Role of subunit IV in the cytochrome b-c1 complex from Rhodobacter sphaeroides.
Rhodobacter sphaeroides mutants lacking subunit IV (M(r) = 14,384) of the cytochrome b-c1 complex (representative mutant strain, RS delta IV-2) have been constructed by site-specific recombination between the wild-type genomic subunit IV structural gene (fbcQ) and a suicide plasmid containing a defective fbcQ sequence. RS delta IV-2 gives rise to a photosynthetically competent phenotype after a period of adaptation. The chemical compositions, spectral properties, and cytochrome b-c1 complex activities in subunit IV-deficient chromatophores from adapted RS delta IV-2 are similar to those in wild-type chromatophores. However, the apparent Km for Q2H2 for the b-c1 complex in subunit IV-deficient chromatophores from adapted RS delta IV-2 cells is about four times higher than that in chromatophores from wild-type cells. The cytochrome b-c1 complex activity in subunit IV-deficient chromatophores of adapted RS delta IV-2 cells is more labile to detergent treatment than that from wild-type cells. The specific activities of dodecylmaltoside-solubilized fractions of RS delta IV-2, based on cytochrome b, are only one-fourth that of the untreated chromatophores. Introducing a wild-type fbcQ operon on a stable low copy number plasmid, pRK415, into RS delta IV-2 restores photosynthetic growth behavior, the apparent Km value for Q2H2, and tolerance to detergent treatment to that of wild-type cells. Cytochrome b-c1 complex purified from adapted RS delta IV-2 contains only three subunits. It has only 25% of the activity of the four-subunit enzyme. This low activity is accompanied by an increase of the apparent Km for Q2H2 from 3 to 13 microM, suggesting that subunit IV may be involved in quinone binding in addition to its structural role.
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Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078.
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D001483:Base Sequence; D003227:Conjugation, Genetic; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; D015247:Deoxyribonuclease HindIII; D003902:Detergents / Q000494:pharmacology; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D046911:Macromolecular Substances; D016296:Mutagenesis; D010957:Plasmids; D012242:Rhodobacter sphaeroides / Q000737:chemistry* / Q000235:genetics; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D014169:Transformation, Bacterial
D003902:Detergents; D046911:Macromolecular Substances; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; D015247:Deoxyribonuclease HindIII; D014450:Electron Transport Complex III