Fluorescent electrochemistry : towards controlled redox-switching of a single metalloprotein

Abstract

The availability of single-molecule fluorescence detection techniques has brought about a breakthrough in the optical studies of biomolecular properties and functions by enabling the study of molecules one at a time. In this thesis, single-molecule detection is used to obtain detailed information of electron transfer reactions at the interface of a copper protein and a gold electrode. The approach is based on Förster resonance energy transfer (FRET) in a suitably labeled redox protein, where a site-specifically attached, fluorescent dye-label (the donor) and the redox center (the acceptor, with a characteristic absorption spectrum) form a FRET pair. The FRET efficiency depends on the overlap integral of emission and absorption bands of the donor and acceptor, respectively. Thus, changes in the absorbance upon reduction or oxidation of the protein can be monitored via changes in the fluorescence intensity of the covalently attached label. Fluorescence detection provides a much enhanced sensitivity compared to absorbance measurements, down to the single-molecule level.

In the present work, azurin was labeled with Cy5, the latter forming the donor in a FRET pair with the Cu redox center of the protein, acting as the acceptor.

In the work described in this thesis, it was shown that fluorescence-detected electrochemistry and chemical control of the redox state-changes of a metalloprotein provide excellent methods to explore the kinetic and thermodynamic mechanisms of redox proteins at the single-molecule level. Especially, fluorescence detected electrochemistry may be used for further investigation of protein‐electrode or protein-protein interactions. The methods can be easily applied to many other redox proteins or enzymes, and have potential for applications in fluorescence‐based biosensors and molecular electronics.