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Biochim Biophys Acta Gen Subj. 2017 Feb;1861(2):157-167. doi: 10.1016/j.bbagen.2016.11.016. Epub 2016 Nov 13.

Interdomain electron transfer in cellobiose dehydrogenase is governed by surface electrostatics.

Author information

1
BioCeV - Institute of Microbiology, The Czech Academy of Sciences, Prumyslova 595, 252 50 Vestec, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43 Prague, Czech Republic.
2
BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France; IPHC, CNRS, UMR7178, 67087 Strasbourg, France.
3
Department of Food Sciences and Technology, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
4
BioCeV - Institute of Microbiology, The Czech Academy of Sciences, Prumyslova 595, 252 50 Vestec, Czech Republic.
5
BioCeV - Institute of Microbiology, The Czech Academy of Sciences, Prumyslova 595, 252 50 Vestec, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43 Prague, Czech Republic. Electronic address: pman@biomed.cas.cz.

Abstract

BACKGROUND:

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.

METHODS:

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.

RESULTS:

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.

CONCLUSIONS:

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.

GENERAL SIGNIFICANCE:

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.

KEYWORDS:

Cellobiose dehydrogenase; Direct electron transfer; Electrostatic interaction; Hydrogen/deuterium exchange; Ion mobility; Mass spectrometry

PMID:
27851982
DOI:
10.1016/j.bbagen.2016.11.016
[Indexed for MEDLINE]

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