The steady state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K12. The reduction of single-electron acceptors

Abstract

The kinetic characteristics of the diaphorase activities associated with the NADH-dependent nitrite reductase (EC 1.6.6.4) from Escherichia coli have been determined. The values of the apparent maximum velocity are similar for the reduction of Fe(CN)6(3)-and mammalian cytochrome c by NADH. These reactions may therefore have the same rate-limiting step. NAD+ activates NADH-dependent reduction of cytochrome c, and the apparent maximum velocity for this substrate increases more sharply with the concentration of NAD+ than for hydroxylamine. The simplest explanation is that NAD+ activation of hydroxylamine reduction derives solely from activation of steps involved in the reduction of cytochrome c, a flavin-mediated reaction, but these steps are only partly rate-limiting for the reduction of hydroxylamine. At 0.5 mM-NAD+, the apparent maximum velocity was 2.3 times higher for 0.1 mM-cytochrome c as substrate than for 100 mM-hydroxylamine, suggesting that the rate-limiting step during hydroxylamine reduction is a step that is not involved in cytochrome c reduction. A scheme is proposed that can account for the pattern of variation with [NAD+] of the Michaelis-Menten parameters for hydroxylamine and for NADH with hydroxylamine or cytochrome c as oxidized substrate.

Nitrate respiration in relation to facultative metabolism in enterobacteria.

“…This soluble enzyme is composed of a single polypeptide species of 88 kDa (72). It resembles the assimilatory nitrite reductase from N. crassa in its cofactors (flavin adenine dinucleotide, sulfhydryl, Fe-S clusters, and siroheme) and its presumed reaction mechanism (92,175,176).…”

The respiratory chains of Escherichia coli.

“…The soluble NADH:nitrite reductase is less well characterized. It has recently been purified (231) and found to contain noncovalently bound FAD, at least one binuclear Fe-S center, and one siroheme per monomer (see also references 55,229,230). As isolated, the enzyme is a dimer of two identical monomers of Mr 88,000 (85,231).…”

Electron-spin-resonance studies of the NADH-dependent nitrite reductase from Escherichia coli K12

“…A kinetic mechanism of the NADH-dependent reductase has been proposed from studies of the reduction of NO2-, hydroxylamine and various single-electron acceptors by NADH (Jackson et al, 1981b(Jackson et al, , 1982. We here present e.s.r.…”