COVALENT ATTACHMENT OF FAD TO THE YEAST SUCCINATE-DEHYDROGENASE FLAVOPROTEIN REQUIRES IMPORT INTO MITOCHONDRIA, PRESEQUENCE REMOVAL, AND FOLDING

Succinate dehydrogenase (EC 1.3.99.1) in the yeast Saccharomyces cerev isiae is a mitochondrial respiratory chain enzyme that utilizes the co factor, FAD, to catalyze the oxidation of succinate and the reduction of ubiqinone. The succinate dehydrogenase enzyme is a heterotetramer c omposed 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 investi gated the attachment of the FAD cofactor with the use of an antiserum that specifically recognizes FAD and hence, can discriminate between a po- and holoflavoproteins. Cofactor attachment, both in vivo and in vi tro, occurs within the mitochondrial matrix once the presequence has b een cleaved. FAD attachment is stimulated by, but not dependent upon, the presence of the iron-sulfur subunit and citric acid cycle intermed iates such as succinate, malate, or fumarate. Furthermore, this modifi cation does not occur with C-terminally truncated flavoprotein subunit s that are fully competent for import. Taken together, these data sugg est that cofactor addition occurs to an imported protein that has fold ed sufficiently to recognize both FAD and its substrate.