STRUCTURE/FUNCTION OF PHOTOSYNTHETIC CYTOCHROME COMPLEX

能量传递细胞色素b6 f复合物的结构与功能 和光系统II反应中心固有的细胞色素B-559将 被研究。 细胞色素b6和细胞色素f 多肽已确定。 (i)的地形特征 细胞色素b6 f复合物将通过扩展这些研究来完成， Rieske铁硫蛋白和亚基IV。 中的定向 膜将使用极性表位的蛋白酶敏感性来确定 用于肽导向抗体和类囊体的免疫金标记 不同侧面的囊泡。 (ii)结晶研究将 继续对b6 f复合物的成分，细胞色素f， 结晶，细胞色素F-质体蓝素的共结晶， Rieske铁硫蛋白和细胞色素的结晶 B6 F复合物使用洗涤剂增溶的纯B6 F的单分散制剂 二聚络合物 (iii)细胞色素b6血红素bn在细胞凋亡中的作用 跨膜光系统I循环磷酸化途径及其作为一个分支 电子传递链中与电子给体连接的点 膜的n-侧将通过测量 ATP合成通过单次闪光对血红素bn的还原状态， 以及磷酸化对抑制剂NQNO的依赖性， 存在独特地增加细胞色素b6还原的幅度， 闪 (iv)对抑制剂NQNO的抑制剂抗性突变体将是 分离自蓝细菌，聚球藻属PCC 7002和7942，和 通过对petD和petB基因进行测序来确定它们的基因座。 (v)定点 Arg-86和Thr-187残基的诱变将解决（a） 和（B）位于血红素bp附近的独特带电残基的影响 插入在螺旋IV中的两个His残基之间的额外残基 细胞色素b6光谱、氧化还原性质及血红素间电子 传输速率 (vi)细胞色素B-559 psbE基因产物在肿瘤细胞中的作用 在管腔上结合氧气释放所必需的锰或钙 (p-)将通过在膜的另一侧插入终止密码子来测试膜的另一侧。 跨膜疏水结构域。 B-559 psbF的要求 将通过在其基因中插入终止早期（G1 n-3）来检测基因产物。 (vii)细胞色素B-559作为PSII替代供体的作用 反应中心在光抑制和应激反应，和（viii）的作用 类囊体细胞色素在脂肪酸去饱和途径中的作用 的高度不饱和类囊体膜将进行研究。

The structure and function of the energy-transducing cytochrome b6f complex and cytochrome b-559 intrinsic to the photosystem II reaction center will be studied. The topography of the cytochrome b6 and cytochrome f polypeptide has been determined. (i) The topographical characterization of the cytochrome b6f complex will be completed by extending these studies to the Rieske iron-sulfur protein and subunit IV. The orientation in the membrane will be determined using protease sensitivity of polar epitopes for peptide-directed antibodies, and immunogold labeling of thylakoid vesicles of different sidedness. (ii) Crystallization studies will be continued on the components of the b6f complex, cytochrome f that has been crystallized, co-crystallization of cytochrome f- plastocyanin, crystallization of the Rieske iron-sulfur protein, and of the cytochrome b6f complex using a detergent-solubilized monodisperse preparation of pure dimeric complex. (iii) The function of heme bn of cytochrome b6 in the trans-membrane photosystem I cyclic phosphorylation pathway and as a branch point in the electron transport chain connecting with electron donors on the n-side of the membrane will be assayed by measuring the dependence of ATP synthesis by a single light flash on the state of reduction of heme bn, as well as the dependence of phosphorylation on the inhibitor NQNO whose presence uniquely increases the amplitude of cytochrome b6 reduction by a flash. (iv) Inhibitor-resistant mutants to the inhibitor NQNO will be isolated from the cyanobacteria, Synechococcus sp. PCC 7002 and 7942, and their loci determined by sequencing petD and petB genes. (v) Site-directed mutagenesis of the Arg-86 and Thr-187 residues would address (a) the role of a unique charged residue located near heme bp and (b) the effect of an extra residue inserted between the two His residues in helix IV of cytochrome b6 on its spectral and redox properties and interheme electron transfer rate. (vi) The role of the cytochrome b-559 psbE gene product in binding manganese or calcium essential for oxygen evolution on the lumen (p-) side of the membrane will be tested by inserting a stop codon after the trans-membrane hydrophobic domain. The requirement for the b-559 psbF gene product will be tested by inserting a stop early (G1n-3) in its gene. (vii) A role for cytochrome b-559 as an alternative donor to the PSII reaction center in photoinhibition and stress response, and (viii) the role of thylakoid cytochromes in the pathway of desaturation of the fatty acids of the highly unsaturated thylakoid membrane will be studied.