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

[FeFe]-Hydrogenases are structurally diverse enzymes that catalyze reversible H₂ 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]²⁺-[Fe^II-Fe^I], or H_ox, functions in H₂ binding and oxidation, or by proton-coupled reduction initiates H₂ evolution. In contrast, in Clostridium pasteurianum [FeFe]-hydrogenase III (CpIII) the resting state of the H-cluster is fully oxidized, [4Fe-4S]²⁺-[Fe^II-Fe^II], or H_ox+1. To begin to understand if H_ox+1 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_ox+1 and either equilibrated under 1 atm of H₂ or reduced with sodium dithionite, resulted in a mixture of reduced states including H_ox (E_m⁸ = -407 mV), H_trans-like [4Fe-4S]⁺-[Fe^II-Fe^II] (E_m⁸ = -418 mV), H_red [4Fe-4S]⁺-[Fe^II-Fe^I], and H_redH+ [4Fe-4S]²⁺-[Fe^I-Fe^I] (E_m⁸ = -455-480 mV). Under H₂ the population of the H_trans-like state was >20-fold higher than H_ox, implicating a role in CpIII catalysis. Unlike other enzymes, there was no spectral evidence of fully reduced states, such as H_sredH+ ([4Fe-4S]⁺-[Fe^I-Fe^I]) or H_hyd ([4Fe-4S]⁺-[Fe^II-Fe^II]-H^-). Thus, while the H-cluster states of CpIII encompass most of the catalytic intermediates, it is either unable to form H_sredH+ and H_hyd, 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 H₂ production.