Detection and characterization of the lignin peroxidase compound II - Veratryl alcohol cation radical complex

Lignin peroxidases (LiP) from the white-rot fungus Phanerochaete chrysosporium oxidize veratryl alcohol (VA) by two electrons to veratryl aldehyde, although the VA cation radical (VA(.+)) is an intermediate [Khindaria, A., et al. (1995) Biochemistry 34, 6020-6025]. It was speculated, on the basis of kinetic evidence, that VA(.+) can form a catalytic complex with Lip compound II. We have used low-temperature EPR to provide direct evidence for the formation of the complex. The EPR spectrum of VA(.+) obtained at 4 K was explained by a model for coupling between the oxoferryl moiety of the heme (S = 1) and VA(.+) (S = 1/2) similar to the model proposed for an oxyferryl and a porphyrin pi cation radical of horseradish peroxidase. The coupling constant suggested that VA(.+) was equally ferro-and antiferromagnetically coupled to the oxoferryl moiety. The spectrum was simulated with g(perpendicular to) only marginally greater than g(parallel to). This was surprising since the only other known organic radical coupled to the heme iron in a peroxidase is the tryptophan cation radical in cytochrome c peroxidase which exhibits a g tensor with g(parallel to) greater than g(perpendicular to). Spin concentration analysis suggested that the 1 mel of VA(.+) was coupled to the oxoferryl moiety per mole of enzyme. The VA(.+) signal 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(.+) and the oxoferryl moiety strongly advocates calling this species (VA(.+) and LiP compound II) a catalytic complex.