Dept. of Biochemistry and Molecular Biology
Oregon Health & Science University
Cellobiose dehydrogenase (CDH) is a unique extracellular flavocytochrome, produced under cellulolytic conditions by the white-rot fungus Phanerochaete chrysosporium. This enzyme consists of a flavin domain and a b-type cytochrome domain connected via a short linker peptide. This enzyme catalyzes the oxidation of cellobiose to cellobionolactone, which is followed by the transfer of electrons to an electron acceptor, either directly by the flavin domain or via the cytochrome domain. The residues Met65, His114, and His163 were targeted for alanine mutation to identify the axial heme ligands. The spectral and kinetic characteristics of H114A were similar to that of wild-type CDH. In contrast, M65A and H163A yielded mainly 70-kDa flavoproteins and very little of the whole 90-kDa flavocytochromes. This strongly suggests Met65 and His163 as ligands, and in the absence of either of the ligands the cytochrome domain is unstable and susceptible to degradation. To further investigate the role of Met65 in modulating the reactivity of the cytochrome domain, a second CDH variant of Met65, M65H, was produced. The flavin reactivity is retained in M65H, but the inter-domain electron transfer is essentially abolished, most likely due to a decrease in the redox potential of the heme. The electronic absorption and resonance Raman spectroscopy data support a bishistidyl coordination. The tertiary structure of the cytochrome M65H variant shows the first example of an iron-N[superscript Î´] His bond in a b-type heme protein. The residues His689 and N732 in the flavin domain were substituted with GIn, Asn, Glu, Asp, Val, Ala and/or His to examine the role of these residues in catalysis. An over WOO-fold decrease in reactivity with cellobiose is observed for all H689 variants, suggesting that this residue acts as a general base in catalysis. The oxidation and binding of substrate by the different N732 variants varies over one to three orders of magnitude, suggesting a role for N732 in the productive binding of the substrate. In addition, the proximity of a neutral residue, such as N732, to the H689 and isoalloxazine ring appears to modulate the acidity of the general base and the electrostatic environment around the flavin ring, respectively.
OGI School of Science and Engineering
Rotsaert, Frederik A.J., "Structure-functions studies on the flavocytochrome cellobiose dehydrogenase from Phanerochaete Chrysosporium" (2003). Scholar Archive. 17.