Abstract
Neurospora crassa nitrite reductase (Mr = 290,000) catalyzes the NAD(P)H-dependent 6-electron reduction of nitrite to ammonia via flavin and siroheme prosthetic groups. Homogeneous N. crassa nitrite reductase has been prepared employing conventional purification methods followed by affinity chromatography on blue dextran-Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of homogeneous nitrite reductase reveals a single subunit band of Mr = 140,000. Isoelectric focusing of dissociated enzyme followed by sodium dodecyl sulfate-gel electrophoresis in the second dimension yields a single subunit spot with an isoelectric point at pH 6.8-6.9. Two-dimensional thin layer chromatography of acid-hydrolyzed nitrite reductase treated with 5-dimethylaminoaphthalene-1-sulfonyl chloride yields a single reactive NH2-terminal corresponding to glycine. An investigation of the prosthetic groups of nitrite reductase reveals little or no flavin associated with the purified protein, although exogenously added FAD is required for activity in vitro. An iron content of 9-10 Fe eq/mol suggests the presence of nonheme iron in addition to the siroheme moieties. Amino acid analysis yields 43 cysteinyl residues and sulfhydryl reagents react with 50 thiol eq/mol of nitrite reductase. The non-cysteinyl sulfur content, determined as 8.1 acid-labile sulfide eq/mol, is presumably associated with nonheme iron to form iron-sulfur centers. We conclude that N. crassa nitrite reductase is a homodimer of large molecular weight subunits housing an electron transfer complex of FAD, iron-sulfur centers, and siroheme to mediate the reduced pyridine nucleotide-dependent reduction of nitrite to ammonia.
Highlights
Neurospora crassa nitrite reductase (Mr = 290,000) soluble, sulfhydryl-containing metalloflavoprotein which mecatalyzes the NAD(P)H-dependent6-electronreduction diatesthe 2-electron transfer sequence (9,and references of nitrite toammonia via flavin and sirohemeprosthetic therein): groups
Bacterial nitrite reductasesare FAD-dependent metalloproteins and t-ypicallyutilize NADH as electron donor (10, 11).The enzyme from photosynthetic organisms is characterized as a low molecular weight (60,000-70,000) FADindependentmetalloprotein for which reduced ferredoxin serves as electron donor (12).Nitrite reductasesfrom the fungi A . nidulans (13) and N . crassa (14)can accept electronsfrom eitherNADH orNADPH.Thenitrite reductase complex from N . crassa is a large molecular weight (290,000)electrontransport system which requires FADfor activity in vitro and which catalyzes the stoichiometric reduction of nitrite to ammonia using 3 eq of reduced pyridine nucleotide to provide required for activity in vitro
We conclude that N. crassa nitrite reductase is a homodimer of large molecular weight subunits housing an electron transfer complex of FAD, iron-sulfur centers, and siroheme to mediate the reduced pyridine nucleotide-dependent reduction of nitrite toammonia
Summary
A nitrate reductase-deficient strain of N. crassa, nit-1, allele 34547 (Fungal Genetics StockCenter No 5 4 ) , wasgrown as described previously (18). To obtain homogeneous nitrite reductase, asingle aliquot of Fraction 6was chromatographed on a Sepharose4B column (1.5X 30 cm; bed volume, 50 ml) previously equilibrated with 10 mM potassium phosphate buffer, pH 7.0, containing 5 mM EDTA, 5 mM cysteine, 10% glycerol, and 2 mM sodium sulfite. The dissociation of Fraction 8 eluates in guanidine HCl was performed according to Weber et al (26).The treated protein samples were dialyzed overnight against 10 mM sodium phosphate buffer, pH 7.2, containing 0.1% SDS and resolved on 5% polyacrylamide gels as described above. NADPH, NADH, NADP, FAD, FMN, blue dextran,Sepharose 4B, RNA polymerase, phosphorylase a, 4,4'-dithiodipyridine, 5,sdithiobis(2-nitrobenzoic acid), 5-dimethylaminonaphthalene-l-~ulfonyl chloride, and standarddansyl amino acids were purchased from Sigma; ultrapure guanidine HCI and urea were from Schwarz/Mann; all materials for the preparation of polyacrylamide gels were from Bio-Rad; /&galactosidase was from Worthington Biochemicals; and rabbit muscle was aldolase from Calbiochem. All other chemicals were reagent grade and used as supplied
Sign-in/Register to view highlights & summary 
Published Version
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 callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
