The Experts below are selected from a list of 312 Experts worldwide ranked by ideXlab platform
Steven D. Aust - One of the best experts on this subject based on the ideXlab platform.
-
Detection and characterization of the lignin peroxidase compound II-Veratryl Alcohol cation radical complex.
Biochemistry, 1997Co-Authors: Aditya Khindaria, Guojun Nie, Steven D. AustAbstract:Lignin peroxidases (LiP) from the white-rot fungus Phanerochaete chrysosporium oxidize Veratryl Alcohol (VA) by two electrons to Veratryl aldehyde, although the VA cation radical (VA•+) is an intermediate [Khindaria, A., et al. (1995) Biochemistry 34, 6020−6025]. It was speculated, on the basis of kinetic evidence, that VA•+ can form a catalytic complex with LiP compound II. We have used low-temperature EPR to provide direct evidence for the formation of the complex. The EPR spectrum of VA•+ obtained at 4 K was explained by a model for coupling between the oxoferryl moiety of the heme (S = 1) and VA•+ (S = 1/2) similar to the model proposed for an oxyferryl and a porphyrin π cation radical of horseradish peroxidase. The coupling constant suggested that VA•+ was equally ferro- and antiferromagnetically coupled to the oxoferryl moiety. The spectrum was simulated with g⊥ only marginally greater than g∥. This was surprising since the only other known organic radical coupled to the heme iron in a peroxidase is...
-
Stabilization of the Veratryl Alcohol Cation Radical by Lignin Peroxidase
Biochemistry, 1996Co-Authors: Aditya Khindaria, Isao Yamazaki, Steven D. AustAbstract:Lignin peroxidase (LiP) catalyzes the H2O2-dependent oxidation of Veratryl Alcohol (VA) to Veratryl aldehyde, with the enzyme-bound Veratryl Alcohol cation radical (VA.+) as an intermediate [Khindaria et al. (1995) Biochemistry 34, 16860-16869]. The decay constant we observed for the enzyme generated cation radical did not agree with the decay constant in the literature [Candeias and Harvey (1995) J. Biol. Chem. 270, 16745-16748] for the chemically generated radical. Moreover, we have found that the chemically generated VA.+ formed by oxidation of VA by Ce(IV) decayed rapidly with a first-order mechanism in air- or oxygen-saturated solutions, with a decay constant of 1.2 x 10(3) s-1, and with a second-order mechanism in argon-saturated solution. The first-order decay constant was pH- independent suggesting that the rate-limiting step in the decay was deprotonation. When VA.+ was generated by oxidation with LiP the decay also occurred with a first-order mechanism but was much slower, 1.85 s-1, and was the same in both oxygen- and argon-saturated reaction mixtures. However, when the enzymatic reaction mixture was acid-quenched the decay constant of VA.+ was close to the one obtained in the Ce(IV) oxidation system, 9.7 x 10(2) s-1. This strongly suggested that the LiP-bound VA.+ was stabilized and decayed more slowly than free VA.+. We propose that the stabilization of VA.+ may be due to the acidic microenvironment in the enzyme active site, which prevents deprotonation of the radical and subsequent reaction with oxygen. We have also obtained reversible redox potential of VA.+/VA couple using cyclic voltammetery. Due to the instability of VA.+ in aqueous solution the reversible redox potential was measured in acetone, and was 1.36 V vs normal hydrogen electrode. Our data allow us to propose that enzymatically generated VA.+ can act as a redox mediator but not as a diffusible oxidant for LiP-catalyzed lignin or pollutant degradation.
