Substituent effects on the electron transfer reactivity of hydroquinones with laccase blue copper

Abstract

Stopped-flow kinetic studies of the anaerobic reduction of Rhus vernicifera laccase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) type 1 copper by 25 mono- and disubstituted hydroquinones-(H 2Q-X) have been performed at 25°C and pH 7.0 in 0.5 M phosphate. All of the data are compatible with a mechanism involving rapid enzyme-substrate complex formation followed by rate-limiting intra-complex electron transfer. ES complex formation constants ( Q p ) for many substrates are strikingly insensitive to the electronic characteristics of the substituent X, falling within the range 5–50 M −1. It is shown that this result may be accounted for if only the singly ionized forms of the substituted hydroquinones are bound by the enzyme. All of the substrate exhibiting exceptionally high Q p values ( >50 M −1 ) have X groups capable of functioning as ligands; substituents with lone pairs of electrons may facilitate enzyme-substrate complex formation by enabling hydroquinone to function as a bidentate bridging ligand between the type 2 and type 3 copper sites. Intra-complex electron transfer rate constants for most substrates and remarkably insensitive to the thermodynamic driving force for the oxidation of H 2Q-X to the corresponding semiquinone, the average value for ten substrates being 30 ± 10 s −1 . The electron transfer reactivity of polyphenols with laccase blue copper therefore appears to be controlled largely by protein-dependent activation requirements rather than by the oxidizability of the substrate.