Operando Electron Paramagnetic Resonance for Elucidating the Electron Transfer Mechanism of Coenzymes

One of the most important challenges in chemistry with direct implication in biochemistry is probing the mechanism of electron transfer originating from biological molecules. On the basis of protein film voltammetry, mediated electron transfer and molecular adsorption followed by heterogeneous catalysis result in similar responses for steady-state currents; both processes increase the Faradaic current at a low overpotential. This is typical of NAD-dependent alcohol dehydrogenase (ADH), an oxidoreductase enzyme that uses the interconversion of NAD(+)/NADH coenzyme to catalyze the oxidation of alcohol to aldehyde. We propose a setup based on operando electron paramagnetic resonance (EPR) spectroscopy to investigate the NADH/NAD(+) redox reaction and introduce how to probe free electrons on a carbon electrode surface and correlate them with the electrocatalytic mechanism. Since knowledge of the g-factor may provide information about the electronic structure of the paramagnetic center at the carbon surface, it was found that the concentration of unpaired free electrons responds to both applied overpotential and NADH oxidation, enabling measurement of the in situ dynamics of the electron transfer reaction. A new correlation for the spin concentration reveals an increasing number of free unpaired electrons with increasing applied overpotential and NADH oxidation, which corroborates the controversial hypothesis that quinone groups act as electrocatalysts and not as redox mediators toward the oxidation of NADH to NAD(+). Furthermore, operando EPR provides useful information in probing the electron transfer dynamics on a carbon surface and may be extended to other chemical systems involving electron transfer reactions.