Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium

Binding properties of lignin peroxidase (LiP) from the basidiomycete Phaner ochaete chrysosporium against a synthetic lignin (dehydrogenated polymeriza te, DHP) were studied with a resonant mirror biosensor. Among several ligni nolytic enzymes, only LiP specifically binds to DHP. Kinetic analysis revea led that the binding was reversible, and that the dissociation equilibrium constant was 330 mu M. The LiP-DHP interaction was controlled by the ioniza tion group with a pK(a) of 5.3, strongly suggesting that a specific amino a cid residue plays a role in lignin binding. A one-electron transfer from DH P to oxidized intermediates LiP compounds I and II (LiPI and LiPII) was cha racterized by using a stopped-flow technique, showing that binding interact ions of DHP with LiPI and LiPII led to saturation kinetics. The dissociatio n equilibrium constants for LiPI-DHP and LiPII-DHP interactions were calcul ated to be 350 and 250 mu M, and the first-order rate constants for electro n transfer from DHP to LiPI and to LiPII were calculated to be 46 and 16 s( -1), respectively. These kinetic and spectral studies strongly suggest that LiP is capable of oxidizing lignin directly at the protein surface by a lo ng-range electron transfer process. A close look at the crystal structure s uggested that LiP possesses His-239 as a possible lignin-binding site on th e surface, which is linked to Asp-238. This Asp residue is hydrogen-bonded to the proximal His-176. This His-Asp...proximal-His motif would be a possi ble electron transfer route to oxidize polymeric lignin.