Breton Jacques, Xu Wu, Diner Bruce A, Chitnis Parag R
Service de Bioénergétique, CEA-Saclay, 91191 Gif-sur-Yvette, France.
Biochemistry. 2002 Sep 17;41(37):11200-10. doi: 10.1021/bi0262404.
The extent of delocalization of the positive charge in the oxidized dimer of chlorophyll (Chl) constituting P700, the primary electron donor of photosystem I (PSI), has been investigated by analyzing the perturbation upon P700(+) formation of infrared (IR) vibrational modes of the two His axial ligands of the two P700 Chl molecules. Fourier transform IR (FTIR) difference spectra of the photooxidation of P700 in PSI core complexes isolated from Synechocystis sp. PCC 6803 isotopically labeled either globally with (15)N or more specifically with (13)C on all the His residues reveal isotopic shifts of a differential signal at 1102/1108 cm(-)(1). This signal is assigned to a downshift upon P700(+) formation of the predominantly C(5)-Ntau imidazole stretching mode of His residue(s). The amplitude of this signal is reduced by approximately half in FTIR spectra of Synechocystis mutants in which His PsaB 651, the axial ligand to one of the two Chl molecules in P700, is replaced by Cys, Gln, or Leu. These observations provide further evidence that the positive charge in P700(+) is essentially delocalized over the two Chl molecules, in agreement with a previous FTIR study in which the frequency of the vibrational modes of the 9-keto and 10a-ester C=O groups of the two Chl's in P700, P700(+), and (3)P700 were firmly established for the first time [Breton, J., et al. (1999) Biochemistry 38, 11585-11592]. Only limited perturbations of the amplitude and frequency of the 9-keto and 10a-ester C=O bands of the P700 Chl are elicited by the mutations. On the basis of comparable mutational studies of the primary electron donor in purple bacteria, these perturbations are attributed to small molecular rearrangements of the Chl macrocycle and substituents caused by the repositioning of the P700 dimer in the new protein cavity generated by the mutations. It is proposed that the perturbation of the FTIR spectra upon mutation of a His axial ligand of the P700 Chl recently reported in Chlamydomonas reinhardtii [Hastings, G., et al. (2001) Biochemistry 40, 12943-12949] can be explained by the same effect without the need for a new assignment of the C=O bands of P700. The distribution of charge/spin in P700(+) and (3)P700 determined by FTIR spectroscopy is discussed in relation with the contrasting interpretations derived from recent magnetic resonance experiments.
通过分析光系统I（PSI）的原初电子供体P700中构成氧化二聚体的叶绿素（Chl）正电荷的离域程度，研究了两个P700 Chl分子的两个His轴向配体的红外（IR）振动模式在P700（+）形成时的扰动。从集胞藻属PCC 6803分离的PSI核心复合物中P700光氧化的傅里叶变换红外（FTIR）差谱，在所有His残基上用（15）N整体同位素标记或更具体地用（13）C标记，揭示了在1102/1108 cm（-）（1）处差异信号的同位素位移。该信号归因于His残基的主要C（5）-Nτ咪唑伸缩模式在P700（+）形成时的下移。在His PsaB 651（P700中两个Chl分子之一的轴向配体）被Cys、Gln或Leu取代的集胞藻突变体的FTIR光谱中，该信号的幅度降低了约一半。这些观察结果进一步证明P700（+）中的正电荷基本上在两个Chl分子上离域，这与先前的FTIR研究一致，在该研究中首次确定了P700、P700（+）和（3）P700中两个Chl的9-酮基和10a-酯C=O基团的振动模式频率[Breton，J.等人（1999年）《生物化学》38，11585 - 11592]。P700 Chl的9-酮基和10a-酯C=O带的幅度和频率仅受到有限的扰动。基于对紫色细菌中原初电子供体的类似突变研究，这些扰动归因于由突变产生的新蛋白质腔中P700二聚体重新定位引起的Chl大环和取代基的小分子重排。有人提出，莱茵衣藻最近报道的P700 Chl的His轴向配体突变时FTIR光谱的扰动[Hastings，G.等人（2001年）《生物化学》40，12943 - 12949]可以用相同的效应来解释，而无需对P700的C=O带进行新的归属。讨论了通过FTIR光谱法测定的P700（+）和（3）P700中的电荷/自旋分布与最近磁共振实验得出的对比解释的关系。
