The effects of p-hydroxymercuribenzoic acid modification and heat treatment on the CuA reduction potential of cytochrome c oxidase.

p-Hydroxymercuribenzoic acid modification of cytochrome c oxidase converts the CuA center into a type 2 copper site while heat treatment of the oxidase in lauryl maltoside can transform CuA almost stoichiometrically (greater than 90%) to a blue type 1 copper site. These modifications of the protein have previously been shown to have a profound effect on the dioxygen reduction and proton pumping activities of the enzyme (Li, P. M., Morgan, J. E., Nilsson, T., Ma, M., and Chan, S. I. (1988) Biochemistry 27, 7538; Nilsson, T., Gelles, J., Li, P. M., and Chan, S. I. (1988) Biochemistry 27, 296; Sone, N., and Nicholls, P. (1984) Biochemistry 23, 6550). In this work, the intrinsic reduction potentials and the midpoint reduction potentials of the "CuA" site in both these modified oxidases have been measured under various conditions in order to clarify the intramolecular electron transfer pathways in these systems. The study reveals that the CuA intrinsic reduction potential decreases by almost 150 mV upon p-hydroxymercuribenzoic acid modification whereas it increases by 100 mV upon heat treatment. In addition, the redox interactions between CuA and the remaining metal centers are perturbed upon CuA modification. It is argued that these results bear on the role of CuA in the proton-pumping paradigm of cytochrome c oxidase.