Peculiarities of cyanide binding to the ba₃-type cytochrome oxidase from the thermophilic bacterium thermus thermophilus

Cytochrome c oxidase of the ba₃-type from Thermus thermophilus does not interact with cyanide in the oxidized state and acquires the ability to bind heme iron ligands only upon reduction. Cyanide complexes of the reduced heme a₃ in cytochrome ba₃ and in mitochondrial aa ₃-type cytochrome oxidase are similar spectroscopically, but the a₃²⁺ -CN complex of cytochrome ba₃ is strikingly tight. Experiments have shown that the K_dvalue of the cytochrome ba₃ complex with cyanide in the presence of reductants of the enzyme binuclear center does not exceed 10^-8 M, which is four to five orders of magnitude less than the K_dof the cyanide complex of the reduced heme a₃ of mitochondrial cytochrome oxidase. The tightness of the cytochrome ba₃ complex with cyanide is mainly associated with an extremely slow rate of the ligand dissociation (k _off ≤ 10^-7 sec^-1), while the rate of binding (k_on ∼ 10² M^-1-sec^-1) is similar to the rate observed for the mitochondrial cytochrome oxidase. It is proposed that cyanide dissociation from the cytochrome ba₃ binuclear center might be hindered sterically by the presence of the second ligand molecule in the coordination sphere of Cu_B²⁺. The rate of cyanide binding with the reduced heme a₃ does not depend on p ^H in the neutral area, but it approaches linear dependence on H ⁺ activity in the alkaline region. Cyanide binding appears to be controlled by protonation of an enzyme group with pK_a = 8.75.