Aditya Khindaria

Reductive Dehalogenation of Aliphatic Halocarbons by Lignin Peroxidase of Phanerochaete chrysosporium.

Contamination of soils and aquifers by aliphatic halocarbons is a serious environmental pollution problem. We report here the novel observation that the halocarbons trichloroethylene (TCE) and CCl 4 were mineralized by Phanerochaete chrysosporium under aerobic conditions. Ligninolytic cultures of this white rot fungus mineralized 20.3% of 10 ppm TCE and 18.8% of 10 ppm CCl 4 in 9 days. These chemicals were not mineralized by nonligninolytic cultures of P. chrysosporium, indicating that lignin peroxidases play an important role in the mineralization of these chemicals. In a previous study, we reported lignin peroxidase-catalyzed reductive dehalogenation of CCl 4 with the resultant formation oftrichloromethyl radical. We have extended this study and report here reductive dehalogenation of CHCl 3 , CH 2 Cl 2 , TCE, and 1,1,1-trichloroethane. Dehalogenation was catalyzed by a reductive reaction system containing lignin peroxidase, veratryl alcohol, EDTA or oxalate, H 2 O 2 , and the halocarbon with phenyl N-tert-butylnitrone as a spin trap for electron spin resonance detection of the resulting radicals. Since all the components of the reductive system with oxalate as an electron donor are excreted by P. chrysosporium, we propose that this mechanism may be involved in the degradation of these halocarbons by the fungus.

EPR detection and characterization of lignin peroxidase porphyrin pi-cation radical.

Lignin peroxidase (LiP) from Phanerochaete chrysosporium catalyzes the H2O2 dependent one- and two-electron oxidations of substrates. The catalytic cycle involves the oxidation of ferric-LiP by H2O2 by two electrons to compound I, which is an oxoferryl heme and a free radical. It has been speculated that the unpaired electron is in a pi delocalized porphyrin radical. However, no direct evidence for the presence of the free radical has been reported. We present electron paramagnetic resonance (EPR) detection and characterization of compound I of LiP. The LiP compound I EPR signal is different than those reported previously for compound I of horseradish peroxidase and chloroperoxidase. However, the EPR signal of compound I of LiP (axial g tensor extending from gperpendicular = 3.42 to gparallel approximately 2) is very similar to the EPR signals of compound I of ascorbate peroxidase and catalase from Micrococcus lysodeikticus, in which the radical has been identified as a porphyrin pi-cation radical. On the basis of the analysis of our data and comparison with the earlier published results for compounds I of other peroxidases, we interpret the LiP compound I signal by a model for exchange coupling between an S = 1 oxyferryl [Fe = O]2+ moiety and a porphyrin pi-cation radical (S = 1/2) [Schulz, C.E., et al. (1979) FEBS Lett. 103, 102-105]. The exchange coupling is characterized by ferromagnetic rather than an antiferromagnetic interaction between the two species. The ferric-Lip EPR signal suggests that the iron in the heme is in near perfect orthogonal symmetry and provides additional evidence of the ferromagnetic interaction between the oxoferryl iron center and the porphyrin pi-cation radical.

Reductions catalyzed by a quinone and peroxidases from Phanerochaete chrysosporium.

A quinone produced from veratryl alcohol by lignin peroxidase from the white rot fungus Phanerochaete chrysosporium was tested for its ability to mediate reduction. The quinone (2-hydroxymethyl-5-methoxy-1,4-benzoquinone), reduced chemically or by cellobiose:quinone reductase isolated from cultures of the fungus, mediated the reduction of cytochrome c in reactions containing either Mn(III), a manganese-dependent peroxidase, Mn(II) and H2O2, or lignin peroxidase and H2O2. Formation of the semiquinone, the species responsible for reducing cytochrome c, was observed by electron spin resonance spectroscopy in these reactions. The production of the quinone was observed in the extracellular fraction of cultures grown under nutrient nitrogen-deficient conditions (2.4 mM ammonium tartrate) for over 10 days, starting on Day 2, but not under nutrient nitrogen-sufficient conditions. These results suggest that a quinone produced by lignin peroxidase can serve as a physiological mediator of reductive reactions catalyzed by the fungal peroxidases.