Kinetic solvent viscosity effects reveal a protein isomerization in the reductive half-reaction of Neurospora crassa class II nitronate monooxygenase.

Nitronate monooxygenase from Neurospora crassa (NcNMO) is an FMN-dependent enzyme that oxidizes nitronates. The enzyme belongs to Group H of flavin monooxygenases. Previous biochemical and mechanistic studies on the enzyme showed that NcNMO oxidizes both anionic nitronates and neutral nitroalkanes, a feature distinguishing NcNMO from bacterial and other fungal NMOs which are active exclusively on nitronates. Recently, NMOs have been shown to oxidize propionate 3-nitronate (P3N), a toxic nitro acid present in legumes, fungi, and leaf beetles; P3N is an irreversible inhibitor of succinate dehydrogenase causing anywhere from neurological disorders to death in livestock and humans. In this study, we report the first kinetic investigation of NcNMO with P3N and its conjugated acid 3-nitropropionic acid (3NPA), and a mechanistic investigation with 3NPA using kinetic solvent viscosity effects. The kcat value with P3N (300 s-1) was 7-times larger than with 3NPA and the kcat/KP3N value (1,700,000 M-1s-1) was ~500-times larger than the kcat/K3NPA value, consistent with P3N being a faster and better substrate than 3NPA. The normalized kcat and kcat/K3NPA values showed inverse hyperbolic dependences on the relative solvent viscosity, consistent with an internal isomerization of the enzyme-substrate complex. A similar inverse hyperbolic pattern of the normalized kred value for the rate constant of flavin reduction determined anaerobically in a stopped-flow spectrophotometer suggested that the internal enzyme isomerization occurs before the flavin reduction step in the reductive half-reaction.