Cellobiose Dehydrogenase from the Fungi Phanerochaete chrysosporiumand Humicola insolens

Cellobiose dehydrogenases (CDH) were purified from cellulose-grown cultures of the fungi Phanerochaete chrysosporiumand Humicola insolens. The pH optimum of the cellobiose-cytochrome coxidoreductase activity ofP. chrysosporiumCDH was acidic, whereas that of H. insolensCDH was neutral. The absorption spectra of the two CDHs showed them to be typical hemoproteins, but there was a small difference in the visible region. Limited proteolysis between the heme and flavin domains was performed to investigate the cofactors. There was no difference in absorption spectrum between the heme domains ofP. chrysosporiumand H. insolensCDHs. The midpoint potentials of heme at pH 7.0 were almost identical, and no difference in pH dependence was observed over the range of pH 3–9. The pH dependence of cellobiose oxidation by the flavin domains was similar to that of the native CDHs, indicating that the difference in the pH dependence of the catalytic activity between the two CDHs is because of the flavin domains. The absorption spectrum of the flavin domain fromH. insolensCDH has absorbance maxima at 343 and 426 and a broad absorption peak at 660 nm, whereas that of P. chrysosporiumCDH showed a normal flavoprotein spectrum. Flavin cofactors were extracted from the flavin domains and analyzed by high-performance liquid chromatography. The flavin cofactor fromH. insolenswas found to be a mixture of 60% 6-hydroxy-FAD and 40% FAD, whereas that from P. chrysosporiumCDH was normal FAD. After reconstitution of the deflavo-proteins it was found that flavin domains containing 6-hydroxy-FAD were clearly active but their cellobiose oxidation rates were lower than those of flavin domains containing normal FAD. Reconstitution of flavin cofactor had no effect on the optimum pH. From these results, it is concluded that the pH dependence is not because of the flavin cofactor but is because of the protein molecule.