Cao J C, Vermaas W F
Department of Physiology and Biophysics, University of Illinois, Urbana 61801-3793.
Biochim Biophys Acta. 1991 Aug 23;1059(2):171-80. doi: 10.1016/s0005-2728(05)80202-6.
Bicarbonate (HCO3-) causes a significant and reversible stimulation of anion-inhibited electron flow in photosystem II of higher plants and cyanobacteria. To test if selected arginine (Arg) residues are involved in the binding of HCO3-, we utilized oligonucleotide-directed mutagenesis to construct Synechocystis sp. PCC 6803 mutants carrying mutations in Arg residues in the D2 protein. Measurements of oxygen evolution showed that the D2 mutants R233Q (arginine-233----glutamine) and R251S (arginine-251----serine) were 10-fold more sensitive to formate than the wild type. The formate concentration giving half-maximal inhibition of the steady-state oxygen evolution rate was 48 mM, 4.5 mM and 4 mM for the wild type, R233Q and R251S, respectively. Measurements of oxygen evolution in single-turnover flashes confirm that the mutants are more sensitive to formate than the wild type. Measurements of chlorophyll a fluorescence decay kinetics after the second saturating actinic flash indicated that, after formate treatment, the halftime of QA- oxidation was decreased by approximately a factor of 2, 4 and 6 in the wild type, R251S and R233Q, respectively. The recombination rate between QA- and S2 was approx. 2-fold slower in R251S and R233Q than in the wild type. In the presence of 100 mM sodium formate, reactivation of the Hill reaction by bicarbonate showed that the wild type had an apparent Km for bicarbonate of 0.5 mM, while the Km values for R233Q and R251S were 1.4 and 1.5 mM, respectively. We suggest that Arg-233 and Arg-251 in the D2 polypeptide contribute to stabilization of HCO3- binding in Photosystem II.
碳酸氢根（HCO3-）可显著且可逆地刺激高等植物和蓝细菌光系统II中阴离子抑制的电子流动。为了测试特定的精氨酸（Arg）残基是否参与HCO3-的结合，我们利用寡核苷酸定向诱变构建了集胞藻属PCC 6803突变体，这些突变体的D2蛋白中的Arg残基发生了突变。放氧测量结果表明，D2突变体R233Q（精氨酸-233→谷氨酰胺）和R251S（精氨酸-251→丝氨酸）对甲酸盐的敏感性比野生型高10倍。使稳态放氧速率受到半数抑制的甲酸盐浓度，野生型为48 mM，R233Q为4.5 mM，R251S为4 mM。单周转闪光下的放氧测量结果证实，这些突变体对甲酸盐的敏感性高于野生型。第二次饱和光化闪光后叶绿素a荧光衰减动力学的测量结果表明，经甲酸盐处理后，野生型、R251S和R233Q中QA-氧化的半衰期分别下降了约2倍、4倍和6倍。QA-与S2之间的重组速率在R251S和R233Q中比野生型慢约2倍。在100 mM甲酸钠存在的情况下，碳酸氢根对希尔反应的再激活表明，野生型对碳酸氢根的表观Km值为0.5 mM，而R233Q和R251S的Km值分别为1.4 mM和1.5 mM。我们认为，D2多肽中的精氨酸-233和精氨酸-251有助于稳定光系统II中HCO3-的结合。
