The Role of the Supernumerary Subunit of Rhodobactersphaeroides Cytochrome bc ₁ Complex

Abstract

The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group,is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc ₁ complex.This subunit is involved in Q binding and the structural integrity of the complex. When thecytochrome bc ₁ complex is photoaffinity labeled with [³H]azido-Q derivative, radioactivity isfound in subunits IV and I (cytochrome b), indicating that these two subunits are responsiblefor Q binding in the complex. When the subunit IV gene (fbcQ) is deleted from the R.sphaeroides chromosome, the resulting strain (RSΔIV) requires a period of adaptation beforethe start of photosynthetic growth. The cytochrome bc ₁ complex in adapted RSΔIVchromatophores is labile to detergent treatment (60–75% inactivation), and shows a four-fold increasein the K _m for Q₂H₂. The first two changes indicate a structural role of subunit IV; the thirdchange supports its Q-binding function. Tryptophan-79 is important for structural andQ-binding functions of subunit IV. Subunit IV is overexpressed in Escherichia coli as a GSTfusion protein using the constructed expression vector, pGEX/IV. Purified recombinant subunitIV is functionally active as it can restore the bc ₁ complex activity from the three-subunit corecomplex to the same level as that of wild-type or complement complex. Three regions in thesubunit IV sequence, residues 86–109, 77–85, and 41–55, are essential for interaction withthe core complex because deleting one of these regions yields a subunit completely or partiallyunable to restore cytochrome bc ₁ from the core complex.