The covalent attachment of FAD to the flavoprotein of Saccharomyces cerevisiae succinate dehydrogenase is not necessary for import and assembly into mitochondria.
Succinate dehydrogenase of the bacterial or inner mitochondrial membrane catalyses the oxidation of succinate to fumarate and directs reducing equivalents into the electron-transport chain. The enzyme is also able to catalyse the reverse reaction, the reduction of fumarate to succinate. The enzyme is composed of four subunits. These subunits include a catalytic dimer composed of a flavoprotein subunit with a covalently bound FAD, and an iron-sulfur protein subunit with three different iron-sulfur centres, which is anchored to the membrane by two smaller integral membrane proteins. The FAD moiety is attached to the flavoprotein subunit by an 8 alpha-[N(3)-histidyl]FAD linkage at a conserved histidine residue, His90 of the Saccharomyces cerevisiae succinate dehydrogenase. By mutating His90 to a serine residue, we have constructed a flavoprotein subunit that is unable to covalently bind FAD. The mutant flavoprotein is targeted to mitochondria, translocated across the mitochondrial membranes, and is assembled with the other subunits where it binds FAD non-covalently. The resulting holoenzyme has no succinate-dehydrogenase activity but retains fumarate reductase activity. The covalent attachment of FAD is therefore necessary for succinate oxidation but is dispensable for both fumarate reduction and for the import and assembly of the flavoprotein subunit.
Department of Biochemistry, University of Alberta, Canada.
K M
KM
Robinson
R A
RA
Rothery
J H
JH
Weiner
B D
BD
Lemire
D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism*; D005650:Fumarates / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013385:Succinate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013386:Succinates / Q000378:metabolism; D019802:Succinic Acid
D005420:Flavoproteins; D005650:Fumarates; D007506:Iron-Sulfur Proteins; D013386:Succinates; D005182:Flavin-Adenine Dinucleotide; C032005:fumaric acid; D019802:Succinic Acid; D013385:Succinate Dehydrogenase