Purification and Properties of Electron-transferring Flavoprotein from Peptostreptococcus elsdenii

Abstract

An electron-transferring flavoprotein (ETF) which couples the oxidation of NADH to the reduction of butyryl coenzyme A dehydrogenase has been purified in large quantities from the anaerobic bacterium Peptostreptococcus elsdenii. The purified protein appears to be homogeneous on the basis of polyacrylamide disc gel electrophoresis, thin layer isoelectric focusing, sedimentation velocity, and the Sephadex G-100 elution profile. Preliminary evidence suggests that ETF is also found in a complex with another flavoprotein. ETF has a molecular weight of 72,000 to 75,000 by Sephadex G-100 chromatography and is a dimer of two nonidentical subunits of approximate molecular weights of 33,000 and 41,000 determined by sodium dodecyl sulfate disc gel electrophoresis. Based on amino acid analyses, there are 2 moles of FAD per mole of protein of molecular weight 75,600. Possibly each subunit is associated with 1 FAD. The protein-bound FAD is 2.4 times more fluorescent than free FAD. Both tyrosine and tryptophan contribute to the protein fluorescence of ETF. Although the major prosthetic group of ETF is FAD, the isolated protein contains variable amounts of 6-hydroxy-7,8-dimethyl-10-(ribityl-5′-ADP)-isoalloxazine and 7-methyl-8-hydroxy-10-(ribityl-5′-ADP)-isoalloxazine. The isolated protein binds 30% more FAD to give enhanced activity and marked changes in the absorption spectrum. The spectrum of the oxidized isolated ETF is also drastically changed when the protein is maintained for long periods in its reduced form before subsequent reoxidation. In both cases, the major change is a large increase in the absorption around 400 nm. Apo-ETF has been prepared by treatment of the native protein with guanidine HCl. Catalytic activity is restored to the apoprotein by addition of FAD but not FMN. The reconstituted ETF also has increased absorption around 400 nm and decreased absorption at 450 nm compared to the isolated ETF. Unlike many other flavoprotein dehydrogenases which stabilize the neutral, blue flavin semiquinone and do not react with sulfite, ETF stabilizes the red, anionic semiquinone and also reacts with sulfite. Anaerobic titration of ETF with NADH or sodium dithionite resulted in a 2-electron reduction per protein-bound FAD, indicating that there are no colorless oxidation-reduction active groups present.