Publications

• Detection and characterization of the lignin peroxidase compound II-veratryl alcohol cation radical complex.

  Khindaria A., Nie G., Aust S.D.

  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 ... >> More

  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 pi 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 perpendicular only marginally greater than g parallel. This was surprising since the only other known organic radical coupled to the heme iron in a peroxidase is the tryptophan cation radical in cytochrome c peroxidase which exhibits a g tensor with g parallel greater than g perpendicular. Spin concentration analysis suggested that the 1 mol of VA*+ was coupled to the oxoferryl moiety per mole of enzyme. The VA.+ signal decayed with a first-order decay constant of 1.76 s-1, in close agreement with the earlier published decay constant of 1.85 s-1 from room-temperature EPR studies. The exchange coupling between VA.+ and the oxoferryl moiety strongly advocates calling this species (VA.+ and LiP compound II) a catalytic complex. << Less

  Biochemistry 36:14181-14185(1997) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Veratryl alcohol oxidation by lignin peroxidase.

  Khindaria A., Yamazaki I., Aust S.D.

  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 ( ... >> More

  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. << Less

  Biochemistry 34:16860-16869(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Lignin peroxidase compounds II and III. Spectral and kinetic characterization of reactions with peroxides.

  Wariishi H., Marquez L., Dunford H.B., Gold M.H.

  Stopped-flow techniques were used to investigate the kinetics of the reaction of lignin peroxidase compounds II and III (LiPII and LiPIII) with peroxides. Rate data were obtained from single-turnover experiments under pseudo-first-order conditions. LiPII reacts with H2O2 or peracetic acid (AcOOH) ... >> More

  Stopped-flow techniques were used to investigate the kinetics of the reaction of lignin peroxidase compounds II and III (LiPII and LiPIII) with peroxides. Rate data were obtained from single-turnover experiments under pseudo-first-order conditions. LiPII reacts with H2O2 or peracetic acid (AcOOH) to form a modified LiPIII, designated as LiPIII*, via a biphasic reaction. During the first phase, LiPIII is formed as an intermediate. Kinetic analysis also indicates a LiPII-peroxide complex. The first-order dissociation rate constants for the reaction of LiPII with H2O2 and AcOOH are 7.9 +/- 0.5 and 4.9 +/-0.6 s-1, respectively. The rate of the H2O2 reaction is approximately 500 times the rate of the comparable reaction with horseradish peroxidase, suggesting it is physiologically significant. The activation energy for the formation of LiPIII is 23 kJ mol-1. During the second phase, the intermediate LiPIII is converted to LiPIII*, confirmed by analyzing the reaction of exogenously prepared LiPIII with peroxides. The second-order rate constants for the reaction of LiPIII with H2O2 and AcOOH are (3.7 +/- 0.2) x 10(2) M-1 s-1 and (2.9 +/-0.2) x 10(2) M-1 s-1, respectively. The conversion of LiPIII to LiPIII* is reversible; the first-order rate constant for the reverse reaction is approximately (6.6 +/-0.6) x 10(-2) s-1. The rates of both LiPIII and LiPIII* formation decrease markedly above pH 4.0. The pH dependence of these reactions is controlled by a heme-linked ionizable group of pK alpha congruent to 4.2. << Less

  J Biol Chem 265:11137-11142(1990) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Two substrate interaction sites in lignin peroxidase revealed by site-directed mutagenesis.

  Doyle W.A., Blodig W., Veitch N.C., Piontek K., Smith A.T.

  It has been shown recently that Trp171 of lignin peroxidase (LiP) is hydroxylated at the Cbeta position [Blodig, W., Doyle, W. A., Smith, A. T., Winterhalter, K., Choinowski, T., and Piontek, K. (1998) Biochemistry 37, 8832-8838]. Comparative experiments, carried out on both wild-type fungal and r ... >> More

