¹H-HYSCORE Reveals Structural Details at the Fe(II) Active Site of Taurine:2-Oxoglutarate Dioxygenase

Proton Hyperfine Sublevel Correlation (¹H-HYSCORE) experiments have been used to probe the ligation structure of the Fe(II) active site of taurine:2-oxoglutarate dioxygenase (TauD), a non-heme Fe(II) hydroxylase. To facilitate Electron Paramagnetic Resonance (EPR) experiments, Fe(II) derivatives of the enzyme were studied using nitric oxide as a substitute for molecular oxygen. The addition of NO to the enzyme yields an S = 3/2 {FeNO}⁷ paramagnetic center characterized by nearly axial EPR spectra with g_⊥ = 4 and g_|| = 2. Using results from (i) an X-ray crystallographic study of TauD crystallized under anaerobic conditions in the presence of both cosubstrate 2-oxoglutarate and substrate taurine, (ii) a published theoretical description of the {FeNO}⁷ derivative of this form of the enzyme, and (iii) previous ²H-Electron Spin Echo Envelope Modulation (ESEEM) studies, we were able to assign the proton cross peaks detected in orientation-selected ¹H-HYSCORE spectra. Discrete contributions from the protons of two coordinated histidine ligands were resolved. If substrate taurine is absent from the complex, orientation-selective HYSCORE spectra show cross peaks that are less resolved and when combined with information obtained from continuous wave EPR, support an alternate binding scheme for 2-oxoglutarate. HYSCORE studies of TauD in the absence of 2-oxoglutarate show additional ¹H cross peaks that can be assigned to two distinct bound water molecules. In addition, ¹H and ¹⁴N cross peaks that arise from the coordinated histidine side chains show a change in NO coordination for this species. For all of the TauD species, ¹H hyperfine couplings and their orientations are sensitive to the detailed electronic structure of the {FeNO}⁷ center.