Application of bifunctional reagents for immobilization of proteins on a carbon electrode surface: Oriented immobilization of photosynthetic reaction centers

Abstract

A new kind of bifunctional reagent was used to immobilize covalently monolayers of photosynthetic reaction centers (RCs) on a carbon electrode surface. Condensed aromatic rings were used as an anchor group for chemisorption on the basal-plane surface of a pyrolytic graphite electrode and chemically active functional groups were used to immobilize the RCs covalently via the amino acid residues of the protein. The RCs were randomly immobilized via lysine residuals when the bifunctional reagent activated for the reaction with amino groups was applied. An oriented immobilization of the RCs via the cysteine residual located at their accepting side was achieved when an electrode surface activated for thiol binding was used. A dramatic difference in the photoinduced currents was observed for different orientations of the RCs immobilized on the electrode surface. The small separation between the quinone sites inside the RCs and the electrode surface in the case of oriented RCs provides efficient non-diffusional electron transfer, and application of an additional solubilized electron transfer mediator does not affect the photocurrent. Electrochemical oxidation of the immobilized electron transfer mediator was shown to be the limiting step of photocurrent formation and a quantum efficiency of ca. 60% (for the absorbed light) was calculated for the photocurrent generation. In the case of randomly oriented RCs the photocurrent was much smaller, but it could be increased by application of a diffusionally mobile electron transfer mediator.