Reduced Nicotinamide Adenine Dinucleotide Phosphate-Sulfite Reductase of Enterobacteria

Abstract

Abstract NADPH-sulfite reductase (EC 1.8.1.2), an enzyme which catalyzes the 6-electron reduction of sulfite to sulfide, has been purified to homogeneity, according to electrophoretic and ultracentrifugal criteria, from Escherichia coli B. The enzyme, molecular weight 670,000, contains four FMN, four 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. The absorption spectrum of sulfite reductase shows maxima at 278, 386, 455, 587, and 714 nm, the latter three bands being partially bleached by NADPH or dithionite. The enzyme exhibits electron paramagnetic resonance signals with g values of 6.7, 5.25, and 2.0, characteristic of 3 to 4 moles of a high spin Fe3+-heme per mole of enzyme, and this signal is reduced in intensity by addition of NADPH. The hemetype chromophore, which appears to be characteristic of assimilatory sulfite-reducing enzymes in a wide variety of organisms, is extractable with acetone-0.015 n HCI and is highly polar; its chromatographic behavior is clearly different from that of protohemin. Both the enzyme and the extracted heme-type chromophore form spectrally distinct complexes with typical heme ligands (CO, cyanide, hydroxide, and pyridine) in which the Soret band maxima are at unusually short wave lengths with low Soret to α-band absorbance ratios. Formation and decay of the enzyme-CO complex is slow in comparison to other hemoproteins. These data indicate that E. coli sulfite reductase is a self-contained complex of electron transport carriers including a heme-type component which, by virtue of its spectrophotometric, solubility, and chromatographic properties, is clearly distinguishable from previously described hemes.