Site-directed mutagenesis of the heme axial ligands in the hemoflavoenzymecellobiose dehydrogenase

Cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium is an extra cellular 90-kDa hemoflavoenzyme, organized into an N-terminal heme domain a nd a C-terminal flavin domain. The amino acid residues Met65 and His114 or His163 were suggested to be heme iron ligands, Mutations of these residues were made and mutant proteins were characterized. H114A mutant cultures pro duce a stable hemoflavoenzyme with spectral and kinetic characteristics sim ilar to those of wild-type CDH. The M65A and H163A transformants secrete a 90-kDa hemoflavoenzyme, which oxidizes cellobiose in the presence of 2,6-di chlorophenol-indophenol (DCPIP), but is unable to reduce cytochrome c. The heme domains of the M65A and H163A CDH variants are, however, unstable and susceptible to degradation, both yielding a 70-kDa cellobiose-oxidizing fla voenzyme, The spectral and kinetic characteristics of these truncated varia nts suggest that they contain only their respective flavin domains, The yie ld of the 90-kDa proteins was low and the proteins could not be purified to homogeneity; however, absorption spectra indicate that the 90-kDa proteins do contain the heme domain. Like the truncated flavoenzymes, the 90-kDa va riants reduce DCPIP but are unable to transfer electrons to cytochrome c, i n contrast to wild-type CDH, These findings suggest that H163 and M65 are t he axial heme ligands and that both ligands are required for the reactivity and structural integrity of the heme domain. (C) 2001 Academic Press.