Structure of cytochrome b6/f deduced from its function

细胞色素b6/f复合体接受被光系统II还原的胞质喹酚的电子，将电子传递到光系统I，并在类囊体膜上形成H^+梯度，同时形成ATP。在本研究项目中，我们从蓝藻的类囊体膜中纯化了该复合体，致力于开发简单的纯化工艺，并获得了较高的产率。我们还研究了该络合物的底物(苯醌)专一性，并估算了其结合口袋的活性部位的结构。我们对现有的方法进行了两点改进：1)在NaBR处理过程中同时对类囊体膜进行超声处理，以去除所有外源蛋白；2)加入脱氧胆酸盐和庚基硫代葡萄糖苷来溶解络合物。通过这一改进，可以在较窄的硫酸铵浓度范围内有效地沉淀该络合物，并可进一步纯化。四甲基对苯二酚通常用于活性测定。三甲基对苯二酚也被发现是有效的。然而，二甲基对苯二酚或二氯对苯二酚(DCBQ)不能作为络合物的电子供体。从DCBQ是光系统II最有效的电子受体来看，B6/f络合物的苯醌结合口袋的结构与光系统II的Q_B位结构有很大的不同。

Cytochrome b6/f complex accepts electrons from plastoquinol which was reduced by photosystem II.The complex transfers electrons to photosystem I and forms H^+ gradient across the thylakoid membranes to form ATP simultaneously. In this research project, we have purified the complex from thylakoid membranes of a cyanobacterium focussing on development of simple purification procedure and its high yield. We also have investigated the substrate (quinone) specificity of the complex and estimated the structure of active site of the quinone binding pocket.The complex was purified from a thermophilic cyanobacterium, Synechococcus vulcanus. We have improved on the existing procedure by the following two points : 1) thylakoid membranes were sonicated simultaneously during NaBr treatment to remove all the extrinsic proteins and 2) deoxycholate was added together with heptylthioglucoside to solubilize the complex. By this improvement, the complex was effectively precipitated in a narrow range of ammonium sulfate concentration and could be purified further.Tetramethyl-p-benzoquinone is normally used for activity measurement. Trimethyl-p-benzoquinone was found to be effective as well. Dimethyl-p-benzoquinones or dichloro-p-benzoquinones (DCBQ) were, however, not avtive as electron donor to the complex. Judging from the fact that DCBQ is the most effective electron acceptor for photosystem II,the structure of quinone binding pocket of b6/f complex is rather different from that of Q_B site of photosystem II.