Direct Electron-Transfer Anisotropy of a Site-Specifically Immobilized Cellobiose Dehydrogenase

Abstract

To study the direct electron transfer (DET) of the multi-cofactor enzyme cellobiose dehydrogenase (CDH) in regard to its orientation on an electrode surface, a recently published, maleimide-based immobilization method was used in combination with sitedirected mutagenesis to establish different orientations on an electrode surface. CDH from Myriococcum thermophilum was chosen for this study, because its protein structure is resolved and the factors influencing the movement of its mobile cytochrome domain (CYT) are established. Seven CDH variants with a surface exposed cysteine residue in different spatial positions were generated for site-specific maleimide coupling. Surface plasmon resonance and cyclic voltammetry showed that all CDH variants, but not the wild-type CDH, bound covalently to gold electrodes or glassy carbon electrodes and were catalytically active. For DET, the CYT domain needs to move from the closed-state conformation, where it obtains an electron from the catalytic FAD cofactor to the open state where it can donate an electron to the electrode. We therefore hypothesized that the mobility of the CYT domain and its distance to the electrode is central for DET. We found that the uniform spatial orientations of CDH influenced DET as follows: an orientation of the two-domain enzyme on the side, with CYT in proximity to the electrode resulted in high DET currents. Orientations with a bigger distance between CYT and the electrode, or orientations where CYT could not swing back to the dehydrogenase domain (DH) to form the closed enzyme conformation, reduced DET. In the latter case calcium ions, that stabilize the closed conformation of CDH, fully recovered DET. The study demonstrates, that a mobile CYT domain can compensate unfavorable orientations of the catalytic domain to a great extent and allows CDH as a multi-cofactor enzyme to transfer electrons even in awkward orientations. The mobile CYT domain reduces the anisotropy of DET, which is also essential for CDH's physiological function as an extracellular, electron transferring enzyme.

Voltammetry and Single‐Molecule In Situ Scanning Tunnelling Microscopy of the Redox Metalloenzyme Human Sulfite Oxidase

“…The reason for this observation remains unknown and is perhaps associated with freezing of the open/closed conformational change on such electrode surfaces. [41] This pattern follows observations for cSO on cysteamine SAMs on polycrystalline gold electrodes. [32] As noted, 8-AOT SAMs on Au(111)-electrode surfaces give "fair" quality CVs, both with an anodic and a cathodic feature, in the absence of sulfite, as well as attractive electrocatalytic voltammograms (Figure 4), reflecting a good combination of positive surface charge densities, SAM structural flexibility, and facile interfacial ET.…”

“…No electrocatalytic voltammetry was observed for h SO on Au(111)/cysteamine SAMs in the presence of sulfite (data not shown). The reason for this observation remains unknown and is perhaps associated with freezing of the open/closed conformational change on such electrode surfaces [41] . This pattern follows observations for cSO on cysteamine SAMs on polycrystalline gold electrodes [32] …”

“…5 indicates that the reductive step limits the overall ET, i. e. the rate of heme reduction is much lower than the rate of oxidation. Such a difference is most likely caused by intramolecular ET between Mo and heme, [30,41] triggered by the conformational change of h SO as rate determining, but only on 8‐AOT and not on cysteamine.…”

“…5 indicates that the reductive step limits the overall ET, i. e. the rate of heme reduction is much lower than the rate of oxidation. Such a difference is most likely caused by intramolecular ET between Mo and heme, [30,41] triggered by the conformational change of hSO on the AOT modified electrode exhibits no catalytic behaviour in the absence of sulfite, but a well-defined electrocatalytic signal in the enzyme substrate saturation range around the heme group equilibrium potential in the presence of variable concentrations of SO 3…”

Enzyme-Based Biosensors: Tackling Electron Transfer Issues

“…The current generated by the enzyme-modified electrodes in the presence of the cellobiose was dependent on the site-specific orientation of the enzyme-mutants ( Figure 13 F). In a similar approach [ 168 ], the same enzyme was immobilized onto a gold electrode by placing cysteine residues only around the CDH DH domain in order to study the influence of CDH CYT domain mobility on the ET rate ( Figure 14 A,B). For DET, the CDH CYT domain needs to move from the closed-state conformation, where it obtains an electron from the catalytic CDH DH to the open state where it can donate an electron to the electrode.…”