A Histidine-Mediated, Pendulum-Like Proton Transport Mechanism Is Required for the High Catalytic Activity of [FeFe]-Hydrogenases

Abstract [FeFe]-hydrogenases are molecular hydrogen (H2) converting enzymes that employ a hexanuclear iron complex, the H-cluster, as the catalytic cofactor, and a proton transfer pathway (PTP) that enables efficient proton-coupled electron transfer (PCET). Recent phylogenetic analyses have revealed different groups of [FeFe]-hydrogenases. Although only very few members of other groups have been characterized, current knowledge suggests that high catalytic activity is predominantly associated with group A members. Here, we show that metal ions inhibit group A [FeFe]-hydrogenases by binding to three specific residues at the entrance of the PTP. Exchanging residue H565 of Clostridium pasteurianum CpI results in a metal-insensitive protein variant with wild-type-like activity. In contrast, exchanging S320 and H569 in CpI, and their counterparts in additional group A [FeFe]-hydrogenases results in enzymes with markedly decreased activities and large overpotential requirements. These features are consistent with important roles of both residues in catalytic proton transfer. CpI structures reveal that H569, locally anchored by E278, can be present in two conformations, such that we propose a histidine-dependent pendulum mechanism for exchanging protons between the bulk solvent and the PTP, which is well supported by theoretical calculations. In doing so, our study broadens understanding of how histidine mobility, governed by neighboring residues in the vicinity, contributes to the fundamental concept of PCET.