title: Active site of cytochrome cbb3.
abstract: 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.
authors.affiliation: Helsinki Bioenergetics Group, Program for Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, P. O. Box 65 (Viikinkaari 1), 00014 Helsinki, Finland. virve.rauhamaki@helsinki.fi
authors.forename: Virve
authors.initials: V
authors.lastname: Rauhamäki
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Bloch
authors.forename: Michael I
authors.initials: MI
authors.lastname: Verkhovsky
authors.forename: Mårten
authors.initials: M
authors.lastname: Wikström
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry; D001692:Biological Transport; D002384:Catalysis; D020134:Catalytic Domain; D003300:Copper / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D007477:Ions; D016296:Mutagenesis; D009154:Mutation; D010084:Oxidation-Reduction; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000378:metabolism
chemical_list: D007477:Ions; D011522:Protons; D005973:Glutamine; D001224:Aspartic Acid; D006418:Heme; D003300:Copper; C414443:cbb3 oxidase; D003576:Electron Transport Complex IV