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 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 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. It has been hypothesized that protonation of C499 determines the H-cluster intermediate HoxH. 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 HoxH state in a manner comparable to wild-type CpI enzyme. In contrast, the CpI variant E279D – previously shown to target the catalytic PTP toward [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.