Electrostatic influence of QA reduction on the IR vibrational mode of the 10a-ester C==O of HA demonstrated by mutations at residues Glu L104 and Trp L100 in reaction centers from Rhodobacter sphaeroides.
Electrostatic influence of QA reduction on the IR vibrational mode of the 10a-ester C==O of HA demonstrated by mutations at residues Glu L104 and Trp L100 in reaction centers from Rhodobacter sphaeroides.
复制标题
QA 还原对 HA 10a-酯 C==O IR 振动模式的静电影响通过球形红杆菌反应中心残基 Glu L104 和 Trp L100 的突变证明。
DOI：
10.1021/bi962871k
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Williams,JC
中科院分区：
文献类型：
--
作者：
Breton,J;Nabedryk,E;Allen,JP;Williams,JC
球形红杆菌反应中心(RCS)的初级对苯二酚(QA)的光诱导QA/QAFTIR差谱在1750-1715 cm-1之间有一组复杂的差分带。其中几个特征在频率上对应于细菌叶绿素电子受体(HA)光还原的HA-/HAFTIR差谱中漂白的谱带。由于来自HA的10a-酯CO和质子化羧酸的侧链可能在这一光谱区有贡献，因此在Trp L100和Glu L104设计了突变，这两个突变被认为分别与HA的10a-酯和V环上的9-酮基羰基形成氢键。将野生型(WT)RCS在1H2O和2H2O中的QA/QA谱与RCS在L100[WF(L100)]、L104[EL(L104)]Glu到Leu突变[EL(L104)/WF(L100)]的突变[EL(L104)/WF(L100)]的QA/QA谱进行比较。与WT相比，突变体在1800−1400 cm-1频率范围内的光谱表现出有限的扰动，表明突变没有显著的结构变化。WT RCS光谱中1732 cm-1附近的部分差分信号在EL(L104)中下移了≈7 cm-1，而在WF(L100)中上移了≈11 cm-1。差动信号的这种上移归因于与色氨酸L100的氢键断裂所引起的HA的10a-酯CO的频率变化。WT RCS的1H2O--2H2O双差分光谱在1730 cm-1处为正信号，在1724 cm-1处为负信号，这在EL(L104)和双突变体的相应光谱中不存在，暗示Glu L104参与了QA-/QA光谱的变化。在WF(L100)的1H2O-负-2H2O光谱中，这一差分信号在频率和幅度上被强烈修改，表明它不对应于Glu L104侧链的CO模在QA还原时的直接响应。相反，9-酮基CO与Glu L104的氢键的扰动是为了引起HA的V环上电子密度的变化，从而改变与V环部分共轭的10a-酯CO的频率。由于Glu L104到Leu的突变而失去了与HA的9-酮CO的氢键，或者通过Glu L104上的1H/2H交换改变了氢键的强度，似乎就产生了这种影响。因此，1700 cm~(-1)以上的QA/QA谱主要来自HA的10a-酯CO的贡献，大部分微分信号被分配给Hain的10a-酯CO对QA还原的响应的小的频率下移。信号的复杂性意味着HA的10a-酯CO的构象和氢键的结构不均一，这可能与电子转移动力学中观察到的功能不均一有关。傅立叶变换红外光谱结果表明，QA的还原会对QA附近的化学基团的分子振动产生明显的静电效应。他们还证明，在实验范围内，在pH为7的QA光还原过程中观察到的质子吸收[McPherson，P.H.，Okamura，M.Y.，&Feher，G.(1988)Biochim]。生物物理学[Acta934,348−368]不涉及RC的可交换羧基。
The light-induced QA-/QAFTIR difference spectrum of the photoreduction of the primary quinone (QA) in reaction centers (RCs) fromRhodobactersphaeroidesexhibits a set of complex differential bands between 1750 and 1715 cm-1. Several of these features correspond in frequency to bands that bleach in the HA-/HAFTIR difference spectra of the photoreduction of the bacteriopheophytin electron acceptor (HA). Since the 10a-ester CO from HAand the side chains of protonated carboxylic acids would be expected to contribute in this spectral region, mutations were designed at Trp L100 and Glu L104, which have been proposed to form hydrogen bonds to the 10a-ester and the 9-keto carbonyls on ring V of HA, respectively. The QA-/QAspectra measured in1H2O and2H2O of RCs from wild type (WT) were compared to those of RCs with the mutation Trp to Phe at L100 [WF(L100)], Glu to Leu at L104 [EL(L104)], or both mutations [EL(L104)/WF(L100)]. The spectra of the mutants in the 1800−1400 cm-1frequency range exhibit only limited perturbations compared to those of WT, indicating the absence of significant structural changes due to the mutations. Part of a differential signal centered around 1732 cm-1in the spectrum of WT RCs is downshifted by ≈7 cm-1in EL(L104), while it is upshifted by ≈11 cm-1in WF(L100). This upshift of the differential signal is assigned to the frequency change of the 10a-ester CO of HAinduced by the rupture of the hydrogen bond with Trp L100. The1H2O-minus-2H2O double-difference spectrum of WT RCs exhibits a characteristic differential signal positive at 1730 cm-1and negative at 1724 cm-1that is absent in the corresponding spectra of EL(L104) and of the double mutant, implicating Glu L104 in the QA-/QAspectral changes. This differential signal is strongly modified in frequency and amplitude in the1H2O-minus-2H2O spectrum of WF(L100), indicating that it does not correspond to a direct response of the CO mode of the Glu L104 side chain upon QAreduction. Instead, perturbation of the hydrogen bond of the 9-keto CO with Glu L104 is proposed to induce a change of electron density on ring V of HA, thereby altering the frequency of the 10a-ester CO that is in partial conjugation with ring V. The loss of the hydrogen bond to the 9-keto CO of HAdue to the Glu L104 to Leu mutation or the alteration of the strength of the hydrogen bond by1H/2H exchange on Glu L104 appears to produce such effects. Thus, the QA-/QAspectra above 1700 cm-1are dominated by contributions from the 10a-ester CO of HA, with most of the differential signals assigned to a small frequency downshift of the 10a-ester CO of HAin response to QAreduction. The complexity of the signals implies a structural heterogeneity of the conformation and hydrogen bonding of the 10a-ester CO of HA, which may be related to the functional heterogeneity observed in electron transfer kinetics. The present FTIR results show that the reduction of QAcan induce a pronounced electrostatic effect on molecular vibrations of chemical groups located about 10 Å away from QA. They also demonstrate that, within experimental limits, the proton uptake observed at pH 7 upon QAphotoreduction [McPherson, P. H., Okamura, M. Y., & Feher, G. (1988)Biochim. Biophys.Acta934, 348−368] involves none of the exchangeable carboxylic groups of the RC.