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

摘要

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 H_2 through a unique metal-locofactor, 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-duster 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 H_2 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 v_(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.