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 experiment al 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 cytochr ome alpha (true k(cat)) by changing the detergent surrounding cytochro me c oxidase. In most detergent environments, the rate of electron inp ut into cytochrome c is not faster than the rate of electron efflux fr om cytochrome alpha. The relatively slow rate of electron input result s 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-af finity site is saturated with cytochrome c, the steady-state reduction level of cytochrome alpha defines V-max,V-1, which is the maximum vel ocity of the high-affinity phase. The remaining fractional oxidation l evel 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 cytochro me a and the bimetallic center, i.e., k(cat). We also were able to eva luate the true k(cat) of cytochrome c oxidase in each detergent enviro nment directly from the steady-state reduction levels without any of t he complications introduced by the analysis of the polarographic kinet ic data. By comparison of the steady-state reduction levels of the red ox centers with the polarographically measured kinetics, we conclude t hat the second kinetic phase is present only when cytochrome alpha can not be fully reduced by the primary high affinity site pathway. Theref ore, the biphasic cytochrome c kinetics of bovine heart cytochrome c o xidase should not be thought of as a necessary and essential part of t he enzymatic mechanism. Rather, the biphasic kinetics are a direct con sequence of limited electron input into cytochrome c from the artifici al electron donor TMPD.