Role of protonatable groups of bovine heart bc1 complex in ubiquinol binding and oxidation

Abstract

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 ≈ 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 ≈ 9.2) decreases the catalytic rate by diminishing the redox potential of the iron-sulfur (Fe-S) cluster. The protonation of the pKa ≈ 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.