Site-directed mutagenesis of the heme axial ligands in the hemoflavoenzyme cellobiose dehydrogenase.
Cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium is an extracellular 90-kDa hemoflavoenzyme, organized into an N-terminal heme domain and 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 produce a stable hemoflavoenzyme with spectral and kinetic characteristics similar 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-dichlorophenol-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 flavoenzyme. The spectral and kinetic characteristics of these truncated variants suggest that they contain only their respective flavin domains. The yield 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 variants reduce DCPIP but are unable to transfer electrons to cytochrome c, in contrast to wild-type CDH. These findings suggest that H163 and M65 are the axial heme ligands and that both ligands are required for the reactivity and structural integrity of the heme domain.
Rotsaert
F A
FA
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, Oregon 97006-8921, USA.
Li
B
B
Renganathan
V
V
Gold
M H
MH
D019943:Amino Acid Substitution; D002237:Carbohydrate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002482:Cellulose / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D008024:Ligands; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D020075:Phanerochaete / Q000201:enzymology* / Q000378:metabolism
D008024:Ligands; D006418:Heme; D006639:Histidine; D002482:Cellulose; D008715:Methionine; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase