Abstract
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.
Highlights
Identification of Amino Acid Residues Involved in Structural and Ubiquinone-binding Functions of Subunit IV of the Cytochrome bCI Complex from Rhodobacter sphaeroides*
Mutational effects on the structural role of subunit IV are indicated by a delay in photosynthetic growth and by a decrease in the cytochrome bel complex activity in chromatophores upon detergent treatment
RSIVd(l13124), 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
Summary
Q, ubiquinone; Q2H2' 2,3-dimethoxy-5methyl-6-geranyl-1,4-benzoquinol; DM, dodecyl J3-maltoside; PAGE, polyacrylamide gel electrophoresis. Introducing a wild-type fbcQ operon on a stable low copy number plasmid, pRK415, into RS~IV restores photosynthetic growth behavior, the apparent Km for Q2H2' and tolerance to detergent treatment comparable with that of wild-type cells. These results suggest that subunit IV has Q binding, as well as a structural, role in the cytochrome complex of photosynthetic grown cells. Km for Q2H2 for the cytochrome bCl complex of these mutants, amino acid residues important for the Q-binding and structural functions of subunit IV were revealed. We report the generation and characterization of these subunit IV mutants
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