# Hydrogenase
**Hydrogenase** is a metalloenzyme that catalyzes the reversible interconversion of molecular hydrogen, protons, and electrons, H₂ ⇌ 2H⁺ + 2e⁻, using earth-abundant metals rather than precious metals such as platinum[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC6414374/) • [2](https://www.sciencedirect.com/topics/neuroscience/hydrogenase). The name was proposed in the 1930s by Stephenson and Stickland for the enzyme system responsible for hydrogen metabolism in *Escherichia coli*[3](https://pure.uva.nl/ws/files/3505968/20576_UBA002000594_06.pdf). Hydrogenases occur mainly in Archaea and Bacteria, with a few examples in Eucarya, and they play a central role in microbial energy metabolism, either providing energy, reducing cofactors, or dispersing reducing equivalents generated through anaerobic processes[4](https://pubmed.ncbi.nlm.nih.gov/11524134/) • [1](https://pmc.ncbi.nlm.nih.gov/articles/PMC6414374/).
| Key fact | Detail |
|---|---|
| Reaction | H₂ ⇌ 2H⁺ + 2e⁻, catalyzed reversibly under ambient conditions and neutral pH[1](https://pmc.ncbi.nlm.nih.gov/articles/PMC6414374/) • [5](https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/celc.202400700) |
| Classes | Three phylogenetically distinct classes, [NiFe], [FeFe], and [Fe] hydrogenases, defined by the metal composition of the active site[4](https://pubmed.ncbi.nlm.nih.gov/11524134/) • [2](https://www.sciencedirect.com/topics/neuroscience/hydrogenase) |
| Catalytic bias | [NiFe] enzymes are generally biased toward H₂ oxidation; [FeFe] enzymes toward H₂ production, with turnover frequencies up to 10,000 H₂ s⁻¹[5](https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/celc.202400700) • [6](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919214/) |
| Unusual ligands | The active-site iron carries nonprotein diatomic ligands, carbon monoxide and cyanide, in both [NiFe] and [FeFe] enzymes[2](https://www.sciencedirect.com/topics/neuroscience/hydrogenase) |
| Oxygen sensitivity | Most hydrogenases are rapidly inactivated by O₂; [FeFe] enzymes are usually damaged irreversibly, while [NiFe] enzymes mostly react reversibly[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC6212873/) |
| O₂-tolerance mechanism | O₂-tolerant membrane-bound [NiFe] hydrogenases carry a unique [4Fe–3S] proximal cluster coordinated by six cysteines that stores two electrons for rapid recovery from O₂ attack[8](https://www.nature.com/articles/nrmicro2940) • [9](https://pubmed.ncbi.nlm.nih.gov/27493211/) |
| Applications | Electrocatalysis, fuel cells, and biohydrogen production, limited chiefly by O₂ sensitivity[10](https://pmc.ncbi.nlm.nih.gov/articles/PMC10657181/) • [11](https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/bit.27136) |
## Classes and active sites
Hydrogenases are divided into [NiFe], [FeFe], and [Fe] classes according to the metal content of the catalytic center, and the classes are phylogenetically unrelated, a case of convergent evolution of the same chemical reaction[2](https://www.sciencedirect.com/topics/neuroscience/hydrogenase) • [1](https://pmc.ncbi.nlm.nih.gov/articles/PMC6414374/). The catalytic core of [NiFe] hydrogenases is a heterodimeric protein, often with additional subunits, and the active site is a dinuclear Ni–Fe center carrying CO and CN⁻ ligands[4](https://pubmed.ncbi.nlm.nih.gov/11524134/) • [9](https://pubmed.ncbi.nlm.nih.gov/27493211/). In both [NiFe] and [FeFe] enzymes, electrons are transferred through chains of iron–sulfur centers, whereas [Fe] hydrogenases have no iron–sulfur centers and catalyze a specific reaction that uses H₂ rather than reversible H₂ oxidation[2](https://www.sciencedirect.com/topics/neuroscience/hydrogenase).
The [FeFe] active site, the H-cluster, is a hexanuclear iron complex consisting of a conventional [4Fe–4S] cluster linked to a unique [2Fe] subcluster bearing two CN⁻ ligands, three CO ligands, and a dithiolate bridge with a central atom X whose identity is ambiguous in X-ray structures; a nitrogen assignment is supported by the assembly of active enzyme after incorporation of a synthetic [2Fe] subcluster with an azadithiolate bridge into apoenzyme[12](https://ora.ox.ac.uk/objects/uuid%3A3c06b35b-9fd6-41a8-ae5d-97bb3a3adc98/files/m8fecd9da57d1e5dc999ff7b1e14073d9) • [6](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919214/). Assembling this cofactor requires a dedicated maturation machinery of three conserved proteins, HydE, HydG, and HydF, first identified in *Chlamydomonas reinhardtii* and since found in all organisms containing a [FeFe] hydrogenase[7](https://pmc.ncbi.nlm.nih.gov/articles/PMC6212873/).
