Oxidative degradation of phenanthrene by the ligninolytic fungus Phanerochaete chrysosporium.

The ligninolytic fungus Phanerochaete chrysosporium oxidized phenanthrene and phenanthrene-9,10-quinone (PQ) at their C-9 and C-10 positions to give a ring-fission product, 2,2'-diphenic acid (DPA), which was identified in chromatographic and isotope dilution experiments. DPA formation from phenanthrene was somewhat greater in low-nitrogen (ligninolytic) cultures than in high-nitrogen (nonligninolytic) cultures and did not occur in uninoculated cultures. The oxidation of PQ to DPA involved both fungal and abiotic mechanisms, was unaffected by the level of nitrogen added, and was significantly faster than the cleavage of phenanthrene to DPA. Phenanthrene-trans-9,10-dihydrodiol, which was previously shown to be the principal phenanthrene metabolite in nonligninolytic P. chrysosporium cultures, was not formed in the ligninolytic cultures employed here. These results suggest that phenanthrene degradation by ligninolytic P. chrysosporium proceeds in order from phenanthrene----PQ----DPA, involves both ligninolytic and nonligninolytic enzymes, and is not initiated by a classical microsomal cytochrome P-450. The extracellular lignin peroxidases of P. chrysosporium were not able to oxidize phenanthrene in vitro and therefore are also unlikely to catalyze the first step of phenanthrene degradation in vivo. Both phenanthrene and PQ were mineralized to similar extents by the fungus, which supports the intermediacy of PQ in phenanthrene degradation, but both compounds were mineralized significantly less than the structurally related lignin peroxidase substrate pyrene was.

Ring fission of anthracene by a eukaryote.

Ligninolytic fungi are unique among eukaryotes in their ability to degrade polycyclic aromatic hydrocarbons (PAHs), but the mechanism for this process is unknown. Although certain PAHs are oxidized in vitro by the fungal lignin peroxidases (LiPs) that catalyze ligninolysis, it has never been shown that LiPs initiate PAH degradation in vivo. To address these problems, the metabolism of anthracene (AC) and its in vitro oxidation product, 9,10-anthraquinone (AQ), was examined by chromatographic and isotope dilution techniques in Phanerochaete chrysosporium. The fungal oxidation of AC to AQ was rapid, and both AC and AQ were significantly mineralized. Both compounds were cleaved by the fungus to give the same ring-fission metabolite, phthalic acid, and phthalate production from AQ was shown to occur only under ligninolytic culture conditions. These results show that the major pathway for AC degradation in Phanerochaete proceeds AC----AQ----phthalate + CO2 and that it is probably mediated by LiPs and other enzymes of ligninolytic metabolism.

Cytochrome oxidase from thermophilic bacterium PS3 contains a fourth protein subunit.

Monoclonal antibodies prepared against subunits II and IV of beef heart cytochrome oxidase were found to cross-react with thermophilic bacterial PS3 oxidase. Each individual antibody affects the enzymatic activity. "Western" blot analyses showed that subunit II antibodies of beef heart recognized subunit II of PS3 and subunit IV antibody likewise recognized a fourth protein subunit on slab gels. This fourth subunit previously thought to be a contaminant or a degradation product has a molecular weight of about 10,500 on SDS-gels, and appears to exist in stoichiometric amount. We have extracted this subunit from slab gels and compared its amino acid composition with that of subunit III.

Two monoclonal antibody lines directed against subunit IV of cytochrome oxidase: a study of opposite effects.

Two monoclonal lines of antibodies were isolated with specificities against the amino half of Subunit IV of beef heart cytochrome oxidase. The lines had nonoverlapping epitopes. Both bound to the matrix face of membranous oxidase, neither bound to the cytoplasmic face. One line (QA4/C4) stimulated electron transfer in soluble or membranous oxidase, while the other (QA4) inhibited that activity by both oxidase preparations. These effects on electron transfer activity were not altered by the inclusion or omission of detergent. ATP depressed the binding of either antibody to either soluble or membranous oxidase. In the absence of ATP, QA4/C4 stimulated electron transfer only in the high affinity phase of cytochrome c oxidation (with decreased KM and increased Vmax), causing slight inhibition in the low affinity phase (with decreased KM). In the presence of ATP, QA4/C4 abolished the high affinity phase, but did not alter the ATP influence on the low affinity phase. In the absence of ATP, antibodies of line QA4 abolished the low affinity phase, leaving a high affinity phase similar to that induced by ATP. In the presence of ATP, QA4 abolished the high affinity phase, leaving a low affinity phase similar to that seen with ATP alone. This behavior is consistent with the dissection of two catalytic sites for cytochrome c and more than one ATP affector site.