Proton and electron transfer to the secondary quinone (QB) in bacterial reaction centers: the effect of changing the electrostatics in the vicinity of QB by interchanging asp and glu at the L212 and L213 sites.
Proton and electron transfer to the secondary quinone (QB) in bacterial reaction centers: the effect of changing the electrostatics in the vicinity of QB by interchanging asp and glu at the L212 and L213 sites.
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质子和电子转移到细菌反应中心的仲醌 (QB)：通过交换 L212 和 L213 位点的 asp 和 glu 来改变 QB 附近静电的效果。
DOI：
10.1021/bi971192m
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Okamura,MY
中科院分区：
文献类型：
--
作者：
Paddock,ML;Feher,G;Okamura,MY
细菌反应中心(RC)通过促进结合的苯醌分子QB的光诱导二次还原和质子化，在光合作用能量转换中起着核心作用。位于Qb附近的两个羧酸残基Asp-L213和Glu-L212先前被证明对质子转移到Qb是重要的。在这项工作中，通过定点突变检测了Glu在L213替换Asp和Asp在L212替换Glu的能力。构建了天冬氨酸和谷氨酸在两个位点之间交换的单突变体和双突变体。电子转移速率常数kBD(D+QAQB-→DQAQB)和kAB(2)(DQA-QB-+H+→DQA(QBH)-)被发现被天冬氨酸/谷氨酸取代改变。这两种速率在RCS和Asp两个部位都是最快的(∼10倍)，在两个部位用Glu最慢(∼50倍)，而羧酸交换相对不变。如果天冬氨酸主要是电离的，而谷氨酸在两个部位都主要是质子化的(pH 7.5)，这些变化可以解释。电荷重组kBD表明观察到的谷氨酸和天冬氨酸的∼5pka单位不同。通过负电酸和qB-之间的强烈静电相互作用(∼3−4 pka单位)对kBD的模拟表明，在∼2−3单位的任一位置，天冬氨酸的固有pKafor比Glu低。所有突变型RCS的kAB(2)反应机理与天然RCS相同。利用为自然RCS提出的两步模型[Graige，M.S.，Paddock，M.L.，Bruce，J.M.，Feher，G.，&Okamura，M.Y.(1996)J.Am]，定量解释了静电环境对AB(2)的影响化学。用简单的模型计算了对苯二酚/对苯二酚的转化率，结果表明，QB位的最佳静电势与天然−中的结果相近。
The bacterial reaction center (RC) plays a central role in photosynthetic energy conversion by facilitating the light induced double reduction and protonation of a bound quinone molecule, QB. Two carboxylic acid residues, Asp-L213 and Glu-L212, located near QB, were previously shown to be important for proton transfer to QB. In this work, the ability of Glu to substitute for Asp at L213 and Asp to substitute for Glu at L212 was tested by site-directed mutagenesis. Both single mutants and a double mutant in which Asp and Glu were exchanged between the two sites were constructed. The electron transfer rate constantskBD(D+QAQB-→ DQAQB), andkAB(2)(DQA-QB-+ H+→ DQA(QBH)-), that are known to be sensitive to the energy of the QB-state, were found to be altered by Asp/Glu substitutions. Both rates were fastest (∼10-fold) in RCs with Asp at both sites, slowest with Glu at both sites (∼50-fold) and relatively unchanged by the caboxylic acid exchange. These changes could be explained if Asp was predominantly ionized and Glu was predominantly protonated at both sites (pH 7.5). The charge recombinationkBDsuggests an observed ∼5 pKaunit difference of Glu over Asp. Modeling ofkBDby strong electrostatic interactions (∼3−4 pKaunits) among negatively charged acids and QB-indicated a lower intrinsic pKafor Asp compared to Glu at either site of ∼2−3 units. The mechanism of thekAB(2)reaction was determined to be the same in all mutant RCs as for native RCs. A quantitative explanation of the effect of the electrostatic environment onkAB(2)was obtained using the two-step model proposed for native RCs [Graige, M. S., Paddock, M. L., Bruce, J. M., Feher, G., & Okamura, M. Y. (1996)J. Am. Chem. Soc.118, 9005−9016] which involves fast protonation of the semiquinone followed by rate-limiting electron transfer.Using simple models for the quinone/quinol conversion rate, it is shown that the optimal electrostatic potential for the QBsite is close to that found in native RCs.