Abstract:

The evolution of oxidative metabolism has shaped life on Earth, driving biodiversification from anaerobic microbes to complex aerobic organisms. However, dioxygen is a double-edged sword—essential for life yet a contributor to aging, oxidative stress, and cellular damage. Metalloenzymes, particularly cambialistic enzymes, harness dioxygen by incorporating different metal ions within a conserved protein scaffold, tuning their activity through primary coordination sphere ligands and secondary noncovalent interactions that control substrate binding and selectivity. Despite extensive studies on quercetin dioxygenase (QD)—an enzyme that cleaves the flavonoid quercetin—its mechanistic intermediates remain elusive due to their transient nature, and the identities of the native metal cofactors are still debated. Investigations into metal-substituted QDs have revealed variations in catalytic activity, prompting further exploration of metal-based functional switching. This work presents a series of artificial metalloproteins designed to mimic QD, stabilize reactive intermediates, and elucidate key structure-function relationships through crystallographic, spectroscopic, and thermodynamic studies, offering insights into both natural and engineered oxidation chemistry.