Cellobiose dehydrogenase (CDH) from wood degrading fungi represents a subclass of oxidoreductases with unique properties. Consisting of two domains exhibiting interdomain electron transfer, this is the only known flavocytochrome involved in wood degradation. High resolution structures of the separated domains were solved, but the overall architecture of the intact protein and the exact interface of the two domains is unknown. Recently, it was shown that divalent cations modulate the activity of CDH and its pH optimum and a possible mechanism involving bridging of negative charges by calcium ions was proposed. Here we provide a structural explanation of this phenomenon confirming the interaction between negatively charged surface patches and calcium ions at the domain interface.

• MeSH
• fungální proteiny chemie   MeSH
• karbohydrátdehydrogenasy chemie   MeSH
• Sordariales enzymologie   MeSH
• terciární struktura proteinů MeSH
• transport elektronů fyziologie   MeSH
• vápník chemie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• celobiosa metabolismus   MeSH
• cytochromy metabolismus   MeSH
• deuterium metabolismus   MeSH
• elektrony MeSH
• flaviny metabolismus   MeSH
• fungální proteiny metabolismus   MeSH
• glykosylace MeSH
• houby metabolismus   MeSH
• karbohydrátdehydrogenasy metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• oxygenasy se smíšenou funkcí metabolismus   MeSH
• polysacharidy metabolismus   MeSH
• proteinové domény MeSH
• proteolýza MeSH
• sekvence aminokyselin MeSH
• statická elektřina MeSH
• transport elektronů fyziologie   MeSH
• vodík metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH