Galactose oxidase (GAO) is a mononuclear copper enzyme that catalyzes the stereospecific oxidation of primary alcohols to their corresponding aldehydes. A distinctive feature of GAO is its radical-containing Cys228-Tyr272 radical cofactor, covalently linked and coordinated to the Cu(II) center.1 The exceptional longevity of this radical defies the conventional expectation that radical species are transient and highly reactive, making GAO a fascinating system for studying radical stabilization in biology. Although the catalytic mechanism of GAO has been extensively characterized, the molecular basis for the remarkable stability of its metal-bound radical cofactor remains unclear. Prior mutagenesis studies have implicated several residues, including Tyr272, Tyr495, Trp290, and Tyr405, in stabilizing the radical species; however, traditional site-directed mutagenesis replaces the entire residue, often complicating interpretation. To address this limitation, we employ genetic code expansion to introduce noncanonical amino acids with tailored electronic properties at key positions. We previously demonstrated that the substitution of Tyr272 with fluorinated and chlorinated analogs modulates C–H, C–F, and C–Cl bond cleavages, providing insight into radical tuning without loss of activity.2 Building on this work, we now substitute Tyr495 with F₂-Tyr, Cl₂-Tyr, and NH₂-Tyr to probe its charge-transfer interactions with the Cys228-Tyr272-Cu(II) center. Our results illuminate the electronic factors underlying the “permanent” yet selectively reactive nature of this cofactor, offering new perspectives on radical stabilization in metalloenzymes.
Acknowledgment
This work is supported by the NSF award CHE-2204225.