Resonance Raman spectroscopic studies of cellobiose dehydrogenase from Phanerochaete chrysosporium.
Cellobiose dehydrogenase (CDH), an extracellular hemoflavoenzyme produced by cellulose-degrading cultures of Phanerochaete chrysosporium, oxidizes cellobiose to cellobionolactone. The enzyme contains one 6-coordinate, low-spin b-type heme and one FAD cofactor per monomeric protein. In this work, resonance Raman (RR) spectra are reported for the oxidized, reduced, and deflavo forms of CDH as well as the individual flavin and heme domains of the enzyme obtained by peptide proteolysis. The RR spectra of the flavin and heme groups of CDH were assigned by comparison to the spectra of other hemoflavoenzymes and model compounds. Proteolytic cleavage of the CDH domains had only a minimal spectroscopic effect on the vibrational modes of the heme and FAD cofactors. Excitation of the oxidized CDH holoenzyme at 413 or 442 nm resulted in photoreduction of the heme. However, the same excitation wavelength used on the deflavo form of the enzyme or on the heme domain alone did not cause photoreduction, indicating that photoinitiated electron transfer requires the FAD cofactor. These observations suggest an enzymatic mechanism whereby reducing equivalents obtained from the oxidation of cellobiose are transferred from the FAD to the heme. A similar mechanism has been proposed for flavocytochrome b2 of Saccharomyces cerevisiae which oxidizes lactate to pyruvate (A. Desbois et al., 1989, Biochemistry 28, 8011-8022).
Cohen
J D
JD
Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland 97291-1000, USA.
Bao
W
W
Renganathan
V
V
Subramaniam
S S
SS
Loehr
T M
TM
D001487:Basidiomycota / Q000201:enzymology*; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry*; D004579:Electron Transport; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D010084:Oxidation-Reduction; D011485:Protein Binding; D013059:Spectrum Analysis, Raman
D005656:Fungal Proteins; D005182:Flavin-Adenine Dinucleotide; D006418:Heme; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase