Role of protonatable groups of bovine heart bc(1) complex in ubiquinol binding and oxidation.
The pH dependence of the initial reaction rate catalyzed by the isolated bovine heart ubiquinol-cytochrome c reductase (bc1 complex) varying decylbenzoquinol (DBH) and decylbenzoquinone (DB) concentrations was determined. The affinity for DBH was increased threefold by the protonation of a group with pKa = 5.7 +/- 0.2, while the inhibition constant (Ki) for DB decreased 22 and 2.8 times when groups with pKa = 5.2 +/- 0.6 and 7.7 +/- 0.2, respectively, were protonated. This suggests stabilization of the protonated form of the acidic group by DBH binding. Initial rates were best fitted to a kinetic model involving three protonatable groups. The protonation of the pKa approximately 5.7 group blocked catalysis, indicating its role in proton transfer. The kinetic model assumed that the deprotonation of two groups (pKa values of 7.5 +/- 0.03 and approximately 9.2) decreases the catalytic rate by diminishing the redox potential of the iron-sulfur (Fe-S) cluster. The protonation of the pKa approximately 7.5 group also decreased the reaction rate by 80-86%, suggesting its role as acceptor of a proton from ubiquinol. The lack of effect on the Km for DBH when the pKa 7.5-7.7 group is deprotonated suggests that hydrogen bonding to this residue is not the main factor that determines substrate binding to the Qo site. The possible relationship of the pKa 5.2-5.7 and pKa 7.5-7.7 groups with Glu272 of cytochrome b and His161 of the Fe-S protein is discussed.
Covián
R
R
Departamento de Bioquímica, Instituto Nacional de Cardiología, México. covianraul@correa.unam.mx
Moreno-Sánchez
R
R
D000818:Animals; D002384:Catalysis; D002417:Cattle; D014450:Electron Transport Complex III / Q000378:metabolism*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D011485:Protein Binding; D011522:Protons; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000378:metabolism*
D011522:Protons; D014451:Ubiquinone; D014450:Electron Transport Complex III; C003741:ubiquinol