Oxidation of anionic nitroalkanes by flavoenzymes, and participation of superoxide anion in the catalysis.

The reactivities of anionic nitroalkanes with 2-nitropropane dioxygenase of Hansenula mrakii, glucose oxidase of Aspergillus niger, and mammalian D-amino acid oxidase have been compared kinetically. 2-Nitropropane dioxygenase is 1200 and 4800 times more active with anionic 2-nitropropane than D-amino acid oxidase and glucose oxidase, respectively. The apparent Km values for anionic 2-nitropropane are as follows: 2-nitropropane dioxygenase, 1.61 mM; glucose oxidase, 16.7 mM; and D-amino acid oxidase, 11.1 mM. Anionic 2-nitropropane undergoes an oxygenase reaction with 2-nitropropane dioxygenase and glucose oxidase, and an oxidase reaction with D-amino acid oxidase. In contrast, anionic nitroethane is oxidized through an oxygenase reaction by 2-nitropropane dioxygenase, and through an oxidase reaction by glucose oxidase. All nitroalkane oxidations by these three flavoenzymes are inhibited by Cu and Zn-superoxide dismutase of bovine blood, Mn-superoxide dismutases of bacilli, Fe-superoxide dismutase of Serratia marcescens, and other O2-. scavengers such as cytochrome c and NADH, but are not affected by hydroxyl radical scavengers such as mannitol. None of the O2-. scavengers tested affected the inherent substrate oxidation by glucose oxidase and D-amino acid oxidase. Furthermore, the generation of O2-. in the oxidation of anionic 2-nitropropane by 2-nitropropane dioxygenase was revealed by ESR spectroscopy. The ESR spectrum of anionic 2-nitropropane plus 2-nitropropane dioxygenase shows signals at g1 = 2.007 and g11 = 2.051, which are characteristic of O2-.. The O2-. generated is a catalytically essential intermediate in the oxidation of anionic nitroalkanes by the enzymes.