-
The Effect of Veratryl Alcohol on Manganese Oxidation by Lignin Peroxidases
Archives of Biochemistry and Biophysics, 1996Co-Authors: Greg R. J. Sutherland, Aditya Khindaria, Steven D. AustAbstract:Abstract The extracellular peroxidase isozymes secreted by the white rot fungus Phanerochaete chrysosporium have been classified as manganese peroxidases (isozymes H3, H4, H5, and H9) and lignin peroxidases (isozymes H1, H2, H6, H7, H8, and H10). Recently we reported that lignin peroxidase isozyme H2 can also oxidize Mn 2+ (Khindaria et al., 1995, Biochemistry 34, 7773–7779). This lignin peroxidase isozyme oxidized Mn 2+ with both of the enzyme intermediates, compound I and compound II, at the same rates as manganese peroxidase isozyme H4. The results of single-turnover kinetic studies have now demonstrated that compound I of the other lignin peroxidase isozymes (H1, H6, H7, H8, and H10) also readily oxidized Mn 2+ , but that the rate of Mn 2+ oxidation by compound II was extremely slow. Compound III rapidly formed in the presence of Mn 2+ , oxalate, and H 2 O 2 . However, upon the addition of Veratryl Alcohol, the results indicate that Veratryl Alcohol served to reduce compound II. Under such conditions, compound III did not accumulate, and a steady-state rate of Mn 2+ oxidation was observed. The rate of Mn 2+ oxidation was the same as for the reduction of compound II by Veratryl Alcohol. The dependence of the rate of Mn 2+ oxidation on the concentration of Veratryl Alcohol was consistent with a mechanism in which Mn 2+ is oxidized by compound I and Veratryl Alcohol is oxidized by compound II. Therefore, under physiologically relevant conditions, in which both Veratryl Alcohol and Mn 2+ are present, all lignin peroxidase isozymes would be capable of oxidizing Mn 2+ to Mn 3+ which can serve as a diffusible oxidant.
-
The Effect of Veratryl Alcohol on Manganese Oxidation by Lignin Peroxidases
Archives of biochemistry and biophysics, 1996Co-Authors: Greg R. J. Sutherland, Aditya Khindaria, Steven D. AustAbstract:The extracellular peroxidase isozymes secreted by the white rot fungus Phanerochaete chrysosporium have been classified as manganese peroxidases (isozymes H3, H4, H5, and H9) and lignin peroxidases (isozymes H1, H2, H6, H7, H8, and H10). Recently we reported peroxidase isozyme H2 can also oxidize Mn2+ (Khindaria et al., 1995, Biochemistry 34, 7773-7779). This lignin peroxidase isozyme oxidized Mn2+ with both of the enzyme intermediates, compound I and compound II, at the same rates as manganese peroxidase isozyme H4. The results of single-turnover kinetic studies have now demonstrated that compound I of the other lignin peroxidase isozymes (H1, H6, H7, H8, and H1O) also readily oxidized Mn2+, but that the rate of Mn2+ oxidation by compound II was extremely slow. Compound III rapidly the presence of Mn2+, oxalate, and H2O2. However, upon the addition of Veratryl Alcohol, the results indicated that Veratryl Alcohol served to reduce compound II. Under such conditions, compound III did not accumulate, and a steady-state rate of Mn2+ oxidation was observed. The rate of Mn2+ oxidation was the same as for the reduction of compound II by Veratryl Alcohol. The dependence of the rate of Mn2+ oxidation on the concentration of Veratryl Alcohol was consistent with a mechanism in which Mn2+ is oxidized by compound I and Veratryl oxidized by compound II. Therefore, under physiologically relevant conditions, in which both Veratryl Alcohol and Mn2+ are present, all lignin peroxidase isozymes would be capable of oxidizing Mn2+ to Mn3+ which can serve as a diffusible oxidant.
-
Veratryl Alcohol oxidation by lignin peroxidase.