## Catalytic bias and performance
The two main bimetallic classes split the work of hydrogen metabolism. Most [NiFe] hydrogenases are of the hydrogen uptake type, biased toward H₂ oxidation, while [FeFe] hydrogenases mainly produce molecular hydrogen[2](https://www.sciencedirect.com/topics/neuroscience/hydrogenase). [FeFe] enzymes reach turnover frequencies of up to 10,000 H₂ s⁻¹, and [FeFe]-hydrogenases show H₂ release activity of up to 8,000 μmol H₂ min⁻¹ mg⁻¹, an efficiency attributed to the design of the H-cluster[5](https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/celc.202400700) • [7](https://pmc.ncbi.nlm.nih.gov/articles/PMC6212873/). In a comparative table of hydrogen production rate, [NiFe] hydrogenases run about 100-fold lower than [FeFe] enzymes, and nitrogenases 3–4 fold lower than [FeFe] enzymes[6](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919214/).
## Oxygen sensitivity and tolerance
Oxygen is the main obstacle to using hydrogenases. Most hydrogenases isolated so far, and all the highly active ones, are inhibited by O₂, and there is no single parameter such as one rate constant that quantifies an overall sensitivity, because the reactions of hydrogenases with O₂ vary greatly between and within families[13](https://pmc.ncbi.nlm.nih.gov/articles/PMC11023054/). Dioxygen irreversibly damages the active site of many [FeFe] hydrogenases, in a complex multistep reaction in which reactive oxygen species form and cause dissociation of the neighboring [4Fe–4S] sub-cluster; [NiFe] active sites are instead oxidized into inactive species, called "ready" and "unready", that can be distinguished by how quickly they reactivate under reducing conditions[13](https://pmc.ncbi.nlm.nih.gov/articles/PMC11023054/) • [14](https://www.jbc.org/content/291/41/21563.full.pdf).
A subgroup of [NiFe] hydrogenases, found mainly in aerobic H₂-oxidizing bacteria, sustains H₂ cycling at ambient O₂ levels, which drives the chemolithoautotrophic lifestyle of their hosts[8](https://www.nature.com/articles/nrmicro2940). Their tolerance depends largely on a unique proximal [4Fe–3S] cluster coordinated by six cysteine residues, two more than the standard [4Fe–4S]–4Cys cluster, which can supply two electrons to the active site and enable rapid recovery from O₂ inactivation[8](https://www.nature.com/articles/nrmicro2940) • [9](https://pubmed.ncbi.nlm.nih.gov/27493211/) • [6](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919214/). Notably, oxygen-tolerant, oxygen-sensitive, and other nonstandard [NiFe] hydrogenases employ very similar active sites for catalysis, so the difference lies outside the Ni–Fe center itself[15](https://www.sciencedirect.com/science/article/pii/S000527281300025X).
[Access control](https://www.edgepedia.org/access-control) is a second strategy. A computational comparison of group 1 [NiFe] hydrogenases led to the hypothesis that O₂ sensitivity correlates with a more complex tunnel matrix into the protein, with more openings toward the solvent; in O₂-tolerant [NiFe] enzymes, two conserved hydrophobic residues (Val and Leu) at the end of the hydrophobic channel are replaced by the larger Ile and Phe[13](https://pmc.ncbi.nlm.nih.gov/articles/PMC11023054/) • [6](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919214/). [Carbon monoxide](https://www.edgepedia.org/carbon-monoxide), a competitive inhibitor that uses the same gas channel as O₂, can be used to measure access rates electrochemically[13](https://pmc.ncbi.nlm.nih.gov/articles/PMC11023054/).
Oxygen tolerance also exists outside the [NiFe] class, though it was long underestimated. For a long time [FeFe] hydrogenases were assumed to lose activity irreversibly and very quickly on O₂ exposure, but characterization of novel enzymes has shown that sensitivity is highly variable across the class and several oxygen-stable examples exist. In *Clostridium beijerinckii* SM10 (CbA5H), a conformational change allows a conserved cysteine to bind the H-cluster directly, forming a protected inactive state, and the heterotetrameric bifurcating enzyme DfHnd from *Desulfovibrio fructosovorans* is likewise protected from oxygen damage[16](https://pmc.ncbi.nlm.nih.gov/articles/PMC8924675/). The [Fe] hydrogenases are reported as resistant to O₂[6](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919214/).
