UNCOMPETITIVE SUBSTRATE-INHIBITION AND NONCOMPETITIVE INHIBITION BY 5-N-UNDECYL-6-HYDROXY-4,7-DIOXOBENZOTHIAZOLE (UHDBT) AND 2-N-NONYL-4-HYDROXYQUINOLINE-N-OXIDE (NQNO) IS OBSERVED FOR THE CYTOCHROME BO(3) COMPLEX - IMPLICATIONS FOR A Q(H-2)-LOOP PROTON TRANSLOCATION MECHANISM

The cytochrome bo(3) ubiquinol oxidase complex from Escherichia coli c ontains two binding sites for ubiquinone(ol) (UQ(H-2)). One of these b inding sites, the ubiquinol oxidation site, is clearly in dynamic equi librium with the UQ(H-2) pool in the membrane. The second site has a h igh affinity for ubiquinone (UQ), stabilizes a semiquinone species, an d is located physically close to the low-spin heme b component of the enzyme. The UQ molecule in this site has been proposed to remain stron gly bound to the enzyme during enzyme turnover and to act as a cofacto r facilitating the transfer of electrons from the substrate ubiquinol to heme b [Sato-Watanabe et al. (1994) J. Biol. Chem. 269, 28908-28912 ]. In this paper, the steady-state turnover of the enzyme is examined in the presence and absence of inhibitors (UHDBT and NQNO) that appear to be recognized as ubisemiquinone analogs. It is found that the kine tics are accounted for best by a noncompetitive inhibitor binding mode l. Furthermore, at high concentrations, the substrates ubiquinol-1 and ubiquinol-2 inhibit turnover in an uncompetitive fashion. Together, t hese observations strongly suggest that there must be at least two UQ( H-2) binding sites that are in rapid equilibrium with the UQ(H-2) pool under turnover conditions. Although these data do not rule out the po ssibility that a strongly bound UQ molecule functions to facilitate el ectron transfer to heme b, they are more consistent with the behavior expected if the two UQ(H-2) binding sites were to function in a Q(H-2) -loop mechanism (similar to that of the cytochrome bc(1) complex) as o riginally proposed by Musser and co-workers [(1993) FEBS Lett. 327, 13 1-136]. In this model, ubiquinol is oxidized at one site and ubiquinon e is reduced at the second site. While the structural similarities of the heme-copper ubiquinol and cytochrome c oxidase complexes suggest t he possibility that these two families of enzymes translocate protons by similar mechanisms, the current observations indicate that the Q(H- 2)-loop proton translocation mechanism for the heme-copper ubiquinol o xidase complexes should be further investigated and experimentally tes ted.