Piotr K. Grzyska

Insight into the mechanism of an iron dioxygenase by resolution of steps following the FeIV═O species

Iron oxygenases generate elusive transient oxygen species to catalyze substrate oxygenation in a wide range of metabolic processes. Here we resolve the reaction sequence and structures of such intermediates for the archetypal non-heme FeII and α-ketoglutarate-dependent dioxygenase TauD. Time-resolved Raman spectra of the initial species with 16O18O oxygen unequivocally establish the FeIV═O structure. 1H/2H substitution reveals direct interaction between the oxo group and the C1 proton of substrate taurine. Two new transient species were resolved following FeIV═O; one is assigned to the νFeO mode of an FeIII─O(H) species, and a second is likely to arise from the vibration of a metal-coordinated oxygenated product, such as FeII─O─C1 or FeII─OOCR. These results provide direct insight into the mechanism of substrate oxygenation and suggest an alternative to the hydroxyl radical rebinding paradigm.

Measuring the Orientation of Taurine in the Active Site of the Non-Heme Fe(II)/α-Ketoglutarate-Dependent Taurine Hydroxylase (TauD) Using Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy

The position and orientation of taurine near the non-heme Fe(II) center of the α-ketoglutarate (α-KG)-dependent taurine hydroxylase (TauD) was measured using Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy. TauD solutions containing Fe(II), α-KG, and natural abundance taurine or specifically deuterated taurine were prepared anaerobically and treated with nitric oxide (NO) to make an S = 3/2 {FeNO}(7) complex that is suitable for robust analysis with EPR spectroscopy. Using ratios of ESEEM spectra collected for TauD samples having natural abundance taurine or deuterated taurine, (1)H and (14)N modulations were filtered out of the spectra and interactions with specific deuterons on taurine could be studied separately. The Hamiltonian parameters used to calculate the amplitudes and line shapes of frequency spectra containing isolated deuterium ESEEM were obtained with global optimization algorithms. Additional statistical analysis was performed to validate the interpretation of the optimized parameters. The strongest (2)H hyperfine coupling was to a deuteron on the C1 position of taurine and was characterized by an effective dipolar distance of 3.90 ± 0.25 Å from the {FeNO}(7) paramagnetic center. The principal axes of this C1-(2)H hyperfine coupling and nuclear quadrupole interaction tensors were found to make angles of 26 ± 5 and 52 ± 17°, respectively, with the principal axis of the {FeNO}(7) zero-field splitting tensor. These results are discussed within the context of the orientation of substrate taurine prior to the initiation of hydrogen abstraction.

Metal and substrate binding to an Fe(II) dioxygenase resolved by UV spectroscopy with global regression analysis

The addition of divalent metal ions or substrate taurine to TauD, an alpha-ketoglutarate-dependent dioxygenase, alters its UV absorption, as clearly observed by monitoring the proteins difference spectra. Binding of metal ions leads to a decrease in absorption at approximately 297 nm and modulation of other features. A separate signature with enhanced absorption at approximately 295 nm is identified for binding of taurine. These narrow ( approximately 700 cm(-1)) and intense ( approximately 0.5mM(-1) cm(-1)) spectral changes are attributed to ligand-induced protein conformational changes affecting the environment of aromatic residues. The changes in the UV difference spectra were exploited to assess directly the thermodynamics and kinetics of ligand interactions in wild-type TauD and selected variants. This approach holds promise as a new tool to probe ligand-induced conformational changes in a wide range of other proteins. Experimental and quantification approaches for a reliable analysis of protein absorption below 320 nm are presented.