A Zundel ion in the catalytic proton transfer pathway of [FeFe]-hydrogenase

[FeFe]-hydrogenases are metalloenzymes that catalyze the interconversion of protons, electrons, and molecular hydrogen (H₂). Their active site cofactor consists of a [4Fe-4S] cluster ([4Fe]_H) and a diiron site ([2Fe]_H), forming the so-called H-cluster. In this work, the putative regulatory proton transfer pathway (PTP) toward the [4Fe]_H cluster of [FeFe]-hydrogenase CpI from Clostridium pasteurianum is characterized by X-ray crystallography, infrared spectroscopy, and quantum mechanical (QM) calculations. The trajectory consists of asparagine N160, glutamine Q195, and several protein-bound water molecules that might function as a PTP toward cysteine C499 at the [4Fe]_H cluster. We have hypothesized that protonation of C499 determines the H-cluster intermediate H_oxH (M. Senger et al., Phys. Chem. Chem. Phys., 2018, 20, 3128-3140). The crystal structures of protein variants N160L and Q195L now confirm that the putative regulatory PTP is disrupted. However, infrared spectroscopy reveals that all variants accumulate the H_oxH state in a manner comparable to wild-type CpI. In contrast, the CpI variant E279D - previously shown to target the catalytic PTP toward [2Fe]_H - is found to enrich the H_oxH state independently of reducing agents. This indicates that the determinants of H_oxH are located in the catalytic PTP, which emphasizes the importance of H_oxH during catalysis and provides evidence against any involvement of the putative regulatory PTP in hydrogen turnover. Supported by QM calculations, a model is proposed in which a conserved water cluster adjacent to E279 is protonated to form a Zundel ion (H₅O₂⁺). Our results paint a new picture of the H-cluster in the H_oxH state and yield important insight into the catalytic mechanism of [FeFe]-hydrogenases.