  It has been shown recently that Trp171 of lignin peroxidase (LiP) is hydroxylated at the Cbeta position [Blodig, W., Doyle, W. A., Smith, A. T., Winterhalter, K., Choinowski, T., and Piontek, K. (1998) Biochemistry 37, 8832-8838]. Comparative experiments, carried out on both wild-type fungal and recombinant LiP isoenzyme H8 (LiPH8), indicate that the process of hydroxylation is autocatalytic and that Trp171 may be implicated in catalysis. The role of this residue has therefore been examined using site-directed mutagenesis to obtain recombinant enzymes with Trp171 substituted by Phe or Ser (W171F and W171S LiPH8, respectively). The wild-type recombinant enzyme (LiPH8) was analyzed in solution using 1H NMR spectroscopy and its integrity confirmed prior to the kinetic and spectroscopic characterization of LiPH8 mutants. A charge neutralization mutation in the "classical heme edge" substrate access channel of LiP, in which Glu146 was substituted by Gly (E146G LiPH8), showed substantial activity with respect to veratryl alcohol (VA) oxidation and a marked (2.4 pH units) increase in pKa for the oxidation of a negatively charged difluoroazo dye. More surprisingly, the Trp171 LiPH8 mutants W171F and W171S LiPH8 were found to have lost all activity with VA as substrate, and compounds I and II were unable to react with VA. Both mutants, however, retained substantial activity with two dye substrates. These data provide the first direct evidence for the existence of two distinct substrate interaction sites in LiP, a heme-edge site typical of those encountered in other peroxidases and a second, novel site centered around Trp171 which is required for the oxidation of VA. Stopped-flow kinetic studies showed that all the mutants examined reacted normally with hydrogen peroxide to give a porphyrin cation radical (compound I). However, the rapid phase of spontaneous compound I reduction (2.3 s-1), typical of wild-type LiP, was absent in the Trp171 mutants, strongly suggesting that an electron-transfer pathway must exist within the protein leading from the heme to a surface site in close proximity to Trp171. The kinetic competence of such a pathway is dependent on interaction of the enzyme with VA, at or near Trp171. << Less

  Biochemistry 37:15097-15105(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Stabilization of the veratryl alcohol cation radical by lignin peroxidase.

  Khindaria A., Yamazaki I., Aust S.D.

  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 genera ... >> More

  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. << Less

  Biochemistry 35:6418-6424(1996) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of the oxycomplex of lignin peroxidases from Phanerochaete chrysosporium: equilibrium and kinetics studies.

  Cai D.Y., Tien M.

  The oxycomplexes (compound III, oxyperoxidase) of two lignin peroxidase isozymes, H1 (pI = 4.7) and H8 (pI = 3.5), were characterized in the present study. After generation of the ferroperoxidase by photochemical reduction with deazoflavin in the presence of EDTA, the oxycomplex is formed by mixin ... >> More

  The oxycomplexes (compound III, oxyperoxidase) of two lignin peroxidase isozymes, H1 (pI = 4.7) and H8 (pI = 3.5), were characterized in the present study. After generation of the ferroperoxidase by photochemical reduction with deazoflavin in the presence of EDTA, the oxycomplex is formed by mixing ferroperoxidase with O2. The oxycomplex of isozyme H8 is very stable, with an autoxidation rate at 25 degrees C too slow to measure at pH 3.5 or 7.0. In contrast, the oxycomplex of isozyme H1 has a half-life of 52 min at pH 4.5 and 29 min at pH 7.5 at 25 degrees C. The decay of isozyme H1 oxycomplex follows a single exponential. The half-lives of lignin peroxidase oxycomplexes are much longer than those observed with other peroxidases. The binding of O2 to ferroperoxidase to form the oxycomplex was studied by stopped-flow methods. At 20 degrees C, the second-order rate constants for O2 binding are 2.3 X 10(5) and 8.9 X 10(5) M-1 s-1 for isozyme H1 and 6.2 X 10(4) and 3.5 X 10(5) M-1 s-1 for isozyme H8 at pH 3.6 and pH 6.8, respectively. The dissociation rate constants for the oxycomplex of isozyme H1 (3.8 Z 10(-3) s-1) and isozyme H8 (1.0 X 10(-3) s-1) were measured at pH 3.6 by CO trapping. Thus, the equilibrium constants (K, calculated from kon/koff) for both isozymes H1 (7.0 X 10(7) M-1) and H8 (6.2 X 10(7) M-1) are higher than that of myoglobin (1.9 Z 10(6) M-1).(ABSTRACT TRUNCATED AT 250 WORDS) << Less

  Biochemistry 29:2085-2091(1990) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.