Responses of the a3 component of cytochrome c oxidase to substrate and ligand addition.

We have previously described a transient high spin ferric heme species in cytochrome c oxidase (EC 1.9.3.1) which represent a3+(3) (Beinert, H. and Shaw, R.W.(1977) Biochim. Biophys. Acta 462, 12u--130), and can be detected and quantitatively determined by EPR. We have now used out ability to generate this species to study reactions of a3+(3) with substrates and ligands and also responses to pH changes. This was accomplished by multiple rapid mixing and freezing techniques in conjunction with low temperature EPR and optical reflectance spectroscopies. The substrates used were O2 and ferrocytochrome c and the ligands cyanide, sulfide, azide and carbon monoxide. Contrary to the oxidized, resting form of the enzyme, the transient high spin species of a3+(3) reacts within less than 10 ms stoichiometrically with cyanide and sulfide and at a slower rate with azide. The transient a3+(3) species responds to O2 and CO by changes in signal size or shape, although no oxidoreduction is involved, indicating that a3+(3) registers the presence of these gases. The high spin signal of the transient species is readily abolished by ferrocytochrome c or on raising the pH. Decreasing the pH induces a shift from the rhombic towards the axial component of the signal. Since the responses to CO and pH are analogous for the rhombic transient species to those observed with the rhombic high spin ferric heme species produced on partial reduction, it is suggested that the rhombic signals represent a3+(3) in either case. In all these experiments, in which EPR detectable a3+(3) was observed in large yield, no extra signals for copper or correspondingly increased intensity in the copper signal at g = 2 were seen. The relationship is discussed of the obviously reactive transient species of a3+(3) to other 'activated' species that have been reported and to the oxidized resting form of the enzyme, which is known to react only slowly with ligands and to respond sluggishly to substrate.