Spectroscopic and kinetic properties of the oxidized intermediates of lignin peroxidase from Phanerochaete chrysosporium.
Stopped-flow rapid scan techniques were used to obtain a spectrum of nearly homogeneous lignin peroxidase compound I (LiPI) under pseudo-first order conditions at the unusually low pH optimum (3.0) for the enzyme. The LiPI spectrum had a Soret band at 407 nm with approximately 60% reduced intensity and a visible maximum at 650 nm. Under steady-state conditions a Soret spectrum for lignin peroxidase compound II (LiPII) was also obtained. The Soret maximum of LiPII at 420 nm was only approximately 15% reduced in intensity compared to native LiP. Transient state kinetic results confirmed the pH independence of LiPI formation over the pH range 3.06-7.39. The rate constant was (6.5 +/- 0.2) x 10(5) M-1 S-1. Addition of excess veratryl alcohol to LiPI resulted in its reduction to LiPII with subsequent reduction of LiPII to the native enzyme. Reactions of LiPI and LiPII with veratryl alcohol exhibited marked pH dependencies. For the LiPI reaction the rate constants ranged from 2.5 x 10(6) M-1 S-1 at pH 3.06 to 4.1 x 10(3) M-1 S-1 at pH 7.39; for the LiPII reaction, 1.6 x 10(5) M-1 S-1 (pH 3.06) to 2.3 x 10(3) M-1 S-1 (pH 5.16). These single turnover experiments demonstrate directly that the pH dependence of these reactions dictates the overall pH dependence of this novel enzyme. These results are consistent with the one-electron oxidation of veratryl alcohol to an aryl cation radical by LiPI and by LiPII.
Marquez
L
L
Department of Chemistry, University of Alberta, Edmonton, Canada.
Wariishi
H
H
Dunford
H B
HB
Gold
M H
MH
D000363:Agaricales / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000378:metabolism*; D010544:Peroxidases; D013053:Spectrophotometry
D007527:Isoenzymes; D010544:Peroxidases; C042858:lignin peroxidase; D010105:Oxygenases