Structural Origin of the Large Redox-Linked Reorganization in the 2-Oxoglutarate Dependent Oxygenase, TauD.

2-Oxoglutarate (2OG)-dependent oxygenases catalyze a wide range of chemical transformations via C-H bond activation. Prior studies raised the question of whether substrate hydroxylation by these enzymes occurs via a hydroxyl rebound or alkoxide mechanism and highlighted the need to understand the thermodynamic properties of transient intermediates. A recent spectroelectrochemical investigation of the 2OG-dependent oxygenase, taurine hydroxylase (TauD), revealed a strong link between the redox potential of the Fe(II)/Fe(III) couple and conformational changes of the enzyme. In this study, we show that the redox potential of wild-type TauD varies by 468 mV between the reduction of 2OG-Fe(III)-TauD (-272 mV) and oxidation of 2OG-Fe(II)-TauD (+196 mV). We use active site variants to investigate the structural origin of the redox-linked reorganization and the contributions of the metal-bound residues to the dynamic tuning of the redox potential of TauD. Time-dependent redox titrations show that reorganization occurs as a multistep process. Transient optical absorption and infrared spectroelectrochemistry show that substitution of any metal ligand alters the kinetics and thermodynamics of the reorganization. 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 high redox potentials of the metal. The D101Q and H255Q variants also suppress the conformational change, supporting their involvement in the structural rearrangement. Similar redox-linked conformational changes are observed in another 2OG dependent oxygenase, ethylene-forming enzyme, indicating that dynamic structural flexibility and the associated thermodynamic tuning may be a common phenomenon in this family of enzymes.

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

2-Oxoglutarate (2OG)-dependent dioxygenases catalyze C-H activation while performing a wide range of chemical transformations. In contrast to their heme analogues, non-heme iron centers afford greater structural flexibility with important implications for their diverse catalytic mechanisms. We characterize an in situ structural model of the putative transient ferric intermediate of 2OG:taurine dioxygenase (TauD) by using a combination of spectroelectrochemical and semiempirical computational methods, demonstrating that the Fe(III/II) transition involves a substantial, fully reversible, redox-linked conformational change at the active site. This rearrangement alters the apparent redox potential of the active site between -127 mV for reduction of the ferric state and +171 mV for oxidation of the ferrous state of the 2OG-Fe-TauD complex. Structural perturbations exhibit limited sensitivity to mediator concentrations and potential pulse duration. Similar changes were observed in the Fe-TauD and taurine-2OG-Fe-TauD complexes, thus attributing the reorganization to the protein moiety rather than the cosubstrates. Redox-difference infrared spectra indicate a reorganization of the protein backbone in addition to the involvement of carboxylate and histidine ligands. 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.

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

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 E1/2 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.