Abstract
1. A methyl viologen-linked sulfite reductase from spinach leaves was cleaved into two protein fractions. Both fractions were required for the oxidation of reduced methyl viologen with sulfite and the formation of sulfide under the assay conditions used. One fraction could be replaced by bovine serum albumin, by several other crystalline proteins, by thiol and disulfide compounds, or by yeast RNA. 2. The other fraction, sulfite reductase, has been purified 492-fold. The enzyme was homogeneous by ultracentrifugation but not by electrophoresis with polyacrylamide gel or cellulose acetate. The molecular weight of the enzyme was estimated to be 84,000 from gel filtration. The enzyme contained 0.76 g atom of iron per 84,000 g of protein. Negligible amounts of flavin are observed, indicating that flavin is not directly involved in catalysis of sulfite reduction. 3. Sulfite reductase, in the oxidized form, has absorption maxima at 279, 404, and 589 mµ. The last two maxima were shifted and diminished upon reduction with sodium borohydride and partially restored by sulfite. Absorption at 404 and 589 mµ was modified by cysteine, cyanide, and carbon monoxide. 4. Prior incubation of the reduced enzyme with sulfite prevented inhibition by p-chloromercuribenzoate and carbon monoxide. The inhibition of sulfite reductase by carbon monoxide was reversed by light. Thus, the sulfite reductase has some of the properties of a hemoprotein. 5. After cleavage, sulfite reductase was inactivated by reduced methyl viologen unless albumin, cysteine, cystine, sulfite, or RNA were present. The possible role of these compounds in prevention of reductase inactivation by methyl viologen is discussed. 6. After cleavage, the enzyme ceased to catalyze the reduction of nitrite by methyl viologen and catalyzed only the reduction of sulfite and hydroxylamine. The ratio of these activities remained constant through the later purification steps, and it is concluded that hydroxylamine reduction, but not nitrite reduction, is catalyzed by sulfite reductase.
Highlights
The purified methyl viologen-linked sulfite reductase induces the oxidation of reduced methyl viologen by hydroxylamine and by sulfite, as is true of the NADPH-linked enzyme (l-3)
The ratio of the bleaching rate of reduced methyl viologen with hydroxylamine to sulfite is high at an early step of enzyme purification but is constant in later fractionation steps (Table I)
In addition to the constant ratio of hydroxylamine- and sulfite-reducing activity at later stages of purification, both activities of the enzyme were inhibited by reduced methyl viologen, CMB, cyanide, and carbon monoxide (Tables VI, IX, and X)
Summary
Hydroxylamine, and nitrite reductase activities were determined by measurement of the initial rate of bleaching of reduced methyl viologen upon addition of the electron acceptor. The apparatus used, permitting addition of reagents under anaerobic conditions, has been described [8]. One unit of reductase activity for sulfite, hydroxylamine, or nitrite is defined as the amount of enzyme which oxidizes 1 pmole of reduced met.hyl viologen per min at 25”. XuZJite-reducing Activity-The reaction mixture contained, in a total volume of 1.5 ml, 100 MM potassium phosphate buffer, Issue of September 25, 1969. S. Bandurslci pH 7.75, 1 mM potassium sulfite, 0.27 mM methyl viologen (40 to 50% in the reduced form), 2 mg of crystalline bovine serum albumin, and enzyme
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
