Séminaire Anna Aldinio Colbachini

14/06/2023 - 11:00 - Jacques Sénez

Exploiting active transport for studying redox enzymes in protein film electrochemistry

Protein Film Electrochemistry (PFE) is a powerful technique to learn about various aspects of redox enzymes kinetics and energetics. The enzyme is immobilized on an electrode which acts as a substitute for its natural redox partner. When the electrode potential is appropriate, the enzyme catalyses the redox reaction, resulting in a flow of electrons between the substrate and the electrode. The magnitude of the current generated is proportional both to the amount of enzyme electrically connected to the electrode (gamma) and its catalytic activity (kcat). Electrochemical cells that actively transport the substrate at the electrode are used to prevent its depletion next to the enzyme. As a result, PFE allows to measure (as long as gamma is known) absolute estimates of kcat and to elaborate models that reproduce its evolution as function of instant changes in the experimental conditions (e.g. the pH, the potential, the concentration of a substrate/inhibitor…). In practice this is not always possible due to limitations of the technique.
I will present two works that go beyond the existing setups by benefitting from the mass transport inside the electrochemical cell. Concerning the first work, we observed that Hydrogenase-1 from E. coli (H2 –> 2H+ + 2e-) spontaneously adsorbs on polished graphite electrodes, when they are rotating, even from sub-nanomolar solutions of enzyme. By demonstrating the process is mass-transport limited we could provide an estimation of gamma (else impossible to determine, for that enzyme, by PFE), and thus of kcat.
The second work consists in the development of an original electrochemical cell (the WTE cell) to control the concentrations in solution (e.g. a substrate/inhibitor) over time, with improvedflexibility, and its application to an enzyme. The design is based on an impinging jet: concentrations are tuned thanks to the inline mixing of different buffers.

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