Detection and Characterization of the Lignin Peroxidase Compound II−Veratryl Alcohol Cation Radical Complex

Abstract

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 only marginally greater than g parallel. 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 greater than g perpendicular. Spin concentration analysis suggested that the 1 mol 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.

Structure and Action Mechanism of Ligninolytic Enzymes

“…At pH 6.0, the second-order rate constant for LiP-catalyzed oxidation of VA is similar to that of β-O-4 dimer (6.7×10 3 and 6.5×10 3 M −1 s −1 , respectively) [81]. The radical cation formed in the first reduction by LiP-I exists as a complex with LiP-II, which is catalytically active on a second VA molecule to form an aldehyde [91]. The VA .+ generated in the reduction step decays by deprotonation at C a , a typical reaction of alkyaromatic radical cations, to form veratraldehyde (Fig.…”

Mechanism for Oxidation of High-Molecular-Weight Substrates by a Fungal Versatile Peroxidase, MnP2

“…While MnP2 directly oxidized Poly R-478 and RNase A (17), LiP formed enzyme-cation radical complexes with VA to oxidize them (18). It is likely that the different surrounding environments of the redox active center reflect the dependence on or independence from VA in the oxidation of these compounds by the two peroxidases.…”

“…In P. chrysosporium LiP isozyme H8 (PcLiPH8) a point mutational analysis demonstrated that an exposed tryptophan residue (W171) was crucial for VA oxidation (9,37). It was suggested that oxidized VA formed a cation radical complex with the enzyme (18) and that this enzyme-cation radical complex oxidized RNase A (36).…”

Mechanistic studies on the effect of veratryl alcohol on the lignin peroxidase catalyzed oxidation of pyrogallol red in reversed micelles

“…Studies in aqueous solution [9,10,14,[24][25][26][27][28] indicated that addition of VA could greatly enhance the degradation efficiency of aromatic compound catalyzed by LiP. How does VA work in a reversed micellar medium?…”