# O2 Activation at an Enzymatic Diiron Site: Bridging Ligand Substitutions Alter Diferric‐(Hydro)peroxo States

## TL;DR

This paper shows how changing bridging ligands in a diiron enzyme can alter its structure and reactivity with oxygen, enabling the formation of different (hydro)peroxo intermediates.

## Contribution

The study demonstrates that bridging ligand substitutions in sulerythrin can generate all three structural subclasses of diferrous active sites and modulate O2 reactivity.

## Key findings

- Replacing bridging carboxylates in sulerythrin shifts coordination from 1,3- to 1,1-carboxylate, shortening the Fe–Fe distance.
- Modifying carboxylate bridges alters the nature of diferric (hydro)peroxo intermediates formed upon O2 reaction.
- Sulerythrin can be engineered to produce various stable (hydro)peroxo intermediates for further study.

## Abstract

A variety of non‐heme diiron enzymes employ a conserved 2‐His‐4‐carboxylate motif to coordinate a dinuclear Fe site and activate dioxygen for diverse types of reactions. Two of the carboxylate residues act as bridging ligands between the Fe ions. As the type and coordination geometry of the bridging ligands in the diferrous state are thought to modulate reactivity, they were used to group diiron oxygenases into three structural subclasses. Here, we use the small diiron‐enzyme sulerythrin as a model to demonstrate that replacements of the bridging carboxylate amino acids allow us to decrease the distance between the two Fe ions, change the coordination of the bridging ligands from 1,3‐carboxylates to 1,1‐carboxylates and generate all three structural subclasses of diferrous active sites within the same protein scaffold. In addition to the known classes, we generated a coordination mode containing two 1,1‐carboxylate bridges. The resulting changes in the Fe coordination also alter the nature of the diferric (hydro)peroxo intermediates formed upon reaction with O2. Finally, we show that modulating the carboxylate bridges influences the reactivity of sulerythrin with O2. We establish sulerythrin as a versatile platform to engineer distinct diFe centers by a few exchanges, producing various stable (hydro)peroxo intermediates for further studies.

Substituting the bridging carboxylates in diFe sulerythrin shifts the bridging ligand coordination from 1,3‐carboxylate to 1,1‐carboxylate, yielding all three diferrous bridging motifs in one protein. This shortens the Fe–Fe distance and increases reactivity with O2. O2 exposure triggers Fe rearrangements and traps two distinct diferric (hydro)peroxo intermediates.