The reaction of primate cytochromes c with cytochrome c oxidase. Analysis of the polarographic assay.

Cytochrome c Oxidase: Biphasic Kinetics Result from Incomplete Reduction of Cytochrome a by Cytochrome c Bound to the High-Affinity Site

“…Increasing the concentration of TMPD increases the steady-state reduction level of both cytochrome c and cytochrome a (refer to Figures 2 and 3), a result that is in agreement with both Kimelberg and Nicholls (1969) and Morgan and Wikstrom (1991). These increased reduction levels are, of course, due to the rate-limiting pseudo-firstorder reduction of tightly bound cytochrome c by TMPD (Osheroff et al, 1983;this study). However, at any given concentration of TMPD, the fractional reduction of cytochrome a is not constant and also depends upon the solubilizing detergent.…”

“…This indicates that Vmax,total is directly proportional to £cat and permits evaluation of the second-order rate constant for reduction of cytochrome c by TMPD from the inverse of the slope of the inset to Figure 5. The value of k\ -(9.5 ± 0.5) x 104 M-1 s-1 obtained by this method is intermediate between the value obtained by Hill and Nicholls (1980) (1.3 x 105 M-1 s~') and that obtained by Osheroff et al (1983) (2.2 x 104 M-1 s-1)-Direct comparison of Vmax,tot with the steady-state evaluation of /rcat (Table 2) is also quite good, as one would expect from the linear relationships in Figure 5.…”

“…Pan et al (1993) report first-order rate constants of > 105 s-1 for cytochrome c -CuA and 2.3 x 104 s-1 for CuAcytochrome a; Pan et al (1991) report a first-order rate constant of 2600 s-1 for the oxidation of tightly bound cytochrome c. The present data support a rapid redox equilibrium between tightly bound cytochrome c and cytochrome a since the ratio of [cytochrome ared/cytochrome aox] to [cytochrome cred/cytochrome cox] remained constant in almost all experiments (refer to Figure 4). However, as proposed by Osheroff et al (1983), it is not the rate of electron transfer from tightly bound cytochrome c to cytochrome a that is rate limiting; it is the rate of electron transfer into tightly bound cytochrome c that is insufficient to maintain both cytochrome c and cytochrome a in a fully reduced state. Reduction of tightly bound cytochrome c by TMPD is relatively slow with a bimolecular rate constant, k\, of (0.95-1.3) x 105 M-1 s-1 [this study and Hill and Nicholls (1980)], a rate that corresponds to a pseudo-first-order rate constant of only 100 s-1 with 0.7-1.0 mM TMPD.…”

“…(2) Electrons could enter by way of the tightly bound cytochrome c if direct electron transfer from soluble, reduced cytochrome c to bound, oxidized cytochrome c occurs. (3) Electrons could enter via the high-affinity site if additional cytochrome c increases the off rate of oxidized cytochrome c from this site [model of Wilms et al (1981); Antalis & Palmer, 1982;Osheroff et al, 1983;Nalecz et al, 1983;Speck et al, 1984;Bolli et al, 1985a,b]. According to any of these models, the second kinetic phase occurs only if pathway result in the further reduction of cytochrome a; i.e., the low-affinity kinetic phase can occur only in environments that support high enzymatic turnover.…”

“…One possible explanation for the biphasic kinetics is the inability of electron transfer through a single, tightly bound cytochrome c to fully reduce cytochrome a. Cytochrome c, cytochrome a, and Cua are all known to be partially oxidized during steady-state turnover when the artificial electron donor Af.AUV'./V'-tetramethyl-p-phenylenediamine (TMPD)1 is used as the electron donor (Thomstrom et al, 1988; Morgan & Wikstrom, 1991;Alleyne et al, 1992;Nicholls, 1993). Incomplete reduction of these redox centers is probably due to the rate-limiting reduction of tightly bound cytochrome c by TMPD (Osheroff et al, 1983). In fact, both the magnitude of the high-affinity phase, Vmax,i, and the percent reduction of the redox centers are dependent upon the concentration of TMPD (Ferguson-Miller et al, 1978;Morgan & Wikstrom, 1991).…”