Active site of cytochrome cbb3.

Cytochrome cbb(3) is the most distant member of the heme-copper oxidase family still retaining the following major feature typical of these enzymes: reduction of molecular oxygen to water coupled to proton translocation across the membrane. The thermodynamic properties of the six redox centers, five hemes and a copper ion, in cytochrome cbb(3) from Rhodobacter sphaeroides were studied using optical and EPR spectroscopy. The low spin heme b in the catalytic subunit was shown to have the highest midpoint redox potential (E(m)(,7) +418 mV), whereas the three hemes c in the two other subunits titrated with apparent midpoint redox potentials of +351, +320, and +234 mV. The active site high spin heme b(3) has a very low potential (E(m)(,7) -59 mV) as opposed to the copper center (Cu(B)), which has a high potential (E(m)(,7) +330 mV). The EPR spectrum of the ferric heme b(3) has rhombic symmetry. To explain the origins of the rhombicity, the Glu-383 residue located on the proximal side of heme b(3) was mutated to aspartate and to glutamine. The latter mutation caused a 10 nm blue shift in the optical reduced minus oxidized heme b(3) spectrum, and a dramatic change of the EPR signal toward more axial symmetry, whereas mutation to aspartate had far less severe consequences. These results strongly suggest that Glu-383 is involved in hydrogen bonding to the proximal His-405 ligand of heme b(3), a unique interaction among heme-copper oxidases.