Refining the Substrate-Cofactor Disposition Model of Hyoscyamine 6β-Hydroxylase Catalysis Using Hyoscyamine Analogs

Hyoscyamine 6β-hydroxylase (H6H) is a mononuclear nonheme iron and 2-oxoglutrate dependent oxidase responsible for the biosynthesis of the tropane alkaloid scopolamine. H6H is an ideal model system to study the nonheme iron enzymes as it has dual functions catalyzing both C6-hydroxylation of hyoscyamine and epoxidation of the resulting alcohol. Previous crystallographic and spectroscopic studies have led to a model where substrate disposition relative to the intermediary iron complexes determines the reaction outcome. Herein, a number of H6H substrate analogs with cyclopropyl, methylidene, fluoro, methoxy and trifluoromethoxy substituents among others at C6 and C7 are assayed with H6H to determine whether the disposition model accurately predicts the reaction outcome. The results suggest that additional factors, which include H-bonding interactions that stabilize intermediary hydroxy-ferric complexes against rebound, are likely at play in determining the fate of the individual substrate radicals. These effects are both important and expected to correlate with substrate-cofactor disposition.