When the inhibitor tells more than the substrate: the cyanide-bound state of a carbon monoxide dehydrogenase

Abstract

Carbon monoxide dehydrogenase (CODH) is a key enzyme for reversible CO interconversion. To elucidate structural and mechanistic details of CO binding at the CODH active site (C-cluster), cyanide is frequently used as an iso-electronic substitute and inhibitor. However, previous studies revealed conflicting results on the structure of the cyanide-bound complex and the mechanism of cyanide-inhibition. To address this issue in this work, we have employed IR spectroscopy, crystallography, site directed mutagenesis, and theoretical methods to analyse the cyanide complex of the CODH from Carboxydothermus hydrogenoformans (CODHII_Ch). IR spectroscopy demonstrates that a single cyanide binds to the Ni ion. Whereas the inhibitor could be partially removed at elevated temperature, irreversible degradation of the C-cluster occurred in the presence of an excess of cyanide on the long-minute time scale, eventually leading to the formation of [Fe(CN)₆]⁴⁻ and [Ni(CN)₄]²⁻ complexes. Theoretical calculations based on a new high-resolution structure of the cyanide-bound CODHII_Ch indicated that cyanide binding to the Ni ion occurs upon dissociation of the hydroxyl ligand from the Fe₁ subsite of the C-cluster. The hydroxyl group is presumably protonated by Lys563 which, unlike to His93, does not form a hydrogen bond with the cyanide ligand. A stable deprotonated ε-amino group of Lys563 in the cyanide complex is consistent with the nearly unchanged CN stretching in the Lys563Ala variant of CODHII_Ch. These findings support the view that the proton channel connecting the solution phase with the active site displays a strict directionality, controlled by the oxidation state of the C-cluster.