208] Methyl viologen- and ferredoxin-linked sulfite reductases (spinach)

Abstract

Publisher Summary This chapter discusses the methods of preparation of methyl viologen- and ferredoxin-linked Sulfite Reductases (Spinach). Sulfite reductase from plants catalyzes the reduction of sulfite to sulfide, using reduced methyl viologen or ferredoxin as electron donor. The ferredoxin-linked enzyme also has methyl viologen-linked activity; however, methyl viologen-linked enzyme does not couple to reduced ferredoxin. The assay is based on the initial rate of oxidation of zinc reduced methyl viologen at 604 mμ following addition of sulfite. During purification, the enzyme is separated into two protein fractions, both of which are required for activity. However, one of these fractions can be replaced by bovine serum albumin; therefore, the reaction mixture contains albumin. An alternative method of assay is based on measurement of hydrogen uptake in the presence of methyl viologen and hydrogenase. The purified enzyme catalyzes the oxidation of reduced methyl viologen by sulfite and by hydroxylamine. Nitrite, cytochrome c, thiosulfate, cystine, cysteic acid, cysteine sulfinic acid, lipoamide, oxidized glutathione, and S-sulfocysteine do not induce oxidation of reduced methyl viologen.