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 membra ne catalyses the oxidation of succinate to fumarate and directs reduci ng equivalents into the electron-transport chain. The enzyme is also a ble to catalyse the reverse reaction, the reduction of fumarate to suc cinate. The enzyme is composed of four subunits. These subunits includ e a catalytic dimer composed of a flavoprotein subunit with a covalent ly 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 flavopro tein subunit by an 8 alpha-[N(3)-histidyl]FAD linkage at a conserved h istidine residue, His90 of the Saccharomyces cerevisiae succinate dehy drogenase. By mutating His90 to a serine residue, we have constructed a flavoprotein subunit that is unable to covalently bind FAD. The muta nt flavoprotein is targeted to mitochondria, translocated across the m itochondrial membranes, and is assembled with the other subunits where it binds FAD non-covalently. The resulting holoenzyme has no succinat e-dehydrogenase activity but retains fumarate reductase activity. The covalent attachment of FAD is therefore necessary for succinate oxidat ion but is dispensable for both fumarate reduction and for the import and assembly of the flavoprotein subunit.