The focus of this proposal is to expand the biocatalytic use of cytochrome P450 monooxygenase variants to include nitration and denitration of aromatic substrates, which to date has not been reported. Specifically, P450-BM3 (BM3) will be used in this study since it has been re-engineered to bind and transform a variety of abiotic substrates. The specific aims for this proposal are to: (1) Develop and evolve existing BM3 variants to utilize reactive nitrogen species to nitrate phenol, (2) Optimize the regioselectivity, product distribution, and substrate scope of BM3 mediated nitration, (3) Develop and evolve existing BM3 variants to function as a denitrase on 2-nitrophenol, and (4) Expand the substrate and reaction scope of denitration mediated by BM3 variants. Initially, existing BM3 libraries in the Arnold will be screened for nitration and denitration chemistries, and high throughput colorimetric screens have been designed to rapidly assay new enzyme activity. Additional mutant enzyme panels for the initial screening will be generated using site directed mutagenesis of key residues followed by directed evolution. Subsequent characterization of the resulting products and biochemical properties of the optimized variants will be used to prepare second-generation enzymes. Finally, the substrate scope for both nitration and denitration reactions will be expanded to small molecules (e.g. explosives, herbicides, fluorophores), pharmaceuticals, and biomolecules bearing the nitro functional group. If necessary, BM3 enzymes can be subjected to further rounds of directed evolution to optimize substrate binding and catalytic activity. Successful accomplishment of these aims will provide a fundamental insight into the use of P450 for new chemistries and will result in enzymatic tools that can be used for synthetic applications, environmental remediation, and diagnostics.