2-Oxoglutarate (2OG)-dependent dioxygenases catalyze C-H activation while performing a wide range of chemical transformations making their method of action and thermodynamic properties of great interest to industrial synthesis. In contrast to their heme analogues, non-heme iron centers afford greater structural flexibility with important implications for their diverse catalytic mechanisms. Unfortunately, the non-heme and less accessible active sites of these enzymes makes it a challenge to study them. To counteract this issue, we develop a method that uses electrochemical mediators and combines normal pulse spectrovoltammetry (NPSV) with Fourier transform infrared (FTIR) for detection and subsequent global spectral regression analysis to resolve the structural and thermodynamic properties simultaneously. We develop comprehensive semiemipirical kinetic simulation models to investigate the thermodynamic and kinetic limitations of mediators/analyte interactions. These methods are first validated using methylene green and thionine acetate as mediators and myoglobin (Mb) as the analyte. Both the E½ and unbiased redox difference FTIR spectra of the Fe(II)/Fe(III) redox couple of Mb in reduction and oxidation NPSV modes were in good agreement with those reported earlier by independent techniques. The modeling effort yielded a flexible computational tool capable of quantitatively predicting the redox response in mediated electrochemical studies and defining its limitations. These methods are used to characterize an in situ structural model of the putative transient ferric intermediate of 2OG:taurine dioxygenase (TauD), demonstrating that the FeIII/II transition involves a substantial, fully reversible, redox-linked conformational change at the active site. This rearrangement changes the apparent redox potential of the active site between -272 mV for reduction of the ferric state and 196 mV for oxidation of the ferrous state of the 2OG-Fe-TauD complex resulting in a maximal observed redox hysteresis in the wild type enzyme of 468 mV. Quantitative modeling of the transient redox response using two alternative reaction schemes across a variety of experimental conditions strongly supports the proposal for intrinsic protein reorganization as the origin of the experimental observations. We use H99A, D101Q, H255Q, and Y73I variants of TauD to investigate the structural origin of the redox-linked reorganization and the relative contributions of the active site residues to the dynamic tuning of the redox potential of TauD. Extended time-dependent redox titrations show that, in all cases, reorganization occurs as a multi-step process, with individual phases exhibiting different sensitivities to ligand substitutions. The H99A variant shows the largest net redox change relative to the wild type protein, suggesting that redox-coupled protonation of H99 is required for TauD to support highly positive potentials. The effect of the D101Q substitution suggests that changes in the metal coordination of the carboxylate group may be secondary to changes involving H99 and are required for the ensuing reorganization steps. The H255Q substitution inhibits the conformational change, providing evidence for its involvement in the structural rearrangement. An investigation of the pD sensitivity of wild type TauD exposes a protonation event at the active site of TauD most likely attributable to H99 or H255. Ultimately, we propose H99 is protonated in the ferrous form of TauD and forms a hydrogen bond with the protein backbone. Oxidation of the enzyme results in the loss of this hydrogen bond allowing movement in the H99-T100-D101 chain so that D101 can form a bidentate ligand with the ferric iron center.
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