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.

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