MUTANTS OF UBIQUINOL CYTOCHROME-C2 OXIDOREDUCTASE RESISTANT TO Q0 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 Q(o) 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 Q(o) site [Robertson, D. E., Davidsion, 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 Q(o) site or kinetic impairment to catalysis. Mutants selected for myxothiazole or mucidin resistance, inducing weakening of inhibitor binding, all displayed impaired rates of Q(o) 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 Q(pool) with the Q(o) site. Consideration of the mutational substitutions and their structural positions along with comparisons with the Q(A) and Q(B) sites of the photosynthetic reaction center suggests a model for the structure of the Q(o) site.