Fourier Transform Infrared Spectrovoltammetry and Quantitative Modeling of Analytes in Kinetically Constrained Redox Mixtures

Abstract

Redox-active analytes that do not support direct electron transfer on the electrode, such as proteins with buried redox centers, pose challenges to characterization of their structural and thermodynamic properties. Investigations of indirect transitions in analytes supported by complex redox mixtures require a careful balance between kinetic limitations and spectral interference from the mediators. Using methylene green and thionine acetate as redox mediators and myoglobin as the analyte, we demonstrate that normal pulse spectrovoltammetry (NPSV) with Fourier transform infrared (FT-IR) detection and subsequent global spectral regression analysis can resolve structural and thermodynamic properties simultaneously with little a priori information. Both the E 1/2 and unbiased redox difference FT-IR spectra of the Fe­(II)/Fe­(III) redox couple of myoglobin in reduction and oxidation NPSV modes were in good agreement with those reported earlier by independent techniques. The thermodynamic and kinetic limitations of mediators/analyte interactions were investigated using comprehensive semiempirical kinetic simulation models. This modeling effort yielded a flexible computational tool capable of quantitatively predicting the redox response in mediated electrochemical studies and defining its limitations, thus greatly expanding the range and precision of the formal mediator/analyte concentration ratio rule.

Strongly Coupled Redox-Linked Conformational Switching at the Active Site of the Non-Heme Iron-Dependent Dioxygenase, TauD

“…Such bias can be reduced or eliminated at higher FCN concentrations in both models in agreement with the observed TauD Ox  ; however, the two models predicted an increasingly divergent response of the apparent reduction potential with increasing FCN. The observed TauD Rd  showed distinctly better agreement with model [2] than with [1].The complete removal of FCN as a mediator resulted in several characteristic features in from the imbalance between the reduction and oxidation rates31 and could be used to further discriminate between models [1] and [2](Fig. 8, Fig.…”

“…Fe-TauD is redox inactive on the electrode without mediators (MG, TA, and FCN), each of which is also described by its own ΔStot , E½, and n. 31 The close similarities between the reduction and oxidation FTIR spectra in all three complexes show that the observed redox processes are fully reversible. The spectra were dominated by vibrational changes in the 1550-1700 cm -1 region, outside of which only a vibration at ~1400 cm -1 was consistently observed in all forms.…”

“…Results of fitting  to Nernstian profiles with n = 1 are shown in Table S1. The appearance of a large redox hysteresis in all three forms of TauD contrasts with the behavior of Mb using the same OTTLE cell, electrode, and mediators (except FCN), 31 raising the possibility that the shift in potentials originates from the difference in the specific interactions of reduced and oxidized forms of the mediator with the protein moiety rather than ½ A E of the metal. This possibility was excluded using Zn 2+ -substituted TauD, 20 which was found to be redox inactive ( Fig.…”

“…NPSV data were analyzed by a non-linear global spectral regression (GSR) 35 and kinetic simulations were performed using KinESim 36 packages for Igor Pro. 31 The solution rate constants ksol for the reaction of MG and TA with Fe(III)-TauD were determined as previously described for myoglobin (Mb). 31 The ksol for the oxidation of Fe(II)-TauD by FCN was determined anaerobically while stirring against 100 µM FCN in 25 mM Tris buffer, pH 8.…”

Strongly Coupled Redox-Linked Conformational Switching at the Active Site of the Non-Heme Iron-Dependent Dioxygenase, TauD

“…3). In the single state model [1], identical to what was used for Mb, 31 TauD exists in a simple redox equilibrium with a single redox potential, ½ development of an apparent hysteresis between -60 and +140 mV in the reduction and oxidation modes, respectively, and the appearance of minor redox transitions ( Fig. 5 and Table S4) A different response can be expected using [2] in the absence of FCN, because protein isomerization provides an alternative pathway for oxidation of Aʹ via A.…”