Oxidative polymerization of ribonuclease A by lignin peroxidase from Phanerochaete chrysosporium - Role of veratryl alcohol in polymer oxidation

The mechanism of lignin peroxidase (LiP) was examined using bovine pancreat ic ribonuclease A (RNase) as a polymeric lignin model substrate. SDS/PAGE a nalysis demonstrates that an RNase dimer is the major product of the LiP-ca talyzed oxidation of this protein. Fluorescence spectroscopy and amino acid analyses indicate that RNase dimer formation is due to the LiP-catalyzed o xidation of Tyr residues to Tyr radicals, followed by intermolecular radica l coupling. The LiP-catalyzed polymerization of RNase is strictly dependent on the presence of veratryl alcohol (VA). In the presence of 100 mu M H2O2 , relatively low concentrations of RNase and VA, together but not individua lly, can protect LiP from H2O2 inactivation. The presence of RNase strongly inhibits VA oxidation to veratraldehyde by LiP; whereas the presence of VA does not inhibit RNase oxidation by LiP. Stopped-flow and rapid-scan spect roscopy demonstrate that the reduction of LIP compound I (LiPI) to the nati ve enzyme by RNase occurs via two single-electron steps. At pH 3.0, the red uction of LiPI by RNase obeys second-order kinetics with a rate constant of 4.7 x 10(4) M-1 . s(-1), compared to the second-order VA oxidation rate co nstant of 3.7 x 10(5) M-1 . s(-1). The reduction of LiP compound II (LiPII) by RNase also follows second-order kinetics with a rate constant of 1.1 x 10(4) M-1 . s(-1), compared to the first-order rate constant for LiPII redu ction by VA. When the reductions of LiPI and LiPII are conducted in the pre sence of both VA and RNase, the rate constants are essentially identical to those obtained with VA alone. These results suggest that VA is oxidized by LiP to its cation radical which, while still in its binding site, oxidizes RNase.