Breton J, Bibikova M, Oesterhelt D, Nabedryk E
Section de Bioénergétique, Département de Biologie Cellulaire et Moléculaire, CEA/Saclay, Gif-sur-Yvette, France.
Biochemistry. 1999 Aug 31;38(35):11541-52. doi: 10.1021/bi990927f.
The light-induced Fourier transform infrared (FTIR) difference spectra corresponding to the photoreduction of either the HA bacteriopheophytin electron acceptor (HA-/HA spectrum) or the QA primary quinone (QA-/QA spectrum) in photosynthetic reaction centers (RCs) of Rhodopseudomonas viridis are reported. These spectra have been compared for wild-type (WT) RCs and for two site-directed mutants in which the proposed interactions between the carbonyls on ring V of HA and the RC protein have been altered. In the mutant EQ(L104), the putative hydrogen bond between the protein and the 9-keto C=O of HA should be affected by changing Glu L104 to a Gln. In the mutant WF(M250), the van der Waals interactions between Trp M250 and the 10a-ester C=O of HA should be modified. The characteristic effects of both mutations on the FTIR spectra support the proposed interactions and allow the IR modes of the 9-keto and 10a-ester C=O of HA and HA- to be assigned. Comparison of the HA-/HA and QA-/QA spectra leads us to conclude that the QA-/QA IR signals in the spectral range above 1700 cm-1 are largely dominated by contributions from the electrostatic response of the 10a-ester C=O mode of HA upon QA photoreduction. A heterogeneity in the conformation of the 10a-ester C=O mode of HA in WT RCs, leading to three distinct populations of HA, appears to be related to differences in the hydrogen-bonding interactions between the carbonyls of ring V of HA and the RC protein. The possibility that this structural heterogeneity is related to the observed multiexponential kinetics of electron transfer and the implications for primary processes are discussed. The effect of 1H/2H exchange on the QA-/QA spectra of the WT and mutant RCs shows that neither Glu L104 nor any other exchangeable carboxylic residue changes appreciably its protonation state upon QA reduction.
报道了与绿脓杆菌光合反应中心（RCs）中HA细菌脱镁叶绿素电子受体的光还原（HA-/HA光谱）或QA初级醌（QA-/QA光谱）相对应的光诱导傅里叶变换红外（FTIR）差光谱。已对野生型（WT）RCs以及两个定点突变体的这些光谱进行了比较，在这两个突变体中，HA环V上的羰基与RC蛋白之间的假定相互作用已被改变。在突变体EQ(L104)中，蛋白与HA的9-酮C=O之间的假定氢键应会因将Glu L104变为Gln而受到影响。在突变体WF(M250)中，Trp M250与HA的10a-酯C=O之间的范德华相互作用应会被改变。两种突变对FTIR光谱的特征性影响支持了所提出的相互作用，并使得能够对HA和HA-的9-酮和10a-酯C=O的红外模式进行归属。HA-/HA和QA-/QA光谱的比较使我们得出结论，在1700 cm-1以上光谱范围内的QA-/QA红外信号在很大程度上由QA光还原时HA的10a-酯C=O模式的静电响应贡献主导。WT RCs中HA的10a-酯C=O模式构象的异质性导致出现三种不同的HA群体，这似乎与HA环V的羰基与RC蛋白之间氢键相互作用的差异有关。讨论了这种结构异质性与观察到的电子转移多指数动力学相关的可能性以及对初级过程的影响。1H/2H交换对WT和突变体RCs的QA-/QA光谱的影响表明，在QA还原时，Glu L104和任何其他可交换的羧基残基均未明显改变其质子化状态。
