Evidence for formation of the veratryl alcohol cation radical by lignin peroxidase

Khindaria, Aditya; Grover, Thomas A.; Aust, Steven D.

doi:10.1021/bi00018a003

Cited by 39 publications

(38 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The most controversial argument was found in the LiP reaction mechanism for the oxidation of recalcitrant substrates (16)(17)(18)(19)(20). Once widely accepted, VA cation radical mediation mechanisms are now questioned (33)(34)(35).…”

Section: Discussionmentioning

confidence: 99%

Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium

Johjima

Itoh

Kabuto

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionmentioning

confidence: 99%

Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium

Johjima

Itoh

Kabuto

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…This indicates that under physiological conditions, the enzyme is not saturated with veratryl alcohol and that phenolic substrates would be just as readily oxidized directly by the enzyme as they would be by the veratryl alcohol cation radical. Furthermore, there are also no data indicating that the recently detected veratryl alcohol cation radical is stable and diffusible (31). Thus, it does not seem likely that veratryl alcohol can be acting at a distance as a diffusible oxidant.…”

Section: Reaction Of Guaiacol In Incubations Containingmentioning

confidence: 95%

Complexes of Adenovirus with Polycationic Polymers and Cationic Lipids Increase the Efficiency of Gene Transfer in Vitro and in Vivo

Fasbender

Zabner²,

Chillón

et al. 1997

Journal of Biological Chemistry

248

218

View full text Add to dashboard Cite

We have investigated the lignin peroxidase-catalyzed oxidation of guaiacol and the role of veratryl alcohol in this reaction by steady-state and pre-steady-state methods. Pre-steady-state kinetic analyses demonstrated that guaiacol is a good substrate for both compounds I and II, the two-and one-electron oxidized enzyme intermediates, respectively, of lignin peroxidase. The rate constant for the reaction with compound I is 1.2 ؋ 10 6 M ؊1 s ؊1. The reaction of guaiacol with compound II exhibits a K d of 64 M and a first-order rate constant of 17 s ؊1 . Oxidation of guaiacol leads to tetraguaiacol formation. This reaction exhibits classical Michaelis-Menten kinetics with a K m of 160 M and a k cat of 7.7 s ؊1. Veratryl alcohol, a secondary metabolite of ligninolytic fungi, is capable of mediating the oxidation of guaiacol. This was shown by steady-state inhibition studies. Guaiacol completely inhibited the oxidation of veratryl alcohol, whereas veratryl alcohol had no corresponding inhibitory effect on guaiacol oxidation. In fact, at low guaiacol concentrations, veratryl alcohol stimulated the rate of guaiacol oxidation. These results collectively demonstrate that veratryl alcohol can serve as a mediator for phenolic substrates in the lignin peroxidase reaction.This study investigates the ability of 3,4-dimethoxybenzyl (veratryl) alcohol to mediate the lignin peroxidase-catalyzed oxidation of guaiacol. Lignin peroxidases are hemeproteins secreted by the white rot fungus Phanerochaete chrysosporium during secondary metabolism (1, 2). These isozymes catalyze the oxidation of lignin and a large number of phenolic and non-phenolic substrates (3, 4). The catalytic cycle of lignin peroxidase is similar to that of other peroxidases (5, 6) where ferric enzyme is first oxidized by H 2 O 2 to generate the twoelectron oxidized intermediate, compound I (7). Compound I is then reduced by one electron donated by a substrate molecule, yielding the 1-electron oxidized enzyme intermediate, compound II, and a free radical product. The catalytic cycle is completed by the one-electron reduction of compound II by a second substrate molecule.In the absence of a reducing substrate, the enzyme can undergo a series of reactions with H 2 O 2 to form compound III, oxyperoxidase (6,8). It is also well documented that prolonged incubation of enzyme with H 2 O 2 in the absence of a reducing substrate such as veratryl alcohol can cause irreversible inactivation of the enzyme (9). In the presence of veratryl alcohol, however, lignin peroxidase undergoes multiple turnovers without any detectable inactivation. Because veratryl alcohol is normally produced by ligninolytic cultures of P. chrysosporium (10), workers have proposed that its physiological function is to protect the enzyme from H 2 O 2 -dependent inactivation (11).An alternate role for veratryl alcohol in lignin biodegradation has been proposed by Harvey et al. (12). These workers observed that substrates that are not oxidized by lignin peroxidase such as anisyl alcohol and 4-methoxyman...

