Modeling the Bacterial Photosynthetic Reaction Center. 4. The Structural, Electrochemical, and Hydrogen-Bonding Properties of 22 Mutants of <i>Rhodobacter sphaeroides</i>

Site-directed mutagenesis has been employed by a number of groups to produce mutants of bacterial photosynthetic reaction centers, with the aim of tuning their operation by modifying hydrogen-bond patterns in the close vicinity of the “special pair” of bacteriochlorophylls P ≡ PLPM. Direct X-ray structural measurements of the consequences of mutation are rare. Attention has mostly focused on effects on properties such as carbonyl stretching frequencies and midpoint potentials to infer indirectly the induced structural modifications. In this work, the structures of 22 mutants of Rhodobacter sphaeroides have been calculated using a mixed quantum-mechanical molecular-mechanical method by modifying the known structure of the wild type. We determine (i) the orientation of the 2a-acetyl groups in the wild type, FY(M197), and FH(M197) series mutants of the neutral and oxidized reaction center, (ii) the structure of the FY(M197) mutant and possible water penetration near the special pair, (iii) that significant protein chain distortions are required to assemble some M160 series mutants (LS(M160), LN(M160), LQ(M160), and LH(M160) are considered), (iv) that there is competition for hydrogen-bonding between the 9-keto and 10a-ester groups for the introduced histidine in LH(L131) mutants, (v) that the observed midpoint potential of P for HL(M202) heterodimer mutants, including one involving also LH(M160), can be correlated with the change of electrostatic potential experienced at PL, (vi) that hydrogen-bond cleavage may sometimes be induced by oxidation of the special pair, (vii) that the OH group of tyrosine M210 points away from PM, and (viii) that competitive hydrogen-bonding effects determine the change in properties of NL(L166) and NH(L166) mutants. A new technique is introduced for the determination of ionization energies at the Koopmans level from QM/MM calculations, and protein-induced Stark effects on vibrational frequencies are considered.多个研究团队已采用定点诱变（site-directed mutagenesis）技术构建细菌光合反应中心突变体，旨在通过修饰“特殊对”（special pair）细菌叶绿素（bacteriochlorophylls）P ≡ PLPM近邻区域的氢键模式，调控其功能运作。直接通过X射线结构测定表征诱变引发的结构变化的研究较为罕见。 此前的相关研究多聚焦于羰基伸缩振动频率、中点电位等性质的变化，以此间接推断诱变所诱导的结构改变。 本研究通过修饰已知的野生型（wild type）结构，采用量子力学-分子力学联用（mixed quantum-mechanical molecular-mechanical, QM/MM）方法，计算得到了球形红杆菌（Rhodobacter sphaeroides）22株突变体的结构。本研究明确了以下八项核心结论：(i) 中性与氧化态反应中心的野生型、FY(M197)及FH(M197)系列突变体中2a-乙酰基的取向；(ii) 解析FY(M197)突变体的结构，以及特殊对区域潜在的水分子渗透情况；(iii) 组装部分M160系列突变体（本研究涵盖LS(M160)、LN(M160)、LQ(M160)及LH(M160)）时，需要发生显著的蛋白质主链构象畸变；(iv) LH(L131)突变体中，引入的组氨酸（histidine）残基会与9-酮基及10a-酯基发生氢键结合竞争；(v) HL(M202)异二聚体（heterodimer）突变体（其中一株同时携带LH(M160)突变）的P位点实测中点电位，可与PL区域的静电势变化建立关联；(vi) 特殊对的氧化作用有时可引发氢键断裂；(vii) 酪氨酸（tyrosine）M210的羟基朝向远离PM的一侧；(viii) 竞争性氢键结合效应决定了NL(L166)与NH(L166)突变体的性质变化。 本研究还提出了一种基于QM/MM计算确定库普曼斯（Koopmans）能级电离能的新方法，并探讨了蛋白质诱导的斯塔克（Stark）效应对振动频率的影响。