Biomedical EPR at the University of Essex

Biomedical EPR Facility
at the University of Essex

We are open for new collaborations, contact Dr Dima Svistunenko for details

We are constantly looking for new PhD students and postdoctoral fellows

The Facility. All biological processes involve redox chemical reactions, that is the the reactions in which electrons are being transferred from one compound to another. When a state of a biomolecule is formed with an odd number of electrons, it is called paramagnetic and can be detected by the method of Electron Paramagnetic Resonance (EPR) spectroscopy. Many biological and biomedical problems are addressed in the School's research with the help of the Biomedical EPR Facility.

The magnet of our Bruker EMX continuous wave EPR spectromete

About EPR. Different paramagnetic centres, such as transition metal ions and free radicals, are characterised by very specific EPR spectrum signatures. In addition, local microenvironment of the centres also has a measurable effect on the spectrum. Therefore a lot of structural information about paramagnetic intermediates of redox reactions can be extracted from the EPR spectra. By following the changes of the EPR spectra over the time of the reaction, important kinetic information can be obtained which may constitute a basis for a reaction mechanism modelling.

A moving picture illustration of how EPR works is available here. One can also practice, in a playful way, in producing different lineshapes of an EPR spectrum by using this clickable EPR spectra simulation tool.

The 4122 SP Bruker high quality resonator is being attached to the Oxford Instruments cryostate for low temperature EPR measurements.

The Six Form student Deborah Asante analyses EPR spectra of cytoglobin.

Scope of Research. Since its foundation in 1996, the Facility has contributed to many publications on a broad range of subjects: the oxidative stress in animals and plants, the role of nitric oxide in a variety of pathological conditions, electron transfer processes in proteins and enzymes, the role of protein bound free radicals and high valence haem states in the peroxidative reactions, the search for new blood substitutes, iron metabolism is cells.

We are always open for new biologically oriented research themes and would be happy to establish new links with the scientists who might be interested in using our equipment and our expertise.

A Bruker Variable Temperature control unit adapted for step-wise annealing of short lived paramagnetic centres.

Our home-made machine for freezing samples on a cold metal surface with an Update Instrument rapid freeze-quench system for controlled mixing of up to four liquids.

Techniques and methods available:

  • Room temperature EPR measurements including kinetic studies
  • Low temperature (from 4 K) measurements
  • Overlapping spectra deconvolution into individual EPR signals
  • Spectra subtraction with correction for frequency
  • Making EPR sample by the rapid freeze-quenching of the reaction mixtures (by traditional isopentane method as well as by the new method employing cooled metal surface)
  • Spin trapping of protein bound radicals and of reactive oxygen species
  • EPR spectra saturation analysis
  • EPR spectra simulation
  • Tyrosyl radical spectra simulation with employment of TRSSA
  • Annealing of short lived species in frozen samples at variable temperatures (100 K - 270 K)
  • Kinetic studies in the samples frozen "slowly" at variable time after the reaction starts (from 6 s)
  • Kinetic studies with the use of the freeze-quench apparatus (from 40 ms)

Our Biomedical EPR Facility includes:

  • a Bruker EMX (X-band) continuous wave EPR spectrometer
  • a 4103 TM Bruker resonators which can accommodate a flat cell or an AquaX cell for liquid samples (t>0°C)
  • an AquaX cell (4-bore) for high sensitivity liquid phase measurements
  • a 4122 SP Bruker high quality spherical resonator (for low temperature and room temperature measurements)
  • an Oxford Instruments liquid helium system for the low temperature measurements
  • a computerised PID (proportional-integral-derivative) controller of temperature that provides an economy helium usage
  • a 4117 MX Bruker Dielectric Mixing Resonator for studying transient free radicals in a continuous flow set-up
  • a pump to be used with 4117 MX
  • an Update Instrument rapid freeze-quench system for making EPR samples in the time range from 5 ms reaction time
  • a customised Bruker temperature control unit for step-wise annealing of short-lived transient paramagnetic species.
  • a home made freeze-quench system for freezing samples on a cold metal surface

