# Mechanistic Intermediates in Copper Oxygenases and Oxidases

> **NIH NIH R01** · STANFORD UNIVERSITY · 2024 · $507,594

## Abstract

Mechanistic intermediates in copper oxygenases and oxidases:
Copper oxygenases and oxidases have a wide range of functions including melanin and siderophore
biosynthesis, neurotransmitter regulation, iron metabolism, and proton pumping for ATP synthesis.
Understanding their reaction mechanisms is both of fundamental importance and has significant downstream
applications in health, biotechnology, and catalysis. There has been much mechanistic speculation based on
structures, model complexes, and calculations. However, the key to determine the enzyme mechanisms and
thus utilize and control their reactions is by trapping catalytic intermediates and defining their structures and
reactivities. Our research combines enzyme kinetics to trap intermediates, a range of spectroscopies to define
these intermediates, and electronic structure calculations correlated to experiments to elucidate reaction
mechanisms. Our focus is on the three classes of copper oxygenases and oxidases. The antiferromagnetically
“coupled” binuclear Cu enzymes include catechol oxidases (CaOx), tyrosinases (Ty), and o-aminophenol
oxidases (AOx) that all utilize the same µ-η2-η2 CuII2O22− intermediate to perform their functions. In Progress,
trapping the ternary intermediate of OxyTy with phenol substrate bound has defined the Ty monooxygenase
mechanism, and our studies are now directed toward determining the mechanisms of the CatOx’s and the AOx’s
and how these enzymes are tuned for their selectivity. The “non-coupled” binuclear Cu enzymes include
dopamine β-monooxygenase that converts dopamine to norepinephrine, the insect homolog tyramine β-
monooxygenase, and peptidylglycine ⍺-hydroxylating monooxygenase. In this class, the two Cu’s are separated
by 11Å resulting in the lack of magnetic coupling. A major point to address is whether co-substrate binding
induces a conformational change to bring the Cu’s together for coupled binuclear O2 activation or if the 11Å
structure is active. If the latter, important issues will be explored including whether O2 activation by a single Cu
(a CuII–O2− intermediate) is able to perform H-atom abstraction and the timing of electron transfer from the 11Å
Cu required to complete the reaction and avoid reactive oxygen species (ROS). The third class is the multicopper
(MCOs) and heme–copper oxidases (HCOs) that reduce O2 to H2O, using different active site structures for
different functions. The MCOs use a trinuclear Cu cluster for efficient oxidation of substrates, while the HCOs
use their binuclear site to pump protons across a membrane for ATP synthesis. Studies on the MCOs are far
along in defining their mechanism of O2 reduction, their role in Fe metabolism with control of ROS, and coupling
to electrodes for fuel cell applications. For the HCOs, intermediates are available that would elucidate the O–O
bond cleavage mechanism and the active site structural changes that enable proton pumping. However, the
dominant spectral features of the hemes have ...

## Key facts

- **NIH application ID:** 10977593
- **Project number:** 2R01DK031450-44A1
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** EDWARD I SOLOMON
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $507,594
- **Award type:** 2
- **Project period:** 1982-01-01 → 2028-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10977593

## Citation

> US National Institutes of Health, RePORTER application 10977593, Mechanistic Intermediates in Copper Oxygenases and Oxidases (2R01DK031450-44A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10977593. Licensed CC0.

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