Cyanide Binding to [FeFe]-Hydrogenase Stabilizes the Alternative Configuration of the Proton Transfer Pathway

Abstract

Hydrogenases are H₂ converting enzymes that harbor catalytic cofactors in which iron (Fe) ions are coordinated by biologically unusual carbon monoxide (CO) and cyanide (CN^- ) ligands. Extrinsic CO and CN^- , however, inhibit hydrogenases. The mechanism by which CN^- binds to [FeFe]-hydrogenases is not known. Here, we obtained crystal structures of the CN^- -treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum. The high resolution of 1.39 Å allowed us to distinguish intrinsic CN^- and CO ligands and to show that extrinsic CN^- binds to the open coordination site of the cofactor where CO is known to bind. In contrast to other inhibitors, CN^- treated crystals show conformational changes of conserved residues within the proton transfer pathway which could allow a direct proton transfer between E279 and S319. This configuration has been proposed to be vital for efficient proton transfer, but has never been observed structurally.

Keywords: Cyanide; Hydrogen Bonds; Hydrogenase; Proton Transfer Pathway; X-Ray Diffraction.