Irreversible Oxidation of Ferricytochrome <i>c </i>by Lignin Peroxidase

Sheng, Dawei; Gold, Michael H.

doi:10.1021/bi972198e

Cited by 18 publications

(14 citation statements)

References 53 publications

(114 reference statements)

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“…Previously, we used Cc 2+ and Cc 3+ as polymeric lignin model substrates in order to explore further the role(s) of VA in the LiP catalytic mechanism [21, 22]. In the present study, we have used RNase as a lignin model polymer for several reasons: (a) The molecular mass of bovine pancreatic RNase A (13 688) is considerably greater than that of chemically synthesized lignins (3–7 kDa).…”

Section: Discussionmentioning

confidence: 99%

“…Because RNase A does not contain free cysteine residues, the formation of intermolecular disulfide linkages between LiP‐oxidized Cys residues would not occur. The reduction in the Met content of the oxidized monomers and dimers probably is due to the LiP‐catalyzed oxidation of Met residues to methionine sulfoxide and methionine sulfone, as is observed with the LiP‐catalyzed oxidation of Cc 3+ [22]. The oxidation of Met would not lead to the polymerization of RNase.…”

Section: Discussionmentioning

confidence: 99%

“…X‐ray crystallographic studies demonstrate that the heme in LiP is buried, ruling out a direct interaction between the heme and polymeric substrates [11]. Therefore, we have examined the LiP oxidation of model protein polymeric substrates, including ferrocytochrome c (Cc 2+ ) [21] and ferricytochrome c (Cc 3+ ) [22], and the role of VA in these oxidations, in order to better understand the mechanism of LiP in the depolymerization of lignin. However, transient‐state kinetic studies using these model compounds are difficult, since the strong Soret absorptions of Cc 2+ and Cc 3+ overlap with that of LiP.…”

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confidence: 99%

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Oxidative polymerization of ribonuclease A by lignin peroxidase from Phanerochaete chrysosporium

Sheng

Gold

1999

European Journal of Biochemistry

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The mechanism of lignin peroxidase (LiP) was examined using bovine pancreatic ribonuclease A (RNase) as a polymeric lignin model substrate. SDS/PAGE analysis demonstrates that an RNase dimer is the major product of the LiP-catalyzed oxidation of this protein. Fluorescence spectroscopy and amino acid analyses indicate that RNase dimer formation is due to the LiP-catalyzed oxidation of Tyr residues to Tyr radicals, followed by intermolecular radical coupling. The LiP-catalyzed polymerization of RNase is strictly dependent on the presence of veratryl alcohol (VA). In the presence of 100 mm H 2 O 2 , relatively low concentrations of RNase and VA, together but not individually, can protect LiP from H 2 O 2 inactivation. The presence of RNase strongly inhibits VA oxidation to veratraldehyde by LiP; whereas the presence of VA does not inhibit RNase oxidation by LiP. Stopped-flow and rapid-scan spectroscopy demonstrate that the reduction of LiP compound I (LiPI) to the native enzyme by RNase occurs via two single-electron steps. At pH 3.0, the reduction of LiPI by RNase obeys second-order kinetics with a rate constant of 4.7 £ 10 4 m ±1´s±1 , compared to the second-order VA oxidation rate constant of 3.7 £ 10 5 m ±1´s±1 . The reduction of LiP compound II (LiPII) by RNase also follows second-order kinetics with a rate constant of 1.1 £ 10 4 m ±1´s±1 , compared to the first-order rate constant for LiPII reduction by VA. When the reductions of LiPI and LiPII are conducted in the presence of both VA and RNase, the rate constants are essentially identical to those obtained with VA alone. These results suggest that VA is oxidized by LiP to its cation radical which, while still in its binding site, oxidizes RNase.Keywords: cation radical, hydrogen peroxide, lignin peroxidase, ribonuclease, veratryl alcohol.Lignin is a heterogeneous, phenylpropanoid polymer that constitutes 20±30% of woody plant cell walls [1]. White-rot basidiomycete fungi are primarily responsible for initiating the depolymerization of lignin, which is a key step in the earth's carbon cycle [2±4]. The best-studied lignin-degrading fungus, Phanerochaete chrysosporium, secretes two types of extracellular heme peroxidases, manganese peroxidase and lignin peroxidase (LiP), which, along with an H 2 O 2 -generating system, are the major extracellular components of its lignin degradative system [2±6]. Nucleotide sequences of a number of lip cDNA and genomic clones [7±10], as well as X-ray crystal structures [11±13], demonstrate that important catalytic residues, including the proximal and distal His, the distal Arg, and an H-bonded Asp, are all conserved within the heme pocket of LiP. Although the catalytic cycle of LiP is similar to that of other plant and fungal peroxidases [2,14±16], LiP has several unique features, including an apparently high redox potential [2,3,17] and a low pH optimum of < 3.0 [16,18].In the presence of veratryl (3,4-dimethoxybenzyl) alcohol (VA), a secondary metabolite that is secreted by P. chrysosporium, LiP slowly depolymerizes synthetic...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Oxidative polymerization of ribonuclease A by lignin peroxidase from Phanerochaete chrysosporium

