Fig. 5: Generation of surface-immobilized EPR-active nitroxide radical intermediate (STEMPO•) under OCP in the absence and presence of MBA.
The potential was held at +0.6 V such that only the EPR-active species (STEMPO•) was present, and then stepped to +1.1 V such that EPR-silent (STEMPO+) was generated exclusively. The current was monitored at OCP over time and the experiment was performed with and without substrate. EPR spectra were recorded throughout the chronoamperometry-OCP measurement. a, Double integrals of EPR spectra showing regeneration of STEMPO• in the absence of substrate (black dots) and in the presence of 1 mM (red dots) and 5 mM (blue dots) MBA after potential steps at +0.6 V and +1.1 V versus SHE, for 100 s each. b, STEMPO• regeneration rates as a function of substrate concentration (14, 92, 137 and 162 µM s−1 for 1, 5, 10 and 20 mM MBA, respectively; the colours of the data points match the data in a and in Supplementary Figure 14), deduced from a first-order relaxation fit of the variation of STEMPO• concentration over time, as quantified from the EPR data. The solid line shows a fit using the Michaelis–Menten model with Michaelis–Menten constant KM = 6.8 ± 1.3 and rate constant for chemical catalysis kcat = 0.22 s−1 (see main text). Extended Data Fig. 8 provides results with glycerol as the substrate at pH 8.0 and Extended Data Fig. 9 the corresponding data for MBA at pH 7.3 (also Supplementary Section 6).
