Abstract
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).
Highlights
Movement of the head domain of the iron-sulfur protein (ISP)1 during bc1 catalysis
Photosynthetic Growth Behaviors of Mutants Carrying Cysteine Substitutions in the Interface between Cytochrome b and the Head Domain of ISP—Three pairs of amino acid residues: Ala185(cytb)/Lys70(ISP), Ile326(cytb)/Gly165(ISP), and Thr386(cytb)/Lys164(ISP) were selected for mutation to cysteines. These choices were based on the three-dimensional structural model of the four-subunit cytochrome bc1 complex of R. sphaeroides (Fig. 1A) constructed with coordinates from bovine cytochromes b and c1, ISP, and subunit XII [21]
For a cysteine pair mutant to be useful in this study, the engineered cysteine positions must not be critical for cytochrome bc1 complex activity
Summary
Materials—Cytochrome c (horse heart, type III) was purchased from Sigma. N-Dodecyl--D-maltoside and N-octyl--D-glucoside were from Anatrace. 2,3-Dimethoxy-5-methyl-6-geranyl-1,4-benzoquinol (Q2H2) was prepared in our laboratory as previously reported [11]. Because R. sphaeroides cells contain four types of endogenous plasmids, the isolated plasmids lack the purity and concentration needed for direct sequencing. For photosynthetic growth of the plasmid-bearing R. sphaeroides BC17 cells, an enriched Sistrom’s medium containing 5 mM glutamate and. To assay ubiquinol-cytochrome c reductase activity, chromatophores, ICM, or purified cytochrome bc complexes were diluted with 50 mM Tris-Cl, pH 8.0, containing 200 mM NaCl to a final concentration of cytochrome b of 5 M. Determination of pH-induced Reduction and Oxidation of ISP and Cytochrome c1 in the Partially Reduced Wild Type and Mutant bc Complexes—The wild type or mutant bc complex was diluted in 3 ml of 20 mM Tris-Cl buffer, pH 8.0, containing 200 mM NaCl and 0.01% dodecylmaltoside. The cytochrome c1 partially reduced wild type and mutant bc complexes were prepared and used for the absorption and CD measurements.
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