Studies on the proton pumping in mitochondrial electrotransfer system.

A. A.细胞色素C氧化酶.完全氧化化的形式：X射线扩散斑点的完全氧化的形式已经达到1.65 A决议。在房间温度下提供了2.30 A分辨率的X射线结构，在液体硝基温度下提供了2.0 A分辨率。A peroxide bridging between FeイD2a3イD2 and CuイD2BイD2 in the OイD22イD2 reduction site and a covalent linkage between His242 and Tyr244 are seen at 2. 30 A resolution。两个独立的氢键网络将O-YD 22-YD 2减少站点与矩阵空间连接。完全减少的表格：The OイイD22イイD2 reduction site in the fully reduced form does not have any peroxide， giving a trigonal planer coordination to CuイイD2BイイD2イイD11+イエD1，the stability of which results in the unusual stability of the Fe_D2a3_其中一个居民居住地（Asp 51）迁移将在夹层空间暴露出大块水相位。在完全氧化的状态下，Asp 51通过氢键网络连接到基质空间，这样Asp 51就可以从基质空间中提取一个保护器。因此，Asp 51是质子泵的站点。其他研究： Redox分级实验展示了两个额外的电子接收器站点，目前在四个Redox活性金属站点中准备好了完全氧化的状态。关于减少的FTIR结果显示，一个碳基组是不同的，最有可能成为Asp 51。这些结果对X射线结构分析有很强的支持。NADH-ubiquinone氧化物还原酶的可复制性是一种显著的改进。As the first step for elucidation of the reaction mechanism of this enzyme， the initial steady state of the enzyme reaction has been analyzed to reveal an ordered sequential mechanism with ubiquione （Qy-D21y-D2）-NADH-NAD D1+ Qy-D1-Qy-D21y-D21y-D2， as the order of substrate bindings and product releasings。

A. Cytochrome c Oxidase.Fully oxidized form : X-ray diffraction spots for the fully oxidized form were obtained up to 1.65 A resolution. The diffraction patterns have provided x-ray structures at 2.30 A resolution at room temperature and at 2.0 A resolution at liquid nitrogen temperature. A peroxide bridging between FeィイD2a3ィエD2 and CuィイD2BィエD2 in the OィイD22ィエD2 reduction site and a covalent linkage between His242 and Tyr244 are seen at 2.30 A resolution. Two independent hydrogen bond networks connect the OィイD22ィエD2 reduction site with matrix space.Fully reduced form : The OィイD22ィエD2 reduction site in the fully reduced form does not have any peroxide, giving a trigonal planer coordination to CuィイD2BィエD2ィイD11+ィエD1, the stability of which results in the unusual stability of the FeィイD2a3ィエD2ィイD12+ィエD1-OィイD22ィエD2. One of aspartate residue (Asp51) migrates to be exposed to the bulk water phase in the intermembrane space. Asp51 in the fully oxidized state is connected to the matrix space by a hydrogen bond network so that Asp51 can take up protons from the matrix space. Thus, that Asp51 is the site for proton pumping.Other studies : Redox titration experiments showed that 2 extra electron acceptor sites are present besides the four redox active metal sites in the fully oxidized state as prepared. FTIR results showed that on reduction, one carboxyl group is dissociated, which is most likely to be that of Asp51. These results strongly support the above x-ray structural analysis.B. NADH-ubiquinone oxidoreductaseReproducibility of the purification method has been improved significantly. As the first step for elucidation of the reaction mechanism of this enzyme, the initial steady state of the enzyme reaction has been analyzed to reveal an ordered sequential mechanism with ubiquione (QィイD21ィエD2)-NADH-NADィイD1+ィエD1-QィイD21ィエD2HィイD22ィエD2, as the order of substrate bindings and product releasings.