# Post-Fixation Nitrogen Cycle Metalloenzymology

> **NIH NIH R35** · CORNELL UNIVERSITY · 2020 · $332,431

## Abstract

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...

## Key facts

- **NIH application ID:** 9995516
- **Project number:** 5R35GM124908-04
- **Recipient organization:** CORNELL UNIVERSITY
- **Principal Investigator:** Kyle M Lancaster
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $332,431
- **Award type:** 5
- **Project period:** 2017-09-15 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9995516

## Citation

> US National Institutes of Health, RePORTER application 9995516, Post-Fixation Nitrogen Cycle Metalloenzymology (5R35GM124908-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9995516. Licensed CC0.

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