Hydrogenase - Structural Classification

Structural Classification

Until 2004, hydrogenases were classified according to the metals thought to be at their active sites; three classes were recognized: iron-only, nickel-iron, and "metal-free". In 2004, Thauer et al. showed that the metal-free hydrogenases in fact contain iron. Thus, those enzymes previously called "metal-free" are now named -hydrogenases, since this protein contains only a mononuclear Fe active site and no iron-sulfur clusters, in contrast to the -enzymes. In some -hydrogenases, one of the Ni-bound cysteine residues is replaced by selenocysteine. On the basis of sequence similarity, however, the - and -hydrogenases should be considered a single superfamily.

• The -hydrogenases are heterodimeric proteins consisting of small (S) and large (L) subunits. The small subunit contains three iron-sulfur clusters while the large subunit contains the active site, a nickel-iron centre which is connected to the solvent by a molecular tunnel. Periplasmic, cytoplasmic, and cytoplasmic membrane-bound hydrogenases have been found. The -hydrogenases, when isolated, are found to catalyse both H₂ evolution and uptake, with low-potential multihaem cytochromes such as cytochrome c₃ acting as either electron donors or acceptors, depending on their oxidation state.

• • The novel hydrogenases of Ralstonia eutropha are unlike typical hydrogenases because they are tolerant to oxygen and carbon monoxide.

• The hydrogenases containing Fe-S clusters and no metal other than iron are called -hydrogenases (-H₂ases). Three families of -H₂ases are recognized:

• • (I) cytoplasmic, soluble, monomeric -H₂ases, found in strict anaerobes such as Clostridium pasteurianum and Megasphaera elsdenii. They are extremely sensitive to inactivation by dioxygen (O₂) and catalyse both H₂ evolution and uptake.

• • (II) periplasmic, heterodimeric -H₂ases from Desulfovibrio spp., which can be purified aerobically and catalyse mainly H₂ oxidation.

• • (III) soluble, monomeric -H₂ases, found in chloroplasts of green alga Scenedesmus obliquus, catalyses H₂ evolution. The ferredoxin functions as natural electron donor linking the enzyme to the photosynthetic electron transport chain.

- and -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 metal is coordinated by carbon monoxide (CO) and cyanide (CN-) ligands.

• 5,10-methenyltetrahydromethanopterin hydrogenase (EC 1.12.98.2) found in methanogenic Archaea contains neither nickel nor iron-sulfur clusters but an iron-containing cofactor that was recently characterized by X-ray diffraction.