A Voltammetric Perspective of Multi-Electron and Proton Transfer in Protein Redox Chemistry: Insights From Computational Analysis of Escherichia coli HypD Fourier Transformed Alternating Current Voltammetry

Abstract

This paper explores the impact of pH on the mechanism of reversible disulfide bond (CysS-SCys) reductive breaking and oxidative formation in Escherichia coli hydrogenase maturation factor HypD, a protein which forms a highly stable adsorbed film on a graphite electrode. To achieve this, low frequency (8.96 Hz) Fourier transformed alternating current voltammetric (FTACV) experimental data was used in combination with modelling approaches based on Butler-Volmer theory with a dual polynomial capacitance model, utilizing an automated two-step fitting process conducted within a Bayesian framework. We previously showed that at pH 6.0 the protein data is best modelled by a redox reaction of two separate, stepwise one-electron, one-proton transfers with slightly “crossed” apparent reduction potentials that incorporate electron and proton transfer terms (Eapp20 > Eapp10). Remarkably, rather than collapsing to a concerted two-electron redox reaction at more extreme pH, the same two-stepwise one-electron transfer model with Eapp20 > Eapp10 continues to provide the best fit to FTACV data measured across a proton concentration range from pH 4.0 to pH 9.0. A similar, small level of crossover in reversible potentials is also displayed in overall two-electron transitions in other proteins and enzymes, and this provides access to a small but finite amount of the one electron reduced intermediate state.

Electrochemistry of Lipophilic Redox Enzymes Associated with a Reversible Follow‐up Chemical Reaction – Theoretical Consideration in Cyclic Staircase Voltammetry

“…In a recent work of Bond et al. [28], experimental features similar as those discussed in current work have been reported by redox enzymes undergoing transformation in two successive electron transfer steps. As each measurement in voltametric techniques requires application of a defined bias, this will lead to disturbance of reversible conditions of any electrochemical system coupled with chemical equilibrium.…”

Electron inventory of the iron-sulfur scaffold complex HypCD essential in [NiFe]-hydrogenase cofactor assembly

“…This redox cascade has been proposed to provide reducing equivalents for the synthesis and attachment of ligands to the cofactor precursor, in agreement with our infrared data and the faradaic currents measured upon oxidation and reduction in disulfide motif SS2. Formation and excision of SS2 on the HypCD complex from E. coli is indicative of a two-electron reaction that comprises two consecutive one-electron transfer steps [ 56 , 57 ]. Our data now confirm the redox activity of the [4Fe-4S] cluster and suggest an electron confurcation mechanism that involves two one-electron transfer steps to form the reduced disulfide motif SS2 red ( Figure 8B ).…”

Practical Guide to Large Amplitude Fourier-Transformed Alternating Current Voltammetry─What, How, and Why

“…We and others have found FTacV to be a particularly powerful method in the bioelectrochemical characterization of redox-active metalloenzymes and proteins. 20,21,23,[34][35][36]50 Other examples illustrating the power of this technique can be found in studies of heterogeneous and homogeneous redox catalysis, with highimpact work on fuel cells 25 and carbon dioxide reduction catalysis. 37,51 As is described in detail in previous work, 6,10,17,18,52−54 it is possible to analyze data from FTacV experiments using simulation approaches in order to determine precise mechanistic details (number of electrons, precise midpoint potentials, electron transfer kinetic regimes, etc.).…”

“…Initial method developments focused on small amplitude (typically 5 mV or less) periodic perturbations, resulting in a detailed quantitative analysis of the fundamental (first) and second harmonic components. − After decades of method evolution, which has shifted onto the use of large amplitudes (50–250 mV) allowing access to higher order harmonics, − the FTacV technique can now be performed using commercially available electrochemical workstations . Thus, a wider community of experimentalists who do not need to be experts in building instrumentation or software development can access this technique.…”