Electron-spin-resonance studies of the NADH-dependent nitrite reductase from Escherichia coli K12.
The NADH-dependent nitrite reductase of Escherichia coli, which contains sirohaem, flavin, non-haem iron and labile sulphide, was examined by low-temperature e.s.r. spectroscopy. The enzyme, stored in the presence of nitrite and ascorbate, gave the spectrum of a nitrosyl derivative, with hyperfine splitting due to the nitrosyl nitrogen. On removal of these reagents, a series of signals centred around g = 6 was observed, typical of high-spin ferric haem. Cyanide converted this into a low-spin form. On reduction of the enzyme with NADH, an axial spectrum at g = 1.92, 2.01 was observed. The temperature-dependence of this signal is indicative of a [2Fe-2S] iron-sulphur cluster. The midpoint potential of this cluster was estimated to be -230 +/- 15 mV by two independent methods. Reduction of the enzyme with dithionite yielded further signals, which are at present unidentified, at g = 2.1-2.28. No signals were observed that could be assigned to a [4Fe-4S] cluster, such as is found in other sulphite reductases and nitrite reductases that contain sirohaem.
D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D006418:Heme / Q000031:analogs & derivatives / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D050898:Nitrite Reductase (NAD(P)H); D009572:Nitrite Reductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011190:Potassium Cyanide / Q000494:pharmacology; D013455:Sulfur / Q000378:metabolism; D013885:Thiosulfates / Q000494:pharmacology
D013885:Thiosulfates; C023692:nitrosyl-siroheme complex; D006418:Heme; D013455:Sulfur; D007501:Iron; D009247:NADH, NADPH Oxidoreductases; D009572:Nitrite Reductases; D050898:Nitrite Reductase (NAD(P)H); C017717:sodium thiosulfate; D011190:Potassium Cyanide