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

Abstract

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 FADH2 in the catalytic flavodehydrogenase domain of CDH to haem b 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 > 3 mm, 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 haem b 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.

Structural insight into the calcium ion modulated interdomain electron transfer in cellobiose dehydrogenase

“…When visualizing these results on the homology models of the two domains (Figs. 3A and 4A), it is apparent that the influenced protein regions are located around or close to the proposed domain interaction interface [4,6]. Indeed, we did not observe any important changes on the opposite sides of the two domains (Figs.…”

“…Therefore, we monitored the protein alone (in 50 mM MOPS pH 7.4) or in the presence of 30 mM calcium ions. Calcium was chosen to be a representative of divalent alkali earth metal cations as the other ions tested showed essentially the same behavior [6]. To distinguish between the role of divalent ions and the increase in ionic strength itself, a control utilizing monovalent potassium ions was also performed.…”

“…The lack of divalent cation selectivity together with the requirement for their rather high concentration (mM) and with the differences between the levels of high pH IET activation among individual CDHs pointed again on the possible elimination of negative charges by calcium cations via the cation bridging effect. This hypothesis was further explored by molecular modeling and domain docking and suggested an explanation by highlighting much higher number of possible divalent cation interacting residues in Mt CDH in contrast to other CDHs from Phanerochaete sordida or Corynascus thermophilus [6].…”

“…Interestingly, the effect of calcium differed between individual cellobiose dehydrogenases from various sources which pointed on a relation to the individual protein sequence/structure rather than to the binding of calcium by a specific site in CDH structure. A recent study explored this phenomenon in detail and screened CDHs from twelve different fungi [6]. It was shown, that any divalent alkali metal cation (regardless its atomic radius or electronegativity) at concentrations above 3 mM increases the IET of most cellobiose dehydrogenases.…”

Polysaccharide oxidation by lytic polysaccharide monooxygenase is enhanced by engineered cellobiose dehydrogenase

“…Myriococcum thermophilum ) CDH ( Ch CDH) used in this study has a ca. 200 times lower turnover number for O 2 than for flavin‐dependent 1,4‐benzoquinone reduction . Still, under physiological conditions, CDH might provide catalytically relevant amounts of H 2 O 2 for LPMOs, which have a µ m affinity for this cosubstrate .…”