Examination of the Reaction of Escherichia coli Cytochrome bd-I in the Fully Reduced State with Cyanide Using Absorption and Circular Dichroism Spectroscopy

We have earlier begun to investigate the reaction of isolated solubilized dithionite-reduced cytochrome <i>bd</i>-I of <i>Escherichia coli</i> with cyanide [Borisov, V. B., and Arutyunyan, A. M. (2024) The fully reduced terminal oxidase <i>bd</i>-I isolated from <i>Escherichia coli</i> binds cyanide, <i>J. Inorg. Biochem.</i>, <b>259</b>, 112653]. The present work is a continuation of this study. Using absorption and CD spectroscopy, the following new results were obtained: (i) The membrane form of the fully reduced 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 studies according to which the <i>bd</i>-I oxidase located in native membranes, in the fully reduced state does not bind cyanide. (ii) CO added to the cyano adduct of both membrane and isolated solubilized forms of the fully reduced cytochrome <i>bd</i>-I displaces cyanide, resulting in the formation of the CO/enzyme complex. 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 upon CO addition to the enzyme not pre-treated with cyanide. CO and cyanide compete for the binding to the same site in the oxidase (heme <i>d</i>²⁺). Being a stronger ligand, CO "wins" the competition with cyanide. (iii) The effect of cyanide on the optical activity of dithionite-reduced cytochrome <i>bd</i>-I was studied. The CD spectra of the enzyme obtained before and after cyanide treatment indicate that the formation of the cyano adduct of heme <i>d</i>²⁺ leads to a significant weakening of the excitonic interactions between heme <i>d</i>²⁺ and heme <i>b</i>₅₉₅²⁺. Schemes of the interaction of cyanide and CO in the presence of excess cyanide with the enzyme active site are proposed.