How [FeFe]-Hydrogenase Facilitates Bidirectional Proton Transfer

Hydrogenases are metalloenzymes that catalyse the interconversion 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, understanding of the catalytic proton transfer is poor. We were able to identify the amino acid residues forming a proton transfer pathway between active site cofactor and bulk solvent; however, the exact mechanism of catalytic proton transfer remained inconclusive. Here, we employ in situ IR difference spectroscopy on the [FeFe]-hydrogenase from Chlamydomonas reinhardtii evaluating dynamic changes in the hydrogen-bonding network upon catalytic proton transfer. Our analysis allows for a direct, molecular unique assignment to individual amino acid residues. We found that transient protonation changes of arginine and glutamic acid residues facilitate bidirectional proton transfer in [FeFe]-hydrogenases.