Cellobiose dehydrogenase from the fungi Phanerochaete chrysosporium and Humicola insolens - A flavohemoprotein from Humicola insolens contains 6-hydroxy-FAD as the dominant active cofactor

Cellobiose dehydrogenases (CDH) were purified from cellulose-grown cultures of the fungi Phanerochaete chrysosporium and Humicola insolens, The pH opt imum of the cellobiose cytochrome c oxidoreductase activity of P, chrysospo rium CDH was acidic, whereas that of H, insolens CDH was neutral. The absor ption spectra of the two CDHs showed them to be typical hemoproteins, but t here was a small. difference in the visible region. Limited proteolysis bet ween the heme and flavin domains was performed to investigate the cofactors , There was no difference in absorption spectrum between the heme domains o f P, chrysosporium and H, insolens CDHs. The midpoint potentials of heme at pH 7.0 were almost identical, and no difference in pH dependence was obser ved 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 t wo CDHs is because of the flavin domains. The absorption spectrum of the fl avin domain from H, insolens CDH has absorbance maxima at 343 and 426 and a broad absorption peak at 660 nm, whereas that of P, chrysosporium CDH show ed a normal flavoprotein spectrum. Flavin cofactors were extracted from the flavin domains and analyzed by high-performance liquid chromatography. The flavin cofactor from H. insolens was found to be a mixture of 60% 6-hydrox y-FAD and 40% FAD, whereas that from P, chrysosporium CDH was normal FAD, A fter reconstitution of the deflavo-proteins it was found that flavin domain s containing B-hydroxy-FAD were clearly active but their cellobiose oxidati on rates were lower than those of flavin domains containing normal FAD, Rec onstitution 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 flavi n cofactor but is because of the protein molecule.