CO-Bridged H-Cluster Intermediates in the Catalytic Mechanism of [FeFe]-Hydrogenase CaI

The [FeFe]-hydrogenases ([FeFe] H₂ases) catalyze reversible H₂ activation at the H-cluster, which is composed of a [4Fe-4S]_H subsite linked by a cysteine thiolate to a bridged, organometallic [2Fe-2S] ([2Fe]_H) subsite. Profoundly different geometric models of the H-cluster redox states that orchestrate the electron/proton transfer steps of H₂ bond activation have been proposed. We have examined this question in the [FeFe] H₂ase I from Clostridium acetobutylicum (CaI) by Fourier-transform infrared (FTIR) spectroscopy with temperature annealing and H/D isotope exchange to identify the relevant redox states and define catalytic transitions. One-electron reduction of H_ox led to formation of H_redH⁺ ([4Fe-4S]_H ²⁺-Fe^I-Fe^I) and H_red′ ([4Fe-4S]_H ¹⁺-Fe^II-Fe^I), with both states characterized by low frequency μ-CO IR modes consistent with a fully bridged [2Fe]_H. Similar μ-CO IR modes were also identified for H_redH⁺ of the [FeFe] H₂ase from Chlamydomonas reinhardtii (CrHydA1). The CaI proton-transfer variant C298S showed enrichment of an H/D isotope-sensitive μ-CO mode, a component of the hydride bound H-cluster IR signal, H_hyd. Equilibrating CaI with increasing amounts of NaDT, and probed at cryogenic temperatures, showed H_redH⁺ was converted to H_hyd. Over an increasing temperature range from 10 to 260 K catalytic turnover led to loss of H_hyd and appearance of H_ox, consistent with enzymatic turnover and H₂ formation. The results show for CaI that the μ-CO of [2Fe]_H remains bridging for all of the "H_red" states and that H_redH⁺ is on pathway to H_hyd and H₂ evolution in the catalytic mechanism. These results provide a blueprint for designing small molecule catalytic analogs.