Spectroelectrochemical investigations of stoichiometry and oxidation-reduction potentials of cytochrome c oxidase components in the presence of carbon monoxide: the "invisible" copper.

Spectroelectrochemical studies are presented for the carbon monoxide complex of isolated, purified cytochrome c oxidase (EC 1.9.3.1) in solutions saturated with carbon monoxide. The results indicate a stoichiometry of three equivalents per oxidase-carbon monoxide complex molecule. Formal reduction potentials (Eo) of the two copper and one heme component at pH 7.0 were obtained by means of quantitative absorbance-charge titrations in the absence and presence of cytochrome c, and by means of a Nernstian "Minnaert" plot in the presence of cytochrome c. Analysis of the absorbance-charge curves from these titrations gave an indirect determination of the high potential, "invisible" copper component. The copper potentials in the carbon monoxide complex were found to be relatively unchanged with respect to those of the native enzyme. The Eo values obtained were: high potential ("invisible") copper (340 +/- 20 mV (NHE)), low potential copper (190 +/- 20 mV), and low potential heme (250 +/- 10 mV).