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The Armstrong Research Group
Inorganic Chemistry Laboratory

Third floor ICL, rooms: T7–T12, T17
Phone: T12 (Fraser’s office): +44 (0)1865 272647
e-mail: fraser.armstrong@chem.ox.ac.uk




Hydrogenases

Hydrogenases are enzymes that catalyse the reversible oxidation of H2 to protons at close to the thermodynamic potential. The reaction takes place at a bimetallic active site consisting of Fe atoms ('[FeFe]-hydrogenases') or one Ni and one Fe atom ('[NiFe]-hydrogenases'), coordinated by biologically unusual CO and CN&ndash ligands.

[NiFe]-Hydrogenases

The [NiFe]-hydrogenases typically consist of an heterodimer, with the active site contained in the larger of the two subunits and an [FeS]-cluster electron relay system running through the smaller subunit, although multimeric proteins have been isolated. The size of both the large and small subunits tends to be consistent across different enzymes, at approximately 60 kDa and 30 kDa, respectively. By acting in sequence, the [FeS]-clusters provide an electron-transfer relay system, allowing electron 'hopping' through the protein matrix. Gas channels may provide a route for H2 to travel between the active site and the exterior of the protein, and a number of residues capable of being easily (de)protonated form a proton transfer pathway.

[NiFe]-hydrogenases exist in a complex variety of spectroscopically-distinct states. Most [NiFe]-hydrogenases react with O2 under electron deficient conditions to form a state that is slow to reactivate under reductive conditions. This is generally described as the 'Unready' state (the term Unready is indicative of the long time required to reactivate it); however, Unready is in fact a general term describing two interconvertible states known as Ni-A (or Niu*) and Ni-SU (or Niu-S). In the Unready state, a peroxide ligand is belived to bind in a bridging position between the Ni and Fe atoms. Under oxidising potentials, [NiFe]-hydrogenases undergo anaerobic inactivation to form the 'Ready' state (this state is also formed by reaction with O2 under electron-rich conditions). In this state, a hydroxide ligand is bound in the bridging position. Analogously with the Unready state, the Ready state consistes of distinct substates; however the Ready state reactivates much faster than the Unready state. [NiFe]-hydrogenases also react with inhibitors such as CO and sulphide.

[FeFe]-Hydrogenases

In many respects, the [FeFe]-hydrogenases are comparable to the [NiFe]-hydrogenases. Thei also possess one or more [FeS]-clusters, although the proximal cluster is ligated to the active site, which is known as the 'H-cluster'. Both monomoric and dimeric [FeFe]-hydrogenases are known. Analogously with the [NiFe]-hydrogenases, the [FeFe]-hydrogenases can access a number of spectroscopically distinct active-site states. Specifically, two 'active' states are formed (known as Hox and Hred respectively. Further oxidation of the Hox state produces the inactive state known as Hoxinact. The active states of the enzyme are susceptible to reaction with small molecules such as CO and O2, whereas the inactive Hoxinact state is not known to react with CO or O2.

Technological Applications

Hydrogenases have a number of potential applications in technology. Three particular fields of interest in the Armstrong group are the application of hydrogenases as the anode catalysts in enzyme fuel cells, the harnessing of the H2 oxidation reaction to provide a source of low-potential electrons for use in reduction reactions, and the enzymatic photo-generation of H2.

A ribbon representation of the X-ray determined structure of the [NiFe]-hydrogenase from D. fructosovorans
A ribbon representation of the X-ray determined structure of the [NiFe]-hydrogenase from D. fructosovorans


The [NiFe]-active site
The structure of the [NiFe]-active site


The [FeFe]-active site
The structure of the [FeFe]-active site

Selected Relevent Publications