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

Abstract

[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.