KINETICS AND MECHANISM FOR THE BINDING OF HCN TO CYTOCHROME-C-OXIDASE

The kinetics of cyanide binding to cytochrome c oxidase were systemati cally studied as a function of [HCN], [oxidase], pH, ionic strength, t emperature, type and concentration of solubilizing detergent, and mono mer-dimer content of oxidase. On the basis of these results a minimum reaction mechanism is proposed in which the spectrally visible rapid a nd slow cyanide binding reactions are two consecutive first-order reac tions, not parallel reactions with different conformers of cytochrome c oxidase. The fast reaction (k(obs)') follows saturation type kinetic s to form an HCN complex that subsequently undergoes a slow reaction ( k(obs)''). The fast k(obs)' reaction is independent of ionic strength but is strongly dependent upon pH. Two pK values were evaluated from t he bell-shaped rate versus pH profile; one is due to an ionizable grou p on the protein (pK(a) = 7.45), while the other is that of HCN (pK(HC N) = 9.15). Therefore, oxidase is reactive toward HCN only when the gr oup on the protein is unprotonated. The slow k(obs)'' reaction is not a reaction of oxidase with either CN- or HCN; in fact, the product for med by the fast k(obs)' reaction, the oxidase-HCN complex, still under goes the slow k '' process even if all of the excess KCN is removed. T he apparent rate constant of the slower phase (k(obs)'') is independen t of all the variations done in this study, and it probably correspond s to either a slow conformational change in the protein or a change in ligand coordination at one of the metal centers after HCN binds to th e bimetallic center of oxidase. Based upon the bell-shaped pH dependen ce of the fast phase and the pH independence of the slow phase, the me chanism also predicts that a single conformer of cytochrome c oxidase can exhibit either monophasic or biphasic cyanide binding kinetics dep ending upon the pH. At either very low or very high pH, the two rates become comparable in magnitude, which makes the reaction appear to be monophasic even though both reactions still occur. The amount of monom eric or dimeric oxidase only slightly affects the magnitude of k(obs)' and k(obs)'' values, and both processes are clearly present in both t ypes of oxidase.