EPR STUDIES OF WILD-TYPE AND SEVERAL MUTANTS OF CYTOCHROME-C-OXIDASE FROM RHODOBACTER-SPHAEROIDES - GLU(286) IS NOT A BRIDGING LIGAND IN THE CYTOCHROME A(3) CU-B CENTER

Wild-type and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides were characterized by EPR spectroscopy. A pH-induced g12 signal, seen previously in mammalian cytochrome oxidase and assigned t o the presence of a bridging carboxyl ligand in the bimetallic cytochr ome a(3)-Cu-B site, is found also in the bacterial enzyme. Mutation of glutamate-286 to glutamine inactivates the enzyme but does not affect this signal, demonstrating that the carboxyl group of this residue is not the bridging ligand. Three mutants, M106Q, located one helix turn below a histidine ligand to cytochrome a, and T352A as well as F391Q, located close to the bimetallic center, are shown to affect dramatica lly the low-spin heme signal of cytochrome a. These mutants are essent ially inactive, suggesting that these three mutations result in altera tions to cytochrome a that render the oxidase non-functional.