STUDY OF QB-STABILIZATION IN HERBICIDE-RESISTANT MUTANTS FROM THE PURPLE BACTERIUM RHODOPSEUDOMONAS-VIRIDIS

The pH dependences of the rate constants of P+Q(B)- (k(BP)) and P+Q(A)- (k(AP)) charge recombination decays have been studied by flash-induced absorbance change technique, in chromatophores of three herbicide-resistant mutants from Rhodopseudomonas (Rps.) viridis, and compared to the wild type. P, Q(A), and Q(B) are the primary electron donor and the primary and the secondary quinone acceptors, respectively. The triazine resistant mutants T1 (Arg L217 --> His and Ser L223 --> Ala), T3 (Phe L216 --> Ser and Val M263 --> Phe), and T4 (Tyr L222 --> Phe), all mutated in the Q(B) binding pocket of the reaction center, have previously been characterized (Sinning, I., Michel, H., Mathis, P., & Rutherford, A. W. (1989) Biochemistry 28, 5544-5553). The pH dependence curves of k(BP) in T4 and the wild type are very close. This confirms that the sensitivity toward DCMU of T4 is mainly due to a structural rearrangement in the QB pocket rather than to a change in the charge distribution in this part of the protein. In T3, a 6-fold increase of k(AP) is observed (k(AP) = 4200 +/- 300 s-1 at pH 8) compared to that of the wild type (k(AP) = 720 +/- 50 s-1 at pH 8). We propose that the Val M263 --> Phe mutation induces a free energy decrease between P+Q(A)- and P+I- (DELTA-G-degrees IA) (I is the primary electron acceptor) of about 49 meV. The very different pH dependence of k(AP) in T3 suggests a substantial change in the Q(A) pocket. The 2.5 times increase of k(AP) above pH 9.5 in the wild type is no longer detected in T3. Instead, a decrease of k(AP) is observed above pH 9.5 (k(AP) = 5100 +/- 300 s-1 at pH 9.5 and k(AP) = 3700 +/- 300 s-1 at pH 11). Since in Rps. viridis the k(AP) variations reflect the changes of DELTA-G-degrees IA, it seems that the protonatable group(s) involved in the increase of k(AP) in the wild type above pH 9.5 has shifted closer to I- than to Q(A)- in T3. The pH dependence of kBp in T3 is also very different from that of the wild type. The 6-fold increase observed in the wild type in the pH range 5.5-8 is no longer detected in T3. We suggest that the Phe L216 --> Ser mutation has an overall effect of shifting to lower pH the p(K) of the group (p(K) approximately 6.5) involved in the DELTA-G-degrees BA (free energy difference between P+Q(B)- and P+Q(A)-) variations at low pH in the wild type. The temperature dependences of k(AP), k(BP), and K2, the Q(A)-Q(B)- <--> Q(A)Q(B)- equilibrium constant, have been determined in T3 and the wild type. At pH 8, the energy barrier between Q(A)- and Q(B)- is substantially increased in T3 (DELTA-G-degrees BA = -0.224 +/- 0.015 eV) compared to that of the wild type (DELTA-G-degrees BA = -0.131 + 0.015 eV). The relative contribution of enthalpic and entropic terms to DELTA-G-degrees BA is very different in T3 and the wild type. In T1, above pH 7, Q(B)- is destabilized compared to the wild type. Assuming that this effect is mainly due to the absence of the positive charge present on Arg L217, we suggest that the apparent p(K) of His L217 in T1 is 8.3 +/- 0.2. The K2 values in T1, T4, and wild type have been compared with the previously measured relative binding affinities of Q(B) (Q50's). The Q(B) binding pocket of the wild type looks well designed for a simultaneous optimization of K2 and Q50's.