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 <i>S</i> = 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, ¹H and ¹⁴N 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 ²H 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 C₁–²H 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

HYSCORE Analysis of the Effects of Substrates on Coordination of Water to the Active Site Iron in Tyrosine Hydroxylase

Tyrosine hydroxylase is a mononuclear non-heme iron monooxygenase found in the central nervous system that catalyzes the hydroxylation of tyrosine to yield l-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. Catalysis requires the binding of tyrosine, a tetrahydropterin, and O₂ at an active site that consists of a ferrous ion coordinated facially by the side chains of two histidines and a glutamate. We used nitric oxide as a surrogate for O₂ to poise the active site iron in an <i>S</i> = ³/₂ {FeNO}⁷ form that is amenable to electron paramagnetic resonance (EPR) spectroscopy. The pulsed EPR method of hyperfine sublevel correlation (HYSCORE) spectroscopy was then used to probe the ligands at the remaining labile coordination sites on iron. For the complex formed by the addition of tyrosine and nitric oxide, TyrH/NO/Tyr, orientation-selective HYSCORE studies provided evidence of the coordination of one H₂O molecule characterized by proton isotropic hyperfine couplings (<i>A</i>_iso = 0.0 ± 0.3 MHz) and dipolar couplings (<i>T</i> = 4.4 and 4.5 ± 0.2 MHz). These data show complex HYSCORE cross peak contours that required the addition of a third coupled proton, characterized by an <i>A</i>_iso of 2.0 MHz and a <i>T</i> of 3.8 MHz, to the analysis. This proton hyperfine coupling differed from those measured previously for H₂O bound to {FeNO}⁷ model complexes and was assigned to a hydroxide ligand. For the complex formed by the addition of tyrosine, 6-methyltetrahydropterin, and NO, TyrH/NO/Tyr/6-MPH₄, the HYSCORE cross peaks attributed to H₂O and OH^– for the TyrH/NO/Tyr complex were replaced by a cross peak due to a single proton characterized by an <i>A</i>_iso of 0.0 MHz and a dipolar coupling (<i>T</i> = 3.8 MHz). This interaction was assigned to the N₅ proton of the reduced pterin

Pulsed EPR Study of Amino Acid and Tetrahydropterin Binding in a Tyrosine Hydroxylase Nitric Oxide Complex: Evidence for Substrate Rearrangements in the Formation of the Oxygen-Reactive Complex

Tyrosine hydroxylase is a nonheme iron enzyme found in the nervous system that catalyzes the hydroxylation of tyrosine to form l-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. Catalysis requires the binding of three substrates: tyrosine, tetrahydrobiopterin, and molecular oxygen. We have used nitric oxide as an O₂ surrogate to poise Fe­(II) at the catalytic site in an <i>S</i> = ³/₂, {FeNO}⁷ form amenable to EPR spectroscopy. ²H-electron spin echo envelope modulation was then used to measure the distance and orientation of specifically deuterated substrate tyrosine and cofactor 6-methyltetrahydropterin with respect to the magnetic axes of the {FeNO}⁷ paramagnetic center. Our results show that the addition of tyrosine triggers a conformational change in the enzyme that reduces the distance from the {FeNO}⁷ center to the closest deuteron on 6,7-²H-6-methyltetrahydropterin from >5.9 Å to 4.4 ± 0.2 Å. Conversely, the addition of 6-methyltetrahydropterin to enzyme samples treated with 3,5-²H-tyrosine resulted in reorientation of the magnetic axes of the <i>S</i> = ³/₂, {FeNO}⁷ center with respect to the deuterated substrate. Taken together, these results show that the coordination of both substrate and cofactor direct the coordination of NO to Fe­(II) at the active site. Parallel studies of a quaternary complex of an uncoupled tyrosine hydroxylase variant, E332A, show no change in the hyperfine coupling to substrate tyrosine and cofactor 6-methyltetrahydropterin. Our results are discussed in the context of previous spectroscopic and X-ray crystallographic studies done on tyrosine hydroxylase and phenylalanine hydroxylase

Characterization of Water Coordination to Ferrous Nitrosyl Complexes with <i>fac</i>-N₂O, <i>cis</i>-N₂O₂, and N₂O₃ Donor Ligands

