Biochemistry zyxwvuts 1995,34, zyxwvu 10009- 10018 10009 Kinetics and Mechanism for the Binding of HCN to Cytochrome c Oxidase? Markandeswar Panda and Neal C. Robinson* Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7760 Received April 25, 1995; Revised Manuscript Received June 5, 1995@ ABSTRACT: The kinetics of cyanide binding to cytochrome zyxwvu c oxidase were systematically studied as a function of [HCN], [oxidase], pH, ionic strength, temperature, type and concentration of solubilizing detergent, and monomer-dimer content of oxidase. On the basis of these results a minimum reaction mechanism is proposed in which the spectrally visible rapid and slow cyanide binding reactions are two consecutive first-order reactions, not parallel reactions with different conformers of cytochrome c oxidase. The fast reaction zyxwvutsrqp (k’obs) follows saturation type kinetics 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 the bell-shaped rate versus pH profile; one is due to an ionizable group on the protein (pK, = 7.43, while the other is that of HCN (~KHCN = 9.15). Therefore, oxidase is reactive toward HCN only when the group on the protein is unprotonated. The slow k”&s reaction is not a reaction of oxidase with either CN- or HCN; in fact, the product formed by the fast k’obs reaction, the oxidase-HCN complex, still undergoes the slow k” process even if all of the excess KCN is removed. The apparent rate constant of the slower phase (k”obs) is independent of all the variations done in this study, and it probably corresponds 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 the bimetallic center of oxidase. Based upon the bell-shaped pH dependence of the fast phase and the pH independence of the slow phase, the mechanism also predicts that a single conformer of cytochrome c oxidase can exhibit either monophasic or biphasic cyanide binding kinetics depending 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 monomeric or dimeric oxidase only slightly affects the magnitude of krobs and k”&s values, and both processes are clearly present in both types of oxidase. Cytochrome c oxidase (EC 1.9.3.1) is a multisubunit enzyme of the mitochondrial respiratory chain that transfers electrons from cytochrome c to dioxygen. Its function is to couple these electron transfers to the translocation of protons across the inner membrane (Wikstrom et al., 1981; Naqui & Chance, 1986; Papa et al., 1987; V b n g h d , 1988; Kadenbach et al., 1987; Beinert, 1988; Copeland & Chan, 1989; Capaldi, 1990; Azzi & Muller, 1990; Malmstrom, 1990a; Saraste, 1990). Mammalian cytochrome c oxidase comprises 13 dissimilar polypeptide subunits (Zhang & Capaldi, 1988) and four redox components: two heme A groups and two coppers (Wikstrom et al., 1981; Naqui & Chance, 1986; Papa et al., 1987; ViinngArd, 1988; Kadenbach et al., 1987; Copeland & Chan, 1989; Malmstrom, 1990b). The two heme A groups are associated with subunit I and are known as cytochromes a and a3 (Holm et al., 1987). One of the coppers, CUB, and cytochrome a3 form a bimetallic center that is the oxygen binding site (Wikstrom et al., 1981; Naqui & Chance, 1986; Papa et al., 1987; Vannghd, 1988; Kadenbach et al., 1987; Beinert, 1988; Copeland & Chan, 1989; Capaldi, 1990; Azzi & Muller, 1990; Malmstrom, 1990a; Saraste, 1990; Zhang & Capaldi, 1988). The other copper, CUA,is associated with subunit I1 (Malmstrom, 1990b; Holm et al., 1987) and participates in electron transfers from bound cytochrome c to cytochrome a. ’ This work has been supported by a grant from National Institutes * Author to whom correspondence should be addressed. @ Abstract published in Advance ACS Abstracts, July 15, 1995. of Health (GM 24795). The oxygen binding site of cytochrome c oxidase has been probed with two common inhibitors of respiration, carbon monoxide and cyanide. Both inhibitors bind at the binuclear cytochrome zyxwv as-& site but react quite differently. Bound CO can be oxidized to CO2 and subsequently released from the binuclear center (Young & Caughey, 1986), whereas CN- (or HCN) is irreversibly bound to the binuclear center (Malmstrom, 1990). The mechanism of CO ligand binding to cytochrome c oxidase is complex but follows well-defined kinetics (Woodruff et al., 1991; HallCn et al., 1994; Geor- giadis et al., 1994; Basu et al., 1994; Einarsd6ttir et al., 1995; Adelroth et al., 1995). However, a unified mechanism that explains the widely divergent cyanide binding data is not available. Nevertheless, the chemistry of cyanide binding to cytochrome c oxidase is quite well understood from a variety of spectroscopic techniques, i.e., Cu X-ray (Scott et al., 1985), EXAFS (Naqui et al., 1984), EPR (Baker et al., 1987; Schoonover & Palmer, 1991; Day et al., 1993; Lodder & Van Gelder, 1994), resonance Raman (Schoonover et al., 1988; Larsen et al., 1989), infrared (Yoshikawa & Caughey, 1990; Caughey et al., 1993; Tsubaki ’& Yoshikawa, 1993; Li & Palmer, 1993), UV-visible (Fabian & Malmstrom, 1989; Berka et al., 1993; Gullo et al., 1993; Lodder & Van Gelder, 1994) and magnetic circular dichroism (Babcock et al., 1976; Papadopoulous et al., 1991; Schoonover & Palmer, 1991). The major complication in understanding the cyanide binding is the fact that the kinetics are sometimes monophasic (Hill & Robinson, 1986; Baker et al., 1987; Schoonover & 0006-2960/95/0434- 10009$09.00/0 0 1995 American Chemical Society