Active site of cytochrome cbb3

Abstract

Cytochrome cbb3 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 cbb3 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 (Em,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 b3 has a very low potential (Em,7 -59 mV) as opposed to the copper center (CuB), which has a high potential (Em,7 +330 mV). The EPR spectrum of the ferric heme b3 has rhombic symmetry. To explain the origins of the rhombicity, the Glu-383 residue located on the proximal side of heme b3 was mutated to aspartate and to glutamine. The latter mutation caused a 10 nm blue shift in the optical reduced minus oxidized heme b3 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 b3, a unique interaction among heme-copper oxidases. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.