PMID:7030314
| Citation |
Jackson, RH, Cornish-Bowden, A and Cole, JA (1981) Prosthetic groups of the NADH-dependent nitrite reductase from Escherichia coli K12. Biochem. J. 193:861-7 |
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| Abstract |
A substantially improved purification of Escherichia coli NADH-dependent nitrite reductase was obtained by purifying it in presence of 1 mM-NO2- and 10 microM-FAD. The enzyme was obtained in 20% yield with a maximum specific activity of 1.04 kat . kg-1: more than 95% of this sample subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis migrated as a single band of protein. This highly active enzyme contained one non-covalently bound FAD molecule, and, probably, 5 Fe atoms and 4 acid-labile S atoms per subunit. No FMN, covalently bound flavin or Mo was detected. The spectrum of the enzyme shows absorption maxima at 386, 455, 530 and about 575 nm with a shoulder at 480--490 nm. The Soret-band/alpha-band absorbance ratio is about 4:1. These spectral features are characteristic of sirohaem, apart from the maximum at 455nm, which is attributed to flavin. The enzyme also catalyses the NADH-dependent reduction of horse heart cytochrome c, 2,6-dichlorophenol-indophenol and K3Fe(CN)6. The presence of sirohaem in E. coli nitrite reductase explains the apparent identity of the cysG and nirB gene of E. coli and inability of hemA mutants to reduce nitrite. |
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| Keywords |
Chemical Phenomena; Chemistry; Escherichia coli/enzymology; Flavins/analysis; Heme/analysis; Iron/analysis; NADH, NADPH Oxidoreductases/isolation & purification; Nitrite Reductase (NAD(P)H); Nitrite Reductases/isolation & purification; Nitrite Reductases/metabolism; Spectrophotometry |
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