Enzymes in action: Fluorescence monitoring of single redox enzyme molecules.

In this project we focus on the mechanism and kinetics of enzyme reactions by fluorescence detected voltammetry and electrochemistry at the single-molecule level. In particular, we will investigate mechanisms of intramolecular and intermolecular electron transfer, and the exchange of electrons between electrodes and surface-immobilized proteins and enzymes. In the latter case we will develop methods of vectorial electron transfer, with control over orientation, distance, and coupling. By site-selective labeling we can address redox changes of individual cofactors in a multi-center enzyme, singling out individual steps of intramolecular electron transfer. We will take advantage of recently developed methods for monitoring enzyme activity with unsurpassed sensitivity by sensitized fluorescence. These developments open up new vistas for basic studies and for potential applications, where the advances in fluorescence detection and imaging are brought to bear on the field of enzymology. The main objective of the project is to monitor enzyme activity with ultimate sensitivity, down to individual turn-over events at the single-molecule level: 1. This requires monitoring of redox enzyme turn-over by fluorescence detection. 2. Single-molecule detection will be applied to study the dynamics of enzyme reactions 3. To realize the full potential of fluorescence detection, the research will involve: a. Protein engineering of suitable binding sites for fluorescent labeling. b. Preparation and characterization of fluorescently labeled redox proteins. c. Surface assembly of labeled proteins of interest on transparent electrodes. d. Engineering of molecular linkers to optimize electron transfer to the electrode. e. Establishment of protocols for fluorescence-detected voltammography. Recent pilot experiments have established the feasibility of the methods proposed here. The enzymes chosen for further study in this project are nitrite reductase (NiR), laccase (SLAC) and cytochrome c oxidase (CCO). The electron transfer partner of NiR is pseudo-azurin, and cytochrome c is the electron donor for cytochrome c oxidase. Laccases are multi-copper oxidases that are able to oxidize a range of organic chemicals, notably (poly)phenol based compounds, under simultaneous reduction of oxygen. The participants in this project have extensive expertise and many years of experience in handling and manipulating these enzyme systems.