H-cluster Intermediates and Catalytic Properties of [FeFe]-Hydrogenase III.

[FeFe]-Hydrogenases are structurally diverse enzymes that catalyze reversible H activation at a catalytic cofactor or H-cluster. The H-cluster is a [4Fe-4S] cubane linked by a cysteine thiolate to a diiron subsite containing unique CO, CN, and dithiomethylamine ligands. The established H-cluster resting state of [4Fe-4S]-[Fe-Fe], or H, functions in H binding and oxidation, or by proton-coupled reduction initiates H evolution. In contrast, in [FeFe]-hydrogenase III (CpIII) the resting state of the H-cluster is fully oxidized, [4Fe-4S]-[Fe-Fe], or H. To begin to understand if H has a role in the mechanism of CpIII, we determined the spectroscopic and redox properties of CpIII H-cluster states under catalytic conditions. CpIII poised in H and either equilibrated under 1 atm of H or reduced with sodium dithionite, resulted in a mixture of reduced states including H ( = -407 mV), H-like [4Fe-4S]-[Fe-Fe] ( = -418 mV), H [4Fe-4S]-[Fe-Fe], and H [4Fe-4S]-[Fe-Fe] ( = -455-480 mV). Under H the population of the H-like state was >20-fold higher than H, implicating a role in CpIII catalysis. Unlike other enzymes, there was no spectral evidence of fully reduced states, such as H ([4Fe-4S]-[Fe-Fe]) or H ([4Fe-4S]-[FeFe]-H). Thus, while the H-cluster states of CpIII encompass most of the catalytic intermediates, it is either unable to form H and H, or these states are highly destabilized in CpIII. Thus, these results demonstrate that catalytic intermediates of reduced CpIII differ from the typical intermediates of other catalytic [FeFe]-hydrogenases and may explain the catalytic preference for H production.