Interaction between the Type-3 Copper Protein Tyrosinase and the Substrate Analogue p-Nitrophenol Studied by NMR

The interaction of the monooxygenating type-3 copper enzyme Tyrosinase (Ty) from Streptomyces antibioticus with its inhibitor p-nitrophenol (pnp) was studied by paramagnetic NMR methods. The pnp binds to oxidized Ty (Tymet) and its halide (F-, Cl-) bound derivatives with a dissociation constant in the mM range. The Cu2 bridging halide ion is not displaced upon the binding of pnp showing that the pnp does not occupy the Cu2 bridging position. The binding of pnp to Tymet or TymetCl leads to localized changes in the type-3 (Cu−His3)2 coordination geometry reflecting a change in the coordination of a single His residue that, still, remains coordinated to Cu. The binding of pnp to TymetCl causes a decrease in the Cu2 magnetic exchange parameter −2J from 200 cm-1 in the absence to 150 ± 10 cm-1 in the presence of pnp. From the 1H and 2D NMR relaxation parameters of pnp bound to Tymet, a structural model of pnp coordination to the Ty type-3 center could be derived. The model explains the absence of hydroxylase activity in the closely related type-3 copper protein catechol oxidase. The relevance of the experimental findings toward the Ty catalytic mechanism is discussed.