Spectroelectrochemical study of the cytochrome a site in carbon monoxide inhibited cytochrome c oxidase.

The reduction potential of the cytochrome a site in the carbon monoxide derivative of beef heart cytochrome c oxidase has been studied under a variety of conditions by thin-layer spectroelectrochemistry. The reduction potential exhibits no ionic strength dependence and only a 9 mV/pH unit dependence between pH 6.5 and 8.5. The weak pH dependence indicates that protonation of the protein is not stoichiometrically linked to oxidoreduction over the pH range examined. The temperature dependence of the reduction potential implies a relatively large standard entropy of reduction of cytochrome a. The measured thermodynamic parameters for reduction of cyctochrome a are (all relative to the normal hydrogen electrode) delta Go'(25 degrees C) = -6.37 kcal mol-1, delta Ho' = -21.5 kcal mol-1, and delta So' = -50.8 eu. When cytochrome c is bound to the oxidase, the reduction potential of cytochrome a and its temperature dependence are not measurably affected. Under all conditions studied, the cytochrome a site did not exhibit simple Nernstian n = 1 behavior. The titration behavior of the site is consistent with a moderately strong anticooperative interaction between cytochrome a and CuA [Wang, H., Blair, D. F., Ellis, W. R., Jr., Gray, H. B., & Chan, S. I. (1985) Biochemistry (following paper in this issue)].