ROLE OF SUBUNIT-IV IN THE CYTOCHROME-B-C(1) COMPLEX FROM RHODOBACTER-SPHAEROIDES

Rhodobacter sphaeroides mutants lacking subunit IV (M(r) = 14 384) of the cytochrome b-c(1) 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-c(1) complex activities in subunit IV-deficient chromatophores from adapted RS Delta IV-2 are similar to those in wild-type chromatophores. However, the apparent K-m for Q(2)H(2) for the b-c(1) 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-c(1) 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 K-m value for Q(2)H(2), and tolerance to detergent treatment to that of wild-type cells. Cytochrome b-c(1) 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 K-m for Q(2)H(2) from 3 to 13 mu M, suggesting that subunit IV may be involved in quinone binding in addition to its structural role.