Inter-domain electron transfer in cellobiose dehydrogenase: modulation by pH and divalent cations

The flavocytochrome cellobiose dehydrogenase (CDH) is secreted by wood-decomposing fungi, and is the only known extracellular enzyme with the characteristics of an electron transfer protein. Its proposed function is reduction of lytic polysaccharide mono-oxygenase for subsequent cellulose depolymerization. Electrons are transferred from FADH(2) in the catalytic flavodehydrogenase domain of CDH to haemb in a mobile cytochrome domain, which acts as a mediator and transfers electrons towards the active site of lytic polysaccharide mono-oxygenase to activate oxygen. This vital role of the cytochrome domain is little understood, e.g. why do CDHs exhibit different pH optima and rates for inter-domain electron transfer (IET)? This study uses kinetic techniques and docking to assess the interaction of both domains and the resulting IET with regard to pH and ions. The results show that the reported elimination of IET at neutral or alkaline pH is caused by electrostatic repulsion, which prevents adoption of the closed conformation of CDH. Divalent alkali earth metal cations are shown to exert a bridging effect between the domains at concentrations of >3mm, thereby neutralizing electrostatic repulsion and increasing IET rates. The necessary high ion concentration, together with the docking results, show that this effect is not caused by specific cation binding sites, but by various clusters of Asp, Glu, Asn, Gln and the haemb propionate group at the domain interface. The results show that a closed conformation of both CDH domains is necessary for IET, but the closed conformation also increases the FAD reduction rate by an electron pulling effect

Effect of Deglycosylation of Cellobiose Dehydrogenases on the Enhancement of Direct Electron Transfer with Electrodes

Cellobiose dehydrogenase (CDH) is a monomeric extracellular flavocytochrome composed of a catalytic dehydrogenase domain (DHCDH) containing flavin adenine dinucleotide (FAD), a cytochrome domain (CYTCDH) containing heme b, and a linker region connecting the two domains. In this work, the effect of deglycosylation on the electrochemical properties of CDH from Phanerochaete chrysosporium (PcCDH) and Ceriporiopsis subvermispora (CsCDH) is presented. All the glycosylated and deglycosylated enzymes show direct electron transfer (DET) between the CYTCDH and the electrode. Graphite electrodes modified with deglycosylated PcCDH (dPcCDH) and CsCDH (dCsCDH) have a 40-65% higher I-max value in the presence of substrate than electrodes modified with their glycosylated counterparts. CsCDH trapped under a permselective membrane showed similar changes on gold electrodes protected by a thiol-based self-assembled monolayer (SAM), in contrast to PcCDH for which deglycosylation did not exhibit any different electrocatalytical response on SAM-modified gold electrodes. Glycosylated PcCDH was found to have a 30% bigger hydrodynamic radius than dPcCDH using dynamic light scattering. The basic bioelectrochemistry as well as the bioelectrocatalytic properties are presented

Effect of Deglycosylation of Cellobiose Dehydrogenases on the Enhancement of Direct Electron Transfer with Electrodes

Cellobiose dehydrogenase (CDH) is a monomeric extracellular flavocytochrome composed of a catalytic dehydrogenase domain (DH_CDH) containing flavin adenine dinucleotide (FAD), a cytochrome domain (CYT_CDH) containing heme <i>b</i>, and a linker region connecting the two domains. In this work, the effect of deglycosylation on the electrochemical properties of CDH from Phanerochaete chrysosporium (<i>Pc</i>CDH) and Ceriporiopsis subvermispora (<i>Cs</i>CDH) is presented. All the glycosylated and deglycosylated enzymes show direct electron transfer (DET) between the CYT_CDH and the electrode. Graphite electrodes modified with deglycosylated <i>Pc</i>CDH (d<i>Pc</i>CDH) and <i>Cs</i>CDH (d<i>Cs</i>CDH) have a 40–65% higher <i>I</i>_max value in the presence of substrate than electrodes modified with their glycosylated counterparts. <i>Cs</i>CDH trapped under a permselective membrane showed similar changes on gold electrodes protected by a thiol-based self-assembled monolayer (SAM), in contrast to <i>Pc</i>CDH for which deglycosylation did not exhibit any different electrocatalytical response on SAM-modified gold electrodes. Glycosylated <i>Pc</i>CDH was found to have a 30% bigger hydrodynamic radius than d<i>Pc</i>CDH using dynamic light scattering. The basic bioelectrochemistry as well as the bioelectrocatalytic properties are presented

Mediated electron transfer of cellobiose dehydrogenase and glucose oxidase at osmium polymer modified nanoporous gold electrodes.

Nanoporous and planar gold electrodes were utilised as supports for the redox enzymes Aspergillus niger glucose oxidase (GOx) and Corynascus thermophilus cellobiose dehydrogenase (CtCDH). Electrodes modified with hydrogels containing enzyme, Os-redox polymers and the cross-linking agent poly(ethylene glycol)diglycidyl ether (PEGDGE) were used as biosensors for the determination of glucose and lactose. Limits of detection of 6.0 (± 0.4), 16.0 (± 0.1) and 2.0 (± 0.1) μM were obtained for CtCDH modified lactose and glucose biosensors and GOx modified glucose biosensors, respectively, at nanoporous gold electrodes. Biofuel cells comprised of GOx and CtCDH modified gold electrodes were utilised as anodes, together with Myrothecium verrucaria bilirubin oxidase (MvBOD) or Melanocarpus albomyces laccase (rMaLc) as cathodes, in biofuel cells. A maximum power density of 41 μW/cm2 was obtained for a CtCDH/MvBOD biofuel cell in 5 mM lactose and O2 saturated buffer (pH 7.4, 0.1 M phosphate, 150 mM NaCl)