Biochemistry, 1995Co-Authors: Aditya Khindaria, Isao Yamazaki, Steven D. AustAbstract:Lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium catalyzes the H2O2-dependent oxidation of Veratryl Alcohol (VA), a secondary metabolite of the fungus, to Veratryl aldehyde (VAD). The oxidation of VA does not seem to be simply one-electron oxidation by LiP compound I (LiPI) to its cation radical (VA.+) and the second by LiP compound II (LiPII) to VAD. Moreover, the rate constant for LiPI reduction by VA (3 x 10(5) M-1 s-1) is certainly sufficient, but the rate constant for LiPII reduction by VA (5.0 +/- 0.2 s-1) is insufficient to account for the turnover rate of LiP (8 +/- 0.4 s-1) at pH 4.5. Oxalate was found to decrease the turnover rate of LiP to 5 s-1, but it had no effect on the rate constants for LiP with H2O2 or LiPI and LiPII, the latter formed by reduction of LiPI with ferrocyanide, with VA. However, when LiPII was formed by reduction of LiPI with VA, an oxalate-sensitive burst phase was observed during its reduction with VA. This was explained by the presence of LiPII, formed by reduction of LiPI with VA, in two different states, one that reacted faster with VA than the other. Activity during the burst was sensitive to preincubation of LiPI with VA, decaying with a half-life of 0.54 s, and was possibly due to an unstable intermediate complex of VA.+ and LiPII. This was supported by an anomalous, oxalate-sensitive, LiPII visible absorption spectrum observed during steady state oxidation of VA. The first order rate constant for the burst phase was 8.3 +/- 0.2 s-1, fast enough to account for the steady state turnover rate of LiP at pH 4.5. Thus, it was concluded that oxalate decreased the turnover of LiP by reacting with VA.+ bound to LiPII. The VA.+ concentration measured by electron spin resonance spectroscopy (ESR) was 2.2 microM at steady state (10 microM LiP, 250 microM H2O2, and 2 mM VA) and increased to 8.9 microM when measured after the reaction was acid quenched. Therefore, we assumed the presence of two states of VA.+ bound to LiPII, one ESR-active and one ESR-silent. The ESR-silent species, which could be detected after acid quenching, would be responsible for the burst phase. Both of the VA.+ species disappeared in the presence of 5 mM oxalate. The ESR-active species reached a maximum (3.5 microM) at 0.5 mM VA under steady state. From these studies, a mechanism for VA oxidation by LiP is proposed in which a complex of LiPII and VA.+ reacts with an additional molecule of VA, leading to Veratryl aldehyde formation.
Ming Tien - One of the best experts on this subject based on the ideXlab platform.
-
Identification of the Veratryl Alcohol Binding Site in Lignin Peroxidase by Site-Directed Mutagenesis
Biochemical and biophysical research communications, 1998Co-Authors: Katia Ambert-balay, Stephen M. Fuchs, Ming TienAbstract:Abstract Site-directed mutagenesis was used to identify the Veratryl Alcohol binding site of lignin peroxidase. The cDNA encoding isozyme H8 was mutated at Glu146 to both an Ala and a Ser residue. The H8 polypeptide was produced byE. colias inclusion bodies and refolded to yield active enzyme. The wild type recombinant enzyme and the mutants were purified to homogeneity and characterized by steady state kinetics. The kcat is decreased for both mutants of Glu146. The reactivity of mutants (kcat/Km) toward H2O2were not affected. In contrast, the kcat/Km of the mutants for Veratryl Alcohol were decreased by at least half. The oxidation of guaiacol by these mutants were more significantly affected. These results collectively suggest that E146 plays a central role in the binding of Veratryl Alcohol by lignin peroxidase.
-
Oxidation of 4-Methoxymandelic Acid by Lignin Peroxidase MEDIATION BY Veratryl Alcohol
The Journal of biological chemistry, 1997Co-Authors: Ming TienAbstract: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 enzyme-bound Veratryl Alcohol cation radical signal, which is consistent with its reaction at the active site of the enzyme.
