Evidence for electron equilibrium between the two hemes bL in the dimeric cytochrome bc1 complex.
Structural analysis of the dimeric mitochondrial cytochrome bc1 complex suggests that electron transfer between inter-monomer hemes bL-bL may occur during bc1 catalysis. Such electron transfer may be facilitated by the aromatic pairs present between the two bL hemes in the two symmetry-related monomers. To test this hypothesis, R. sphaeroides mutants expressing His6-tagged bc1 complexes with mutations at three aromatic residues (Phe-195, Tyr-199, and Phe-203), located between two bL hemes, were generated and characterized. All three mutants grew photosynthetically at a rate comparable to that of wild-type cells. The bc1 complexes prepared from mutants F195A, Y199A, and F203A have, respectively, 78%, 100%, and 100% of ubiquinol-cytochrome c reductase activity found in the wild-type complex. Replacing the Phe-195 of cytochrome b with Tyr, His, or Trp results in mutant complexes (F195Y, F195H, or F195W) having the same ubiquinol-cytochrome c reductase activity as the wild-type. These results indicate that the aromatic group at position195 of cytochrome b is involved in electron transfer reactions of the bc1 complex. The rate of superoxide anion (O2*) generation, measured by the chemiluminescence of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one hydrochloride-O2* adduct during oxidation of ubiquinol, is 3 times higher in the F195A complex than in the wild-type or mutant complexes Y199A or F203A. This supports the idea that the interruption of electron transfer between the two bL hemes enhances electron leakage to oxygen and thus decreases the ubiquinol-cytochrome c reductase activity.
Gong
Xing
X
Department of Biochemistry & Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
Yu
Linda
L
Xia
Di
D
Yu
Chang-An
CA
D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry*; D007700:Kinetics; D008163:Luminescent Measurements; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D006418:Heme; D014450:Electron Transport Complex III