THE ILE(L229)-]MET MUTATION IMPAIRS THE QUINONE BINDING TO THE Q(B)-POCKET IN REACTION CENTERS OF RHODOBACTER-SPHAEROIDES

A spontaneous mutant (R/89) of photosynthetic purple bacterium Rhodoba cter sphaeroides R-26 was selected for resistance to 200 mu M atrazin. It showed increased resistance to interquinone electron transfer inhi bitors of o-phenanthroline (resistance factor, RF = 20) in UQ(0) recon stituted isolated reaction centers and terbutryne in reaction centers (RF = 55) and in chromatophores (RF = 85). The amino acid sequence of the Q(B) binding protein of the photosynthetic reaction center (the L subunit) was determined by sequencing the corresponding pufL gene and a single mutation was found (Ile(L229) --> Met). The changed amino aci d of the mutant strain is in van der Waals contact with the secondary quinone Q(B). The binding and redox properties of Q(B) in the mutant w ere characterized by kinetic (charge recombination) and multiple turno ver (cytochrome oxidation and semiquinone oscillation) assays of the r eaction center. The free energy for stabilization of Q(A)Q(B)(-) with respect to Q(A)(-)Q(B) was Delta G(AB) = -60 meV and 0 meV in reaction centers and Delta G(AB) = -85 meV and -46 meV in chromatophores of R- 26 and R/89 strains at pH 8, respectively. The dissociation constants of the quinone UQ(0) and semiquinone UQ(0)(-) in reaction centers from R-26 and R/89 showed significant and different pH dependence. The obs erved changes in binding and redox properties of quinones are interpre ted in terms of differential effects (electrostatics and mesomerism) o f mutation on the oxidized and reduced states of Q(B).