EFFECTS OF OVERALL OXIDATION-STATE ON INFRARED-SPECTRA OF HEME A(3) CYANIDE IN BOVINE HEART CYTOCHROME-C-OXIDASE - EVIDENCE OF NOVEL MECHANISTIC ROLES FOR CU-B

Effects of changes in oxidation state at the other metal centers on ox idized heme alpha(3) cyanide of bovine heart cytochrome c oxidase have been investigated. Only one CN- binds, giving Fea33+CN, in fully-oxid ized cytochrome c oxidase and its 1-, 2-, and 3-electron reduction pro ducts. Soret/visible spectra for the heme alpha(3) cyanide are indepen dent of overall redox level, whereas distinct shifts in C-N infrared s tretch band frequency occur upon reduction, reflecting changes in the polarity of the ligand (CN-) environment. Catalysis of O-2 reduction c an be critically dependent upon such changes in polarity at the reduct ion site. These findings indicate that Cu-B, when reduced, exists in t wo forms whose relative stabilities are independent of Fe-a and Cu-A o xidation 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 Cu-B Ligand in respiratory cont rol. Electron equivalents introduced into the enzyme are distributed e qually among Fe-a, Cu-A, and Cu-B, which raises the possibility that a h four electrons used in O-2 reduction are donated via Cu-B(+), which is favorably positioned with respect to Fe-a3 (the O-2 binding site) i n order to carry out this role in electron transfers.