Veratryl alcohol oxidation by lignin peroxidase.
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.
Khindaria
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A
Department of Chemistry and Biochemistry, Utah State University, Logan 84322, USA.
Yamazaki
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Aust
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D001487:Basidiomycota / Q000201:enzymology; D001592:Benzyl Alcohols / Q000378:metabolism*; D001665:Binding Sites; D001673:Biodegradation, Environmental; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008031:Lignin / Q000378:metabolism; D010070:Oxalates / Q000494:pharmacology; D019815:Oxalic Acid; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism*; D013053:Spectrophotometry
D001592:Benzyl Alcohols; D005609:Free Radicals; D010070:Oxalates; D008031:Lignin; D019815:Oxalic Acid; D006861:Hydrogen Peroxide; D010544:Peroxidases; C042858:lignin peroxidase; C042197:veratryl alcohol