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The mechanism of reaction of ferricyanide-pretreated mixed-valence-state membrane-bound cytochrome oxidase with oxygen at 173 K.

ABSTRACT: 1. The results of non-linear optimization studies on the mechanism of reaction of ferricyanide-pretreated mixed-valence-state cytochrome oxidase with O2 at 173 K are presented. The analysis is carried out on data obtained by means of dual-wavelength multi-channel spectroscopy at four wavelength pairs (444-463 nm, 604-630 nm, 608-630 nm and 830-940 nm) and at two O2 concentrations (360 micron and 520 micron). The only model that satisfies the triple requirement of a standard deviation within the standard error of the experimental data, a random distribution of residuals and good determination of the optimized parameters, is a three-intermediate sequential mechanism. 2. On the basis of the optimized values of the relative absorption coefficients of the intermediates at each wavelength obtained from the present paper together with data from optical wavelength scanning and e.p.r. spectroscopy obtained by low-temperature trapping studies, the possible valence states of the metal centres in each of the intermediates are discussed.

SUBMITTER: Clore GM

PROVIDER: S-EPMC1185846 | biostudies-other | 1978 Sep

REPOSITORIES: biostudies-other

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Project description:1. The reaction of the partially reduced mixed-valence state of cytochrome c oxidase (a3+CuA2+a3(2+)COCuB+) with O2 was studied by the rapid-reaction technique of flow-flash spectrophotometry at room temperature. Biphasic absorption records are observed in the time range up to 2 ms in both the Soret and visible spectral regions. The fast-phase rate is O2-concentration-dependent and reaches a pseudo-first-order value of 4.5 X 10(4)s-1 at 680 microM-O2 at 20 degrees C. Under the same conditions the second-phase rate is limited at 6.0 X 10(3)s-1. Kinetic difference spectra of the two species in the Soret region are not markedly different in form, whereas in the visible region two spectroscopically different species are clearly distinguished. 4. The first intermediate has a peak at 595 nm and a trough at 605 nm. The form of this spectrum resembles that seen in low-temperature studies and assigned to an O2-bound form of ferrocytochrome a3. This evidence supports a structure for oxycytochrome c oxidase with O2 bound only to cytochrome a3 and not bridged between cytochrome a3 and CuB. The second intermediate has a difference spectrum with a trough at 592 nm and a peak at 610 nm. Again, the form of this spectrum is similar to that observed during the O2 reaction at low temperature and is though to be a result of electron transfer from the oxidase to bound O2. 5. The oxygen profile of the fast phase suggests that a spectroscopically silent species may precede the formation of compound A. These data represent the first spectroscopic distinction, in the physiological temperature range, between O2 binding and electron transfer during the O2 reaction of mammalian cytochrome c oxidase. 6. A mechanism is presented for the O2 reaction of the mixed-valence state of cytochrome c oxidase involving four intermediate species. Electron transfer during this reaction is slow, relative to that seen with the fully reduced enzyme, and probably accounts for the detectability of the oxyferro species under these conditions.

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The mechanism of reaction of ferricyanide-pretreated mixed-valence-state membrane-bound cytochrome oxidase with oxygen at 173 K.

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