show abstract

“…ESR Quantitation of Cation Radical-The veratryl alcohol cation radical formed from LP was quantitated by acid quench with a threesyringe fast-flow system as described previously (11). The contents of the first two syringes, one containing LP and veratryl alcohol, the other containing H 2 O 2 , were mixed for 0.5 s. The contents were then quenched with 10% HNO 3 .…”

Section: Methodsmentioning

confidence: 99%

“…Harvey's hypothesis was supported by the work of Khindaria et al (11). They were able to generate the radical chemically and also enzymatically.…”

mentioning

confidence: 89%

Oxidation of 4-Methoxymandelic Acid by Lignin Peroxidase

Tien

1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

The mechanism of veratryl alcohol-mediated oxidation of 4-methoxymandelic acid by lignin peroxidase was studied by kinetic methods. For monomethoxylated substrates not directly oxidized by lignin peroxidase, veratryl alcohol has been proposed to act as a redox mediator. Our previous study showed that stimulation of anisyl alcohol oxidation by veratryl alcohol was not due to mediation but rather due to the requirement of veratryl alcohol to complete the catalytic cycle. Anisyl alcohol can react with compound I but not with compound II. In contrast, veratryl alcohol readily reduces compound II. We demonstrate in the present report that the oxidation of 4-methoxy mandelic acid is mediated by veratryl alcohol. Increasing veratryl alcohol concentration in the presence of 2 mM 4-methoxymandelic acid resulted in increased oxidation of 4-methoxymandelic acid yielding anisaldehyde. This is in contrast to results obtained with anisyl alcohol where increased concentrations of veratryl alcohol caused a decrease in product formation. ESR spectroscopy demonstrated that 4-methoxymandelic acid caused a decrease in the enzymebound veratryl alcohol cation radical signal, which is consistent with its reaction at the active site of the enzyme.To degrade the aromatic polymer lignin, the white-rot fungus Phanerochaete chrysosporium secretes H 2 O 2 (1), and two families of H 2 O 2 -utilizing enzymes, the lignin peroxidase (LP) 1 and manganese peroxidases (MnP). The MnPs catalyze the oxidation of Mn 2ϩ to Mn 3ϩ (2) whereas the LPs catalyze the oxidation of phenolic and nonphenolic methoxylated aromatic substrates (3). The catalytic cycle of both peroxidases is similar to that of other peroxidases. The enzyme is first oxidized by H 2 O 2 to form a two-electron-oxidized intermediate, compound I (4). Compound I then returns to the resting ferric enzyme by two sequential one-electron reduction steps producing two, oneelectron-oxidized products. The one-electron oxidized species of the enzyme formed during turnover is referred to as compound II. Unique to these fungal peroxidases is the recalcitrant nature of their substrate (5). Both act on substrates that other peroxidases are not capable of oxidizing.The role and mechanism by which the LP and MnP interact with lignin is yet to be elucidated. For both enzymes, the role of mediators has been proposed. With MnP, the mediation phenomenon is well characterized and widely accepted. Complexed Mn 2ϩ (6, 7) is oxidized by MnP and diffuses away from the active site of the enzyme to oxidize lignin (8). In the case of LP, the role of mediators and the mechanism by which lignin is depolymerized by LP is still unknown. A key component of LP catalysis is veratryl alcohol, a secondary metabolite also produced by ligninolytic cultures of P. chrysosporium (9). Harvey et al. (10) proposed that veratryl alcohol is the mediator for LP as Mn 2ϩ is the mediator for MnP. They proposed that the initial one-electron oxidation of veratryl alcohol forms the cation radical which then diffuses away from the active s...

show abstract

Evidence for formation of the veratryl alcohol cation radical by lignin peroxidase

Cited by 39 publications

References 32 publications

Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium

Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium

Complexes of Adenovirus with Polycationic Polymers and Cationic Lipids Increase the Efficiency of Gene Transfer in Vitro and in Vivo

Oxidation of 4-Methoxymandelic Acid by Lignin Peroxidase

Contact Info

Product

Resources

About