CYTOCHROME-C-OXIDASE - BIPHASIC KINETICS RESULT FROM INCOMPLETE REDUCTION OF CYTOCHROME-A BY CYTOCHROME-C BOUND TO THE HIGH-AFFINITY SITE

The electron-transfer kinetics of cytochrome c oxidase were probed by measuring the reduction levels of bound cytochrome c, cytochrome alpha, and cytochrome alpha(3) during steady-state turnover. Our experimental approach was to measure these reduction levels as a function of (1) the rate of electron input into tightly bound cytochrome c by varying the concentration of TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) and/or cytochrome c and (2) the rate of electron efflux out of cytochrome alpha (true k(cat)) by changing the detergent surrounding cytochrome c oxidase. In most detergent environments, the rate of electron input into cytochrome c is not faster than the rate of electron efflux from cytochrome alpha. The relatively slow rate of electron input results in incomplete reduction of both cytochrome a and cytochrome c bound at the high-affinity site unless k(cat) is very slow. When the high-affinity site is saturated with cytochrome c, the steady-state reduction level of cytochrome alpha defines V-max,V-1, which is the maximum velocity of the high-affinity phase. The remaining fractional oxidation level of cytochrome alpha determines V-max,V-2, the maximum velocity of the low-affinity phase. Therefore, it is the sum V-max,V-1 + V-max,V-2 which defines the maximum rate of electron transfer between cytochrome a and the bimetallic center, i.e., k(cat). We also were able to evaluate the true k(cat) of cytochrome c oxidase in each detergent environment directly from the steady-state reduction levels without any of the complications introduced by the analysis of the polarographic kinetic data. By comparison of the steady-state reduction levels of the redox centers with the polarographically measured kinetics, we conclude that the second kinetic phase is present only when cytochrome alpha cannot be fully reduced by the primary high affinity site pathway. Therefore, the biphasic cytochrome c kinetics of bovine heart cytochrome c oxidase should not be thought of as a necessary and essential part of the enzymatic mechanism. Rather, the biphasic kinetics are a direct consequence of limited electron input into cytochrome c from the artificial electron donor TMPD.