We have previously begun to investigate the reaction of the isolated solubilized dithionite-reduced cytochrome bd-I of Escherichia coli with cyanide Borisov and Arutyunyan (2024) J. Inorg. Biochem., 259, 112653. The present work is a continuation of this study. The following new results were obtained using absorption and CD spectroscopy. 1) The membrane form of the fully reduced (FR) enzyme is also capable of binding cyanide. The apparent dissociation constant and second-order rate constant values are 81.1 ± 7.8 mM KCN and 0.11 ± 0.01 M-1·s-1, respectively. This contradicts the data of other researchers according to which the bd-I oxidase, located in native membranes, in FR-state does not bind cyanide. 2) CO added to the cyano adduct of both membrane and isolated solubilized forms of FR-cytochrome bd-I displaces cyanide, resulting in the formation of a complex of the enzyme with CO. This indicates the reversibility of cyanide binding to the protein. To saturate the oxidase binding site with CO in the presence of 100 mM KCN, much more CO is required than in the case of addition of CO to the enzyme that was not pre-treated with cyanide. CO and cyanide compete with each other for binding to the same site in the oxidase — heme d2+, and CO, being a stronger ligand, wins the competition with cyanide. 3) The effect of cyanide on the optical activity of FR-cytochrome bd-I was studied. The CD spectra of FR-enzyme obtained before and after cyanide treatment indicate that the formation of the cyano adduct of heme d2+ leads to a significant weakening of the excitonic interactions between heme d2+ and heme b5952+. Schemes of interaction of cyanide and CO in the presence of excess cyanide with the active center of FR-enzyme are proposed.
Borisov et al. (Wed,) studied this question.