-
The Roles of Veratryl Alcohol and oxalate in fungal lignin degradation
Journal of Biotechnology, 1997Co-Authors: Laura Schick Zapanta, Ming TienAbstract:Abstract Veratryl Alcohol (3,4-dimethoxybenzyl Alcohol) and oxalate are secondary metabolites of Phanerochaete chrysosporium and other white-rot fungi. Veratryl Alcohol is involved in lignin peroxidase catalysis as a substrate, and oxalate is involved in Mn peroxidase activity in its ability to chelate Mn 2+ . The role of Veratryl Alcohol in lignin degradation has been the subject of numerous studies and considerable debate. Several investigators have proposed that Veratryl Alcohol can act as a redox mediator of lignin degradation. In this mechanism, Veratryl Alcohol is oxidized by lignin peroxidase to form a cation radical. This cation radical then acts as a diffusible oxidant, mediating the oxidation of compounds that are putatively inaccessible to the lignin peroxidase active site, such as the bulky lignin polymer. Other investigators suggested that the short lifetime of the Veratryl Alcohol cation radical would make diffusion from the active site unlikely. Still others have suggested that Veratryl Alcohol is not a mediator at all and that its primary role is protecting lignin peroxidase from inactivation by H 2 O 2 . Recent evidence clearly shows that Veratryl Alcohol does form a cation radical upon oxidation by lignin peroxidase and that it can mediate the oxidation of some substrates. While the possibility of cation radical diffusion exists, it now appears that an enzyme-bound radical would have greater stability. In contrast, diffusible oxidation does play a role in Mn peroxidase activity. Mn peroxidase catalyzes the oxidation of Mn 2+ to Mn 3+ . Mn 3+ is a diffusible oxidant and is capable of oxidizing phenolic substrates. Studies have shown that chelating Mn 3+ with oxalate enhances its reactivity with its organic substrates. Other investigations have shown that Mn peroxidase activity is stimulated by oxalate. This article summarizes current understanding of the roles Veratryl Alcohol and oxalate play in the enzymatic degradation of lignin.
-
Oxidation of Guaiacol by Lignin Peroxidase ROLE OF Veratryl Alcohol
Journal of Biological Chemistry, 1995Co-Authors: Rao S Koduri, Ming TienAbstract:Abstract 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 × 10M s. The reaction of guaiacol with compound II exhibits a K of 64 μM and a first-order rate constant of 17 s. Oxidation of guaiacol leads to tetraguaiacol formation. This reaction exhibits classical Michaelis-Menten kinetics with a K of 160 μM and a k of 7.7 s. 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.
-
kinetic analysis of lignin peroxidase explanation for the mediation phenomenon by Veratryl Alcohol
Biochemistry, 1994Co-Authors: Rao S Koduri, Ming TienAbstract:We investigated the role of Veratryl Alcohol in lignin peroxidase-catalyzed oxidation of anisyl Alcohol with pre-steady-state and steady-state kinetic methods. Veratryl Alcohol has been proposed to act as a redox mediator for substrates that are not directly oxidized by the enzyme. Alternatively, its mediation activity has also been attributed to its ability to protect the enzyme from H 2 O 2 -dependent inactivation. As previously reported, Veratryl Alcohol was able to stimulate the oxidation of anisyl Alcohol. However, this stimulation is not due to mediation or protection of the enzyme. The stimulation can be attributed to the relative reactivity of anisyl Alcohol with compounds I and II of lignin peroxidase. We found that anisyl Alcohol reacts with compound I, but not with compound II
Klaus Piontek - One of the best experts on this subject based on the ideXlab platform.
-
Session C. Biochemistry and enzymologyThe oxidation of Veratryl Alcohol, dimeric lignin models and lignin by lignin peroxidase: The redox cycle revisited
Fems Microbiology Reviews, 1994Co-Authors: Hans E. Schoemaker, Taina Lundell, Annele Hatakka, Klaus PiontekAbstract:The mechanism of oxidation of Veratryl Alcohol and β-0–4 dimeric lignin models is reviewed. Veratryl Alcohol radicals are intermediates in both oxidation pathways. The possible role of the Veratryl Alcohol radical cation as a mediator is discussed. The lignin peroxidase (LIP) redox cycle is analyzed in terms of the Marcus theory of electron transfer. Reduction of both LiP-Compound I (LiP-I) and LiP-Compound II (LiP-II) by Veratryl Alcohol occurs in the endergonic region of the driving force. The reduction of LiP-II has a higher reorganization energy due to the change in spin state and the accompanying conformational change in the protein. It is suggested that a reversible nucleophilic addition of a carbohydrate residue located at the entrance of the active site channel plays a key role in the LiP redox cycle. Moreover. (polymeric) hydroxysubstituted benzyl radicals may reduce LiP-II via long-range electron transfer.
