Investigations on the Role of Proton-Coupled Electron Transfer in Hydrogen Activation by [FeFe]-Hydrogenase

Proton-coupled electron transfer (PCET) is a fundamental process at the core of oxidation-reduction reactions for energy conversion. The [FeFe]-hydrogenases catalyze the reversible activation of molecular H₂ through a unique metallocofactor, the H-cluster, which is finely tuned by the surrounding protein environment to undergo fast PCET transitions. The correlation of electronic and structural transitions at the H-cluster with proton-transfer (PT) steps has not been well-resolved experimentally. Here, we explore how modification of the conserved PT network via a Cys → Ser substitution at position 169 proximal to the H-cluster of Chlamydomonas reinhardtii [FeFe]-hydrogenase (CrHydA1) affects the H-cluster using electron paramagnetic resonance (EPR) and Fourier transform infrared (FTIR) spectroscopy. Despite a substantial decrease in catalytic activity, the EPR and FTIR spectra reveal different H-cluster catalytic states under reducing and oxidizing conditions. Under H₂ or sodium dithionite reductive treatments, the EPR spectra show signals that are consistent with a reduced [4Fe-4S]_H⁺ subcluster. The FTIR spectra showed upshifts of ν_CO modes to energies that are consistent with an increase in oxidation state of the [2Fe]_H subcluster, which was corroborated by DFT analysis. In contrast to the case for wild-type CrHydA1, spectra associated with H_red and H_sred states are less populated in the Cys → Ser variant, demonstrating that the exchange of -SH with -OH alters how the H-cluster equilibrates among different reduced states of the catalytic cycle under steady-state conditions.