EPR studies of wildtype and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides: Glu-286 is not a bridging ligand in the cytochrome a3CuB center
David M. Mitchell, Roland Aasa, Pia Adelroth, Peter Brezinski, Robert B. Gennis & Bo G. Malmstrom FEBS Lett. 374 371-374 (1995).

Summary: 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 to the presence of a bridging carboxyl ligand in the bimetallic cytochrome a3-CuB site is also found 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, T352A, as well as F391Q located close to the bimetallic center are shown to affect dramatically the low-spin heme signal of cytochrome a. These mutants were essentially inactive, suggesting that these three mutations result in alterations to cytochrome a that render the oxidase non-functional.