Studies by molecular biological methods on proton pumping mechanisms of bovine heart and bacterial cytochrome c oxidases

利用我们建立的牛/人杂交细胞色素c氧化酶（CcO）HeLa细胞表达系统，通过定点突变的方法，对CcO的质子传递途径--D途径的功能进行了分析。已经提出了相应的细菌途径来输送泵送和水形成质子，这得到了诱变的支持。我们的分析不支持质子泵送功能。利用振动光谱研究蛋白质的质子转移机理需要对蛋白质进行位点特异性同位素标记，这可以通过无细胞蛋白质合成来实现。我们已经成功地开发了一个E。coli无细胞系统中产生正常细菌CcO。

Function of a putative proton transfer pathway, D-pathway of bovine heart cytochrome c oxidase (CcO) was analyzed by site-directed mutagenesis employing HeLa cell expression system we have developed to produce bovine/human hybrid CcO. Corresponding bacterial pathway has been proposed to convey pumping and water-forming protons, which is supported by mutagenesis. Our analyses did not support the proton pumping function. Study on proton transfer mechanism of proteins by vibrational spectroscopy requires site-specific isotope labeling of proteins, which can be achieved by cell-free protein synthesis. We have successfully developed a E. coli cell free system to produce normal bacterial CcO.