Ananyev Gennady, Nguyen Tuan, Putnam-Evans Cindy, Dismukes G Charles
Princeton University, Department of Chemistry and Princeton Environmental Institute, Princeton, NJ 08544, USA.
Photochem Photobiol Sci. 2005 Dec;4(12):991-8. doi: 10.1039/b507519j. Epub 2005 Nov 8.
The chlorophyll-binding protein CP43 is an inner subunit of the Photosystem II (PSII) reaction center core complex of all oxygenic photoautotrophs. X-Ray structural evidence places the guanidinium cation of the conserved arginine 357 residue of CP43 within a few Angstroms to the Mn(4)Ca cluster of the water-oxidizing complex (WOC) and has been implicated as a possible carbonate binding site. To test the hypothesis, the serine mutant, CP43-R357S, from Synechocystis PCC 6803 was investigated by PSII variable fluorescence (F(v)/F(m)) and simultaneous flash O(2) yield measurements in cells and thylakoid membranes. The R357S mutant assembles PSII-WOC centers, but is unable to grow photoautotrophically. Reconstitution of O(2) evolution by photoactivation and the occurrence of period-four oscillations of F(v)/F(m) establishes that the R357S mutant contains an assembled Mn(4)Ca cluster, but turnover is impaired as seen by an 11-fold larger Kok double miss parameter and faster decay of upper S states. Using pulsed light to avoid photoinactivation, wild-type cells and thylakoid membranes exhibit a 2-4-fold loss in O(2) evolution rate upon partial bicarbonate depletion under multiple turnover conditions, while the R357S mutant is unaffected by bicarbonate. Arginine R357 appears to function in binding a (bi)carbonate ion essential to normal catalytic turnover of the WOC. The quantum yield of electron donation from the WOC into PSII increases with decreasing turnover rate in R357S mutant cells and involves an aborted two-flash pathway that is distinct from the classical four-flash pattern. We speculate that an altered photochemical mechanism for O(2) production occurs via formation of hydrogen peroxide, by analogy to other treatments that retard the kinetics of proton release into the lumen.
叶绿素结合蛋白CP43是所有产氧光合自养生物的光系统II（PSII）反应中心核心复合物的一个内部亚基。X射线结构证据表明，CP43保守精氨酸357残基的胍阳离子位于距水氧化复合物（WOC）的Mn(4)Ca簇几埃的范围内，并且被认为是一个可能的碳酸盐结合位点。为了验证这一假设，通过PSII可变荧光（F(v)/F(m)）以及在细胞和类囊体膜中同时进行闪光O(2)产量测量，对来自集胞藻PCC 6803的丝氨酸突变体CP43-R357S进行了研究。R357S突变体组装了PSII-WOC中心，但无法进行光合自养生长。通过光激活重建O(2)释放以及F(v)/F(m)的四周期振荡的出现，表明R357S突变体包含一个组装好的Mn(4)Ca簇，但周转受到损害，这表现为Kok双失活参数大11倍以及较高S态的更快衰减。在多周转条件下，使用脉冲光以避免光失活，野生型细胞和类囊体膜在部分碳酸氢盐耗尽时O(2)释放速率损失2-4倍，而R357S突变体不受碳酸氢盐影响。精氨酸R357似乎在结合WOC正常催化周转所必需的（双）碳酸根离子中发挥作用。在R357S突变体细胞中，从WOC向PSII的电子供体量子产率随着周转速率的降低而增加，并且涉及一条不同于经典四闪光模式的中断双闪光途径。我们推测，类似于其他延缓质子释放到内腔动力学的处理，通过过氧化氢的形成发生了一种改变的O(2)产生光化学机制。
