Project Summary/Abstract: The proposed research will study the mechanisms and structure-function relationships of novel nitric oxide (NO)-dependent enzymatic activities with relevance to protein nitration, natural product biosynthesis, and nitrosative stress protection in pathogenic bacteria. Exposure of cells to NO results in cell damage including nitration of protein residues such as tyrosine (Tyr) and tryptophan (Trp). Protein nitration can modulate protein function and are biomarkers of some neurodegenerative diseases. Some protein nitration can be catalyzed by metal ions. The mechanisms of metal-mediated nitration pathways are unclear; therefore, Research Direction 1 is to study the mechanisms of metal-catalyzed protein nitration. To better understand these mechanisms, we will study the nitration by cytochromes P450 (CYPs), including that of the enzyme TxtE, a CYP homolog that catalyzes the regiospecific and NO-dependent nitration of Trp to 4-nitrotryptophan (4-NO2-Trp). The observed regiospecificity suggests that diffusible RNS are not produced on pathway. Furthermore, CYP metal-oxo intermediates have been well characterized. Therefore, studies of TxtE have great potential to provide clear mechanistic data on nitration. One challenge is that TxtE nitration intermediates are elusive. Therefore, the 5-year goal of Research Direction 1 is to determine the outer sphere coordination features of TxtE needed to promote substrate nitration and avoid substrate hydroxylation, the latter being canonical CYP activity. Identifying outer sphere interactions that influence TxtE reactivity will provide structure-function insight to devise strategies to trap nitration intermediates. To avoid cell damage from NO, pathogenic Mycobacteria express enzymes to scavenge NO. Research Direction 2 will study activities and mechanisms of metalloenzymes involved in Mycobacterial virulence, including hemerythrin-like proteins (HLPs) found in pathogenic Mycobacteria, including M. tuberculosis. Our preliminary data on HLPs shows that its diferric oxidation state can oxidize NO to nitrite (NO2–) by reductive nitrosylation. Such reactivity is well known for heme enzymes but has never been reported for a non-heme protein. In the presence of O2, HLP reacts with NO to form nitrate (NO3–) and a metalloproduct with a 350-nm absorbance feature. This metalloproduct can be independently generated by reacting the as isolated HLP with peroxynitrite (ONOO–). The novel reactivities of HLP with RNS suggest a role for it in nitrosative stress protection. An uncommon Tyr ligand bound to the HLP non-heme diiron site may facilitate these unusual reactivities. The 5-year goals of Research Direction 2 are to trap and characterize intermediates of RNS reactions with HLP, determine the catalytic activity of HLP, and identify the role of the Tyr ligand. The vision of our program is to discover novel nitrogen biochemistries related to natural product biosynthesis and human health and to pursue en...