STRUCTURE/FUNCTION OF PHOTOSYNTHETIC CYTOCHROME COMPLEX

能量转导细胞色素b6f复合体的结构与功能 而光系统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的形态 对多肽进行了测定。(I)该地区的地形特征 细胞色素b6f复合体将通过将这些研究扩展到 Rieske铁硫蛋白及其亚基IV的定位 膜将用极性表位的蛋白酶敏感性来确定 用于多肽导向的抗体和类囊体的免疫金标记 不同侧面的小泡。(2)结晶研究将是 继续研究b6f络合物的成分，细胞色素f 结晶，共结晶的细胞色素f-塑料蓝蛋白， Rieske铁硫蛋白和细胞色素的结晶 B6F络合物采用洗涤剂增溶单分散法制备纯 二聚体络合物。(Iii)细胞色素b6的血红素bn在 跨膜光系统I的循环磷酸化途径及其分支 电子传输链中与电子供体相连的点 膜的n面将通过测量依赖于 在还原血红素bN的状态下，通过单次闪光合成ATP， 以及磷酸化对其抑制剂NQNO的依赖性 存在独特地增加了细胞色素b6还原的幅度 闪电侠。(Iv)对抑制剂NQNO具有抗药性的突变体将是 从蓝藻中分离到聚球藻。PCC 7002和7942，以及 通过对PETD和PetB基因测序确定了它们的基因座。(V)现场定向 Arg-86和Thr-187残基的突变将解决(A) 位于血红素BP附近的独特带电残基的影响和(B) 在His螺旋IV的两个His残基之间插入额外残基 细胞色素b6的光谱和氧化还原性质及其电子 转移率。(Vi)细胞色素b-559psbE基因产物在 结合内腔放氧所必需的锰或钙 膜的(P-)侧将通过在以下位置插入终止密码子进行测试 跨膜疏水结构域。对B-559PSBF的要求 基因产物将通过在其基因中插入一个早期停止(G1n-3)来进行测试。 (Vii)细胞色素b-559作为PSII的替代供体的作用 反应中心在光抑制和应激反应中的作用以及(Viii) 类囊体细胞色素在脂肪酸去饱和途径中的作用 将对高度不饱和的类囊体膜进行研究。

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.