Confirmation of the involvement of protein domain movement during the catalytic cycle of the cytochrome bc1 complex by the formation of an intersubunit disulfide bond between cytochrome b and the iron-sulfur protein.
To study the essentiality of head domain movement of the Rieske iron-sulfur protein (ISP) during bc(1) catalysis, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with three pairs of cysteines engineered (one cysteine each) on the interface between cytochrome b and ISP, A185C(cytb)/K70C(ISP), I326C(cytb)/G165C(ISP), and T386C(cytb)/K164C(ISP), were generated and characterized. Formation of an intersubunit disulfide bond between cytochrome b and ISP is detected in membrane (intracytoplasmic membrane and air-aged chromatophore), and purified bc(1) complex was prepared from the A185C(cytb)/K70C(ISP) mutant cells. Formation of the intersubunit disulfide bond in this cysteine pair mutant complex is concurrent with the loss of its bc(1) activity. Reduction of this disulfide bond by beta-mercaptoethanol restores activity, indicating that mobility of the head domain of ISP is functionally important in the cytochrome bc(1) complex. The rate of intramolecular electron transfer, between 2Fe2S and heme c(1), in the A185C(cytb)/K70C(ISP) mutant complex is much lower than that in the wild type or in their respective single cysteine mutant complexes, indicating that formation of an intersubunit disulfide bond between cytochrome b and ISP arrests the head domain of ISP in the "fixed state" position, which is too far for electron transfer to heme c(1).
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Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
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D019943:Amino Acid Substitution; D002384:Catalysis; D003545:Cysteine; D003573:Cytochrome b Group / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010788:Photosynthesis; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D012242:Rhodobacter sphaeroides / Q000201:enzymology / Q000235:genetics
D003573:Cytochrome b Group; D004220:Disulfides; D007506:Iron-Sulfur Proteins; D021122:Protein Subunits; D014450:Electron Transport Complex III; D003545:Cysteine