# Mechanisms of Mononuclear non-Heme-Iron Enzymes

> **NIH NIH R35** · PENNSYLVANIA STATE UNIVERSITY, THE · 2022 · $492,537

## Abstract

Project Summary/Abstract
Enzymes that utilize iron-containing cofactors for their activity catalyze a bewildering array of (often very
difficult) chemical reactions that are fundamentally important to central life processes (e.g., DNA biosynthesis
and repair, gene regulation, regulation of epigenetic inheritance, biosyntheses of a plethora of compounds with
antibacterial and antifungal activities). Dysfunction of these enzymes is often associated with the onset of
severe diseases, e.g. cancer, cardiovascular diseases, and diabetes. Strategies to harness the synthetic
potential of these enzymes and to combat diseases associated with their dysfunction involves the rational
manipulation of these processes on a molecular level. A prerequisite for this endeavor is a detailed knowledge
of the underlying reaction mechanisms, in particular how the enzymes control the outcome of their reactions.
The Bollinger/Krebs joint group specializes in combining transient-state rapid kinetic experiments with various
spectroscopic (e.g. stopped-flow absorption, freeze-quench EPR and Mössbauer) and analytical (LC/MS)
methods to monitor metalloenzyme reactions. In the last 15 years, their group has successfully studied many
enzymes that require a mononuclear or a dinuclear non-heme-iron cofactor for activity by trapping and
characterizing key reaction intermediates in their catalytic cycles. In particular, they identified high-spin Fe(IV)-
oxo (ferryl) intermediates in several mononuclear non-heme-iron enzymes, mostly Fe(II)- and 2-oxo-glutarate-
dependent (Fe/2OG) enzymes. The ferryl intermediate initiates substrate oxidation, typically by cleavage of an
aliphatic C-H bond. The outcome of these reactions is diverse and includes hydroxylation (the default
outcome), halogenation, desaturation, epimerization, and heterocyclization reactions. Many of these reactions
are employed in the biosyntheses of medically important natural products. The current focus of research in the
Bollinger/Krebs group aims at deciphering the factors that result in the diverse outcomes. The long-term goal of
this research is to lay the foundation for the rational manipulation of these enzymes for biotechnological
applications. The PI also has a long-standing collaboration with Squire Booker on mechanistic studies of Fe/S
enzymes, in particular those that belong to the superfamily of radical S-adenosylmethionine (RS) enzymes.
These enzymes use a [4Fe-4S] cluster to generate a canonical 5’-deoxy-adenos-5’-yl radical that initiates a
wide variety of substrate oxidations, often by cleavage of an aliphatic C-H bond. The current focus of the
collaborative research efforts on RS enzymes aim at delineating the reaction mechanisms of different reaction
outcomes, viz sulfur insertion, methylation, methylthiolation, and desaturation.

## Key facts

- **NIH application ID:** 10394263
- **Project number:** 5R35GM127079-05
- **Recipient organization:** PENNSYLVANIA STATE UNIVERSITY, THE
- **Principal Investigator:** CARSTEN KREBS
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $492,537
- **Award type:** 5
- **Project period:** 2018-05-01 → 2023-09-04

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10394263

## Citation

> US National Institutes of Health, RePORTER application 10394263, Mechanisms of Mononuclear non-Heme-Iron Enzymes (5R35GM127079-05). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10394263. Licensed CC0.

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