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

> **NIH NIH R01** · STANFORD UNIVERSITY · 2021 · $344,096

## Abstract

Project Summary
Iron active sites play key roles in O2 activation in biology. These are present in mononuclear non-heme Fe
(MNHFe), binuclear non-heme Fe (BNHFe) and heme enzymes, which further divide into different subclasses
(vide infra). We have developed a range of spectroscopic methods that enable the detailed study of the geometric
and electronic structures of the FeII active sites and their oxygen intermediates, the coupling of electronic
structure calculations to these spectroscopic data and the use of calculations supported by experiment in
evaluating reaction coordinates. From our recent progress, formation of an FeIIIO2•- intermediate in the sulfur
oxidizing/oxygenating enzymes (SOx) is stabilized by the substrate sulfur coordination, while for the Rieske
dioxygenases (RDO) generation of this intermediate is uphill but its electrophilic attack is driven by a subsequent
exergonic proton coupled electron transfer (PCET) from the Rieske center. The extradiol dioxygenases (EDO)
also utilize an FeIIIO2•- reactive intermediate in electrophilic attack on a coordinated catecholate substrate. We
now propose to complete the reaction coordinate for the EDOs and determine why these enzymes are selective
in extradiol insertion and cleavage while the intradiol dioxygenases (IDO), which have a ferric active site, are
selective for intradiol cleavage. For both the RDOs and EDOs, an FeIII(OOH) can also be formed but is relatively
unreactive, while in a number of the BNHFe enzymes, peroxide intermediates are formed that are reactive in
electrophilic chemistry. Our focus here is to understand the additional contributions of the second Fe in binuclear
ferric centers that activate peroxide reactivity. For the a-ketoglutarate (aKG) and pterin dependent hydroxylases
(PDH), an FeIV=O intermediate is formed and proceeds to react with substrate. Our research here focuses on
the FeII/O2 reaction mechanisms for the generation of these FeIV=O species and how they enable selective
hydroxylation, desaturation and electrophilic aromatic substitution (EAS). In the BNHFe enzyme soluble methane
monooxygenase (sMMO), the peroxo biferric intermediate goes on to form a high valent Fe2IVO2 intermediate Q
that H-atom abstracts (HAA) from the strong C-H bond of methane. Our focuses here are on determining why
only this binuclear peroxo goes on to form a high valent intermediate, defining the geometric and electronic
structure of Q and understanding the enhanced reactivity of this Fe2IVO2 intermediate relative to the MNHFeIV=O
intermediates. An FeIV=O intermediate is also formed in the heme enzymes both with and without porphyrin
oxidation and is also more reactive in the case of P450 than FeIV=O species in MNHFe sites. Our studies here
are directed toward spectroscopically determining the effects of the porphyrin, its oxidation and of variation of
the trans axial ligand on the FeIV=O bond; all topics thus far mostly addressed through calculations. Our studies
should significa...

## Key facts

- **NIH application ID:** 10146405
- **Project number:** 5R01GM040392-34
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** EDWARD I SOLOMON
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $344,096
- **Award type:** 5
- **Project period:** 1988-07-01 → 2023-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10146405

## Citation

> US National Institutes of Health, RePORTER application 10146405, Spectroscopic Characterization of Oxygen Intermediates in Non-heme and Heme Iron Enzymes (5R01GM040392-34). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10146405. Licensed CC0.

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