Purification and Properties of a Sulfite Reductase From Leaf Tissue

Sulfite reductase (EC 1.8.1.2) has been purified 190-fold from spinach leaves. The enzyme catalyzes the reduction of sulfite to sulfide, with the use of reduced methyl viologen. NADH and NADPH are not capable of replacing reduced methyl viologen. A stoichiometry of 6 molecules of reduced methyl viologen oxidized per molecule of sulfide formed has been found. The enzyme, following treatment with reduced methyl viologen, is sensitive to inhibition by p-chloromercuribenzoate and cyanide. Without treatment with reduced methyl viologen the enzyme is not sensitive to either compound. Inhibition is not relieved by thiol compounds. Sensitivity of the enzyme to p-chloromercuribenzoate is lost in the presence of sulfite, while cyanide sensitivity remains. These results suggest the occurrence of two catalytically active sites, a p-chloromercuribenzoate-sensitive, sulfite-protected site, and a cyanide-sensitive site. Nucleoside di- and triphosphates, irrespective of the kinds of base and sugar, stimulate reductase activity. Pyrophosphate gives the same stimulation as nucleoside diphosphates. This effect is observed in 2-(hydroxymethyl)-2-(N-glycine)-1,3-dihydroxypropane buffer, but not in phosphate buffer.