Binding properties of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium against a synthetic lignin (dehydrogenated polymerizate, DHP) were studied with a resonant mirror biosensor. Among several ligninolytic enzymes, only LiP specifically binds to DHP. Kinetic analysis revealed that the binding was reversible, and that the dissociation equilibrium constant was 330 M. The LiP-DHP interaction was controlled by the ionization group with a pK a of 5.3, strongly suggesting that a specific amino acid residue plays a role in lignin binding. A oneelectron transfer from DHP to oxidized intermediates LiP compounds I and II (LiPI and LiPII) was characterized by using a stopped-f low technique, showing that binding interactions of DHP with LiPI and LiPII led to saturation kinetics. The dissociation equilibrium constants for LiPI-DHP and LiPII-DHP interactions were calculated to be 350 and 250 M, and the first-order rate constants for electron 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 long-range electron transfer process. A close look at the crystal structure suggested that LiP possesses His-239 as a possible lignin-binding site on the surface, which is linked to Asp-238. This Asp residue is hydrogen-bonded to the proximal His-176. This HisAsp⅐⅐⅐proximal-His motif would be a possible electron transfer route to oxidize polymeric lignin.Lignin is the most abundant renewable aromatic polymer and is known as one of the most recalcitrant biomaterials on earth (1, 2). Its degradation plays a key role in the carbon cycle of the biosphere (2-7). Only white-rot basidiomycetes are responsible for the complete mineralization of this polymer. Phanerochaete chrysosporium, the best studied white-rot fungus, secretes two heme peroxidases, lignin peroxidase (LiP) and manganese peroxidase (MnP) under ligninolytic conditions (3-8). Thus, these enzymes have been believed to be involved in triggering lignin biodegradation. MnP oxidizes Mn II to Mn III , and the latter acts as a freely diffusible one-electron oxidizer, nonspecifically reacting with terminal organic substrates such as phenols, thiols, and lignin (3, 8 -12). This nonspecific manner is advantageous for lignin degradation because lignin is such a heterogeneous polymer.LiP is another unique heme peroxidase secreted by P. chrysosporium. It catalyzes a one-electron oxidation of nonphenolic aromatic compounds, forming the aryl cation radical (13,14), suggesting that oxidized intermediates of the enzyme possess a very high redox potential. The mechanism of LiP catalytic action on lignin is still uncertain, because it has not been clear whether LiP can oxidize lignin through a direct interaction or through radical mediation. Veratryl (3,4-dimethoxybenzyl) alcohol (VA), a preferred substrate for LiP, is synthesized de novo by P. chrysosporium under ligninolytic conditions (15). The...
Microperoxidase-11 (MP-11), a heme-containing undecapeptide, derived from horse heart cytochrome c was utilized as a peroxidative catalyst. Catalytic characteristics of MP-11 in hydrophilic organic media were studied using 2,6-dimethoxyphenol as a reducing substrate in a series of organic solvents at various concentrations, indicating that MP-11 was active in water-miscible organic solvents but at least 5% water was compulsory for the catalytic action. Thus, MP-11 was not active in hydrophobic solvents. The pH of the water portion in the media affected the reaction rate. The optimal pH was found to be 9, where a release of protons from either an oxidizing or reducing substrate to the media was facilitated. The decolorization of water-insoluble synthetic dyes by MP-11 in 90% methanol was attempted. MP-11 showed effective decolorization activities against either azo or anthraquinone dyes. The degradation pathway for Solvent Orange 7 was investigated in detail, showing that MP-11 catalyzed the oxidative cleavage of the azo linkage to generate 1,2-naphthoquinone and 2,4-dimethylphenol as key intermediates.
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