Robinson K M, Lemire B D
Medical Research Council of Canada Group in the Molecular Biology of Membranes, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
J Biol Chem. 1996 Feb 23;271(8):4055-60. doi: 10.1074/jbc.271.8.4055.
Succinate dehydrogenase (EC 1.3.99.1) in the yeast Saccharomyces cerevisiae is a mitochondrial respiratory chain enzyme that utilizes the cofactor, FAD, to catalyze the oxidation of succinate and the reduction of ubiqinone. The succinate dehydrogenase enzyme is a heterotetramer composed of a flavoprotein, an iron-sulfur protein, and two hydrophobic subunits. The FAD is covalently attached to a histidine residue near the amino terminus of the flavoprotein. In this study, we have investigated the attachment of the FAD cofactor with the use of an antiserum that specifically recognizes FAD and hence, can discriminate between apo- and holoflavoproteins. Cofactor attachment, both in vivo and in vitro, occurs within the mitochondrial matrix once the presequence has been cleaved. FAD attachment is stimulated by, but not dependent upon, the presence of the iron-sulfur subunit and citric acid cycle intermediates such as succinate, malate, or fumarate. Furthermore, this modification does not occur with C-terminally truncated flavoprotein subunits that are fully competent for import. Taken together, these data suggest that cofactor addition occurs to an imported protein that has folded sufficiently to recognize both FAD and its substrate.
酿酒酵母中的琥珀酸脱氢酶（EC 1.3.99.1）是一种线粒体呼吸链酶，它利用辅因子FAD催化琥珀酸的氧化和泛醌的还原。琥珀酸脱氢酶是一种异源四聚体，由一种黄素蛋白、一种铁硫蛋白和两个疏水亚基组成。FAD共价连接在黄素蛋白氨基末端附近的一个组氨酸残基上。在本研究中，我们使用了一种能特异性识别FAD、从而能够区分脱辅基黄素蛋白和全黄素蛋白的抗血清，来研究FAD辅因子的连接情况。一旦前导序列被切割，辅因子在体内和体外的连接都发生在线粒体基质中。FAD的连接受到铁硫亚基和柠檬酸循环中间产物（如琥珀酸、苹果酸或富马酸）的刺激，但不依赖于它们的存在。此外，对于完全能够导入的C末端截短的黄素蛋白亚基，这种修饰不会发生。综上所述，这些数据表明，辅因子的添加发生在一种已充分折叠以识别FAD及其底物的导入蛋白上。
