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

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 (H2Q-X) have been performed at 25 degrees 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 (Qp) 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 substrates exhibiting exceptionally high Qp values (greater than 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 are remarkably insensitive to the thermodynamic driving force for the oxidation of H2Q-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.