Oxidation of Ferrocytochrome c by Lignin Peroxidase

We demonstrate direct oxidation of ferrocytochrome c by lignin peroxidase (LiP) from the lignin-degrading basidiomycete, Phanerochaete chrysosporium. Steady-state kinetic data fit a peroxidase ping-pong mechanism rather than an ordered bi-bi ping-pong mechanism, suggesting that the reductions of LiP compounds I and II by ferrocytochrome c are irreversible. The pH dependence of the overall reaction apparently is controlled by two factors, the pH dependence of the electron-transfer rate and the pH dependence of enzyme inactivation in the presence of H₂O₂. In the presence of 100 μM H₂O₂, veratryl alcohol (VA) significantly enhanced cytochrome c oxidation at pH 3.0 but had little effect above pH 4.5. In the presence of <10 μM H₂O₂, the stimulating effect of VA on the reaction is greatly diminished. As with cytochrome c peroxidase reactions, LiP oxidation of ferrocytochrome c decreased as the ionic strength increased, implying the involvement of electrostatic interactions between the polymeric substrate and enzyme. The reaction product ferricytochrome c inhibited VA oxidation by LiP in a noncompetitive manner, suggesting that cytochrome c binds to LiP at a site different from the small aromatic substrate binding site. Recent crystallographic studies show that the heme is buried in the LiP protein and unavailable for direct interaction with polymeric substrates, suggesting that electron transfer from ferrocytochrome c to LiP occurs over a relatively long range. The role of VA in this electron-transfer reaction is discussed. This observation of ferrocytochrome c oxidation by LiP, which is stimulated at low pH and high [H₂O₂] by VA, suggests that this system may serve as a useful model for probing the mechanism of electron transfer between the buried heme of the enzyme and other polymeric substrates, such as lignin.