Establishing the kinetic competency of the cationic imine intermediate in nitroalkane oxidase

Michael P. Valley, Shane E. Tichy, Paul F Fitzpatrick

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

The flavoprotein nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes and ketones. Cyanide inactivates the enzyme during turnover in a concentration-dependent fashion. Mass spectrometry of the flavin from enzyme inactivated by cyanide in the presence of nitroethane or nitrohexane shows that a flavin cyanoethyl or cyanohexyl intermediate has formed. At high concentrations of cyanide, inactivation does not consume oxygen. Rapid reaction studies show that formation of the adduct with 2- 2H2-nitroethane shows a kinetic isotope effect of 7.9. These results are consistent with cyanide reacting with a species formed after proton abstraction but before flavin oxidation. The proposed mechanism for nitroalkane oxidase involves removal of a proton from the nitroalkane, forming a carbanion which adds to the flavin N(5). Elimination of nitrite from the resulting adduct would form an electrophilic imine which can be attacked by hydroxide. The present results are consistent with cyanide trapping this electrophilic intermediate.

Original languageEnglish (US)
Pages (from-to)2062-2066
Number of pages5
JournalJournal of the American Chemical Society
Volume127
Issue number7
DOIs
StatePublished - Feb 23 2005
Externally publishedYes

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Imines
Cyanides
Kinetics
Protons
Enzymes
Flavoproteins
Oxidation
Nitrites
Ketones
Aldehydes
Isotopes
Mass spectrometry
Mass Spectrometry
2-nitropropane dioxygenase
Oxidoreductases
Oxygen
4,6-dinitro-o-cresol

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Establishing the kinetic competency of the cationic imine intermediate in nitroalkane oxidase. / Valley, Michael P.; Tichy, Shane E.; Fitzpatrick, Paul F.

In: Journal of the American Chemical Society, Vol. 127, No. 7, 23.02.2005, p. 2062-2066.

Research output: Contribution to journalArticle

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N2 - The flavoprotein nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes and ketones. Cyanide inactivates the enzyme during turnover in a concentration-dependent fashion. Mass spectrometry of the flavin from enzyme inactivated by cyanide in the presence of nitroethane or nitrohexane shows that a flavin cyanoethyl or cyanohexyl intermediate has formed. At high concentrations of cyanide, inactivation does not consume oxygen. Rapid reaction studies show that formation of the adduct with 2- 2H2-nitroethane shows a kinetic isotope effect of 7.9. These results are consistent with cyanide reacting with a species formed after proton abstraction but before flavin oxidation. The proposed mechanism for nitroalkane oxidase involves removal of a proton from the nitroalkane, forming a carbanion which adds to the flavin N(5). Elimination of nitrite from the resulting adduct would form an electrophilic imine which can be attacked by hydroxide. The present results are consistent with cyanide trapping this electrophilic intermediate.

AB - The flavoprotein nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes and ketones. Cyanide inactivates the enzyme during turnover in a concentration-dependent fashion. Mass spectrometry of the flavin from enzyme inactivated by cyanide in the presence of nitroethane or nitrohexane shows that a flavin cyanoethyl or cyanohexyl intermediate has formed. At high concentrations of cyanide, inactivation does not consume oxygen. Rapid reaction studies show that formation of the adduct with 2- 2H2-nitroethane shows a kinetic isotope effect of 7.9. These results are consistent with cyanide reacting with a species formed after proton abstraction but before flavin oxidation. The proposed mechanism for nitroalkane oxidase involves removal of a proton from the nitroalkane, forming a carbanion which adds to the flavin N(5). Elimination of nitrite from the resulting adduct would form an electrophilic imine which can be attacked by hydroxide. The present results are consistent with cyanide trapping this electrophilic intermediate.

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