Subunit IV of Cytochrome bc 1 Complex from Rhodobacter sphaeroides

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.