Spinach nitrite reductase. Purification and properties of a siroheme-containing iron-sulfur enzyme.
Ferredoxin-nitrite reductase (EC 1.7.7.1.) from spinach has been purified to homogeneity with a specific activity of 110 units/mg of protein. The enzyme, Mr = 61,000 has 3 iron atoms (of which one is in siroheme) and 2 labile sulfides, i.e. 1 (Fe2-S2) per molecule, with absorption maxima at 276, 386 (Soret), 573 (alpha), and 690 nm, with an E386 of 3.97 X 10(4) M-1-cm-1, and A276/A386 absorptivity ratio of 1.8. Anaerobic addition of dithionite results in the loss of the 690 nm peak and the splitting of the 573 nm absorption band into two broad peaks at 545 and 585 nm. Reduction by dithionite is enhanced by cyanide (Fig. 7) and requires about 3 electron eq per mol of enzyme. With nitrite or hydroxylamine (substrates of the enzyme), cyanide (a competitive inhibitor with respect to nitrite), or sulfite, the 690 nm absorption band of substrate-free enzyme disappears and the absorbance in the Soret and alpha region are altered. The high spin EPR signals disappear (J. M. Vega, H. Kamin, N. R. Orme-Johnson, and W. H. Orme-Johnson, unpublished observations). Titration permits calculation of 1 mol of nitrite bound/mol of enzyme with a Kdiss of 3.2 X 10(-6) M. Dithionite-reduced enzyme also forms complexes with added nitrite, hydroxylamine, or cyanide, characterized by marked alterations in the 573 (alpha) absorption band. THus, substrates or competitive inhibitors can be bound to the oxidized or reduced enzyme forms. CO inhibits nitrite reductase and forms a complex with reduced enzyme (epsilonmax at 395, 543, and 585 nm). Formation or dissociation of the spectrophotometrically detectable CO complex correlates with inhibition or inhibition-reversal of nitrite reduction catalysis. During steady state turnover with dithionite and nitrite, the enzyme forms a complex with added nitrite with absorption difference maxima at 445, 538, and 580 nm with respect to reduced enzyme. When nearly all substrate is depleted the spectrum of a new species appears, indicating that nitrite reductase may form complexes with nitrogen compounds of more than one oxidation state. Nitrite is stoichiometrically reduced to ammonia without detectable free nitrogen compounds of intermediate reduction state. p-Chloromercuribenzoate (pCMB) inhibits nitrite reductase activity and nitrite partially protects against this inhibition. Titration of native enzyme with the mercurial shows that 6 mol of pCMB can be bound/mol or nitrite reductase. The Soret absorption band of the native nitrite reductase is altered and partially bleached in the pCMB-treated enzyme, and the 573 (alpha) band disappears.
D000596:Amino Acids / Q000032:analysis; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003486:Cyanides; D005415:Flavins / Q000032:analysis; D006418:Heme / Q000032:analysis; D007122:Immunoelectrophoresis; D007501:Iron / Q000032:analysis; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009572:Nitrite Reductases / Q000302:isolation & purification / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D011485:Protein Binding; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013440:Sulfides / Q000032:analysis; D013885:Thiosulfates
D000596:Amino Acids; D002729:Chloromercuribenzoates; D003486:Cyanides; D005415:Flavins; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D013440:Sulfides; D013885:Thiosulfates; D006418:Heme; D007501:Iron; D009247:NADH, NADPH Oxidoreductases; D009572:Nitrite Reductases