91 10 Biochemistry zyxwvu 1991, 30, 91 10-91 16 A. (1985) J. Biol. Chem. 260, 4688-4693. Yamashita, K., Tachibana, Y., Matsuda, Y., Katsunuma, N., Kochibe, N., zyxwvutsrq & Kobata, A. (1988) Biochemistry 27, 5565-5573. Yoshima, H., Takasaki, zyxw S., & Kobata, A. (1980) J. Biol. Yoshima, H., Matsumoto, A., Mizuochi, T., Kawasaki, T., & Chem. 225, 10793-10804. Kobata, A. (1981) J. Biol. Chem. 256, 8476-8484. Study of QB- Stabilization in Herbicide-Resistant Mutants from the Purple Bacterium Rhodopseudomonas viridis Laura Baciou,* Irmgard Sinning,$ and Pierre Sebban*** UPR 407, Bat 24 CNRS, Centres Rlactionnels Photosynthltiques, Gif Sur Yvette 91 198, France, and Max-Planck-Institut zy fur Biophysik, Heinrich- Hoffmann Strasse 7, 0-6000 Frankfurt 71, Germany Received November 29, 1990; Revised Manuscript Received June 27, 1991 ABSTRACT: The pH dependences of the rate constants of P'QB- (kBp) and P'QA- (kM) charge recombination decays have been studied by flash-induced absorbance change technique, in chromatophores of three herbicide-resistant mutants from Rhodopseudomonas (Rps.) uiridis, and compared to the wild type. P, QA, and QB 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 QB 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 kBp 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 kAP is observed (kAp = 4200 f 300 s-' at pH 8) compared to that of the wild type (kAp = 720 zy f 50 s-l at pH 8). We propose that the Val M263 - Phe mutation induces a free energy decrease between P'QA- and P'I- (AGOIA) (I is the primary electron acceptor) of about 49 meV. The very different pH dependence of kAP in T3 suggests a substantial change in the QA pocket. The 2.5 times increase of kAP above pH 9.5 in the wild type is no longer detected in T3. Instead, a decrease of kAP is observed above pH 9.5 (kAp = 5100 f 300 s-l at pH 9.5 and kAP = 3700 f 300 s-* at pH 11). Since in Rps. uiridis the kAp variations reflect the changes of AGOIA, it seems that the protonatable groups@) involved in the increase of kAp in the wild type above pH 9.5 has shifted closer to I- than to QA- 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 pK of the group (pK zyxwv N 6.5) involved in the AGOBA (free energy difference between P'QB- and P+QA-) variations at low pH in the wild type. The temperature dependences of kAP,kBp, and K2, the QA-QB * QAQB- equilibrium constant, have been determined in T3 and the wild type. At pH 8, the energy barrier between QA- and QB- is substantially increased in T3 (AGOBA = -0.224 f 0.015 eV) compared to that of the wild type (AGOBA = -0.131 + 0.015 eV). The relative contribution of enthalpic and entropic terms to AGOBA is very different in T3 and the wild type. In T1, above pH 7, QB- 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 pK of His L217 in T1 is 8.3 f 0.2. The K2 values in T1, T4, and wild type have been compared with the previously measured relative binding affinities of QB (Q50'~). The QB binding pocket of the wild type looks well designed for a simultaneous optimization of K2 and Q50'~. %e absorption of light energy by photosynthetic organisms results in the creation of a transmembrane charge-separated state of the reaction centers, in less than a nanosecond. The photosynthetic reaction center from Rhodopseudomonas (Rps.) uiridis is composed of four polypeptides, the so-called H, M, and L subunits and a tightly bound cytochrome c. The three-dimensional structure of the reaction centers from Rps. viridis and Rhodobacter (Rb.) sphaeroides became known since their successful crystallization and X-ray structure * To whom correspondence should be addressed. *Centres RCctionnels Photosynthetiques. 1 Max-Planck-lnstitut fiir Biophysik. analysis (Allen et al., 1988; Arnoux et al., 1989; Chang et al., 1986; Deisenhofer et al., 1985). The primary charge sepa- ration occurs in these reaction centers between a dimer of bacteriochlorophyll, P, and a quinone, QA, bound to the M polypeptide. The electron present on QA is then transferred to a secondary quinone, QB (bound to the L polypeptide), which can be doubly reduced. QB- is tightly bound to the reaction centers (Wraight, 1981), whereas QB2- is loosely bound and is supposed to leave the reaction center in its quinol state, QBH2, after two protons have been uptaken from the cytoplasm. In vivo, the redox potentials of the quinone molecules are very different compared to their values measured in different solvents (Gunner et al., 1986; Woodbury et al., 0006-2960/91/0430-9110$02.50/0 0 1991 American Chemical Society