Yi Sophia M, Narasimhulu Kuppala V, Samoilova Rimma I, Gennis Robert B, Dikanov Sergei A
Departments of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
J Biol Chem. 2010 Jun 11;285(24):18241-51. doi: 10.1074/jbc.M110.116186. Epub 2010 Mar 29.
Cytochrome aa(3)-600 is one of the principle respiratory oxidases from Bacillus subtilis and is a member of the heme-copper superfamily of oxygen reductases. This enzyme catalyzes the two-electron oxidation of menaquinol and the four-electron reduction of O(2) to 2H(2)O. Cytochrome aa(3)-600 is of interest because it is a very close homologue of the cytochrome bo(3) ubiquinol oxidase from Escherichia coli, except that it uses menaquinol instead of ubiquinol as a substrate. One question of interest is how the proteins differ in response to the differences in structure and electrochemical properties between ubiquinol and menaquinol. Cytochrome bo(3) has a high affinity binding site for ubiquinol that stabilizes a ubi-semiquinone. This has permitted the use of pulsed EPR techniques to investigate the protein interaction with the ubiquinone. The current work initiates studies to characterize the equivalent site in cytochrome aa(3)-600. Cytochrome aa(3)-600 has been cloned and expressed in a His-tagged form in B. subtilis. After isolation of the enzyme in dodecylmaltoside, it is shown that the pure enzyme contains 1 eq of menaquinone-7 and that the enzyme stabilizes a mena-semiquinone. Pulsed EPR studies have shown that there are both similarities as well as significant differences in the interactions of the mena-semiquinone with cytochrome aa(3)-600 in comparison with the ubi-semiquinone in cytochrome bo(3). Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa(3)-600 in comparison with ubiquinone in cytochrome bo(3). In addition, the electronic structure of the semiquinone cyt aa(3)-600 is more shifted toward the anionic form from the neutral state in cyt bo(3).
细胞色素aa(3)-600是枯草芽孢杆菌主要的呼吸氧化酶之一，属于血红素-铜超家族氧还原酶。该酶催化甲萘醌的双电子氧化以及将O(2)四电子还原为2H(2)O。细胞色素aa(3)-600备受关注，因为它是大肠杆菌细胞色素bo(3)泛醇氧化酶的非常接近的同源物，只是它使用甲萘醌而非泛醇作为底物。一个有趣的问题是，这两种蛋白质如何因泛醇和甲萘醌在结构和电化学性质上的差异而有所不同。细胞色素bo(3)有一个对泛醇具有高亲和力的结合位点，可稳定泛半醌。这使得能够使用脉冲电子顺磁共振技术来研究蛋白质与泛醌的相互作用。当前的工作启动了对细胞色素aa(3)-600中对应位点进行表征的研究。细胞色素aa(3)-600已被克隆并以带His标签的形式在枯草芽孢杆菌中表达。在用十二烷基麦芽糖苷分离该酶后，结果表明纯酶含有1当量的甲萘醌-7，并且该酶能稳定甲萘半醌。脉冲电子顺磁共振研究表明，与细胞色素bo(3)中的泛半醌相比，甲萘半醌与细胞色素aa(3)-600的相互作用既有相似之处，也有显著差异。我们的数据表明，与细胞色素bo(3)中的泛醌相比，细胞色素aa(3)-600中甲萘醌的氢键较弱。此外，细胞色素aa(3)-600半醌的电子结构比细胞色素bo(3)中从中性状态更偏向阴离子形式。
