Mamedov Fikret, Rintamäki Eevi, Aro Eva-Mari, Andersson Bertil, Styring Stenbjörn
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, Lund, 221 00, Sweden,
Photosynth Res. 2002;74(1):61-72. doi: 10.1023/A:1020835822266.
Many of the core proteins in Photosystem II (PS II) undergo reversible phosphorylation. It is known that protein phosphorylation controls the repair cycle of Photosystem II. However, it is not known how protein phosphorylation affects the partial electron transport reactions in PS II. Here we have applied variable fluorescence measurements and EPR spectroscopy to probe the status of the quinone acceptors, the Mn cluster and other electron transfer components in PS II with controlled levels of protein phosphorylation. Protein phosphorylation was induced in vivo by varying illumination regimes. The phosphorylation level of the D1 protein varied from 10 to 58% in PS II membranes isolated from pre-illuminated spinach leaves. The oxygen evolution and Q(A) (-) to Q(B)(Q(B) (-)) electron transfer measured by flash-induced fluorescence decay remained similar in all samples studied. Similar measurements in the presence of DCMU, which reports on the status of the donor side in PS II, also indicated that the integrity of the oxygen-evolving complex was preserved in PS II with different levels of D1 protein phosphorylation. With EPR spectroscopy we examined individual redox cofactors in PS II. Both the maximal amplitude of the charge separation reaction (measured as photo-accumulated pheophytin(-)) and the EPR signal from the Q(A) (-) Fe(2+) complex were unaffected by the phosphorylation of the D1 protein, indicating that the acceptor side of PS II was not modified. Also the shape of the S(2) state multiline signal was similar, suggesting that the structure of the Mn-cluster in Photosystem II did not change. However, the amplitude of the S(2) multiline signal was reduced by 35% in PS II, where 58% of the D1 protein was phosphorylated, as compared to the S(2) multiline in PS II, where only 10% of the D1 protein was phosphorylated. In addition, the fraction of low potential Cyt b (559) was twice as high in phosphorylated PS II. Implications from these findings, were precise quantification of D1 protein phosphorylation is, for the first time, combined with high-resolution biophysical measurements, are discussed.
光系统II（PS II）中的许多核心蛋白会发生可逆磷酸化。已知蛋白磷酸化控制着光系统II的修复循环。然而，尚不清楚蛋白磷酸化如何影响PS II中的部分电子传递反应。在此，我们应用可变荧光测量和电子顺磁共振波谱（EPR光谱）来探测PS II中醌受体、锰簇和其他电子传递成分的状态，其中蛋白磷酸化水平受到控制。通过改变光照条件在体内诱导蛋白磷酸化。从预光照菠菜叶中分离出的PS II膜中，D1蛋白的磷酸化水平在10%至58%之间变化。通过闪光诱导荧光衰减测量的放氧量以及从Q(A) (-)到Q(B)(Q(B) (-))的电子传递在所有研究样本中均保持相似。在存在二氯苯基二甲基脲（DCMU）的情况下进行的类似测量，该物质可反映PS II供体侧的状态，也表明在具有不同D1蛋白磷酸化水平的PS II中，放氧复合体的完整性得以保留。利用EPR光谱，我们研究了PS II中的各个氧化还原辅因子。电荷分离反应的最大振幅（以光积累的去镁叶绿素(-)测量）和来自Q(A) (-) Fe(2+)复合体的EPR信号均不受D1蛋白磷酸化的影响，这表明PS II的受体侧未发生改变。此外，S(2)态多线信号的形状也相似，这表明光系统II中锰簇的结构没有变化。然而，与D1蛋白磷酸化水平仅为10%的PS II中的S(2)多线相比，D1蛋白磷酸化水平为58%的PS II中S(2)多线信号的振幅降低了35%。此外，低电位细胞色素b(559)的比例在磷酸化的PS II中是其两倍。本文讨论了这些发现的意义，即首次将D1蛋白磷酸化的精确定量与高分辨率生物物理测量相结合。
