Identification of amino acid residues involved in structural and ubiquinone-binding functions of subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides.
Previous studies established that subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides is involved in structural and ubiquinone-binding functions of the complex. To identify regions or amino acid residues responsible for these functions, deletion, insertion, and substitution mutations at various regions of subunit IV were generated and characterized. Mutational effects on the structural role of subunit IV are indicated by a delay in photosynthetic growth and by a decrease in the cytochrome bc1 complex activity in chromatophores upon detergent treatment. An effect on the ubiquinone-binding function of subunit IV is suggested by an increase in the apparent Km for 2,3-dimethoxy-5-methyl-6-geranyl-1,4-benzoquinol (Q2H2) of the complex. RSIV delta (2-5), in which residues 2-5 are deleted, had photosynthetic growth behavior, tolerance to detergent treatment, and an apparent Km for Q2H2 of its cytochrome bc1 complex similar to those of wild-type or complement cells, indicating that amino acid residues 2-5 are not essential for subunit IV function. RSIV delta (2-11), with residues 2-11 missing, showed a 24-h delay in photosynthetic growth and a 65% inactivation of the cytochrome bc1 complex upon dodecyl maltoside solubilization. However, its apparent Km for Q2H2 was the same as in wild-type cells, indicating that deletion of amino acid residues 6-11 results in loss of the structural but not the ubiquinone-binding function of subunit IV. RSIV delta (113-124), which has 13 amino acid residues deleted from the C terminus, had photosynthetic growth behavior, tolerance to detergent treatment, and ubiquinone-binding kinetics similar to those of wild-type or complement cells, indicating that residues 113-124 are not essential. Point mutants RSIV(W79L) and RSIV(W79F), in which tryptophan 79 was replaced with leucine or phenylalanine, showed a 24-h delay in photosynthetic growth, a decrease of 75% of the cytochrome bc1 complex activity in chromatophores upon detergent solubilization, and a 4-fold increase in the apparent Km for Q2H2, indicating that Trp-79 is essential for the structural and ubiquinone-binding functions of subunit IV.
Chen
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Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078, USA.
Shenoy
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Yu
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D017931:DNA Primers; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000254:growth & development; D017384:Sequence Deletion; D014451:Ubiquinone / Q000378:metabolism*
D017931:DNA Primers; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014451:Ubiquinone; D014450:Electron Transport Complex III