scholarly journals Crystal structures of human 3-hydroxyanthranilate 3,4-dioxygenase with native and non-native metals bound in the active site

2017 ◽  
Vol 73 (4) ◽  
pp. 340-348 ◽  
Author(s):  
Lakshmi Swarna Mukhi Pidugu ◽  
Heather Neu ◽  
Tin Lok Wong ◽  
Edwin Pozharski ◽  
John L. Molloy ◽  
...  
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Inductively Coupled Plasma ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Kynurenine Pathway ◽  
Heme Iron ◽  
Resonance Spectroscopy ◽  
Extradiol Dioxygenase ◽  
Paramagnetic Resonance ◽  
Tryptophan Degradation

3-Hydroxyanthranilate 3,4-dioxygenase (3HAO) is an enzyme in the microglial branch of the kynurenine pathway of tryptophan degradation. 3HAO is a non-heme iron-containing, ring-cleaving extradiol dioxygenase that catalyzes the addition of both atoms of O2to the kynurenine pathway metabolite 3-hydroxyanthranilic acid (3-HANA) to form quinolinic acid (QUIN). QUIN is a highly potent excitotoxin that has been implicated in a number of neurodegenerative conditions, making 3HAO a target for pharmacological downregulation. Here, the first crystal structure of human 3HAO with the native iron bound in its active site is presented, together with an additional structure with zinc (a known inhibitor of human 3HAO) bound in the active site. The metal-binding environment is examined both structurally andviainductively coupled plasma mass spectrometry (ICP-MS), X-ray fluorescence spectroscopy (XRF) and electron paramagnetic resonance spectroscopy (EPR). The studies identified Met35 as the source of potential new interactions with substrates and inhibitors, which may prove useful in future therapeutic efforts.

scholarly journals Engineered holocytochrome c synthases that biosynthesize new cytochromes c

2017 ◽  
Vol 114 (9) ◽  
pp. 2235-2240 ◽  
Author(s):  
Deanna L. Mendez ◽  
Shalon E. Babbitt ◽  
Jeremy D. King ◽  
John D’Alessandro ◽  
Michael B. Watson ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Axial Ligand ◽  
Heme Iron ◽  
Resonance Spectroscopy ◽  
Redox Potentials ◽  
Hydrogen Electrode ◽  
Paramagnetic Resonance ◽  
Axial Ligands ◽  
Circular Dichroïsm

Cytochrome c (cyt c), required for electron transport in mitochondria, possesses a covalently attached heme cofactor. Attachment is catalyzed by holocytochrome c synthase (HCCS), leading to two thioether bonds between heme and a conserved CXXCH motif of cyt c. In cyt c, histidine (His19) of CXXCH acts as an axial ligand to heme iron and upon release of holocytochrome c from HCCS, folding leads to formation of a second axial interaction with methionine (Met81). We previously discovered mutations in human HCCS that facilitate increased biosynthesis of cyt c in recombinant Escherichia coli. Focusing on HCCS E159A, novel cyt c variants in quantities that are sufficient for biophysical analysis are biosynthesized. Cyt c H19M, the first bis-Met liganded cyt c, is compared with other axial ligand variants (M81A, M81H) and single thioether cyt c variants. For variants with axial ligand substitutions, electronic absorption, near-UV circular dichroism, and electron paramagnetic resonance spectroscopy provide evidence that axial ligands are changed and the heme environment is altered. Circular dichroism spectra in far UV and thermal denaturation analyses demonstrate that axial ligand changes do not affect secondary structures and stability. Redox potentials span a 400-mV range (+349 mV vs. standard hydrogen electrode, H19M; +252 mV, WT; −19 mV, M81A; −69 mV, M81H). We discuss the results in the context of a four-step mechanism for HCCS, whereby HCCS mutants such as E159A are enhanced in release (step 4) of cyt c from the HCCS active site; thus, we term these “release mutants.”

