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Stable, site-specific immobilization of redox proteins and enzymes is of interest for the development of biosensors and biofuel cells, where the long-term stability of enzymatic electrodes as well as the possibility of controlling the orientation of the biomolecules at the electrode surface have a great importance. Ideally, it would be desirable to immobilize redox proteins and enzymes in a specific orientation, but still with some flexibility to optimize reaction kinetics. In this work, we establish such an approach by using site-directed mutagenesis to introduce cysteine residues at specific locations on the protein surface and the reaction between the free thiol group and maleimide groups attached to the electrode surface to immobilize the mutated enzymes. Using cellobiose dehydrogen-ase (CDH) as a model system, carbon nanotube electrodes were first covalently modified with maleimide groups following a modular approach based on electrografting of primary amines at the carbon surface and solid-phase synthesis methodology to elaborate the surface-modified electrode. The CDH-modified electrodes were tested for direct electron transfer (DET), showing high catalytic currents as well as excellent long-term storage stability. The key advantage of this method is its great flexibility, as the main components of the modification can be independently varied to change the local environment at the electrode surface and a wide range of redox proteins or enzymes can be specifically engineered to present cysteine residues at their surface for oriented immobilization.

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.

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