Cytochrome c552 and nitrite reduction in Escherichia coli

Abstract

The influence of growth conditions on the synthesis of cytochrome c 552 and the activities of NADH-nitrite oxidoreductase (EC 1.6.6.4) and NADH-cytochrome c oxidoreductase (EC 1.6.99.3) have been studied in Escherichia coli strain K 12. Nitrite added to anaerobic cultures stimulated cytochrome c 552 synthesis as well as nitrite and cytochrome c reductase activities. These were decreased by high concentrations of glucose or amino acids, and were low or absent in aerobically grown cells. When cells were suspended in sucrose and lysozyme (EC 3.2.1.17), and EDTA added, 80% of the cytochrome c 552 was released into solution. The NADH-nitrite oxidoreductase activity remaining in the osmotically sensitive spheres so formed was the same as in untreated cells. Nitrite reductase activity and cytochrome c 552 were also separated by gel filtration, and by (NH 4) 2SO 4 fractionation. When cytochrome-rich fractions were recombined with nitrite reductase active fractions, no stimulation of the rate of NADH oxidation by nitrite was observed. In every experiment cytochrome c reductase and nitrite reductase activities were found in the same fractions in approximately the same ratio. Both reductases showed substrate inhibition with respect to NADH: the pH optima were 7.9 for nitrite reductase and 8.6 for cytochrome c reductase. It is suggested that cytochrome c reductase activity is due to a lack of specificity of nitrite reductase for an electron acceptor, and that cytochrome c 552 is not a physiological intermediate in either reaction. An alternative function for cytochrome c 552 in nitrite reduction has been suggested.