## Engineering and applications
Because O₂ sensitivity has seriously limited the use of recombinant hydrogenases as biotechnological tools, protein engineering aims to improve tolerance. The CpI M4 mutant (CpIᴺ¹⁶⁰ᴰᴵ¹⁹⁷ᵛᴬ²⁸⁰ⱽᴺ²⁸⁹ᴰ) retained 62% activity after exposure to 1% O₂ for 5 minutes, against 23% for the wild type, and the CpIᵀ³⁵⁶ⱽ/ˢ³⁵⁷ᵀ variant showed an equivalent aerobic hydrogen production rate in the presence of 5% O₂[6](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919214/). O₂-tolerant [NiFe] hydrogenases have been found in *Ralstonia eutropha*, *Aquifex aeolicus*, *Escherichia coli*, and *Desulfovibrio fructosovorans*, and the KoHyd3 enzyme from *Klebsiella oxytoca* HP1 showed substantial hydrogen evolution under 10–20% O₂ in the gas phase[6](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919214/).
The main applications are electrocatalytic. [NiFe] hydrogenases have been developed for fuel cells, where their O₂ tolerance can mitigate the need for a gas-impermeable membrane separating the electrodes, opening possibilities for miniaturization[10](https://pmc.ncbi.nlm.nih.gov/articles/PMC10657181/) • [17](https://portlandpress.com/biochemsoctrans/article-pdf/doi/10.1042/BST20230120/950228/bst-2023-0120c.pdf). [FeFe] enzymes are more desirable for electroenzymatic H₂ production because of their high turnover frequencies and at least one surface-exposed redox cofactor, which makes enzyme wiring easier than for enzymes such as glucose oxidase[5](https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/celc.202400700).
## References
1. [Molecular Hydrogen, a Neglected Key Driver of Soil Biogeochemical Processes, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC6414374/)
2. [Hydrogenases, Structure and Function, ScienceDirect Topics (Lubitz & Ogata, Encyclopedia of Biological Chemistry)](https://www.sciencedirect.com/topics/neuroscience/hydrogenase)
3. [Chapter one, Universiteit van Amsterdam thesis on hydrogenase history](https://pure.uva.nl/ws/files/3505968/20576_UBA002000594_06.pdf)
4. [Vignais, Billoud & Meyer (2001). Classification and phylogeny of hydrogenases. FEMS Microbiology Reviews](https://pubmed.ncbi.nlm.nih.gov/11524134/)
5. [Webb et al. (2025). Towards Scalable Electroenzymatic Hydrogen Production with [FeFe]-Hydrogenase. ChemElectroChem](https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/celc.202400700)
6. [Hydrogenase and Nitrogenase: Key Catalysts in Biohydrogen Production, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC9919214/)
7. [Overview of the Maturation Machinery of the H-Cluster of [FeFe]-Hydrogenases with a Focus on HydF, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC6212873/)
8. [Fritsch, Lenz & Friedrich (2013). Structure, function and biosynthesis of O₂-tolerant hydrogenases. Nature Reviews Microbiology](https://www.nature.com/articles/nrmicro2940)
9. [Structure and function of [NiFe] hydrogenases, PubMed](https://pubmed.ncbi.nlm.nih.gov/27493211/)
10. [Developing high-affinity, oxygen-insensitive [NiFe]-hydrogenases as biocatalysts for energy conversion, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC10657181/)
11. [Lu & Koo (2019). O₂ sensitivity and H₂ production activity of hydrogenases—A review. Biotechnology and Bioengineering](https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/bit.27136)
12. [Oxford Research Archive: [FeFe] hydrogenase H-cluster and azadithiolate bridge](https://ora.ox.ac.uk/objects/uuid%3A3c06b35b-9fd6-41a8-ae5d-97bb3a3adc98/files/m8fecd9da57d1e5dc999ff7b1e14073d9)
13. [Outer-sphere effects on the O₂ sensitivity, catalytic bias and catalytic reversibility of hydrogenases, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC11023054/)
14. [Koo et al. (2016). Characterization of [FeFe] Hydrogenase O₂ Sensitivity Using a New, Physiological Approach. Journal of Biological Chemistry](https://www.jbc.org/content/291/41/21563.full.pdf)
15. [Vincent et al. (2013). [NiFe] hydrogenases: A common active site for hydrogen metabolism under diverse conditions. BBA Bioenergetics](https://www.sciencedirect.com/science/article/pii/S000527281300025X)
16. [Fantastic [FeFe]-Hydrogenases and Where to Find Them, PMC](https://pmc.ncbi.nlm.nih.gov/articles/PMC8924675/)
17. [Biochemical Society Transactions: [NiFe]-hydrogenases in electrocatalysis and fuel cells](https://portlandpress.com/biochemsoctrans/article-pdf/doi/10.1042/BST20230120/950228/bst-2023-0120c.pdf)
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*Topic: Encyclopedia › Life and health › Biological foundations › Biochemistry and metabolism › Metabolism and metabolic pathways › Elemental and cofactor metabolism › Trace elements and metalloids › Nickel and cobalt metabolism*
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