Biology subjects

Bawn, M.

Publications and source records attributed to Bawn, M..

4 recordsLinked to original sources

bioRxiv2018

Electron Paramagnetic Resonance Investigation of Nitrite Binding in Myoglobin

It has been proposed that myoglobin (Mb) may act as a nitrite reductase under hypoxic conditions. Any mechanism describing such activity should take into account the binding geometry of the ligand to the heme. Crystal structures of horse-heart Mb and human hemoglobin-nitrite complexes suggest that the anion adopts an uncommon O-nitrito binding mode. Electron Paramagnetic Resonance (EPR) spectroscopy was employed to investigate the nature of nitrite binding to Mb at pH values ranging from 6.5 to 10.8. Results suggest that for ferric Mb at low pH, nitrite binds in the O-bound nitrito mode resulting in a low-spin (LS) iron center. Further a high-spin (HS) iron center is observed at high pH in Mb-Nitrite with spectral values different to that of purely HS-Mb that is proposed to be due to an N-bound nitrite. The yields of these two species were found to be influenced by pH.\n\nBackgroundMyoglobin has been theorized to have a role as a nitrite reductase.\n\nResultsO-bound nitrite produces a low-spin ferric heme complex, whilst at high pH a high-spin species is found proposed to be the N-bound form.\n\nConclusionNitrite may bind to heme in myoglobin via N-nitro or O-nitrito mode.\n\nSignificanceThe mechanism of any nitrite reduction will depend on its binding to the heme cofactor.

biochemistry↗

bioRxiv2018

Pulsed ELDOR Measurement of the Distance Between a Spin-Label and Copper (II) Centre in the Copper Loaded R48C Mutant of N. gonorrhoeae Ferric Binding Protein

Distance determination in proteins and biomolecules using pulsed EPR (electron paramagnetic resonance) techniques is becoming an increasingly popular and accessible technique. PELDOR (pulsed electron-electron double resonance), is a technique designed for distance determination over a nanoscopic scale. Here, ferric binding protein (Fbp) is used to demonstrate the practicability of this technique to Cu (II) Metalloproteins. PELDOR is usually applied to bi-radicals or endogenous radicals, and distance determination using pulsed EPR of metal containing centres in biomolecules has been restricted to relaxation experiments. PELDOR distance measurements between a Cu (II) ion and a nitroxide have previously only been reported for model compounds [1, 2].\n\nFbp as the name suggests usually, contains a Fe (III) ion centre. For the purposes of this investigation the Fe (III) ion was removed and replaced by a Cu (II) ion, after a nitroxide spin-label was added to the Fbp using of site directed spin-labelling (SDSL). PELDOR was then applied to measure the distance between the two centres.\n\nSimulation methods were then employed to fully investigate these data and allow a quantitative interpretation of the copper nitroxide PELDOR data. The observed PELDOR time traces were analysed using DEER analysis[3].

biochemistry↗

bioRxiv2018

216 GHz Electron Paramagnetic Resonance of Mycobacterium Tuberculosis Catalase-Peroxidase: The Role of the Arg418 Residue

The catalase-peroxidase protein from Mycobacterium tuberculosis contains a variety of unique structural features including a covalently-linked three amino acid adduct capable of hosting a tyrosine-based radical. Previous work has demonstrated that the Arg418 residue is essential for the catalse but not the peroxidase activity of the protein and crystallography has indicated the residue to be capable of adopting two conformations relative to the adduct-radical. In the present work the WT and Arg418Leu mutant proteins were investigated using high-field electron magnetic resonance spectroscopy. Different sets of g-values were found for each protein indicating different paramagnetic environments. Quantum chemical calculations of model structures were undertaken to elucidate the geometrical environment of the radical. It is proposed that the two sets of g-values correspond to the two conformations of the Arg418 residue. The implications for the catalytic mechanism are discussed.

biochemistry↗

bioRxiv2018

Molecular Dynamics Investigation of the Role of Residues D137 and S315 to INH Binding in KatG

Introduction Abstract Introduction Materials and Methods Results Discussion References Then Mycobacterium tuberculosis (Mtb) catalase-peroxidase (KatG) protein is a bi-functional enzyme that exists as a homodimer of 80-kDa subunits1 (Figure 1). Each subunit binds one heme cofactor, exhibiting a binding pocket environment and sequence homology that classifies the enzyme as a class I peroxidase. KatG also contains a covalently-linked three amino acid adduct in the distal heme pocket, comprised of W107, Y229 and M255 that is not found in monofunctional peroxidases. Mutagenesis studies have indicated that the adduct is necessary for catalase but not peroxidase activity. Existing crystal structures of KatG show adduct formation in each case and a high degree terti ...

biochemistry↗