2-Nitropropane dioxygenase from Hansenula mrakii is a flavin-dependent enzyme that catalyzes the oxidation of anionic nitroalkanes into the corresponding carbonyl compounds and nitrite, with oxygen as the electron acceptor. Although nitroalkanes are anticipated to be toxic and carcinogenic, they are used widely in chemical industry for a quick and effective way of synthesizing common reagents. Consequently, the biochemical and biophysical analysis of 2-nitropropane dioxyganase has a potential for bioremediation purposes. In this study, recombinant enzyme is purified to high levels, allowing for detailed characterization. The biochemical analysis of 2-nitropropane dioxygenase presented in this study has established that enzyme utilizes alkyl nitronates as substrates by forming an anionic flavosemiquinone in catalysis. The enzyme is inhibited by halide ions, does not contain iron and has a positive charge located close to the N(1)-C(2)=O locus of the isoalloxazine moiety of the FMN cofactor.

Nitronate monooxygenase (E.C. 1.13.11.16), formerly known as 2-nitropropane dioxygenase (EC 1.13.11.32), is a flavin dependent enzyme that catalyzes the oxidation of nitronates to their corresponding carbonyl compounds and nitrite. Despite the fact that the enzyme was first isolated from Neurospora crassa 60 years ago, the biochemical and physiological properties of nitronate monooxygenase have remained largely elusive. This dissertation will present the work that established both the catalytic mechanism and physiological role of the fungal enzyme. The biological and biochemical properties of propionate-3-nitronate, the recently discovered physiological substrate for nitronate monooxygenase, will be extensively reviewed. The nitronate is produced by a variety of variety leguminous plants and fungi and is a potent and irreversible inhibitor of succinate dehydrogenase. Nitronate monooxygenase allows N. crassa to overcome the toxicity of propionate-3-nitronate as demonstrated by in vivo studies of the yeast, which showed that the wild-type can grow in the presence of the toxin whereas a knock out mutant that lacks the gene encoding for the enzyme could not. In addition to establishing the physiological role of nitronate monooxygenase, the work presented here demonstrates that the catalytic mechanism of the enzyme involves the formation of an anionic flavosemiquinone intermediate. This intermediate is stabilized by the protonated form of an active site histidine residue (His-196) that acts as an electrostatic catalyst for the reaction as demonstrated by pH studies of the reductive half reaction of the enzyme. Histidine 196 also serves as the catalytic base for the reaction of the enzyme with nitroethane as substrate as revealed through mutagenesis studies in which the residue was replaced with an asparagine. The kinetic implications of branching of reaction intermediates in enzymatic catalysis are also demonstrated through studies of the kinetic isotope effects of nitronate monooxygenase with 1,1-[2H2]-nitroethane as substrate. Finally the use of competitive inhibitors as a probe of enzyme structure will be presented through a study of the inhibition of nitronate monooxygenase with mono-valent inorganic ions. The dissertation will close with unpublished work on the enzyme and concluding remarks concerning the biochemistry and physiology of nitronate monooxygenase.