How [FeFe]-Hydrogenase Facilitates Bidirectional Proton Transfer

Abstract

Hydrogenases are metalloenzymes that catalyze the conversion of protons and molecular hydrogen, H₂. [FeFe]-hydrogenases show particularly high rates of hydrogen turnover and have inspired numerous compounds for biomimetic H₂ production. Two decades of research on the active site cofactor of [FeFe]-hydrogenases have put forward multiple models of the catalytic proceedings. In comparison, our understanding of proton transfer is poor. Previously, residues were identified forming a hydrogen-bonding network between active site cofactor and bulk solvent; however, the exact mechanism of catalytic proton transfer remained inconclusive. Here, we employ <i>in situ</i> infrared difference spectroscopy on the [FeFe]-hydrogenase from <i>Chlamydomonas reinhardtii</i> evaluating dynamic changes in the hydrogen-bonding network upon photoreduction. While proton transfer appears to be impaired in the oxidized state (<b>Hox</b>), the presented data support continuous proton transfer in the reduced state (<b>Hred</b>). Our analysis allows for a direct, molecular unique assignment to individual amino acid residues. We found that transient protonation changes of glutamic acid residue E141 and, most notably, arginine R148 facilitate bidirectional proton transfer in [FeFe]-hydrogenases.