Functional characterization of the mitochondrial cytochrome b-c1 complex: steady-state kinetics of the monomeric and dimeric forms.
The QH2:cytochrome c oxidoreductase activity of the isolated bovine heart cytochrome b-c1 complex resolved into monomeric and dimeric form was titrated with three different inhibitors of electron transfer, antimycin, myxothiazol, and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). In all cases one inhibitor molecule per cytochrome c1 was found necessary to block completely the activity of both molecular forms of the enzyme. The antimycin-sensitive cytochrome c reduction catalyzed by the b-c1 complex was also studied as a function of increasing concentrations of either cytochrome c or quinol. Double-reciprocal plots of the activity of the monomeric enzyme were found linear either when the concentration of cytochrome c or of quinol derivatives, 2,3-dimetoxy-5-methyl-6-decyl-1,4-benzoquinol (DBH), and 2-methyl-3-undecyl-1,4-naphthoquinol (UNH), was changed. Cytochrome c reductase activity of the dimeric b-c1 complex also showed a linear Lineweaver-Burk plot as a function of cytochrome c concentrations. In contrast to the monomeric enzyme, however, dimers of the b-c1 complex express a clear nonlinear kinetic behavior toward quinol derivatives, with two apparent Km values differing approximately by one order of magnitude (about 3-4 and about 20-30 microM). At saturating quinol concentrations the activity of the dimeric enzyme becomes two to three times higher than that of monomers. The nonlinear kinetic plots were found to be the same at different temperatures and different cytochrome c concentrations. The data suggest that although the monomer of the b-c1 complex appears to be the functional unit of the enzyme, the dimer is more active. A regulatory role of the dimerization process resulting in an increase of the electrons flux through the enzyme is postulated.
Nałecz
M J
MJ
Azzi
A
A
D000818:Animals; D000968:Antimycin A / Q000031:analogs & derivatives / Q000494:pharmacology; D002417:Cattle; D003574:Cytochrome c Group / Q000378:metabolism; D014450:Electron Transport Complex III; D006873:Hydroquinones / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008689:Methacrylates; D008929:Mitochondria, Heart / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D011808:Quinone Reductases / Q000378:metabolism*; D013844:Thiazoles / Q000494:pharmacology
C025993:5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole; D003574:Cytochrome c Group; D006873:Hydroquinones; D046911:Macromolecular Substances; D008689:Methacrylates; D009097:Multienzyme Complexes; D013844:Thiazoles; C032456:antimycin; D000968:Antimycin A; C030517:myxothiazol; D009247:NADH, NADPH Oxidoreductases; D011808:Quinone Reductases; D014450:Electron Transport Complex III