Mutants of ubiquinol-cytochrome c2 oxidoreductase resistant to Qo site inhibitors: consequences for ubiquinone and ubiquinol affinity and catalysis.
Seven single-site mutants in six residues of the cyt b polypeptide of Rhodobacter capsulatus selected for resistance to the Qo site inhibitors stigmatellin, myxothiazol, or mucidin [Daldal, F., Tokito, M.K., Davidson, E., & Faham, M. (1989) EMBO J. 8, 3951-3961] have been characterized by using optical and EPR spectroscopy and single-turnover kinetic analysis. The strains were compared with wild-type strain MT1131 and with the Ps- strain R126 (G158D), which is dysfunctional in its Qo site [Robertson, D.E., Davidson, E., Prince, R.C., van den Berg, W.H., Marrs, B.L., & Dutton, P.L. (1986) J. Biol. Chem. 261, 584-591]. Mutants selected for stigmatellin resistance induced a weakening in the binding of the inhibitor without discernible loss of ubiquinone(Q)/ubiquinol(QH2) binding affinity to the Qo site or kinetic impairment to catalysis. Mutants selected for myxothiazol or mucidin resistance, inducing weakening of inhibitor binding, all displayed impaired rates of Qo site catalysis: The most severe cases (F144L, F144S) displayed loss of affinity for Q, and evidence suggests that parallel loss of affinity for the substrate QH2 was incurred in these strains. The results provide a view of the nature of the interaction of Q and QH2 of the Qpool with the Qo site. Consideration of the mutational substitutions and their structural positions along with comparisons with the QA and QB sites of the photosynthetic reaction center suggests a model for the structure of the Qo site.
Robertson
D E
DE
Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia 19104.
Daldal
F
F
Dutton
P L
PL
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D007700:Kinetics; D008689:Methacrylates; D008961:Models, Structural; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D011090:Polyenes / Q000494:pharmacology; D011487:Protein Conformation; D016354:Rhodobacter capsulatus / Q000378:metabolism; D000073739:Strobilurins; D013844:Thiazoles / Q000494:pharmacology; D014451:Ubiquinone / Q000378:metabolism* / Q000031:analogs & derivatives
D000900:Anti-Bacterial Agents; D003573:Cytochrome b Group; D005231:Fatty Acids, Unsaturated; D008689:Methacrylates; D011090:Polyenes; D000073739:Strobilurins; D013844:Thiazoles; D014451:Ubiquinone; C008556:mucidin; C030517:myxothiazol; C041573:stigmatellin; D014450:Electron Transport Complex III; C003741:ubiquinol