Electron Paramagnetic Resonance and Electron Spin

Authors: Stanisław K Hoffmann Janina Goslar Stefan Lijewski

Publish Date: 2013/02/24

Volume: 44, Issue: 7, Pages: 817-826

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Abstract

Co2+ binding to the nicotinamide adenine dinucleotide NAD+ molecule in water solution was studied by electron paramagnetic resonance EPR and electron spin echo at low temperatures Cobalt is coordinated by NAD+ when the metal is in excess only but even in such conditions the Co/NAD+ complexes coexist with CoH2O6 complexes EPR spinHamiltonian parameters of the Co/NAD+ complex at 6 K are g z  = 201 g x  = 238 g y  = 306 A z  = 94 × 10−4 cm−1 A x  = 33 × 10−4 cm−1 and A y  = 71 × 10−4 cm−1 They indicate the lowspin Co2+ configuration with S = 1/2 Electron spin echo envelope modulation spectroscopy with Fourier transform of the modulated spin echo decay shows a strong coordination by nitrogen atoms and excludes the coordination by phosphate and/or amide groups Thus Co2+ ion is coordinated in pseudotetrahedral geometry by four nitrogen atoms of adenine rings of two NAD+ moleculesNAD+ molecule is a potential binder of metal ions Coordinated metal ions can modify the electron density distribution over the molecule it can also influence a possibility of configuration changes as well as the dynamics of coenzyme binding what may be essential for its biochemical activity in the Wilson disease or a poisoning There exist four potential sites of metallation in NAD+ molecule oxygen atoms of the phosphate groups the amide group of the nicotinamide moiety oxygens of the ribose molecules and nitrogens of adenosine On the basis of kinetic potentiometric and calorimetric studies it was suggested that various metal ions prefer coordination at different sitesNi2+ ions are coordinated by phosphate moiety and interact simultaneously with adenine and nicotinamide rings of NAD+ as inferred from temperature–jump relaxation studies 9 10 11 The VO4+ ion acts as a diphosphate chelator in the acidic range of water solution whereas in the basic range a binding of the ion to the deprotonated hydroxyls of two ribose moieties was suggested 12 The Mn2+ ion was found as chelated by N and O of the nicotinamide group 13 and as bonded to oxygens of all the phosphates in phosphate NAD NADP 14 Cr5+ ions were identified as oxygen bonded to the NAD+ 15 A coordination of Mg2+ to a hydroxyl group of the ribose was studied by the density functional theory DFT calculations 8 Cobalt ion complexation by NAD+ was studied by calorimetry and potentiometry methods and it was suggested that similarly to Ni2+ the Co2+ ions can be chelated by oxygens of the phosphorous group and nitrogens of the adenosine ring 11 The abovementioned coordination modes need a confirmation by a microscopic method as for example the electron paramagnetic resonance EPR or electron spin echo envelope modulation ESEEM spectroscopy which are able to identify the ligand atoms around a paramagnetic central ion Such investigations we have recently performed for identification of Cu2+ binding sites in NAD+ in water solutions 16 We have shown that Cu2+ is coordinated by two hydroxyl oxygen atoms of ribose moieties of two NAD+ molecules and four solvated H2O molecules forming axially deformed octahedral chromophore CuO2H2O4Nicotinamide adenine dinucleotide βNAD+ used in the experiments was purchased from Sigma without additional purification Cobalt nitrate from POCh Gliwice Poland was used after double crystallization from water All the solutions were prepared with deionized and then distilled water for various pH values with excess of ligand or excess of metal with metal ions concentration in the range 85–90 × 10−3 M We have found that above pH 82 a poorly soluble pink color precipitation occurs and the best NAD+ coordination conditions exist in the samples with excess of metal Thus we present the EPR results for samples with the metaltoligand concentration ratio ML = 451 with C Co = 9 × 10−3 M C NAD = 2 × 10−3 MEPR and ESE measurements were performed for the Co/NAD+ system using a Bruker ESP380E FT/CW spectrometer with a loopgap resonator equipped with a helium flow Oxford CF935 cryostat EPR and ESE measurements were done at low temperatures 4–10 K for frozen solutions using glycerol/water solvent This solvent allowed obtaining uniform glasses after rapid freezing at liquid nitrogen Even at such low temperature a saturation effect was easily visible thus to avoid this effect we recorded the spectra at high microwave power attenuation of 45 dB 0007 mW The EPR spectrum was simulated using the Bruker SimFonia routine In pulsed EPR experiments the ESE signal was used for recording the echodetected ED EPR spectra and for observations of the two and threepulse ESE decay The ED EPR spectra are obtained when the ESE amplitude is recorded during magnetic field sweep through the EPR spectrum The twopulse ESE signal was excited by two 24 ns pulses excitation bandwidth of 18 mT with interpulse interval τ = 96–176 ns at 272 mT The maximal echo amplitude was observed for τ = 96 as expected for echo decay modulated by protons The stimulated ESE was generated by three 24 ns pulses with first interpulse interval τ = 96–176 ns and varied the second interval T starting from 96 ns with 8 ns step

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