Reduced Nicotinamide Adenine Dinucleotide Phosphate-Sulfite Reductase of Enterobacteria: V. STUDIES WITH THE ESCHERICHIA COLI HEMOFLAVOPROTEIN DEPLETED OF FLAVIN MONONUCLEOTIDE: DISTINCT ROLES FOR THE FLAVIN ADENINE DINUCLEOTIDE AND FLAVIN MONONUCLEOTIDE PROSTHETIC GROUPS IN CATALYSIS

Abstract

Abstract Escherichia coli NADPH-sulfite reductase (EC 1.8.1.2), molecular weight 670,000, contains 4 FAD, 4 FMN, approximately 16 atoms of non-heme iron-acid-labile sulfide, and 3 to 4 molecules of siroheme per enzyme molecule. NADPH interacts with the flavins, while sulfite interacts with the siroheme component. The sulfite reductase-FMN complex exhibits a dissociation constant (Kdiss) of 10 nm at 25°. All four FMN groups appear to be equivalent. The FAD prosthetic group is bound much more tightly than the FMN. The fluorescence of free FMN is largely quenched when the flavin is enzyme-bound. Sulfite reductase freed of g95% of its FMN while retaining g85% of its FAD was prepared by irradiation of enzyme solutions, in phosphate buffer 30% saturated in ammonium sulfate, with a strong fluorescent light. Such FMN-depleted enzyme preparations can bind FMN with a Kdiss identical with that of the native enzyme. By comparing properties of FMN-depleted enzyme, with and without added FMN, with those of native enzyme, the following conclusions were reached: 1. The FAD prosthetic group of sulfite reductase serves as the sole entry port for electrons of NADPH. Thus, the ability of the enzyme to bind pyridine nucleotide (4.0 [14C]-NADP+ per enzyme molecule; Kdiss = 0.1 mm) and to catalyze electron exchange between pyridine nucleotides (NADPH and 3-acetylpyridine adenine dinucleotide phosphate) is unaffected by the presence or absence of the FMN prosthetic group. The FAD moiety of FMN-depleted enzyme is reducible by NADPH. The FAD is reduced as rapidly by NADPH (k = 190 s-1) as is the most rapidly reducible flavin component of the native (FMN-containing) enzyme. 2. The FMN prosthetic group is required for transfer of electrons from NADPH, via the FAD, either to enzyme-bound siroheme, and thence to sulfite, or to the exogenous electron acceptor cytochrome c. The majority of electrons which are transferred from NADPH to the diaphorase-type acceptors 2,6-dichlorophenolindophenol, ferricyanide, or menadione by sulfite reductase, pass through an FMN-dependent pathway. The Michaelis constant for FMN-dependent reactivation of the NADPH-cytochrome c reductase activity of enzyme from which FMN has been removed either by irradiation or dilution of the enzyme is 8 to 11 nm, a value in good agreement with the Kdiss of the enzyme-FMN complex. Other flavins, including FAD, can substitute for FMN, although FMN exhibited the smallest Km of all flavins tested. The 5'-phosphate group of FMN appears to be quite important for interaction of the flavin with the enzyme as measured by reactivation of enzymatic activity.