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 bH, cyt bL, cyt c1, and the 2Fe2S center, the Qos and Qow domains of the Qo site, and the Qi 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 bH heme is altered by antimycin, consistent with a change in the dihedral angle between the ligating histidine imidazoles, while the spectrum of the cyt bL 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.
Department of Biochemistry and Biophysics, Johnson Research Foundation, Philadelphia, Pennsylvania.
D E
DE
Robertson
H
H
Ding
P R
PR
Chelminski
C
C
Slaughter
J
J
Hsu
C
C
Moomaw
M
M
Tokito
F
F
Daldal
P L
PL
Dutton
D000595:Amino Acid Sequence; D000906:Antibodies; D000911:Antibodies, Monoclonal; D020130:Bacterial Chromatophores / Q000201:enzymology; D015153:Blotting, Western; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D014451:Ubiquinone / Q000032:analysis
D000906:Antibodies; D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D014451:Ubiquinone; D014450:Electron Transport Complex III