A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.

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(D-Glc) and several aldopyranoses at the C2 position to yield the corresponding 2-ketoaldoses and hydrogen peroxide (2). The overall catalytic reaction can be divided into two half-reactions (Scheme 1) obeying a Ping-Pong type mechanism at pH 7 (3): a reductive half-reaction in which the protein-bound flavin receives a hydride equivalent from a sugar substrate to produce the reduced FAD (FADH Ϫ ) and the 2-keto-sugar, and an oxidative half-reaction in which two electrons are transferred from the reduced flavin to O 2 to form H 2 O 2 (2, 3).We reported the first detection of a C4a-hydroperoxyflavin intermediate for a flavoprotein oxidase (4). This flavin intermediate has not been previously observed for flavoprotein oxidases but has been limited to reactions of flavoprotein monooxygenases (5-7). Besides P2O, an intermediate in flavoprotein oxidases has been detected only in the crystalline forms of choline oxidase (1.86 Å) (8) and nitroalkane oxidase (in the presence of cyanide) (9), and in the mutant form, C42S, of an NADH oxidase (10). In the case of glucose oxidase from Aspergillus niger, the generation of a flavin semiquinone-superoxide radical pair using pulse radiolysis resulted in formation of a putative C4a-hydroperoxyflavin intermediate (11). Studies on the P2O reductive half-reaction indicate that P2O binds D-Glc according to a two-step binding mechanism: first an initial complex is formed, followed by an isomerization step to form an active Michaelis ES complex (P2O⅐glucose). Interestingly, this complex shows higher absorbance at 395 nm than does the oxidized enzyme, which is unique among flavoprotein oxidases (3).P2O belongs to the large family of glucose-methanol-choline oxidoreductases (12, 13) with the catalytic side chains His 548 - * This work was supported in part by the Thailand Research Fund throughGrants BRG5180002 (to P. C.), MRG4980117 (to J. S.), and PHD/0151/2547 of the Royal Golden Jubilee Ph.D. program (to M. P.) and by the Faculty of Science, Mahidol University (to P. C.) and from the Faculty of Dentistry Chulalongkorn University (to J. S.). The atomic coordinates and structure factors (codes 3K4B and 3K4C) The abbreviations used are: P2O, pyranose 2-oxidase; ABTS, 2,2Ј-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt; , wavelength; WT, wild type; Mes, 4-morpholineethanesulfonic acid.

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We have documented sensitization to tropical pollen and spores in our population. Its association with atopy suggests that it has a role in allergic diseases in the tropics.