Nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to aldehydes with production of nitrite and hydrogen peroxide. The enzyme has a molecular weight of 47 955 +/- 39, as determined by MALDI-TOF mass spectrometry; under nondenaturing conditions, the aggregation state of the enzyme is best described by a tetramer-dimer self-associating model, with an association constant of (8.5 +/- 4.4) x 10(6) M-1 (pH 7.0 and 4 degreesC). The amino acid composition and the N-terminal amino acid sequence do not match any known protein or open reading frame. The inactive 5-nitrobutyl-1,5-dihydroflavin found in the enzyme as purified was converted to FAD, allowing characterization of the active FAD-containing enzyme. With nitroethane as substrate, the Vmax and Km values are 655 +/- 45 min-1 and 2.9 +/- 0.5 mM at pH 8.0 and 30 degreesC, respectively. One mole of FAD per mole of monomer enzyme is required for catalysis. No activity can be detected with amino acids or alpha-hydroxy acids as substrates. Reversible removal of the FAD cofactor yields inactive enzyme. The properties of the FAD cofactor in nitroalkane oxidase are within the range described for other oxidases. The UV-visible absorbance spectrum of the active enzyme shows maxima at 446, 384, and 274 nm; the extinction coefficient at 446 nm is 11.7 mM-1 cm-1. The neutral form of the flavin semiquinone, with maxima at 536 and 342 nm, is kinetically stabilized. The UV-visible absorbance spectrum of the reduced enzyme is typical of the anionic form of a flavin, with a peak centered at 335 nm. The affinity of the enzyme for sulfite is low (Kd value of 13.8 +/- 0.9 mM at pH 7.0 and 25 degreesC); this result, along with the stabilization of the neutral flavin semiquinone, suggests the presence of a weak positive charge near the N(1)-C(2)=O of FAD. The reduction potential of the enzyme is -367 mV. Benzoate and phenylacetic acid are competitive inhibitors, with Kis values of 5.1 +/- 0.6 and 13.1 +/- 2.3 mM, respectively. Binding of benzoate to nitroalkane oxidase results in spectral changes similar to those observed with d-amino acid oxidase. The absorbance spectrum of the flavin bound to nitroalkane oxidase is pH-dependent, with a pKa value of 8.4.