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Neurospora crassa NAD(P)H-nitrite reductase. Studies on its composition and structure.
J Biol Chem. 1981 Sep 25; 256(18):9711-7.JB

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.

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Authors

Prodouz KN
No affiliation info available
Garrett RH
No affiliation info available

MeSH

Amino AcidsElectron TransportFlavin-Adenine DinucleotideHemeIronIron-Sulfur ProteinsMolecular WeightNADH, NADPH OxidoreductasesNeurosporaNeurospora crassaNitrite Reductases

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

6457037

Citation

Prodouz, K N., and R H. Garrett. "Neurospora Crassa NAD(P)H-nitrite Reductase. Studies On Its Composition and Structure." The Journal of Biological Chemistry, vol. 256, no. 18, 1981, pp. 9711-7.
Prodouz KN, Garrett RH. Neurospora crassa NAD(P)H-nitrite reductase. Studies on its composition and structure. J Biol Chem. 1981;256(18):9711-7.
Prodouz, K. N., & Garrett, R. H. (1981). Neurospora crassa NAD(P)H-nitrite reductase. Studies on its composition and structure. The Journal of Biological Chemistry, 256(18), 9711-7.
Prodouz KN, Garrett RH. Neurospora Crassa NAD(P)H-nitrite Reductase. Studies On Its Composition and Structure. J Biol Chem. 1981 Sep 25;256(18):9711-7. PubMed PMID: 6457037.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Neurospora crassa NAD(P)H-nitrite reductase. Studies on its composition and structure. AU - Prodouz,K N, AU - Garrett,R H, PY - 1981/9/25/pubmed PY - 1981/9/25/medline PY - 1981/9/25/entrez SP - 9711 EP - 7 JF - The Journal of biological chemistry JO - J Biol Chem VL - 256 IS - 18 N2 - 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. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/6457037/Neurospora_crassa_NAD_P_H_nitrite_reductase__Studies_on_its_composition_and_structure_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(19)68821-6 DB - PRIME DP - Unbound Medicine ER -