Electron paramagnetic resonance (EPR) experiments were done on a series of <i>S</i> = ³/₂ ferrous nitrosyl model complexes prepared with chelating ligands that mimic the 2-His-1-carboxylate facial triad iron binding motif of the mononuclear nonheme iron oxidases. These complexes formed a comparative family, {FeNO}⁷(N₂O_<i>x</i>)­(H₂O)_3–<i>x</i> with <i>x</i> = 1–3, where the labile coordination sites for the binding of NO and solvent water were fac for <i>x</i> = 1 and cis for <i>x</i> = 2. The continuous-wave EPR spectra of these three complexes were typical of high-spin <i>S</i> = ³/₂ transition-metal ions with resonances near <i>g</i> = 4 and 2. Orientation-selective hyperfine sublevel correlation (HYSCORE) spectra revealed cross peaks arising from the protons of coordinated water in a clean spectral window from <i>g</i> = 3.0 to 2.3. These cross peaks were absent for the {FeNO}⁷(N₂O₃) complex. HYSCORE spectra were analyzed using a straightforward model for defining the spin Hamiltonian parameters of bound water and showed that, for the {FeNO}⁷(N₂O₂)­(H₂O) complex, a single water conformer with an isotropic hyperfine coupling, <i>A</i>_iso = 0.0 ± 0.3 MHz, and a dipolar coupling of <i>T</i> = 4.8 ± 0.2 MHz could account for the data. For the {FeNO}⁷(N₂O)­(H₂O)₂ complex, the HYSCORE cross peaks assigned to coordinated water showed more frequency dispersion and were analyzed with discrete orientations and hyperfine couplings for the two water molecules that accounted for the observed orientation-selective contour shapes. The use of three-pulse electron spin echo envelope modulation (ESEEM) data to quantify the number of water ligands coordinated to the {FeNO}⁷ centers was explored. For this aspect of the study, HYSCORE spectra were important for defining a spectral window where empirical integration of ESEEM spectra would be the most accurate

Lactate Racemase Nickel-Pincer Cofactor Operates by a Proton-Coupled Hydride Transfer Mechanism

Lactate racemase (LarA) of <i>Lactobacillus plantarum</i> contains a novel organometallic cofactor with nickel coordinated to a covalently tethered pincer ligand, pyridinium-3-thioamide-5-thiocarboxylic acid mononucleotide, but its function in the enzyme mechanism has not been elucidated. This study presents direct evidence that the nickel-pincer cofactor facilitates a proton-coupled hydride transfer (PCHT) mechanism during LarA-catalyzed lactate racemization. No signal was detected by electron paramagnetic resonance spectroscopy for LarA in the absence or presence of substrate, consistent with a +2 metal oxidation state and inconsistent with a previously proposed proton-coupled electron transfer mechanism. Pyruvate, the predicted intermediate for a PCHT mechanism, was observed in quenched solutions of LarA. A normal substrate kinetic isotope effect (<i>k</i>_H/<i>k</i>_D of 3.11 ± 0.17) was established using 2-α-²H-lactate, further supporting a PCHT mechanism. UV–visible spectroscopy revealed a lactate-induced perturbation of the cofactor spectrum, notably increasing the absorbance at 340 nm, and demonstrated an interaction of the cofactor with the inhibitor sulfite. A crystal structure of LarA provided greater resolution (2.4 Å) than previously reported and revealed sulfite binding to the pyridinium C4 atom of the reduced pincer cofactor, mimicking hydride reduction during a PCHT catalytic cycle. Finally, computational modeling supports hydride transfer to the cofactor at the C4 position or to the nickel atom, but with formation of a nickel-hydride species requiring dissociation of the His200 metal ligand. In aggregate, these studies provide compelling evidence that the nickel-pincer cofactor acts by a PCHT mechanism

Induced sputum gene expression of study participants.

†<p>glyceraldehyde 3-phosphate dehydrogenase.</p

Lung function of study participants.

<p>*Data are categorical variables and the percent represents number of participants with concavity or reversibility divided by total number of participants in each group.</p

Simple linear regression between percent neutrophils (independent variable) and percent predicted FEV₁ (dependent variable).

<p>Dots represent individual estimates. Solid line is made to linear best fit model.</p

Exposure-outcome relationships between exhaled concentration of CO and inflammatory markers.

†<p>glyceraldehyde 3-phosphate dehydrogenase.</p>‡<p>Log-linear scale after back-transformation.</p

Characteristics of study participants.

<p>Characteristics of study participants.</p