Project Summary - Bioinorganic Chemistry of Nitrogen The long-term goal of the PI’s research program is to understand how biology uses transition metals to control the speciation of redox-active substrates including reactive or “fixed” nitrogen species (RNS). Reactive nitrogen species serve vital roles in biology. For example, nitric oxide (NO) is a cellular signaling agent that regulates vasodilation in mammalian systems. In a separate context, nitrate (NO3–) can substitute for dioxygen (O2) as the terminal electron acceptor during cellular respiration by bacteria that include human pathogens. This proposal describes a continuation of efforts to elucidate mechanisms of the biogeochemical nitrogen cycle via the study of metalloenzymes as well as model complexes that interconvert RNS. A key knowledge gap that will be addressed through proteomics and enzymology concerns the means by which ammonia oxidizing archaea derive chemical energy from the oxidation of hydroxylamine. The operative enzyme and the product of this reaction remain unknown. Activity guided purification and mass spectrometry will furnish the identity of this globally proliferated nitrogen cycle protein for subsequent characterization by spectroscopy, X-ray crystallography, and kinetics. Further work will explore product selectivity in RNS oxidation biochemistry by heme P460 proteins to determine how NO is selected over nitrous oxide (N2O) to differentiate metabolic from detoxification proteins. The PI will continue to collaborate with leading bioinorganic chemists to understand how transition metals prime RNS for oxidation or reduction and how selectivity in these reactions is achieved. These collaborations will leverage the PI’s expertise in X-ray spectroscopic as well as in other inorganic spectroscopies. Key examples of these collaborations involve site-selective spectroscopic probing of metal atoms in the FeMo cofactor of nitrogenase, the means by which multicopper clusters reduce N2O, and studying the electronic structures and reactivities of Lewis-acid stabilized RNS that have been rendered capable of undergoing redox transformations independent of proton transfer.