Müh F, Williams J C, Allen J P, Lubitz W
Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Technische Universität Berlin, Germany.
Biochemistry. 1998 Sep 22;37(38):13066-74. doi: 10.1021/bi980799f.
It is demonstrated by ENDOR and Special TRIPLE spectroscopy that two distinct radical anion states of the intermediate electron acceptor (I), a bacteriopheophytin, can be freeze-trapped in isolated photosynthetic reaction centers of Rhodobacter sphaeroides. The formation of these states depends on the illumination time prior to freezing and the temperature. The first state, I1.-, is metastable and relaxes irreversibly at T approximately 160 K to the second state, I2.-. Experiments on quinone depleted as well as mutant reaction centers help to exclude the possibility that other cofactors besides the bacteriopheophytin in the A-branch, PhiA, are reduced during the trapping procedure. In particular, two mutants are investigated, in which the hydrogen bonds to PhiA that exist in the wild type are removed. These mutants are EL(L104), in which Glu at position L104 near the 13(1)-keto group of PhiA is replaced by Leu, and WF(L100), in which Trp at position L100 near the 13(2)-methyl ester of PhiA is replaced by Phe. Both mutations have characteristic effects on both I.- states. In addition, the replacement of Thr at position M133 near the 13(1)-keto group of the inactive bacteriopheophytin and of Gly at position M203 near the 13(1)-keto group of the accessory bacteriochlorophyll in the A-branch by Asp causes no changes of the electronic structure of I.-. The two I.- states are interpreted in terms of a reorientation of the 3-acetyl group of PhiA after reduction. Possible implications for the initial charge separation process are discussed.
电子核双共振（ENDOR）和特殊三重态光谱表明，中间电子受体（I）——细菌叶绿素的两种不同的自由基阴离子态可以在球形红杆菌的分离光合反应中心中被冷冻捕获。这些态的形成取决于冷冻前的光照时间和温度。第一种态I1.-是亚稳态的，在约160K的温度下不可逆地弛豫为第二种态I2.-。对醌耗尽的反应中心以及突变反应中心进行的实验有助于排除在捕获过程中除了A分支中的细菌叶绿素PhiA之外的其他辅因子被还原的可能性。特别地，研究了两个突变体，其中野生型中存在的与PhiA的氢键被去除。这些突变体是EL（L104），其中PhiA的13(1)-酮基附近L104位的谷氨酸被亮氨酸取代；以及WF（L100），其中PhiA的13(2)-甲酯附近L100位的色氨酸被苯丙氨酸取代。这两种突变对两种I.-态都有特征性影响。此外，将非活性细菌叶绿素的13(1)-酮基附近M133位的苏氨酸以及A分支中辅助细菌叶绿素的13(1)-酮基附近M203位的甘氨酸替换为天冬氨酸不会引起I.-电子结构的变化。两种I.-态是根据PhiA还原后3-乙酰基的重新取向来解释的。讨论了对初始电荷分离过程的可能影响。
