# Heme/Copper and Heme/Nonheme Iron O(2) and NO Reactivity

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2020 · $322,409

## Abstract

Project Summary
The long-term research objective is to design, synthesize and investigate model compound systems which can
help elucidate fundamental aspects of structure, metal-ligation, spectroscopy and reactivity relevant to the
dioxygen (O2) and nitrogen oxide (NOx) chemistry which occurs in heme-copper oxidases (e.g., CcOs), nitric
oxide reductases (NORs) and related proteins. HCOs and NORs are evolutionarily related enzymes which play
critical roles in cellular processes within aerobic and anaerobic organisms. They have a heme/M (M = Cu or
non-heme Fe) active site that reductively cleaves O2 or couples NO, respectively. The research proposed will
contribute to a better understanding of enzyme structure and mechanism by providing a comprehensive and
fundamental basis relevant to biological processing of O2 and NO that extends even beyond the heme and Cu
metalloprotein sphere. Specific aims include: (A) the study of O–O cleavage chemistry in low-spin heme-(μ-1-
2)-peroxo-Cu complexes by addition of appropriate H+/e– sources, including phenols or derivatives. The large
degree of synthetic variability in porphyrins and copper ligands, along with substrates of varying pKa's and/or
E0, will be utilized in order to meticulously study the factors which lead to successful O2-activation and reductive
cleavage (vs. other pathways) in the context of structure-function relationships of CcO & model systems. (B)
the generation of new heme-peroxo-Cu complexes (with incorporated biomimetic or H-bonding moieties) and
detailed characterization of their structural and electronic properties. (C) the elaboration of two chemical
systems designed to test how CcO (bio)chemistry leads to the actual formation of the copper-ligand His-Tyr
crosslink. Detailed studies will involve new new ligand scaffolds bearing open imidazole N-H sites poised for
covalent coupling to an exogenous phenolic substrate. An oxidative element will be included, such as the
presence of a peroxo group or high-valent iron-oxo species. (D) investigation of chemical systems with input
variations of the heme axial `base' ligand, where heme/NO/O2 coordination chemistry will be studied
mechanistically with regard to peroxynitrite formation and its subsequent decay or substrate reactivity. This
chemistry occurs in NO dioxygenases, enzymes critically involved in cellular NO homeostasis (and cellular
signaling) via amino-acid nitration chemistry. (E) the study of chemistry relevant to NORs, heme/Cu or
heme/non-heme Fe assemblies that enable NO reductive coupling. A clear focus will be on the study of the
formation of putative hyponitrite intermediates, their structures and their reactivity leading to N2O as product.
Such information is key to the understanding of N-N coupling and N-O cleavage chemistries which also involve
protonation events. These processes are of broad interest with respect to other biological metalloenzyme
nitrogen oxide processing. In-hand heme/Cu assemblies also enable this chemis...

## Key facts

- **NIH application ID:** 9980910
- **Project number:** 5R01GM060353-20
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** KENNETH D. KARLIN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $322,409
- **Award type:** 5
- **Project period:** 2000-04-01 → 2022-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9980910

## Citation

> US National Institutes of Health, RePORTER application 9980910, Heme/Copper and Heme/Nonheme Iron O(2) and NO Reactivity (5R01GM060353-20). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9980910. Licensed CC0.

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