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

Abstract

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.

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

“…Following that phase are multiple slower kinetic phases, which cannot be limited by the bimolecular rate and, therefore, are attributed to the changes in the properties of the protein. This behavior contrasts with a simple, monophasic oxidation of ferrous Mb by FCN, 11 or the reduction of ferric myoglobin (Mb) by thionine acetate (TA, Figure S3, left), as expected for a sample with reversible electrochemical behavior. Reactions with Mb quickly reach a redox equilibrium that corresponds to a E 1/2 of −155 mV, in good agreement with the reported value of −153 mV 17 and show little change after the first 5 s. The lack of change in the extent of oxidation of TA by Mb confirms that the sample remains anaerobic over a 1 h measurement and that gradual changes observed in TauD are not due to a slow ingress of atmospheric O 2 .…”

“…These features were previously proposed to arise from redox-linked peptide backbone reorganization. 11 Several other vibrations were assigned to individual metal ligands. The complete loss of the 1558 cm −1 mode in the H99A variant and its retention in the D101Q variant suggest that it arises from the imidazole CC stretching vibration.…”

“…Unexpectedly, our initial investigation of the Fe(II/III) redox couples for these species revealed reversible, redox-linked conformational changes that modulate their redox potentials by at least 300 mV. 11 On the basis of the observed vibrational changes and the magnitudes of the redox hysteresis, we proposed an isomerization that involves more than one primary metal ligand and the reorganization of the protein backbone. In this study, we use site-directed mutagenesis of the primary Fe ligating residues, H99, D101, and H255 (Figure 1), to test this hypothesis and to characterize the contributions of individual ligands to the overall redox response of the active site.…”

G3F: Global, Multidimensional Spectral Regression Analysis

“…A similar approach was later used in the analysis of Raman spectra of methane monooxygenase (Banerjee, Proshlyakov, Lipscomb, & Proshlyakov, 2015). In more recent studies, it was used to obtain completely unknown difference spectra of a redox transition with an unknown potential over a variable polynomial background John, Swain, Hausinger, & Proshlyakov, 2019). In this case, analysis involved two orthogonal parametric vectors (frequency-dependent amplitudes vs. potential-dependent populations).…”

KinESim: Pre-equilibrium kinetic simulation of electrochemical reactions

“…Therefore, it can be used to predict experimental data and to test proposed reaction mechanisms in most chemical processes controlled by a combination of kinetics and thermodynamics. These abilities have recently been demonstrated in a study of how electrochemical mediators affect the observed electrochemical properties of an analyte John, Swain, Hausinger, & Proshlyakov, 2019).…”