-
The oxidation of Veratryl Alcohol, dimeric lignin models and lignin by lignin peroxidase: The redox cycle revisited
FEMS Microbiology Reviews, 1994Co-Authors: Hans E. Schoemaker, Taina Lundell, Annele Hatakka, Klaus PiontekAbstract:The mechanism of oxidation of Veratryl Alcohol and β-0–4 dimeric lignin models is reviewed. Veratryl Alcohol radicals are intermediates in both oxidation pathways. The possible role of the Veratryl Alcohol radical cation as a mediator is discussed. The lignin peroxidase (LIP) redox cycle is analyzed in terms of the Marcus theory of electron transfer. Reduction of both LiP-Compound I (LiP-I) and LiP-Compound II (LiP-II) by Veratryl Alcohol occurs in the endergonic region of the driving force. The reduction of LiP-II has a higher reorganization energy due to the change in spin state and the accompanying conformational change in the protein. It is suggested that a reversible nucleophilic addition of a carbohydrate residue located at the entrance of the active site channel plays a key role in the LiP redox cycle. Moreover. (polymeric) hydroxysubstituted benzyl radicals may reduce LiP-II via long-range electron transfer.
Aditya Khindaria - One of the best experts on this subject based on the ideXlab platform.
-
Detection and characterization of the lignin peroxidase compound II-Veratryl Alcohol cation radical complex.
Biochemistry, 1997Co-Authors: Aditya Khindaria, Guojun Nie, Steven D. AustAbstract:Lignin peroxidases (LiP) from the white-rot fungus Phanerochaete chrysosporium oxidize Veratryl Alcohol (VA) by two electrons to Veratryl aldehyde, although the VA cation radical (VA•+) is an intermediate [Khindaria, A., et al. (1995) Biochemistry 34, 6020−6025]. It was speculated, on the basis of kinetic evidence, that VA•+ can form a catalytic complex with LiP compound II. We have used low-temperature EPR to provide direct evidence for the formation of the complex. The EPR spectrum of VA•+ obtained at 4 K was explained by a model for coupling between the oxoferryl moiety of the heme (S = 1) and VA•+ (S = 1/2) similar to the model proposed for an oxyferryl and a porphyrin π cation radical of horseradish peroxidase. The coupling constant suggested that VA•+ was equally ferro- and antiferromagnetically coupled to the oxoferryl moiety. The spectrum was simulated with g⊥ only marginally greater than g∥. This was surprising since the only other known organic radical coupled to the heme iron in a peroxidase is...
-
Stabilization of the Veratryl Alcohol Cation Radical by Lignin Peroxidase
Biochemistry, 1996Co-Authors: Aditya Khindaria, Isao Yamazaki, Steven D. AustAbstract:Lignin peroxidase (LiP) catalyzes the H2O2-dependent oxidation of Veratryl Alcohol (VA) to Veratryl aldehyde, with the enzyme-bound Veratryl Alcohol cation radical (VA.+) as an intermediate [Khindaria et al. (1995) Biochemistry 34, 16860-16869]. The decay constant we observed for the enzyme generated cation radical did not agree with the decay constant in the literature [Candeias and Harvey (1995) J. Biol. Chem. 270, 16745-16748] for the chemically generated radical. Moreover, we have found that the chemically generated VA.+ formed by oxidation of VA by Ce(IV) decayed rapidly with a first-order mechanism in air- or oxygen-saturated solutions, with a decay constant of 1.2 x 10(3) s-1, and with a second-order mechanism in argon-saturated solution. The first-order decay constant was pH- independent suggesting that the rate-limiting step in the decay was deprotonation. When VA.+ was generated by oxidation with LiP the decay also occurred with a first-order mechanism but was much slower, 1.85 s-1, and was the same in both oxygen- and argon-saturated reaction mixtures. However, when the enzymatic reaction mixture was acid-quenched the decay constant of VA.+ was close to the one obtained in the Ce(IV) oxidation system, 9.7 x 10(2) s-1. This strongly suggested that the LiP-bound VA.+ was stabilized and decayed more slowly than free VA.+. We propose that the stabilization of VA.+ may be due to the acidic microenvironment in the enzyme active site, which prevents deprotonation of the radical and subsequent reaction with oxygen. We have also obtained reversible redox potential of VA.+/VA couple using cyclic voltammetery. Due to the instability of VA.+ in aqueous solution the reversible redox potential was measured in acetone, and was 1.36 V vs normal hydrogen electrode. Our data allow us to propose that enzymatically generated VA.+ can act as a redox mediator but not as a diffusible oxidant for LiP-catalyzed lignin or pollutant degradation.
