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 is constituted by 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) of [FeFe]-hydrogenase CpI from Clostridium pasteurianum toward [4Fe]H is characterized by X-ray crystallography, infrared spectroscopy, protein film electrochemistry, and quantum mechanical (QM) calculations. This PTP consists of asparagine N160, glutamine Q195, and several protein-bound water molecules, which have been hypothesized to function as a route for protons from the protein surface to cysteine C499 at the [4Fe]H cluster. The crystal structures of protein variants N160L and Q195L now confirm that the regulatory PTP is significantly disrupted. However, infrared spectroscopy reveals that all variants accumulate the H-cluster intermediate HoxH in a manner comparable to wild-type CpI enzyme. In contrast, the CpI variant E279D – previously shown to target the catalytic PTP towards [2Fe]H – is found to enrich the HoxH state independently of reducing agents. This indicates that the determinants of HoxH are located in the catalytic PTP, which emphasizes the importance of HoxH during catalysis. Supported by QM calculations of Hox and HoxH, a model is proposed in which a conserved water cluster adjacent to E279 in the catalytic PTP is protonated and forms a Zundel ion (H5O2+). Our results reveal the identity of HoxH state and provide important new insights into the catalytic mechanism of [FeFe]-hydrogenases.