Studies on yeast sulfite reductase I. Purification and characterization

Abstract

1. NADPH-sulfite reductase, catalyzing the reduction of sulfite to sulfide by NADPH, was purified from baker's yeast to an almost homogeneous state. The purified enzyme showed a sedimentation coefficient of 14.8 S. A minimal molecular weight of 350 000 was estimated from its flavin content. 2. The oxidized enzyme was greenish yellow in color, showing absorption peaks at 386, 455 and 587 mμ. The intensity of these peaks was lowered considerably by NADPH or dithionite. 3. The enzyme contained 1 mole each of FMN and FAD per 350 000 g of protein. About 5 atoms of non-heme iron were detected per mole of FMN or FAD. The enzyme also contained a non-flavin chromophore (or chromophores) absorbing at 587 mμ and at about 386 mμ. The 587-mμ chromophore was unstable to treatment causing alterations in protein conformation. Five to six moles of p-chloromercuribenzoate combined with 350 000 g of the enzyme protein. 4. The enzyme catalyzed, besides the NADPH-sulfite reductase activity, the reduction of sulfite by reduced viologen dyes, the reductions by NADPH of nitrite, hydroxylamine, ferricyanide, cytochrome c, quinones, and 2,6-dichlorophenolindophenol, and the reduction of NADP + by reduced methyl viologen. 5. All the NADPH-linked activities were inhibited by NADP +, 2′-AMP and p-chloromercuribenzoate. NADP + and 2′-AMP, however, did not affect the reduction of sulfite by reduced viologen dyes. 6. Cyanide strongly inhibited the NADPH-sulfite reductase activity only when the reaction was started by adding sulfite to the enzyme which had been incubated with NADPH and cyanide. The addition of cyanide to the NADPH-reduced (but not the oxidized) enzyme caused an irreversible conversion to a reddish violet form. This conversion was prevented by the presence of sulfite.