The unrelated active sites of Ni-Fe and Fe-only hydrogenases have several common features: coordination of diatomic ligands to an Fe ion; a vacant coordination site on one of the metal ions representing a possible substrate-binding site; a thiolate-bridged binuclear center; and plausible proton- and electron-transfer pathways and substrate channels. The diatomic coordination to Fe ions makes them low spin and favors low redox states, which may be required for catalysis. Complex electron paramagnetic resonance signals typical of Fe-only hydrogenases arise from magnetic interactions between the [4Fe-4S] cluster and the active site binuclear center. The paucity of protein ligands to this center suggests that it was imported from the inorganic world as an already functional unit.

The binding of carbon monoxide, a competitive inhibitor of many hydrogenases, to the active site of Desulfovibrio fructosovorans hydrogenase has been studied by infrared spectroscopy in a spectroelectrochemical cell. Direct evidence has been obtained of which redox states of the enzyme can bind extrinsic CO. Redox states A, B and SU do not bind extrinsic CO; only after reductive activation of the hydrogenase can CO bind to the active site. Two states with bound extrinsic CO can be distinguished by FTIR. These two states are in redox equilibrium and are most probably due to different oxidation states of the proximal 4Fe-4S cluster. Vibrational frequencies and theoretical quantum mechanics studies (DFT) of this process preclude the possibility of strong bonding of extrinsic CO to the Fe or Ni atoms of the active site. We propose that CO inhibition is caused by weak interaction of the extrinsic ligand with the Ni atom, blocking electron and proton transfer at the active site. A calculated structure with a weakly bound extrinsic CO at Ni has relative CO frequencies in excellent agreement with the experimental ones.

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