-
The Effect of Veratryl Alcohol on Manganese Oxidation by Lignin Peroxidases
Archives of Biochemistry and Biophysics, 1996Co-Authors: Greg R. J. Sutherland, Aditya Khindaria, Steven D. AustAbstract:Abstract The extracellular peroxidase isozymes secreted by the white rot fungus Phanerochaete chrysosporium have been classified as manganese peroxidases (isozymes H3, H4, H5, and H9) and lignin peroxidases (isozymes H1, H2, H6, H7, H8, and H10). Recently we reported that lignin peroxidase isozyme H2 can also oxidize Mn 2+ (Khindaria et al., 1995, Biochemistry 34, 7773–7779). This lignin peroxidase isozyme oxidized Mn 2+ with both of the enzyme intermediates, compound I and compound II, at the same rates as manganese peroxidase isozyme H4. The results of single-turnover kinetic studies have now demonstrated that compound I of the other lignin peroxidase isozymes (H1, H6, H7, H8, and H10) also readily oxidized Mn 2+ , but that the rate of Mn 2+ oxidation by compound II was extremely slow. Compound III rapidly formed in the presence of Mn 2+ , oxalate, and H 2 O 2 . However, upon the addition of Veratryl Alcohol, the results indicate that Veratryl Alcohol served to reduce compound II. Under such conditions, compound III did not accumulate, and a steady-state rate of Mn 2+ oxidation was observed. The rate of Mn 2+ oxidation was the same as for the reduction of compound II by Veratryl Alcohol. The dependence of the rate of Mn 2+ oxidation on the concentration of Veratryl Alcohol was consistent with a mechanism in which Mn 2+ is oxidized by compound I and Veratryl Alcohol is oxidized by compound II. Therefore, under physiologically relevant conditions, in which both Veratryl Alcohol and Mn 2+ are present, all lignin peroxidase isozymes would be capable of oxidizing Mn 2+ to Mn 3+ which can serve as a diffusible oxidant.
-
The Effect of Veratryl Alcohol on Manganese Oxidation by Lignin Peroxidases
Archives of biochemistry and biophysics, 1996Co-Authors: Greg R. J. Sutherland, Aditya Khindaria, Steven D. AustAbstract:The extracellular peroxidase isozymes secreted by the white rot fungus Phanerochaete chrysosporium have been classified as manganese peroxidases (isozymes H3, H4, H5, and H9) and lignin peroxidases (isozymes H1, H2, H6, H7, H8, and H10). Recently we reported peroxidase isozyme H2 can also oxidize Mn2+ (Khindaria et al., 1995, Biochemistry 34, 7773-7779). This lignin peroxidase isozyme oxidized Mn2+ with both of the enzyme intermediates, compound I and compound II, at the same rates as manganese peroxidase isozyme H4. The results of single-turnover kinetic studies have now demonstrated that compound I of the other lignin peroxidase isozymes (H1, H6, H7, H8, and H1O) also readily oxidized Mn2+, but that the rate of Mn2+ oxidation by compound II was extremely slow. Compound III rapidly the presence of Mn2+, oxalate, and H2O2. However, upon the addition of Veratryl Alcohol, the results indicated that Veratryl Alcohol served to reduce compound II. Under such conditions, compound III did not accumulate, and a steady-state rate of Mn2+ oxidation was observed. The rate of Mn2+ oxidation was the same as for the reduction of compound II by Veratryl Alcohol. The dependence of the rate of Mn2+ oxidation on the concentration of Veratryl Alcohol was consistent with a mechanism in which Mn2+ is oxidized by compound I and Veratryl oxidized by compound II. Therefore, under physiologically relevant conditions, in which both Veratryl Alcohol and Mn2+ are present, all lignin peroxidase isozymes would be capable of oxidizing Mn2+ to Mn3+ which can serve as a diffusible oxidant.
