The fully reduced terminal oxidase bd-I isolated from Escherichia coli binds cyanide.

Cytochrome bd-I from Escherichia coli belongs to the superfamily of prokaryotic bd-type oxygen reductases. It contains three hemes, b, b and d, and couples oxidation of quinol by dioxygen with the generation of a proton-motive force. The enzyme exhibits resistance to various stressors and is considered as a target protein for next-generation antimicrobials. By using electronic absorption and MCD spectroscopy, this work shows that cyanide binds to heme d in the isolated fully reduced cytochrome bd-I. Cyanide-induced difference absorption spectra display changes near the heme d α-band, a minimum at 633 nm and a maximum around 600 nm, and a W-shaped response in the Soret region. Apparent dissociation constant (K) of the cyanide complex of heme d is ∼0.052 M. Kinetics of cyanide binding is monophasic, indicating the presence of a single ligand binding site in the enzyme. Consistently, MCD data show that cyanide binds to heme d but not to b or b. This agrees with the published structural data that the enzyme's active site is not a di-heme site. The observed rate of binding (k) increases as the concentration of cyanide is increased, giving a second-order rate constant (k) of ∼0.1 M s.