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 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.
Robinson
K M
KM
Medical Research Council of Canada Group in the Molecular Biology of Membranes, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Lemire
B D
BD
D002952:Citric Acid Cycle; D004792:Enzyme Precursors / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005420:Flavoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D005838:Genotype; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013045:Species Specificity; D013385:Succinate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*
D004792:Enzyme Precursors; D005420:Flavoproteins; D007506:Iron-Sulfur Proteins; D005182:Flavin-Adenine Dinucleotide; D013385:Succinate Dehydrogenase