of methenyl-H4MPT+ to methylene-H4MPT. The hydrogenation of a methenyl-H4MPT+ occurs instead of H2 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.
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. Moreover, hydrogenases may also be involved in membrane-linked energy conservation through the generation of a transmembrane protonmotive force.15There is a possibility that hydrogenases have been responsible for bioremediation of chlorinated compounds. Hydrogenases proficient in H2 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.15
Low overpotential and high catalytic activity of FeFe hydrogenases are accompanied by high O2 sensitivity. It is necessary to engineer them O2-tolerant for use in solar H2 production since O2 is a by-product of water splitting reaction. Past research efforts by various groups worldwide have focused on understanding the mechanisms involved in O2-inactivation of hydrogenases. For instance, Stripp et al. relied on protein film electrochemistry and discovered that O2 first converts into a reactive species at the active site of FeFe hydrogenases, and then damages its 4Fe-4S domain. 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 O2 diffuses through mainly two pathways that are formed by enlargement of and interconnection between cavities during dynamic motion. These works, in combination with other reports, suggest that inactivation is governed by two phenomena: diffusion of O2 to the active site, and destructive modification of the active site.
Despite these findings, research is still underway to engineer oxygen tolerance in hydrogenases. While researchers have found oxygen-tolerant NiFe hydrogenases, they are only efficient in hydrogen uptake and not production21. Bingham et al.'s recent success in engineering FeFe hydrogenase from Clostridium pasteurianum was also limited to retained activity (during exposure to oxygen) for H2 consumption only.
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