Prosthetic groups of the NADH - dependent nitrite reductase from Escherichia coli K 12 Ronald

A substantially improved purification of Escherichia coli NADH-dependent nitrite reductase was obtained by purifying it in the presence of 1 mM-NO2and 10,uM-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 480-490nm. The Soret-band/a-band absorbance ratio is about 4: 1. These spectral features ate characteristic of sirohaem, apart from the maximum at 455 nm, which is attributed to fiavin. 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 genes of E. coli and inability of hemA mutants to reduce nitrite.