DETECTION AND CHARACTERIZATION OF THE LIGNIN PEROXIDASE COMPOUND-II -VERATRYL ALCOHOL CATION-RADICAL COMPLEX

Lignin peroxidases (LiP) from the white-rot fungus Phanerochaete chrys osporium oxidize veratryl alcohol (VA) by two electrons to veratryl al dehyde, although the VA cation radical (VA(.+)) is an intermediate [Kh indaria, A., et al. (1995) Biochemistry 34, 6020-6025]. It was specula ted, on the basis of kinetic evidence, that VA(.+) can form a catalyti c complex with Lip compound II. We have used low-temperature EPR to pr ovide direct evidence for the formation of the complex. The EPR spectr um of VA(.+) obtained at 4 K was explained by a model for coupling bet ween the oxoferryl moiety of the heme (S = 1) and VA(.+) (S = 1/2) sim ilar to the model proposed for an oxyferryl and a porphyrin pi cation radical of horseradish peroxidase. The coupling constant suggested tha t VA(.+) was equally ferro-and antiferromagnetically coupled to the ox oferryl moiety. The spectrum was simulated with g(perpendicular to) on ly marginally greater than g(parallel to). This was surprising since t he only other known organic radical coupled to the heme iron in a pero xidase is the tryptophan cation radical in cytochrome c peroxidase whi ch exhibits a g tensor with g(parallel to) greater than g(perpendicula r to). Spin concentration analysis suggested that the 1 mel of VA(.+) was coupled to the oxoferryl moiety per mole of enzyme. The VA(.+) sig nal decayed with a first-order decay constant of 1.76 s(-1), in close agreement with the earlier published decay constant of 1.85 s(-1) from room-temperature EPR studies. The exchange coupling between VA(.+) an d the oxoferryl moiety strongly advocates calling this species (VA(.+) and LiP compound II) a catalytic complex.