Post-Fixation Nitrogen Cycle Metalloenzymology 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. 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. Of the biochemical pathways that generate these and other reactive nitrogen species, those involving oxidations of nitrogenous substrates are largely uncharacterized at the molecular level. This proposal describes an interdisciplinary research program leveraging molecular biology, biochemistry, inorganic spectroscopy, and quantum chemical calculations to understand in precise detail the mechanisms used by metalloenzymes operative in nitrification––biological ammonia (NH3) and nitrite (NO2–) oxidation. Despite the global scale on which this biochemistry operates and the impacts of nitrification products on the environment and on human health, detailed mechanisms for the operative metalloenzymes are unavailable. Early work from the PI has afforded a revised mechanism used by the nitrification enzyme cytochrome P460 for the step-wise, selective oxidation of hydroxylamine (NH2OH), an intermediate in NH3 oxidation. A key iron-nitrosyl (FeNO) intermediate has since been identified that gates catalysis via axial ligand dissociation. The structural and electronic factors that dictate the conversion between catalytically competent and incompetent forms will be explored to gain insight into similar mechanisms operative in NO-mediated cellular signaling. These studies will directly probe metal-NO bonding using resonance Raman, electron paramagnetic resonance, and X-ray spectroscopy. Work on biological NH2OH oxidation will be extended to previously reported but largely uncharacterized non-heme Fe hydroxylamine oxidases. Understanding of NH2OH-oxidation mechanisms will elucidate means by which nature mediates the intermediacy of a toxic metabolite during cellular energy transduction. A full suite of biophysical characterization will be carried out including X-ray crystallography and Fe-focused spectroscopy. Finally, the mechanism of NO2– oxidation by the integral membrane metalloenzymes nitrite oxidoreductase (NXR) will be studied. No molecular structure is available for NXR, and its complement of metallocofactors is undefined. NXR is thought to mediate multi-electron transfer using multiple iron-sulfur clusters. Spectroelectrochemical probing of NXR will be carried out to afford new understanding of how nature controls multi-step electron flow using chains of metallocofactors. This program proposal offers progress in a number of NIGMS mission areas including the development of s...