Rautter J, Lendzian F, Schulz C, Fetsch A, Kuhn M, Lin X, Williams J C, Allen J P, Lubitz W
Max-Volmer-Institut für Biophysikalische und Physikalische Chemie, Technische Universität Berlin.
Biochemistry. 1995 Jun 27;34(25):8130-43. doi: 10.1021/bi00025a020.
The electronic structure of the cation radical of the primary electron donor was investigated in genetically modified reaction centers of Rhodobacter sphaeroides. The site-directed mutations were designed to add or remove hydrogen bonds between the conjugated carbonyl groups of the primary donor, a bacteriochlorophyll dimer, and histidine residues of the protein and were introduced at the symmetry-related sites L168 His-->Phe, HF(L168), and M197 Phe-->His, FH(M197), near the 2-acetyl groups of the dimer and at sites M160 Leu-->His, LH(M160), and L131 Leu-->His, LH(L131), in the vicinity of the 9-keto carbonyls of the dimer. The single mutants and a complete set of double mutants were studied using EPR, ENDOR, and TRIPLE resonance spectroscopy. The changes in the hydrogen bond situation of the primary donor were accompanied by changes in the dimer oxidation midpoint potential, ranging from 410 to 710 mV in the investigated mutants [Lin, X., Murchison, H. A., Nagarajan, V., Parson, W. W., Williams, J. C. & Allen, J. P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10265-10269]. It was found that the addition or removal of a hydrogen bond causes large shifts of the spin density between the two halves of the dimer. Measurements on double mutants showed that the unpaired electron can be gradually shifted from a localization on the L-half of the dimer to a localization on the M-half, depending on the hydrogen bond situation. As a control, the effects of the different hydrogen bonds on P.+ in the mutant HL(M202), which contains a BChlL-BPheM heterodimer as the primary donor with localized spin on the BChl aL [Bylina, E. J., & Youvan, D. C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7226-7230; Schenck, C. C., Gaul, D., Steffen M., Boxer S. G., McDowell L., Kirmaier C., & Holten D. (1990) in Reaction Centers of Photosynthetic Bacteria (Michel-Beyerle M. E., Ed.) pp 229-238, Springer, Berlin] were studied. In this mutant only small local changes of the spin densities (< or = 10%) in the vicinity of the hydrogen bonds were observed. The effects of the introduced hydrogen bonds on the spin density distribution of the dimer in the mutants are discussed in terms of different orbital energies of the two BChl a moieties which are directly influenced by hydrogen bond formation. The observed changes of the spin density distribution for the double mutants are additive with respect to the single mutations.(ABSTRACT TRUNCATED AT 400 WORDS)
在球形红杆菌的基因工程改造反应中心中，对初级电子供体阳离子自由基的电子结构进行了研究。定点突变的设计目的是在初级供体（细菌叶绿素二聚体）的共轭羰基与蛋白质的组氨酸残基之间添加或去除氢键，这些突变被引入到二聚体2 - 乙酰基附近的对称相关位点L168 His→Phe、HF(L168)和M197 Phe→His、FH(M197)，以及二聚体9 - 酮羰基附近的位点M160 Leu→His、LH(M160)和L131 Leu→His、LH(L131)。使用电子顺磁共振（EPR）、电子核双共振（ENDOR）和三重共振光谱对单突变体和全套双突变体进行了研究。初级供体氢键情况的变化伴随着二聚体氧化中点电位的变化，在所研究的突变体中，该电位范围为410至710毫伏[林，X.，默奇森，H. A.，纳加拉扬，V.，帕森，W. W.，威廉姆斯，J. C.和艾伦，J. P.（1994年）《美国国家科学院院刊》91，10265 - 10269]。发现氢键的添加或去除会导致二聚体两半部分之间的自旋密度发生大幅偏移。对双突变体的测量表明，取决于氢键情况，未成对电子可以从二聚体L半部分的局域化逐渐转移到M半部分的局域化。作为对照，研究了不同氢键对突变体HL(M202)中P.+的影响，该突变体含有BChlL - BPheM异二聚体作为初级供体，其自旋局域在BChl aL上[比林纳，E. J.，& 尤万，D. C.（1988年）《美国国家科学院院刊》85，7226 - 7230；申克，C. C.，高尔，D.，斯特芬，M.，博克瑟，S. G.，麦克道威尔，L.，基尔迈尔，C.，& 霍尔滕，D.（1990年）载于《光合细菌的反应中心》（米歇尔 - 拜耶勒，M. E.编）第229 - 238页，施普林格出版社，柏林]。在这个突变体中，仅观察到氢键附近自旋密度的小的局部变化（≤10%）。根据氢键形成直接影响的两个BChl a部分的不同轨道能量，讨论了引入的氢键对突变体中二聚体自旋密度分布的影响。观察到的双突变体自旋密度分布的变化相对于单突变是可加的。（摘要截断于400字）
