Covalent Attachment of FAD to the Yeast Succinate Dehydrogenase Flavoprotein Requires Import into Mitochondria, Presequence Removal, and Folding

Abstract

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.

Towards the phylogeny of APS reductases and sirohaem sulfite reductases in sulfate-reducing and sulfur-oxidizing prokaryotes

“…lb). Robinson & Lemire (1996) removed the 70 carboxyterminal residues of the flavoprotein subunit of succinate dehydrogenase from Saccharomyces cereuisiae. The residual polypeptide was no longer able to bind FAD.…”

The ribosome regulates flavodoxin folding

“…Due to quenching of FMN fluorescence upon cofactor binding to apoprotein, the slope of fluorescence data arising from FMN bound to protein is less steep than the slope resulting from free FMN. This characteristic is widely used to demonstrate FMN binding (42,43,45). To determine the ratio of FMN to apoprotein, we added trichloroacetic acid (TCA) to F44Y P .…”

“…Off-pathway intermediate formation is also characteristic for the folding of CheY-like proteins, which share the flavodoxin-like fold (33,34). A MG that has been extensively studied is the off-pathway MG (MG off ) of the 179-residue flavodoxin from A. vinelandii (32, [35][36][37][38][39][40][41][42][43].…”

“…fluorescence spectroscopy, as FMN's fluorescence becomes severely quenched upon binding to native apoflavodoxin (42,43).…”

“…This procedure allows the mimicking of late periods during protein translation and enables the elucidation of the stage at which nascent apoflavodoxin is capable of binding FMN. Translational stalling is made possible by attaching a sequence derived from E. coli SecM to the C-terminus of a nascent protein (41,42). The RNCs are isolated from E. coli strain BL21(DE3) Δtig::kan, which lacks chaperone Trigger factor.…”