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

Abstract

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.

Figure 1.

Selected residues at the TauD active site. The peptide segment proposed to be linked to structural rearrangement is highlighted in stick mode. Selected hydrogen bonding interactions (yellow) and water molecules (red) are shown. The carbon atoms of the substrates are shown in orange.

Figure 2.

Catalytic cycle of TauD comparing the hydroxyl radical rebound (gray) and alkoxide-forming (blue) mechanisms. Vertical transitions indicate a change in the oxidation state of the Fe center. Diagonal transitions indicate a change in a protonation state. Red structures show the artificial manipulation of the enzyme used in this study to generate an in situ model of the F3 intermediate.

Figure 3.

Normalized population kinetic traces of the amount of 2OG-Fe(II)-TauD oxidized or 2OG-Fe(III)-TauD reduced after addition into solutions containing various mediators. (A) 2OG-Fe(II)-TauD was titrated into solutions containing 1.4-fold excess electron equivalents of the listed mediators, all in their oxidized forms. (B) 2OG-Fe(III)-TauD was titrated into solutions containing 1.4-fold excess electron equivalents of the listed mediators, all in their reduced forms. Time zero indicates the point at which TauD was added to the sample. The reported E_1/2 values of the mediators (vs Ag/AgCl) are shown in parentheses. Similar results were observed at other redox equivalent ratios (Figure S4).

Figure 4.

Transient changes in the redox potential of 2OG-Fe-TauD and three of its variants. E_Ox and E_Rd values (markers) were calculated using eq 1 from the absorbance data. Top row: oxidation of 2OG-Fe(II)-TauD species by FCN. Bottom row: reduction of 2OG-Fe(III)-TauD species by TA. The uncertainty derived from error propagation is shown by the gray area (see SI). The initial, pre-equilibrium bimolecular reaction phases seen in Figure 3 are not shown. *Absorbance data for the oxidation of the H99A variant indicated that the entire population of H99A TauD was immediately oxidized, allowing for the estimation of only the upper limit of E_Ox assuming 99.8% oxidation of H99A TauD.

Figure 5.

Redox-coupled vibrational changes in WT and variants of 2OG-Fe-TauD. Left: Fe(III) vs Fe(II) redox difference IR-NPSV spectra collected in the reduction (red) and oxidation (blue) modes. Reduction spectra are inverted for the purpose of comparison with the oxidation spectra. Right: Experimental (markers) and fitted (lines) ϕ_Rd (red) and ϕ_Ox (blue) NPSV profiles for the indicated TauD proteins were normalized using ΔA₁₆₈₂ (WT), ΔA₁₆₈₃ (Y73I), ΔA₁₆₆₉ (H99A), ΔA₁₆₅₉ (D101Q), and ΔA₁₆₉₂ (H255Q).

Figure 6.

Redox-coupled reorganization of 2OG-Fe-EFE. (A) Fe(III) vs Fe(II) IR-NPSV redox difference spectra collected in the reduction (red) and oxidation (blue) modes. Reduction spectra are inverted for the purpose of comparison with the oxidation spectra. (B) Experimental (markers) and fitted (lines) ϕ_Rd (red) and ϕ_Ox (blue) NPSV profiles. (C) Transient changes in the E_Ox of EFE upon oxidation by FCN.

Figure 7.

Proposed redox-linked conformational changes in TauD. Conformational changes of H99 upon (de)protonation are exemplified by the rotations indicated by the dashed arrows. See text for details.