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 the removal of radical NO produced during respiratory metabolism, and 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 optimized reaction
conditions for the development of a spectrophotometric NOR activity microassay using NOC-5 for the
provision of NO in solution. We also demonstrate that the assay is 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, but with improved convenience and cost efficiency,
effectively by miniaturization. To our knowledge, this is the first study to present the
quantification of NOR activity in a kinetic microassay format.
