The copper centers of tyramine β-monooxygenase and its catalytic-site methionine variants: an X-ray absorption study

Tyramine β-monooxygenase (TBM) is a member of a family of copper monooxygenases containing two noncoupled copper centers, and includes peptidylglycine monooxygenase and dopamine β-monooxygenase. In its Cu(II) form, TBM is coordinated by two to three His residues and one to two non-His O/N ligands consistent with a [CuM(His)2(OH2)2–CuH(His)3(OH2)] formulation. Reduction to the Cu(I) state causes a change in the X-ray absorption spectroscopy (XAS) spectrum, consistent with a change to a [CuM(His)2S(Met)–CuH(His)3] environment. Lowering the pH to 4.0 results in a large increase in the intensity of the Cu(I)–S extended X-ray absorption fine structure (EXAFS) component, suggesting a tighter Cu–S bond or the coordination of an additional sulfur donor. The XAS spectra of three variants, where the CuM Met471 residue had been mutated to His, Cys, and Asp, were examined. Significant differences from the wild-type enzyme are evident in the spectra of the reduced mutants. Although the side chains of His, Cys, and Asp are expected to substitute for Met at the CuM site, the data showed identical spectra for all three reduced variants, with no evidence for coordination of residue 471. Rather, the K-edge data suggested a modest decrease in coordination number, whereas the EXAFS indicated an average of two His residues at each Cu(I) center. These data highlight the unique role of the Met residue at the CuM center, and pose interesting questions as to why replacement by the cuprophilic thiolate ligand leads to detectable activity whereas replacement by imidazole generates inactive TBM

Electronic Structure Description of the cis-MoOS Unit in Models for Molybdenum Hydroxylases

Copyright © 2008 American Chemical SocietyThe molybdenum hydroxylases catalyze the oxidation of numerous aromatic heterocycles and simple organics and, unlike other hydroxylases, utilize water as the source of oxygen incorporated into the product. The electronic structures of the cis-MoOS units in CoCp2[TpiPrMoVOS(OPh)] and TpiPrMoVIOS(OPh) (TpiPr = hydrotris(3-isopropylpyrazol-1-yl)borate), new models for molybdenum hydroxylases, have been studied in detail using S K-edge X-ray absorption spectroscopy, vibrational spectroscopy, and detailed bonding calculations. The results show a highly delocalized Mo=S pi* LUMO redox orbital that is formally Mo(dxy) with approximately 35% sulfido ligand character. Vibrational spectroscopy has been used to quantitate Mo-Ssulfido bond order changes in the cis-MoOS units as a function of redox state. Results support a redox active molecular orbital that has a profound influence on MoOS bonding through changes to the relative electro/nucleophilicity of the terminal sulfido ligand accompanying oxidation state changes. The bonding description for these model cis-MoOS systems supports enzyme mechanisms that are under orbital control and dominantly influenced by the unique electronic structure of the cis-MoOS site. The electronic structure of the oxidized enzyme site is postulated to play a role in polarizing a substrate carbon center for nucleophilic attack by metal activated water and acting as an electron sink in the two-electron oxidation of substrates.Christian J. Doonan, Nick D. Rubie, Katrina Peariso, Hugh H. Harris, Sushilla Z. Knottenbelt, Graham N. George, Charles G. Young, and Martin L. Kir

Incidence of seizure activity for the seven OBX animals.

<p>Lines show peak seizure number on a given day for each animal. Note that the seizures occurred in clusters rather than being randomly distributed throughout the monitoring period. Three animals had a second seizure cluster (marked by asterisks), data from which were used for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138178#pone.0138178.g003" target="_blank">Fig 3</a>. No seizures were observed in control animals (not shown).</p

Photomicrographs of Nissl stained sagittal sections from a control mouse (left) and an epileptic mouse with a clean removal of the olfactory bulb.

<p>Sections are approximately 0.60 mm lateral to the midline. <b>Lower panels:</b> Photomicrographs from control (left) and epileptic (right) mice showing hippocampus and the CA3 pyramidal cell layer. No overt cell loss, disruption or distortion of the overall structure of the hippocampus was evident in hippocampus of any of the animals. Scale bars: Top, 2 mm; middle, 1 mm; bottom, 25 μm.</p

Scatter plot correlating behavioral seizure severity with seizure duration for individual seizures which occurred in either the first (black circles) or second (red diamonds) seizure cluster.

<p>Data are from the three animals that had two recorded seizure clusters (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138178#pone.0138178.g002" target="_blank">Fig 2</a>). Note the right and upward shift between seizures in the first and second clusters, indicating that seizures in the second cluster were more severe and lasted longer (P<0.001 for both parameters).</p

Scatter plot showing the relationship between seizure frequency and estimated cortical tissue loss in mm³.

<p>A significant correlation between the two parameters was not observed (Pearson product moment, R = -0.218, p = 0.64).</p