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The steady-state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K 12. Nitrite and hydroxylamine reduction.

ABSTRACT: The reduction of both NO2- and hydroxylamine by the NADH-dependent nitrite reductase of Escherichia coli K 12 (EC 1.6.6.4) appears to follow Michaelis-Menten kinetics over a wide range of NADH concentrations. Substrate inhibition can, however, be detected at low concentrations of the product NAD+. In addition, NAD+ displays mixed product inhibition with respect to NADH and mixed or uncompetitive inhibition with respect to hydroxylamine. These inhibition characteristics are consistent with a mechanism in which hydroxylamine binds during catalysis to a different enzyme form from that generated when NAD+ is released. The apparent maximum velocity with NADH as varied substrate increases as the NAD+ concentration increases from 0.05 to 0.7 mM with 1 mM-NO2- or 100 mM-hydroxylamine as oxidized substrate. This increase is more marked for hydroxylamine reduction than for NO2- reduction. Models incorporating only one binding site for NAD can account for the variation in the Michaelis-Menten parameters for both NADH and hydroxylamine with [NAD+] for hydroxylamine reduction. According to these models, activation of the reaction occurs by reversal of an over-reduction of the enzyme by NADH. If the observed activation of the enzyme by NAD+ derives both from activation of the generation of the enzyme-hydroxylamine complex from the enzyme-NO2- complex during NO2- reduction and from activation of the reduction of the enzyme-hydroxylamine complex to form NH4+, then the variation of Vapp. for NO2- or hydroxylamine with [NAD+] is consistent with the occurrence of the same enzyme-hydroxylamine complex as an intermediate in both reactions.

SUBMITTER: Jackson RH

PROVIDER: S-EPMC1163347 | biostudies-other | 1981 Oct

REPOSITORIES: biostudies-other

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

Project description: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.

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Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction.

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Prosthetic groups of the NADH-dependent nitrite reductase from Escherichia coli K12.

Project description: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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Electron-spin-resonance studies of the NADH-dependent nitrite reductase from Escherichia coli K12.

Project description: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.

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Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) does not disproportionate hydroxylamine to ammonia and nitrite, despite a strongly favorable driving force.

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Studies on alkaline phosphatase. Transient-state and steady-state kinetics of Escherichia coli alkaline phosphatase.

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