Interdomain electron transfer in cellobiose dehydrogenase is governed by surface electrostatics

• Published in: Biochimica et biophysica acta. General subjects Vol. 1861; no. 2; pp. 157 - 167
• Main Authors: Kadek, Alan, Kavan, Daniel, Marcoux, Julien, Stojko, Johann, Felice, Alfons K.G., Cianférani, Sarah, Ludwig, Roland, Halada, Petr, Man, Petr
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.02.2017; Elsevier
• Subjects: Amino Acid Sequence; Biochemistry, Molecular Biology; biosensors; Carbohydrate Dehydrogenases - metabolism; Cellobiose - metabolism; Cellobiose dehydrogenase; cellulose; Cytochromes - metabolism; deuterium; Deuterium - metabolism; Direct electron transfer; electron transfer; Electron Transport - physiology; Electrons; Electrostatic interaction; electrostatic interactions; Flavins - metabolism; Fungal Proteins - metabolism; fungi; Fungi - metabolism; Glycosylation; Hydrogen - metabolism; hydrogen bonding; Hydrogen-Ion Concentration; Hydrogen/deuterium exchange; Ion mobility; Life Sciences; Mass spectrometry; microbial fuel cells; Mixed Function Oxygenases - metabolism; neutralization; Polysaccharides - metabolism; Protein Domains; Proteolysis; solvents; Static Electricity; Structural Biology; Cellobiose dehydrogenase; Hydrogen/deuterium exchange; DH; HDX-MS; Ion mobility; CDH; IM-MS; Electrostatic interaction; IET; Direct electron transfer; Mass spectrometry; CYT
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2016.11.016

Cellobiose dehydrogenase (CDH) is a fungal extracellular oxidoreductase which fuels lytic polysaccharide monooxygenase with electrons during cellulose degradation. Interdomain electron transfer between the flavin and cytochrome domain in CDH, preceding the electron flow to lytic polysaccharide monooxygenase, is known to be pH dependent, but the exact mechanism of this regulation has not been experimentally proven so far. To investigate the structural aspects underlying the domain interaction in CDH, hydrogen/deuterium exchange (HDX-MS) with improved proteolytic setup (combination of nepenthesin-1 with rhizopuspepsin), native mass spectrometry with ion mobility and electrostatics calculations were used. HDX-MS revealed pH-dependent changes in solvent accessibility and hydrogen bonding at the interdomain interface. Electrostatics calculations identified these differences to result from charge neutralization by protonation and together with ion mobility pointed at higher electrostatic repulsion between CDH domains at neutral pH. In addition, we uncovered extensive O-glycosylation in the linker region and identified the long-unknown exact cleavage point in papain-mediated domain separation. Transition of CDH between its inactive (open) and interdomain electron transfer-capable (closed) state is shown to be governed by changes in the protein surface electrostatics at the domain interface. Our study confirms that the interdomain electrostatic repulsion is the key factor modulating the functioning of CDH. The results presented in this paper provide experimental evidence for the role of charge repulsion in the interdomain electron transfer in cellobiose dehydrogenases, which is relevant for exploiting their biotechnological potential in biosensors and biofuel cells. [Display omitted] •Cellobiose dehydrogenase and its interdomain interaction were studied in solution.•CDH is extensively O-glycosylated in the linker region.•H/D exchange was used to describe structure changes in CDH at different pH values.•Ion mobility points at higher electrostatic repulsion between domains at neutral pH.•Charge neutralization at acidic pH enables the domain interaction in CDH.