Structural studies of staphylococcal protease. III. Binding of anions to the spin-labeled enzyme

1978 ◽  
Vol 56 (1) ◽  
pp. 7-12
Author(s):  
Hermann Dugas ◽  
Fernand Gaudet ◽  
Paul Leduc
Keyword(s):  
Active Site ◽  
Spin Label ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Chloride Ions ◽  
Resonance Spectroscopy ◽  
Serine Residue ◽  
Paramagnetic Resonance ◽  
Competitive Inhibitors ◽  
Electron Paramagnetic ◽  
Active Site Region

The staphylococcal protease was coupled at the active-site serine residue with a spin-labeled analog of diisopropyl fluorophosphonate and the interaction of competitive inhibitors such as chloride and acetate anions, as well as N-carbobenzoxy-L-glutamic acid (Z-L-Glu), was investigated by electron paramagnetic resonance spectroscopy. It was observed that the addition of chloride ions to the spin-labeled enzyme increased the freedom of motion of the spin label while the presence of acetate ions and Z-L-Glu resulted in an increase in the immobilization of the spin label. These results suggest that these ions bind to the active site region in different ways.

scholarly journals Electron-paramagnetic-resonance spectroscopy of complexes of xanthine oxidase with xanthine and uric acid

1978 ◽  
Vol 171 (3) ◽  
pp. 653-658 ◽  
Author(s):  
R C Bray ◽  
M J Barber ◽  
D J Lowe
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Uric Acid ◽  
Xanthine Oxidase ◽  
Catalytic Reaction ◽  
Active Site ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Resonance Spectroscopy ◽  
Substrate Molecule ◽  
Paramagnetic Resonance ◽  
Electron Paramagnetic

Molybdenum(V) e.p.r. signals from reduced functional milk xanthine oxidase molecules (the Rapid signals), obtained in the presence of purine substrates and products, were further investigated [cf. Bray & Vänngård, (1969) Biochem. J. 114, 725-734; Pick & Bray (1969) Biochem. J. 114, 735-742]. Xanthine forms two complexes with the enzyme that are believed to correspond to different orientations of the substrate molecule in the active site. Only one complex appears to undergo the catalytic reaction. Non-productive complexes, analogous to theone with xanthine, are not formed by 1-methylxanthine or purine. Uric acid forms more than one e.p.r.-detectable complex, one of which is analogous to the non-productive xanthine complex. The computer program used for handing the e.p.r. data is described briefly.

scholarly journals Tauroursodeoxycholic Acid (TUDCA)—Lipid Interactions and Antioxidant Properties of TUDCA Studied in Model of Photoreceptor Membranes

Membranes ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 327
Author(s):  
Michał J. Sabat ◽  
Anna M. Wiśniewska-Becker ◽  
Michał Markiewicz ◽  
Katarzyna M. Marzec ◽  
Jakub Dybas ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Plasma Membranes ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Protective Action ◽  
Antioxidant Properties ◽  
Model Membranes ◽  
Dynamics Simulation ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Tauroursodeoxycholic Acid

Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid containing taurine conjugated with the ursodeoxycholic acid (UDCA), has been known and used from ancient times as a therapeutic compound in traditional Chinese medicine. TUDCA has recently been gaining significant interest as a neuroprotective agent, also exploited in the visual disorders. Among several mechanisms of TUDCA’s protective action, its antioxidant activity and stabilizing effect on mitochondrial and plasma membranes are considered. In this work we investigated antioxidant activity of TUDCA and its impact on structural properties of model membranes of different composition using electron paramagnetic resonance spectroscopy and the spin labeling technique. Localization of TUDCA molecules in a pure POPC bilayer has been studied using a molecular dynamics simulation (MD). The obtained results indicate that TUDCA is not an efficient singlet oxygen (1O2 (1Δg)) quencher, and the determined rate constant of its interaction with 1O2 (1Δg) is only 1.9 × 105 M−1s−1. However, in lipid oxidation process induced by a Fenton reaction, TUDCA reveals substantial antioxidant activity significantly decreasing the rate of oxygen consumption in the system studied. In addition, TUDCA induces slight, but noticeable changes in the polarity and fluidity of the investigated model membranes. The results of performed MD simulation correspond very well with the experimental results.

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