-
Veratryl Alcohol oxidation by lignin peroxidase.
Biochemistry, 1995Co-Authors: Aditya Khindaria, Isao Yamazaki, Steven D. AustAbstract:Lignin peroxidase (LiP) from the white rot fungus Phanerochaete chrysosporium catalyzes the H2O2-dependent oxidation of Veratryl Alcohol (VA), a secondary metabolite of the fungus, to Veratryl aldehyde (VAD). The oxidation of VA does not seem to be simply one-electron oxidation by LiP compound I (LiPI) to its cation radical (VA.+) and the second by LiP compound II (LiPII) to VAD. Moreover, the rate constant for LiPI reduction by VA (3 x 10(5) M-1 s-1) is certainly sufficient, but the rate constant for LiPII reduction by VA (5.0 +/- 0.2 s-1) is insufficient to account for the turnover rate of LiP (8 +/- 0.4 s-1) at pH 4.5. Oxalate was found to decrease the turnover rate of LiP to 5 s-1, but it had no effect on the rate constants for LiP with H2O2 or LiPI and LiPII, the latter formed by reduction of LiPI with ferrocyanide, with VA. However, when LiPII was formed by reduction of LiPI with VA, an oxalate-sensitive burst phase was observed during its reduction with VA. This was explained by the presence of LiPII, formed by reduction of LiPI with VA, in two different states, one that reacted faster with VA than the other. Activity during the burst was sensitive to preincubation of LiPI with VA, decaying with a half-life of 0.54 s, and was possibly due to an unstable intermediate complex of VA.+ and LiPII. This was supported by an anomalous, oxalate-sensitive, LiPII visible absorption spectrum observed during steady state oxidation of VA. The first order rate constant for the burst phase was 8.3 +/- 0.2 s-1, fast enough to account for the steady state turnover rate of LiP at pH 4.5. Thus, it was concluded that oxalate decreased the turnover of LiP by reacting with VA.+ bound to LiPII. The VA.+ concentration measured by electron spin resonance spectroscopy (ESR) was 2.2 microM at steady state (10 microM LiP, 250 microM H2O2, and 2 mM VA) and increased to 8.9 microM when measured after the reaction was acid quenched. Therefore, we assumed the presence of two states of VA.+ bound to LiPII, one ESR-active and one ESR-silent. The ESR-silent species, which could be detected after acid quenching, would be responsible for the burst phase. Both of the VA.+ species disappeared in the presence of 5 mM oxalate. The ESR-active species reached a maximum (3.5 microM) at 0.5 mM VA under steady state. From these studies, a mechanism for VA oxidation by LiP is proposed in which a complex of LiPII and VA.+ reacts with an additional molecule of VA, leading to Veratryl aldehyde formation.
David P. Barr - One of the best experts on this subject based on the ideXlab platform.
-
Effect of superoxide and superoxide dismutase on lignin peroxidase-catalyzed Veratryl Alcohol oxidation.
Archives of Biochemistry and Biophysics, 1994Co-Authors: David P. Barr, Steven D. AustAbstract:Abstract We have shown that superoxide (O ⨪ 2 ) is produced during the oxidation of Veratryl Alcohol by lignin peroxidase (LiP) by the reaction of the Veratryl Alcohol cation radical with hydrogen peroxide (D. B. Barr, M. M. Shah, and S. D. Aust, 1993, J. Biol. Chem. 268, 241-244). Compound III, an inactive form of peroxidases can be formed by reaction of the ferric enzyme with O ⨪ 2 . We therefore studied the effects of OI and superoxide dismutase (SOD) on the Veratryl Alcohol oxidase activity of LiP. SOD enhanced the rate of Veratryl Alcohol oxidation by LiP and Veratryl Alcohol oxidation was inhibited by the addition of KO 2 . Upon the addition of KO 2 , activity was also preceded by a lag period. Under steady-state turnover conditions (i.e., for Veratryl Alcohol oxidation), the addition of KO 2 resulted in the formation of LiP compound III. Compound II of LiP was observed following a time period that correlated with the lag prior to Veratryl aldehyde formation. The extent of the lag preceding Veratryl aldehyde formation increased with increasing concentrations of KO 2 and decreased with increasing concentrations of Veratryl Alcohol. It was postulated that during the lag period the Veratryl Alcohol cation radical reacted with compound III to regenerate the native enzyme. In this process the Veratryl Alcohol cation radical was reduced to Veratryl Alcohol, and thus, no Veratryl aldehyde was detected during the lag period.
