Subunit IV of cytochrome bc1 complex from Rhodobacter sphaeroides. Localization of regions essential for interaction with the three-subunit core complex.

Recombinant subunit IV mutants which identify the regions essential for restoration of bc(1) activity to the three-subunit core complex of Rhodobacter sphaeroides were generated and characterized. Four C-terminal truncated mutants: IV(1-109), IV(1-85), IV(1-76), and IV(1-40) had 100, 0, 0, and 0% of reconstitutive activity of the wild-type IV, indicating that residues 86-109 are essential. IV(1-109) is associated with the core complex in the same manner as the wild-type IV while mutants IV(1-85), IV(1-76), and IV(1-40) do not associate with the core complex, indicating that subunit IV requires its transmembrane helix region (residues 86-109) for assembly into the bc(1) complex. Since GST-IV(86-109) fusion protein has little reconstitutive activity, some region(s) in residues 1-85 are required for bc(1) activity restoration after subunit IV is incorporated into the complex through the transmembrane helix, presumably by interaction with cytochrome b in the core complex. The interacting regions are identified as residues 41-53 and 77-85, since mutants IV(21-109), IV(41-109), IV(54-109), and IV(77-109) had 95, 98, 53, and 53% of the reconstitutive activity of the wild-type IV. These two interacting regions are on the cytoplasmic side of the chromatophore membrane and closed to the DE loop and helix G of cytochrome b, respectively.

The role of the supernumerary subunit of Rhodobacter sphaeroides cytochrome bc1 complex.

The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group, is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc1 complex. This subunit is involved in Q binding and the structural integrity of the complex. When the cytochrome bc1 complex is photoaffinity labeled with [3H]azido-Q derivative, radioactivity is found in subunits IV and I (cytochrome b), indicating that these two subunits are responsible for Q binding in the complex. When the subunit IV gene (fbcQ) is deleted from the R. sphaeroides chromosome, the resulting strain (RSdeltaIV) requires a period of adaptation before the start of photosynthetic growth. The cytochrome bc1 complex in adapted RSdeltaIV chromatophores is labile to detergent treatment (60-75% inactivation), and shows a four-fold increase in the Km for Q2H2. The first two changes indicate a structural role of subunit IV; the third change supports its Q-binding function. Tryptophan-79 is important for structural and Q-binding functions of subunit IV. Subunit IV is overexpressed in Escherichia coli as a GST fusion protein using the constructed expression vector, pGEX/IV. Purified recombinant subunit IV is functionally active as it can restore the bc1 complex activity from the three-subunit core complex to the same level as that of wild-type or complement complex. Three regions in the subunit IV sequence, residues 86-109, 77-85, and 41-55, are essential for interaction with the core complex because deleting one of these regions yields a subunit completely or partially unable to restore cytochrome bc1 from the core complex.