Sheng

Gold

1999

European Journal of Biochemistry

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“…In contrast, P. chrysosporium LiP oxidizes Poly R-478 (12), RNase A (36), and ferricytochrome c (35) only when VA was concomitantly present in the reaction mixture. In P. chrysosporium LiP isozyme H8 (PcLiPH8) a point mutational analysis demonstrated that an exposed tryptophan residue (W171) was crucial for VA oxidation (9,37).…”

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confidence: 92%

Mechanism for Oxidation of High-Molecular-Weight Substrates by a Fungal Versatile Peroxidase, MnP2

Tsukihara

Honda

Sakai

et al. 2008

Appl Environ Microbiol

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Unlike general peroxidases, Pleurotus ostreatus MnP2 was reported to have a unique property of direct oxidization of high-molecular-weight compounds, such as Poly R-478 and RNase A. To elucidate the mechanism for oxidation of polymeric substrates by MnP2, a series of mutant enzymes were produced by using a homologous gene expression system, and their reactivities were characterized. A mutant enzyme with an Ala substituting for an exposing Trp (W170A) drastically lost oxidation activity for veratryl alcohol (VA), Poly R-478, and RNase A, whereas the kinetic properties for Mn 2؉ and H 2 O 2 were substantially unchanged. These results demonstrated that, in addition to VA, the high-molecular-weight substrates are directly oxidized by MnP2 at W170. Moreover, in the mutants Q266F and V166/168L, amino acid substitution(s) around W170 resulted in a decreased activity only for the high-molecular-weight substrates. These results, along with the three-dimensional modeling of the mutants, suggested that the mutations caused a steric hindrance to access of the polymeric substrates to W170. Another mutant, R263N, contained a newly generated N glycosylation site and showed a higher molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Interestingly, the R263N mutant exhibited an increased reactivity with VA and high-molecular-weight substrates. The existence of an additional carbohydrate modification and the catalytic properties in this mutant are discussed. This is the first study of a direct mechanism for oxidation of high-molecular-weight substrates by a fungal peroxidase using a homologous gene expression system.Pleurotus ostreatus, known as the oyster mushroom, is a white rot basidiomycete that degrades plant cell wall lignin effectively (33). It secrets a series of isozymes of extracellular oxidizing enzymes belonging to manganese peroxidase (MnP)

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“…Studies in aqueous solution [9,10,14,[24][25][26][27][28] indicated that addition of VA could greatly enhance the degradation efficiency of aromatic compound catalyzed by LiP. How does VA work in a reversed micellar medium?…”

Section: Introductionmentioning

confidence: 99%

Mechanistic studies on the effect of veratryl alcohol on the lignin peroxidase catalyzed oxidation of pyrogallol red in reversed micelles

Lan

Huang²,

et al. 2007

Open Chemistry

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Abstract:The lignin peroxidase (LiP) catalyzed oxidation of pyrogallol red (PR) in the absence and presence of veratryl alcohol (3,4-dimethoxybenzyl alcohol, VA) was carried out in bis (2-ethylhexyl) sulfosuccinate sodium (AOT)/ polyoxyethylene lauryl ether (Brij30) reversed micelles to elucidate the role of VA. Results indicated that VA could accelerate the LiP catalyzed oxidation of PR, especially at low H 2 O 2 concentrations. Unlike in bulk aqueous medium, the protection of LiP by VA in the present medium was relatively unsubstantial, even at high H 2 O 2 concentrations. Analysis of data from a series of experiments showed that the enhancement of the PR oxidation caused by VA was mainly due to the indirect oxidation of PR by VA +• from the LiP catalyzed oxidation of VA. It was also found that at the same protector concentration (40 µM), VA (the physiological substrate of LiP) was less effective than PR (a phenolic compound) in protecting LiP from the H 2 O 2 derived inactivation. This novel phenomenon deserves further study.

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Irreversible Oxidation of Ferricytochrome c by Lignin Peroxidase

Cited by 18 publications

References 53 publications

Oxidative polymerization of ribonuclease A by lignin peroxidase from Phanerochaete chrysosporium

Oxidative polymerization of ribonuclease A by lignin peroxidase from Phanerochaete chrysosporium

Mechanism for Oxidation of High-Molecular-Weight Substrates by a Fungal Versatile Peroxidase, MnP2

Mechanistic studies on the effect of veratryl alcohol on the lignin peroxidase catalyzed oxidation of pyrogallol red in reversed micelles

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