Abstract
Complex formation between cytochrome c oxidase and cytochrome c perturbs the optical absorption spectrum of heme c and heme a in the region of the alpha-, beta, and gamma-bands. The perturbations have been used to titrate cytochrome c oxidase with cytochrome c. A stoichiometry of one molecule of cytochrome c bound per molecule of cytochrome c oxidase is obtained (1 heme c per heme aa3). In contrast, a stoichiometry of 2:1 was found earlier using a gel-filtration method (Rieder, R., and Bosshard, H.R. (1978) J. Biol. Chem. 253, 6045-6053). From the result of the spectrophotometric titration and from the wavelength position of the perturbation signals it is concluded that cytochrome c oxidase contains only a single binding site for cytochrome c which is close enough to heme a to function as an electron transfer site. The second site detected earlier by the gel-filtration method must be remote from this electron transfer site. Scatchard plots of the titration data are curvilinear, possibly indicating interactions between cytochrome c-binding sites on adjacent monomers of dimeric cytochrome c oxidase. The relationship between cytochrome c binding and the reaction of cytochrome c oxidase with ferrocytochrome c is discussed.
Highlights
Complexformationbetweencytochrome c oxidase the same surface area interacts at the low-affinity binding and cytochromec perturbs the optical absorption specs-ite too [2]
A stoi- Ferricytochrome c and resting oxidase are the products of the chiometry of one molecule of cytochromec bound per enzymic reaction
The dissociation constant for the high-affinity binding measured directly with a series of different ferricytochromesc are indistinguishable within the limit of error from the K,v,alues is close enough to heme a to function as an electron determined for the high-affinity kinetic phase [1,4, 6,8]. transfer site
Summary
Difference Spectra-Fig. 1shows difference spectra due to the interaction of cytochrome c with cytochrome c oxidase. Under the reducing conditions of our experiment, the contribution of ferric heme &.CN to the Soret band at 443 nm is small since ferric heme a3.CN shows a broad double band with maxima around 420 and 430 nm [32] It seems possible, that the442-nm band of the difference spectrum is primarily due to a spectral perturbation of heme a. The concentration of oxidasebound cytochrome c (Sb in Fig. 5 ) is calculated from the absorption difference at 410 nm with At = 6.5 mM"cm" The latter value is obtained from the initial slope of the titration curve, To our surprise, nonlinear Scatchard plots are obtained for the spectrophotometric titration experiments performed at 50 and 100 mM ionic strength. The latter value is obtained from the initial slope of the titration curve, To our surprise, nonlinear Scatchard plots are obtained for the spectrophotometric titration experiments performed at 50 and 100 mM ionic strength. (At 23 and 300 rnM ionic strength the spread of data points is not sufficiently wide for a meaningful analysis,) Despite the nonlinearity of the Scatchard curves a single binding site per heme aa is extrapolated on
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