# Spectroscopic Characterization of Oxygen Intermediates in Non-heme and Heme Iron Enzymes

> **NIH NIH R35** · STANFORD UNIVERSITY · 2022 · $451,685

## Abstract

Project summary
Iron enzymes play major roles in O2 activation in biology. These divide into four classes based on
their active site structures that reflect their mode of O2 activation: the non-cofactor dependent
mononuclear non-heme iron (MNHFe) enzymes, the cofactor (α-ketoglutarate (α-KG) and pterin)
dependent MNHFe enzymes, the binuclear NHFe enzymes and the O2/H2O2 activating heme
enzymes. Crystal structures and oxygen reaction intermediates exist for metalloenzymes in all
four classes. Over the years, we have developed new spectroscopic methods enabling the
detailed study of the NHFeII active sites, and the geometric and electronic structures of their O2
intermediates and have now developed a method to quantitatively study the iron center in the
highly covalent and chromophoric heme environment. Among our accomplishments in the past 5
years are: 1) determined that FeIII-O2- species are the reactive intermediates in all the subclasses
of non-cofactor dependent MNHFe enzymes; 2) defined the O2 reaction coordinates to generate
the FeIV=O intermediates in both the α-KG and pterin dependent enzymes; 3) for the α-KG
dependent subclass, defined the geometric and electronic structures of their FeIV=O enzyme
intermediates and how these direct halogenation over the thermodynamically favored
hydroxylation in the halogenases; 4) showed that in contrast to the MNHFe enzymes,
hydroperoxide intermediates are active in the binuclear NHFe enzymes for direct reaction with
substrates; 5) for methane monooxygenase, where the peroxo-biferric intermediate rapidly
converts to a high-valent 2FeIV-oxo intermediate Q, we have determined the structure of Q (a
topic of current debate) and provided insight into its high reactivity with methane; 6) used the
spectroscopic method we have now developed for iron in heme environments to determine
experimentally the computationally controversial electronic structure of oxyhemoglobin; 7) and
extended this method to analyze active sites with strong Fe-oxo bonds. Our studies are now
directed toward completing the reaction coordinates of the four classes and their subclasses,
understanding O2 activation at the superoxo, peroxo and FeIV-oxo levels, determining the role of
the second iron in the enhanced reactivity of the binuclear NHFe enzymes, and understanding
the differences in the activation and selectivity of high-valent iron-oxo intermediates in
mononuclear NH, binuclear NH and heme enzyme active sites.

## Key facts

- **NIH application ID:** 10396809
- **Project number:** 1R35GM145202-01
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** EDWARD I SOLOMON
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $451,685
- **Award type:** 1
- **Project period:** 2022-05-01 → 2027-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10396809

## Citation

> US National Institutes of Health, RePORTER application 10396809, Spectroscopic Characterization of Oxygen Intermediates in Non-heme and Heme Iron Enzymes (1R35GM145202-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10396809. Licensed CC0.

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