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43763-AC4
Probing the Mechanism of 2-Nitropropane Dioxygenase: A Model for Flavin Semiquinone Intermediates in Catalysis
2-Nitropropane dioxygenase (2NPD; E.C. 1.13.11.32) is a flavin mononucleotide-dependent enzyme that catalyzes the oxidative denitrification of nitroalkanes to the corresponding carbonyl compounds and nitrite (1). Among nitroalkane-oxidizing enzymes, 2NPD is the only one described to date which is capable of utilizing both neutral (nitroalkane) and anionic (nitronate) forms of the substrate. 2NPD is also one of the very few flavin-dependent enzymes reported to date in which a transient, anionic, flavin semiquinone intermediate has been observed in turnover with nitroalkanes or alkyl nitronates (1, 2). For these reasons, 2NPD has emerged as a model system to understand the reactivity of anionic flavosemiquinone intermediates in enzymatic turnover.
Our group has used solvent viscosity, temperature, pH, and kinetic isotope effects, in steady state and pre-steady state, as well as x-ray crystallographic approaches, to gain insights on the chemical mechanism for oxidation of nitroalkanes or nitronates by 2NPD from Neurospora crassa. The results that we obtained thus far have provided detailed mechanistic insights on the chemical mechanism for the oxidation of nitroethane and ethylnitronate catalyzed by the enzyme. Interestingly, 2NPD catalyzes both the oxidative denitrification of nitroalkanes and nitronates to the corresponding carbonyl compounds and the non-oxidative tautomerization between their anionic and neutral forms. During enzymatic turnover with neutral substrates, the rate of oxidative denitrification of nitroalkanes is limited by the cleavage of the substrate CH bond at low pH. With anionic substrates, the non-oxidative protonation of nitronates to yield the corresponding nitroalkanes limits enzymatic turnover at high pH (2).
Current studies suggest that a similar nitro-aci tautomerization occurs with nitroethane as substrate for 2NPD, in which the neutral substrate is deprotonated to form ethylnitronate without involvement of the enzyme-bound flavin cofactor. This reaction has been further characterized by studying the pH dependence of the kinetic isotope effects with 1,1-[2H2]-nitroethane as substrate for the enzyme under steady-state conditions with a stopped-flow spectrophotometer. The corresponding non-enzymatic deprotonation of nitroethane by sodium pyrophosphate has also been studied as a function of pH. Moreover, site-directed mutagenesis studies in which His 196 was replaced with glutamine indicated that this histidine residue is located in the active site of the enzyme and is responsible for the enzyme-catalyzed deprotonation of nitroethane, which initiates the oxidation reaction with neutral substrates. These experiments, and the results that are emerging, set the stage for future mechanistic investigations of the enzyme aimed at the comparison of the mechanism of proton transfer from nitroethane to the flavin or to imidazole in which the contribution of quantum mechanical tunneling will be critically evaluated in the enzymatic and non-enzymatic reactions of oxidation of nitroethane. In a parallel series of experiments in collaboration with Dr. Allen M. Orville at the Brookhaven National Laboratory, our group has obtained good quality crystals of the enzyme both in the free and liganded forms, which diffract to a resolution of 1.7 Å. We are currently working on the elucidation of the three-dimensional structure of the enzyme, which will provide useful structural information to complement the mechanistic data on the enzyme that is obtained in solution.
References
1. Francis, K., Russell, B., and Gadda, G. (2005) Involvement of a flavosemiquinone in the enzymatic oxidation of nitroalkanes catalyzed by 2-nitropropane dioxygenase, J. Biol. Chem. 280, 5195-5204.
2. Francis, K., and Gadda, G. (2006) Probing the chemical steps of nitroalkane oxidation catalyzed by 2-nitropropane dioxygenase with solvent viscosity, pH, and substrate kinetic isotope effects, Biochemistry 45, 13889-13898.
