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 bo vine heart ubiquinol-cytochrome c reductase (be, complex) varying decylbenz oquinol (DBH) and decylbenzoquinone (DB) concentrations was determined. The affinity for DBH was increased threefold by the protonation of a group wit h pK(a) = 5.7 +/- 0.2, while the inhibition constant (K-i) for DB decreased 22 and 2.8 times when groups with pK(a) = 5.2 +/- 0.6 and 7.7 +/- 0.2, res pectively, were protonated. This suggests stabilization of the protonated f orm of the acidic group by DBH binding. Initial rates were best fitted to a kinetic model involving three protonatable groups. The protonation of the pK(a) approximate to 5.7 group blocked catalysis, indicating its role in pr oton transfer. The kinetic model assumed that the deprotonation of two grou ps (pK(a) values of 7.5 +/- 0.03 and approximate to9.2) decreases the catal ytic rate by diminishing the redox potential of the iron-sulfur (Fe-S) clus ter. The protonation of the pK(a) approximate to 7.5 group also decreased t he reaction rate by 80-86%, suggesting its role as acceptor of a proton fro m ubiquinol. The lack of effect on the K-m for DBH when the pK(a) 7.5-7.7 g roup is deprotonated suggests that hydrogen bonding to this residue is not the main factor that determines substrate binding to the Q(o) site. The pos sible relationship of the pK(a) 5.2-5.7 and pK(a) 7.5-7.7 groups with Glu27 2 of cytochrome b and His161 of the Fe-S protein is discussed.