Cyclic Voltammetry Measurements.

Gold electrodes were cleaned and prepared with a self-assembled monolayer of 3-mercapto-1-propanol before cyclic voltammetry was carried out in a three-electrode cell as previously described.³⁰ The reference cell side-arm was filled with the same buffer used for the working cell and sparged with argon before final assembly of the reference electrode. Protein samples were dialyzed into buffer solutions of 0.1 M sodium phosphate at pH 7.4 or pH 6.0 or 0.1 M sodium acetate at pH 4.5. The protein solution (≈ 100 μM) in the working cell was allowed to incubate for over 30 minutes under argon flow to avoid the electrocatalytic reduction of oxygen at the low potential limit (≈ −300 mV in these experiments). All potentials were corrected to the standard hydrogen electrode (SHE) using the relationship SHE = SCE + 243 mV at 22 °C.⁴⁹

The 3-mercapto-1-propanol surface modification and buffer conditions used here are resistant to protein adsorption.⁵⁰ Nevertheless, to limit the possible influence of protein adsorption over time, scans were collected as quickly as possible, starting with the faster scan rates (those over 1 V/s) before performing slower ones. A fresh electrode was also used to repeat measurements at slower scan rates. As an additional control, scans in the absence of protein were used to find the lower potential limit available at each scan rate and pH.

Voltammograms were both analyzed and simulated with software included with the potentiostat (CH Instruments) and DigiElch (Gamry). The modeling software is limited in the complexity of systems amenable to modeling, therefore we have reduced the full mechanistic Scheme 1 to a scheme consisting of only one square (Scheme 2). Instead of differentiating between the Met-ligated species with protonated (Met|TH⁺) and deprotonated (Met|T) “trigger” group, the effects of the protonation equilibrium of the “trigger” group were folded into the composite conformational exchange with an equilibrium constant K_C*, as has been previously done in spectroscopic studies of the alkaline transition.⁵¹ The relationship between the equilibrium constant for this modeled ligand switch, K_C*, and the true conformational equilibrium constant, K_C, is determined by eq 7 and rate constants k_f* and k_b* are related to k_f and k_b as follows: k_f* = k_f × (K_H/(K_H + [H⁺])) and k_b* = k_b. At high scan rates (10 – 50 V/s), positions of signals in voltammograms were not affected by coupling to ligand-switch processes and peak positions were used to find reduction potentials for the Met-ligated (EMet*°′, an equilibrium mixture of Met|TH⁺ and Met|T species) and Lys-ligated (ELys°′) forms of the protein. These values, together with K_C^III* values calculated from experimental pK_C^III and pK_H^III values, were used to find K_C^II* values using Scheme 2.

To model the voltammetry according to Scheme 2, simulations were carried out based on the rate constants k_f^III and k_b^III from spectroscopic measurements and reduction potentials EMet*°′ and ELys°′ from fast scan voltammetry. The separation of peak positions increased as progressively faster scan rates were used^49,52 and heterogeneous interfacial electron exchange constants were adjusted until the simulation matched the experiment. With all the parameters optimized for modeling the voltammetry at the highest and slowest scan rates, simulations were done at scan rates that would correspond to the onset of reversibility for the signal of the Lys-ligated form. The input value for ferrous k_b^II was systematically varied in 0.5 s⁻¹ increments, and the narrowest range of values was found for each variant that replicated the scan rates where reversibility was reached in the experiment.

Comparison of voltammetry to digital simulation was also used to measure the background capacitance (C_d).⁴⁹ The C_d value was then multiplied by the scan rate to find the corresponding contribution to the current response. When necessary, this background current was subtracted from the observed voltammetry using SOAS.⁵³