Studying Direct Electron Transfer by Site‐Directed Immobilization of Cellobiose Dehydrogenase

Abstract

Covalent coupling between a surface-exposed cysteine residue and maleimide groups was used to immobilize variants of Myriococcum thermophilum cellobiose dehydrogenase (MtCDH) at multiwall carbon nanotube electrodes. By introducing individual cysteine residues at particular places on the surface of the flavodehydrogenase domain of the flavocytochrome, we are able to immobilize the different variants in different orientations. Our results show that direct electron transfer (DET) occurs exclusively through the haem b cofactor and that the redox potential of the haem is unaffected by the orientation of the enzyme. Electron transfer between the haem and the electrode is fast in all cases and, at high glucose concentrations, the catalytic currents are limited by the rate of inter-domain electron transfer (IET) between the FAD and the haem. Using ferrocene carboxylic acid as a mediator, we find that the total amount of immobilized enzyme is 4 to 5 times greater than the amount of enzyme that participates in DET. The role of IET in the overall DET catalysed oxidation was also demonstrated by the effects of changing Ca 2 + concentration and by proteolytic cleavage of the cytochrome domain on the DET and MET currents.[a] Dr. the same dimension as the GC disc to restrict the spread of the dispersion, and left to dry at room temperature for 2 d.

Direct Electrochemical Enzyme Electron Transfer on Electrodes Modified by Self-Assembled Molecular Monolayers

“…The cysteine mutant with a surface thiol group made it possible to form stable thioether bonds with maleimide groups via click-chemistry, thereby controlling the orientations of the MtCDH mutant on the electrode surface and revealing efficient electrochemical glucose oxidation. Another example reported by Meneghello and associates clearly indicated that the DET-type bioelectrocatalysis of CDH is highly sensitive to the cysteine residues introduced at particular positions [115]. Experimental results also showed that divalent cations (i.e., Ca 2+ and Mg 2+ ) affect the IET kinetics [23,115].…”

“…Another example reported by Meneghello and associates clearly indicated that the DET-type bioelectrocatalysis of CDH is highly sensitive to the cysteine residues introduced at particular positions [115]. Experimental results also showed that divalent cations (i.e., Ca 2+ and Mg 2+ ) affect the IET kinetics [23,115].…”

Enzyme-Based Biosensors: Tackling Electron Transfer Issues

“…For example, Bartlett et al reported on the covalent coupling between a surface-exposed cysteine residue and maleimide groups to immobilize different variants of Myriococcum thermophilum cellobiose dehydrogenase ( Mt CDH) at multiwall carbon nanotube electrodes [ 166 , 167 ] ( Figure 13 E). By placing individual cysteine residues around the surface of the CDH DH domain of the enzyme, they were able to immobilize the different variants with different orientations ( Figure 13 A–D).…”

“…The definition of all used mutants and their abbreviate names can be found in ref. [ 167 ]. (The figure was adopted from ref.…”

From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes

“…This elegant method combines controlled orientation of the protein at the electrode for ET, sufficient flexibility for activity, and expected enhanced stability. In the case of cellulose dehydrogenase, 40% of initial electroactivity was recovered after 2 months of storage while the wild type enzyme loses progressively its activity within 20 days [245,246]. In addition, controlled orientation allowed investigating mechanistic aspects of the ET.…”