Cytochrome c oxidase

Diphasic kinetics result from incomplete reduction of cytochrome a by cytochrome c bound to the high-affinity site

Jaime Ortega-Lopez, Neal C. Robinson

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

The electron-transfer kinetics of cytochrome c oxidase were probed by measuring the reduction levels of bound cytochrome c, cytochrome a, and cytochrome a 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 a (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 a. 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 a defines V max,1, which is the maximum velocity of the high-affinity phase. The remaining fractional oxidation level of cytochrome a determines V max,2, the maximum velocity of the low-affinity phase. Therefore, it is the sum V max,i + V max,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 a 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.

Original languageEnglish (US)
Pages (from-to)10000-10008
Number of pages9
JournalBiochemistry
Volume34
Issue number31
StatePublished - 1995

Fingerprint

Cytochromes a
Electron Transport Complex IV
Cytochromes c
Electrons
Kinetics
Cats
Detergents
Cytochromes a3
Oxidation-Reduction

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cytochrome c oxidase : Diphasic kinetics result from incomplete reduction of cytochrome a by cytochrome c bound to the high-affinity site. / Ortega-Lopez, Jaime; Robinson, Neal C.

In: Biochemistry, Vol. 34, No. 31, 1995, p. 10000-10008.

Research output: Contribution to journalArticle

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