Hydroubiquinone-Cytochrome c₂ Oxidoreductase from Rhodobacter capsulatus: Definition of a Minimal, Functional Isolated Preparation

The hydroubiquinone-cytochrome c₂ oxidoreductase (cyt bc₁) from Rhodobacter capsulatus has been solubilized according to the dodecyl maltoside method and isolated, and its minimal functional composition has been characterized. We find the complex to be composed of three protein subunits corresponding to polypeptides of cyt b (44 kDa), cyt c₁ (33 kDa), and 2Fe2S cluster (24 kDa). A fourth band sometimes discernable at 22 kDa appears to be an artifact of the Polyacrylamide gel electrophoresis procedure. Its appearance is shown to be derived from the 2Fe2S cluster subunit by the similarity of the binding of subunit-specific monoclonal antibodies and the identical N-terminal sequence of the 24- and 22-kDa bands. The cofactors of cyt bc₁, namely, cyt b_H, cyt b_L, cyt c₁, and the 2Fe2S center, the Q_os and Q_ow domains of the Q_o site, and the Q_i site appear intact as indicated by their optical and EPR spectral signatures, redox properties, and inhibitor binding. The electron paramagnetic resonance spectrum of the cyt b_H heme is altered by antimycin, consistent with a change in the dihedral angle between the ligating histidine imidazoles, while the spectrum of the cyt b_L heme is broadened by stigmatellin. The ubiquinone-10 content is variable, ranging from 0.8 to 3 molecules/cyt bc₁. Activity studies define this three-subunit cyt bc₁ complex as a minimal structure, equipped as the enzyme in the native state and capable of full catalytic activity.