Reduced Nicotinamide Adenine Dinucleotide Phosphate-Sulfite Reductase of Enterobacteria (1974)

Abstract NADPH-sulfite reductase (EC 1.8.1.2) from Escherichia coli is a complex hemoflavoprotein, molecular weight 670,000, containing 4 FMN, 4 FAD, 20 to 21 atoms of iron, 14 to 15 labile sulfides, and 3 to 4 molecules of a novel type of heme per enzyme molecule. This heme has been identified as an octacarboxylic iron-tetrahydroporphyrin (Murphy, M. J., Siegel, L. M., Kamin, H., and Rosenthal, D. (1973) J. Biol. Chem. 248, 2801). The enzyme catalyzes the stoichiometric conversion of sulfite to sulfide at the expense of 3 NADPH. The Km values for sulfite and NADPH are both 4 to 5 µm. Reduced methyl or benzyl viologens can serve as electron donors for sulfite reduction, but NADH cannot. In addition to sulfite reduction, the enzyme catalyzes the NADPH-dependent reduction of a variety of diaphorase acceptors (cytochrome c, ferricyanide, 2,6-dichlorophenolindophenol, menadione, FMN, FAD) as well as NADPH oxidase, NADPH-3-acetylpyridine adenine dinucleotide phosphate transhydrogenase, NADPH-nitrite and -hydroxylamine reductase and reduced methyl viologen-NADP+ reductase activities. All NADPH-dependent activities examined were competitively inhibited by NADP+. Agents which react with the heme prosthetic group, i.e. CO, cyanide, and arsenite, inhibit the reductions of sulfite, nitrite, and hydroxylamine (with either NADPH or reduced methyl viologen as electron donor), while all other activities are unaffected. Cyanide and CO binding to and CO dissociation from the enzyme (determined spectrophotometrically) parallel the respective development and relief of inhibition of NADPH-sulfite reductase activity. Development of inhibition requires the presence of reductant (NADPH) as well as inhibitor, in accord with the observation that CO, cyanide, or arsenite can react with reduced, but not oxidized enzyme. Treatment of enzyme with 1 µm p-chloromercuriphenylsulfonate causes the dissociation of virtually all of the FMN while permitting retention of FAD and heme. This treatment inhibits all pyridine nucleotide-dependent reactions of the enzyme except transhydrogenase and FMN reductase. The methyl viologen-sulfite reductase is unaffected. The development of fluorescence due to FMN release parallels the development of the observed inhibitions. The FAD of the FMN-free enzyme is reducible by NADPH, but the heme is not. If exogenous FMN is added, the heme becomes reducible and all NADPH-dependent activities are restored. We have concluded that electron flow from NADPH to sulfite follows the minimum linear sequence: NADPH → FAD → FMN → heme → sulfite In this scheme, FAD serves as the entry port for electrons from NADPH. It can transfer electrons directly to FMN (internal or external) or to pyridine nucleotides and their analogues. The heme is required for electron transfer to sulfite (and nitrite and hydroxylamine). The FMN is required for electron transfer from the reduced FAD to the heme (and hence to acceptors dependent on the heme) or, more directly, to diaphorase-type acceptors and O2. Reduced methyl viologen can donate electrons to both the FMN and heme. The patterns of inhibition by a variety of salts of the NADPH-cytochrome c and reduced methyl viologen-sulfite reductase reactions are consistent with the hypothesis that these two reactions involve independent portions of the enzyme molecule.