From the Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107 and.
the Department of Biochemistry, University of California, Riverside, California 92521.
J Biol Chem. 2018 Feb 16;293(7):2487-2497. doi: 10.1074/jbc.M117.813170. Epub 2017 Dec 20.
Photoinduced water oxidation at the O-evolving complex (OEC) of photosystem II (PSII) is a complex process involving a tetramanganese-calcium cluster that is surrounded by a hydrogen-bonded network of water molecules, chloride ions, and amino acid residues. Although the structure of the OEC has remained conserved over eons of evolution, significant differences in the chloride-binding characteristics exist between cyanobacteria and higher plants. An analysis of amino acid residues in and around the OEC has identified residue 87 in the D1 subunit as the only significant difference between PSII in cyanobacteria and higher plants. We substituted the D1-Asn residue in the cyanobacterium sp. PCC 6803 (wildtype) with alanine, present in higher plants, or with aspartic acid. We studied PSII core complexes purified from D1-N87A and D1-N87D variant strains to probe the function of the D1-Asn residue in the water-oxidation mechanism. EPR spectra of the S state and flash-induced FTIR spectra of both D1-N87A and D1-N87D PSII core complexes exhibited characteristics similar to those of wildtype PSII core complexes. However, flash-induced O-evolution studies revealed a decreased cycling efficiency of the D1-N87D variant, whereas the cycling efficiency of the D1-N87A PSII variant was similar to that of wildtype PSII. Steady-state O-evolution activity assays revealed that substitution of the D1 residue at position 87 with alanine perturbs the chloride-binding site in the proton-exit channel. These findings provide new insight into the role of the D1-Asn site in the water-oxidation mechanism and explain the difference in the chloride-binding properties of cyanobacterial and higher-plant PSII.
光诱导水氧化在光合作用系统 II（PSII）的放氧复合 物（OEC）是一个复杂的过程，涉及一个四锰-钙簇，该簇被水分子、氯离子和氨基酸残基的氢键网络所包围。尽管 OEC 的结构在亿万年的进化过程中保持不变，但蓝藻和高等植物之间在氯离子结合特性上存在显著差异。对 OEC 及其周围氨基酸残基的分析确定了 D1 亚基中的残基 87 是蓝藻和高等植物 PSII 之间唯一的显著差异。我们用在高等植物中存在的丙氨酸或天冬氨酸替代了蓝藻 sp. PCC 6803（野生型）中的 D1-Asn 残基。我们研究了从 D1-N87A 和 D1-N87D 变体菌株中纯化的 PSII 核心复合物，以探究 D1-Asn 残基在水氧化机制中的功能。D1-N87A 和 D1-N87D PSII 核心复合物的 S 态 EPR 光谱和闪光诱导的 FTIR 光谱均表现出与野生型 PSII 核心复合物相似的特征。然而，闪光诱导的 O 演化研究表明，D1-N87D 变体的循环效率降低，而 D1-N87A PSII 变体的循环效率与野生型 PSII 相似。稳态 O 演化活性测定表明，用丙氨酸替代 D1 残基 87 位会扰乱质子出通道中的氯离子结合位点。这些发现为 D1-Asn 位点在水氧化机制中的作用提供了新的见解，并解释了蓝藻和高等植物 PSII 之间氯离子结合特性的差异。
