Structural studies on the extracellular flavocytochrome cellobiose dehydrogenase from Phanerochaete chrysosporium

Abstract: Microorganisms that degrade lignocellulose play an important role in maintaining the global carbon cycle. Under cellulolytic conditions, the fungus Phanerochaete chrysosporium produces an extracellular flavocytochrome, cellobiose dehydrogenase (CDH), with a proposed role in lignocellulose degradation. CDH consists of 755 amino acids including a C-terminal flavodehydrogenase linked by a peptide hinge to an N-terminal b-type cytochrome. The enzyme catalyses the oxidation of cellobiose to cellobiono-1,5-lactone, followed by transfer of electrons to an electron acceptor, either directly by the flavodehydrogenase domain, or via the cytochrome domain. This thesis presents a structural study on the individual domains of P. chrysosporium cellobiose dehydrogenase.The crystal structure of the cytochrome was determined at 1.9 Å resolution. It folds as a ?-sandwich with the topology of the antibody Fab V(H) domain, and the haem iron is ligated by Met65 and His163. This is only the second example of a b-type cytochrome with this ligation. The haem propionates are surface exposed to facilitate interdomain electron transfer.The structure of a cytochrome Met65His mutant was determined at 1.9 Å resolution. In the mutant, the iron is ligated by the histidyl ? and ? nitrogens, rather than the usual N-?/N-?ligation. This is the first example of a bis-His N-?/N-? coordinated protoporphyrin IX iron. The structure of the flavoprotein domain was determined at 1.5 Å resolution. It is partitioned into an FAD-binding subdomain of ?/?-type and a substrate-binding subdomain consisting of a seven-stranded ?-sheet and six ?-helices. Furthermore, the structure of the flavoprotein with the inhibitor cellobiono-1,5-lactam at 1.8 Å resolution lends support to a hydride-transfer mechanism for the reductive-half reaction of CDH although a radical mechanism cannot be excluded.