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 Hox led to formation of HredH+ ([4Fe–4S]H 2+–FeI–FeI) and Hred′ ([4Fe–4S]H 1+–FeII–FeI), 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 HredH+ 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, Hhyd. Equilibrating CaI with increasing amounts of NaDT, and probed at cryogenic temperatures, showed HredH+ was converted to Hhyd. Over an increasing temperature range from 10 to 260 K catalytic turnover led to loss of Hhyd and appearance of Hox, 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 “Hred” states and that HredH+ is on pathway to Hhyd and H2 evolution in the catalytic mechanism. These results provide a blueprint for designing small molecule catalytic analogs.