Effects of changes in oxidation state at the other metal centers on oxidized heme a3 cyanide of bovine heart cytochrome c oxidase have been investigated. Only one CN- binds, giving Fe3+a3CN, in fully-oxidized cytochrome c oxidase and its 1-, 2-, and 3-electron reduction products. Soret/visible spectra for the heme a3 cyanide are independent of overall redox level, whereas distinct shifts in C-N infrared stretch band frequency occur upon reduction, reflecting changes in the polarity of the ligand (CN-) environment. Catalysis of O2 reduction can be critically dependent upon such changes in polarity at the reduction site. These findings indicate that CuB, when reduced, exists in two forms whose relative stabilities are independent of Fea and CuA oxidation states and, when oxidized, is in only one stable form. These results are consistent with the oxidation of Cu+B triggering proton pumping and with the involvement of a CuB ligand in respiratory control. Electron equivalents introduced into the enzyme are distributed equally among Fea, CuA, and CuB, which raises the possibility that all four electrons used in O2 reduction are donated via Cu+B, which is favorably positioned with respect to Fea3 (the O2 binding site) in order to carry out this role in electron transfers.