Spectrophotometric activity microassay for pure and recombinant cytochrome P450-type nitric oxide reductase

Abstract

Nitric oxide reductase (NOR) of the P450 oxidoreductase family accepts electrons directly from its cofactor, NADH, to reduce two nitric oxide (NO) molecules to one nitrous oxide molecule and water. The enzyme plays a key role in removal of radical NO produced during respiratory metabolism while applications in bioremediation and biocatalysis have been identified. However, a rapid, accurate and sensitive enzyme assay has not yet been developed for this enzyme family. In this study, we optimised reaction conditions for the development of a spectrophotometric NOR activity micro-assay using NOC-5 for the provision of NO in solution. We also demonstrate that the assay was suitable for the quantification and characterization of P450-type NOR. The Km and kcat kinetic constants obtained by this assay were comparable to the values determined by gas chromatography, however with improved convenience and cost efficiency, effectively by miniaturisation. To our knowledge, this is the first study to present the quantification of NOR activity in a kinetic micro-assay format.