Barry B A, Babcock G T
Department of Chemistry, Michigan State University, East Lansing 48824-1322.
Proc Natl Acad Sci U S A. 1987 Oct;84(20):7099-103. doi: 10.1073/pnas.84.20.7099.
In addition to the reaction-center chlorophyll, at least two other organic cofactors are involved in the photosynthetic oxygen-evolution process. One of these cofactors, called "Z," transfers electrons from the site of water oxidation to the reaction center of photosystem II. The other species, "D," has an uncertain function but gives rise to the stable EPR signal known as signal II. Z+. and D+. have identical EPR spectra and are generally assumed to arise from species with the same chemical structure. Results from a variety of experiments have suggested that Z and D are plastoquinones or plastoquinone derivatives. In general, however, the evidence to support this assignment is indirect. To address this situation, we have developed more direct methods to assign the structure of the Z+./D+. radicals. By selective in vivo deuteration of the methyl groups of plastoquinone in cyanobacteria, we show that hyperfine couplings from the methyl protons cannot be responsible for the partially resolved structure seen in the D+. EPR spectrum. That is, we verify by extraction and mass spectrometry that quinones are labeled in algae fed deuterated methionine, but no change is observed in the line shape of signal II. Considering the spectral properties of the D+. radical, a tyrosine origin is a reasonable alternative. In a second series of experiments, we have found that deuteration of tyrosine does indeed narrow the D+. signal. Extraction and mass spectral analysis of the quinones in these cultures show that they are not labeled by tyrosine. These results eliminate a plastoquinone origin for D+.; we conclude instead that D+., and most likely Z+., are tyrosine radicals.
除了反应中心叶绿素外，光合放氧过程中至少还涉及另外两种有机辅助因子。其中一种辅助因子称为“Z”，它将电子从水氧化位点转移到光系统II的反应中心。另一种物质“D”功能尚不明确，但会产生被称为信号II的稳定电子顺磁共振（EPR）信号。Z⁺和D⁺具有相同的EPR光谱，通常认为它们源自具有相同化学结构的物种。各种实验结果表明，Z和D是质体醌或质体醌衍生物。然而，总体而言，支持这一归属的证据是间接的。为了解决这一情况，我们开发了更直接的方法来确定Z⁺/D⁺自由基的结构。通过对蓝细菌中质体醌甲基基团进行体内选择性氘代，我们发现甲基质子的超精细耦合不能解释D⁺的EPR光谱中部分分辨的结构。也就是说，我们通过提取和质谱分析验证了在以氘代甲硫氨酸喂养的藻类中醌被标记，但信号II的线形没有变化。考虑到D⁺自由基的光谱特性，酪氨酸来源是一个合理的替代方案。在第二系列实验中，我们发现酪氨酸的氘代确实使D⁺信号变窄。对这些培养物中醌的提取和质谱分析表明它们没有被酪氨酸标记。这些结果排除了D⁺的质体醌来源；我们转而得出结论，D⁺，很可能还有Z⁺，是酪氨酸自由基。