-
Veratryl Alcohol-dependent production of molecular oxygen by lignin peroxidase.
The Journal of biological chemistry, 1993Co-Authors: David P. Barr, Manish M. Shah, Steven D. AustAbstract:Abstract Veratryl Alcohol- and H2O2-dependent production of oxygen by lignin peroxidase isozyme H2 (LiPH2) from Phanerochaete chrysosporium was investigated. Veratryl Alcohol oxidation by LiPH2 decreased with increasing concentrations of H2O2 while oxygen evolution increased. The absorption spectrum of the LiPH2 in these experiments indicated that it was in the compound II state. We propose that O2 production results from the one electron oxidation of H2O2 by the Veratryl Alcohol cation radical to yield superoxide, as the addition of superoxide dismutase stimulated oxygen production. It has been reported previously that oxygen is consumed in reaction mixtures containing lignin peroxidase, H2O2, Veratryl Alcohol, and oxalate (Popp, J. L., Kalyanaraman, B., and Kirk, T.K. (1990) Biochemistry 29, 10475-10480). In the presence of oxalate, we observed oxygen consumption that was dependent on the H2O2 concentration. The ability of other methoxybenzenes to mediate oxygen production appeared to be related to their redox potential. It was concluded that cation radicals can oxidize H2O2 by one electron which results in the production of superoxide and the evolution of molecular oxygen. Thus, the rates of LiPH2-catalyzed O2 consumption or O2 production are dependent on the relative concentrations of H2O2 and oxalate.
-
On the mechanism of inhibition of the Veratryl Alcohol oxidase activity of lignin peroxidase H2 by EDTA.
The Journal of biological chemistry, 1992Co-Authors: Manish M. Shah, David P. Barr, Thomas A. Grover, Steven D. AustAbstract:Abstract The mechanism of inhibition of the Veratryl Alcohol oxidase activity of lignin peroxidase H2 (LiPH2) by EDTA was investigated. It was found that EDTA was decarboxylated and that cytochrome c, nitro blue tetrazolium, ferric iron, and molecular oxygen were reduced in a reaction mixture containing LiPH2, H2O2, Veratryl Alcohol, and EDTA. The reductive activity observed with LiPH2 followed first order kinetics with respect to the concentration of EDTA. Stoichiometry studies showed that in the presence of sufficient EDTA, 1.7 mol of ferric iron were reduced per mole of H2O2 added to the reaction mixture. Superoxide- and EDTA-derived radicals were detected by ESR spin trapping upon incubation of LiPH2 with H2O2, Veratryl Alcohol, and EDTA. The Km values of Veratryl Alcohol and H2O2 remained the same for both the oxidative and reductive activities of LiPH2. Reductive activity was also observed with LiPH2 and EDTA using other free radical mediators in the place of Veratryl Alcohol, such as 1,4-dimethoxybenzene, 1,2,3- and 1,2,4-trimethoxybenzenes, and 1,2,4,5-tetramethoxybenzene. EDTA reduced the cation radical of 1,2,4,5-tetramethoxybenzene formed by LiPH2 in the presence of H2O2. Hence, it is proposed that the apparent inhibition of the Veratryl Alcohol oxidase activity of LiPH2 by EDTA is due to the reduction of the Veratryl Alcohol cation radical intermediate back to Veratryl Alcohol by EDTA. The reduction of cytochrome c, nitro blue tetrazolium, ferric ion, and molecular oxygen appears to be mediated by the EDTA radical formed by reduction of the Veratryl Alcohol cation radical.
