Biophysical and structural analysis of a novel heme B iron ligation in the flavocytochrome cellobiose dehydrogenase.

The fungal extracellular flavocytochrome cellobiose dehydrogenase (CDH) participates in lignocellulose degradation. The enzyme has a cytochrome domain connected to a flavin-binding domain by a peptide linker. The cytochrome domain contains a 6-coordinate low spin b-type heme with unusual iron ligands and coordination geometry. Wild type CDH is only the second example of a b-type heme with Met-His ligation, and it is the first example of a Met-His ligation of heme b where the ligands are arranged in a nearly perpendicular orientation. To investigate the ligation further, Met65 was replaced with a histidine to create a bis-histidyl ligated iron typical of b-type cytochromes. The variant is expressed as a stable 90-kDa protein that retains the flavin domain catalytic reactivity. However, the ability of the mutant to reduce external one-electron acceptors such as cytochrome c is impaired. Electrochemical measurements demonstrate a decrease in the redox midpoint potential of the heme by 210 mV. In contrast to the wild type enzyme, the ferric state of the protoheme displays a mixed low spin/high spin state at room temperature and low spin character at 90 K, as determined by resonance Raman spectroscopy. The wild type cytochrome does not bind CO, but the ferrous state of the variant forms a CO complex, although the association rate is very low. The crystal structure of the M65H cytochrome domain has been determined at 1.9 A resolution. The variant structure confirms a bis-histidyl ligation but reveals unusual features. As for the wild type enzyme, the ligands have a nearly perpendicular arrangement. Furthermore, the iron is bound by imidazole N delta 1 and N epsilon 2 nitrogen atoms, rather than the typical N epsilon 2/N epsilon 2 coordination encountered in bis-histidyl ligated heme proteins. To our knowledge, this is the first example of a bis-histidyl N delta 1/N epsilon 2-coordinated protoporphyrin IX iron.

Peroxidase-catalyzed oxidation of azo dyes: mechanism of disperse Yellow 3 degradation.

Disperse Yellow 3 [2-(4'-acetamidophenylazo)-4-methylphenol] (DY3) (I) is an important yellow dye used in industry and is also a carcinogen. Earlier we demonstrated that lignin-degrading cultures of white-rot basidiomycete Phanerochaete chrysosporium degrade DY3 to CO2. In this report, we have examined the degradation of DY3 and its naphthol analog, 1-(4'-acetamidophenylazo)-2-naphthol (NDY3) (II) by lignin peroxidase, horseradish peroxidase, and Mn(III)-malonate complex (a manganese peroxidase mimic). Lignin and manganese peroxidases are two extracellular peroxidase produced by ligninolytic cultures of P. chrysosporium and are involved in the degradation of lignin and various other environmental pollutants by this fungus. DY3 oxidation by peroxidases yields 4-methyl-1,2-benzoquinone (III), acetanilide (IV), and a dimer of DY3 (V) as products. NDY3 oxidation yields acetanilide (IV) and 1,2-naphthoquinone (VI). In deuterium incorporation experiments with DY3, 55-67% incorporation of deuterium from dioxane-d8 into acetanilide (IV) is observed. However, when D2O is the donor, deuterium is not incorporated into acetanilide (IV). Based on these results, a mechanism for azo dye degradation is proposed. The H2O2-oxidized forms of a peroxidase oxidize the phenolic ring of DY3, or its analogs, by two electrons to produce a carbonium ion, which is located on the carbon bearing the azo linkage. Water attacks the carbonium ion, producing an unstable intermediate which breaks down to generate 1,2-naphthoquinone (VI) or 4-methyl-1,2-benzoquinone (III) and 4-acetamido-phenyldiazene. O2, H2O2-oxidized peroxidase, or a metal ion, oxidize the phenyldiazene by one electron to produce a phenyldiazene radical, which cleaves homolytically to generate 4-acetamidophenyl radical and molecular nitrogen. The 4-acetamidophenyl radical then abstracts a hydrogen radical from the surroundings to produce acetanilide (IV). DY3 degradation by whole cultures of P. chrysosporium yields acetanilide as the major product. This suggests that lignin peroxidase and manganese peroxidase are involved in the in vivo metabolism of DY3 by P. chrysosporium.