Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II

Abstract

Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate: ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol: fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation.

The unassembled flavoprotein subunits of human and bacterial complex II have impaired catalytic activity and generate only minor amounts of ROS

“…The flavoproteins were constructed with an N-terminal His tag. Previous work showed that a His tag does not interfere with covalent flavinylation of the proteins (27,35). This indicates that the proteins are folded correctly for interaction with the SdhE chaperone and that the His tag does not affect active site preorganization for self-catalytic FAD attachment (9,11,25,27,35).…”

“…One notable difference between the bacterial and mammalian proteins was in their response to their covalent FAD assembly factors. In bacteria, SdhE readily dissociates from either SdhA or FrdA once the covalent FAD linkage has formed (35,63,64). By contrast, SDHAF2 binds very tightly to hSDHA, and mild chaotropic agents were needed to dissociate the tightly bound complex.…”

“…The latter digestion removes the initial portion of the T5 promoter for SDHAF2 so that expression of both genes is from a single T5 promoter. Plasmid pFrdA used to express the tag-free WT E. coli FrdA flavoprotein subunit was constructed as previously described (35).…”

“…The E. coli SdhA and FrdA subunits containing an N-terminal His 6 tag were expressed and purified as previously described (35). The hSDHA was expressed from plasmid pQE-hSDHA/ SDHAF2.…”

“…The eluted fractions containing hSDHA were concentrated as described above and stored at Ϫ80°C. The E. coli FrdA/SdhA proteins were purified as described (35).…”