Reduced Nicotinamide Adenine Dinucleotide Phosphate-Sulfite Reductase of Enterobacteria: IV. THE ESCHERICHIA COLI HEMOFLAVOPROTEIN: SUBUNIT STRUCTURE AND DISSOCIATION INTO HEMOPROTEIN AND FLAVOPROTEIN COMPONENTS

Abstract

Abstract Escherichia coli NADPH-sulfite reductase (EC 1.8.1.2), which catalyzes the 6-electron reduction of sulfite to sulfide, is a complex hemoflavoprotein, molecular weight approximately 700,000, which contains 4 FMN, 4 FAD, 20 to 21 atoms of iron, and 14 to 15 labile sulfides, and 3 to 4 molecules of a novel type of heme per enzyme molecule. This complex structure is composed of only two types of polypeptide chain and has the subunit structure α8β4. α and β each have molecular weights of 54,000 to 60,000, α being slightly larger than β. Sulfite reductase can be dissociated in 5 m urea into a hemoprotein and a flavoprotein component, which can be separated by DEAE-cellulose chromatography. The hemoprotein contains iron, labile sulfide, and the novel sulfite reductase heme, but no flavin. It exists as a β monomer in phosphate buffer, and exhibits reduced methyl viologen-sulfite reductase activity, but no NADPH-dependent activities. The flavoprotein contains FAD and FMN, but no iron, labile sulfide, or heme. It exists as an octamer in phosphate buffer, and exhibits an NADPH-cytochrome c reductase activity (as well as NADPH oxidase, transhydrogenase, and a variety of diaphorase-type activities characteristic of the sulfite reductase holoenzyme), but no NADPH- or reduced methyl viologen-sulfite reductase activity. By mixing the isolated hemoprotein and flavoprotein components in appropriate proportions, an NADPH-sulfite reductase activity can be reconstituted in vitro.