Quantitative Analysis of Flavonoid Binding to Tyrosinase by Quartz Crystal Microbalance and Molecular Dynamics

Tyrosinase is a copper-containing oxidase that catalyzes the rate-limiting steps of melanogenesis and represents an important therapeutic target for the treatment of hyperpigmentation and other melanin-related disorders. Flavonoids are naturally occurring polyphenols that inhibit tyrosinase, yet the molecular basis of their binding remains incompletely understood. In this study, quartz crystal microbalance with dissipation monitoring (QCM-D) was used to quantify the binding kinetics of the water-soluble flavonoids (-)-epicatechin, taxifolin, and rutin toward immobilized tyrosinase. Analysis of the association and dissociation kinetics yielded equilibrium dissociation constants (KD) of the tested flavonoids, and revealed that epicatechin has approximately 4-fold weaker affinity for tyrosinase than taxifolin and rutin. To elucidate the structural origins of these experimentally determined binding affinities, all-atom molecular dynamics simulations were performed to examine flavonoid binding modes, Cu(II) coordination, and protein-ligand interactions within the tyrosinase active site. The simulations reveal that flavonoid binding is governed by proximity to the catalytic Cu(II) center together with stabilizing hydrogen-bonding interactions and that conformational preorganization of the flavonoid scaffold plays a key role in determining binding affinity. Relatively, the weaker affinity of epicatechin arises from reduced structural preorganization of the flavonoid scaffold, resulting in less persistent interactions within the active site. Together, the experimental and computational results provide mechanistic insight into flavonoid recognition by tyrosinase and establish a quantitative framework for evaluating and designing improved tyrosinase inhibitors.