ROLES IN INHIBITOR RECOGNITION AND QUINOL OXIDATION OF THE AMINO-ACIDSIDE-CHAINS AT POSITIONS OF CYT B PROVIDING RESISTANCE TO QO-INHIBITORS OF THE BC1 COMPLEX FROM RHODOBACTER-CAPSULATUS

The substitutions M1401, F144S and L, G152S, T163A and V333A in cytoch rome b of the ubiquinolcytochrome c oxidoreductase (bc1 complex) from Rhodobacter capsulatus provide resistance to the quinol oxidation (Q(o )) inhibitors myxothiazol, mucidin and stigmatellin. Site-directed mut agenesis with degenerate primers was used to define the role of these positions in inhibitor recognition and quinol oxidation, and a collect ion of various substitutions at each of these positions was obtained. The effects of these mutations on quinol oxidation, nature and level o f inhibitor resistance, prosthetic group incorporation and assembly of the complex were analysed. Most of these mutations, unlike those at p osition 158 reported earlier, yielded functional bc1 complexes able to support the photosynthetic growth of R. capsulatus. However, they per turbed steady-state quinol oxidation and inhibitor recognition indicat ing that they are important for the function of the Q(o) site. In part icular, the presence of a methyl group on the beta-carbon (Ile and Val residues) at position 140, the absence of an aromatic ring (Phe, Tyr and Trp residues) at position 144 and the loss of residues with small side chains (Gly and Ala) at position 152 correlated with resistance t o myxothiazol. On the other hand, no myxothiazol resistance was observ ed with the substitutions at positions 163 and 333 suggesting that the y affected solely the recognition of stigmatellin. Five substitutions, M140R, F144H and R, G152P and T163R, yielded photosynthesis-deficient mutants with assembled but impaired bc1 complexes. Unexpectedly, two substitutions at position 163 (T to F or P) yielded mutants lacking th e three subunits of the bc1 complex indicating that this position affe cts its assembly or stability in vivo. These findings are discussed in terms of the contributions of these residues to inhibitor recognition and quinol oxidation at the Q(o) site of the bc1 complex.