Publications of the Biomedical EPR facility

Cooper, C. E.; Schaer, D. J.; Buehler, P. W.; Wilson, M. T.; Reeder, B. J.; Silkstone, G.; Svistunenko, D. A.; Bulow, L., and Alayash, A. I. (2013) Haptoglobin bindng stabilizes hemoglobin ferryl iron and the globin radical on tyrosine 145. Antioxid. Redox Signal., 18, 2264-2273

Blundell, K. L.; Wilson, M. T.; Svistunenko, D. A.; Vijgenboom, E., and Worrall, J. A. (2013) Morphological development and cytochrome c oxidase activity in Streptomyces lividans are dependent on the action of a copper bound Sco protein. Open Biol, 3, 120163.

Svistunenko, D. A.; Worrall, J. A.; Chugh, S. B.; Haigh, S. C.; Ghiladi, R. A., and Nicholls, P. (2012) Ferric haem forms of Mycobacterium tuberculosis catalase-peroxidase probed by EPR spectroscopy: Their stability and interplay with pH. Biochimie, 94, 1274-1280

Reeder, B. J.; Svistunenko, D. A.; Cooper, C. E., and Wilson, M. T. (2012) Engineering tyrosine-based electron flow pathways in proteins: the case of aplysia myoglobin. J. Am. Chem. Soc., 134, 7741-7749

Reeder, B. J., Svistunenko, D. A., and Wilson, M. T. (2011) Lipid binding to cytoglobin leads to a change in haem co-ordination: a role for cytoglobin in lipid signalling of oxidative stress, Biochem. J., 434, 483-492

Thompson, M. K., Franzen, S., Ghiladi, R. A., Reeder, B. J., and Svistunenko, D. A. (2010)Compound ES of dehaloperoxidase decays via two alternative pathways depending on the conformation of the distal histidine, J. Am. Chem. Soc., 132 (49), 17501-17510

Demidchik, V., Cuin, T.A., Svistunenko, D., Smith, S.J., Miller, A. J., Shabala, S., Sokolik, A., and Yurin, V. (2010) Arabidopsis root K+ efflux conductance activated by hydroxyl radicals: single-channel properties, genetic basis and involvement in stress-induced cell death. J. Cell Sci. 123, Issue 9, 1468-1479

Mot, A., Zoltan, K., Svistunenko, D. A., Damian, G, Silaghi-Dumitrescu, R., and Makarov, S. V. (2010) "Super-reduced" iron under physiologically-relevant conditions . Dalton Trans., 39, Issue 6, 1464 - 1466

Svistunenko, D.A., and Jones, G.A. (2009) Tyrosyl radicals in proteins: a comparison of empirical and density functional calculated EPR parameters. Phys. Chem. Chem. Phys., 11, 6600-6613.

Reeder, B.J., M. Grey, R.L. Silaghi-Dumitrescu, D.A. Svistunenko, L. Bulow, C.E. Cooper, and M.T. Wilson, Tyrosine residues as redox cofactors in human hemoglobin: Implications for engineering non toxic blood substitutes. J Biol Chem, 2008. 283(45): p. 30780-30787.

Chauhan, N., J. Basran, I. Efimov, D.A. Svistunenko, H.E. Seward, P.C. Moody, and E.L. Raven, The role of serine 167 in human indoleamine 2,3-dioxygenase: a comparison with tryptophan 2,3-dioxygenase. Biochemistry, 2008. 47(16): p. 4761-9.

Svistunenko, D.A., B.J. Reeder, M.M. Wankasi, R.-L. Silaghi-Dumitrescu, C.E. Cooper, S. Rinaldo, F. CutruzzolĂ , and M.T. Wilson, Reaction of Aplysia limacina metmyoglobin with hydrogen peroxide. Dalton Trans., 2007(8): p. 840-850.

Pipirou, Z., A.R. Bottrill, D.A. Svistunenko, I. Efimov, J. Basran, S.C. Mistry, C.E. Cooper, and E.L. Raven, The reactivity of heme in biological systems: autocatalytic formation of both tyrosine-heme and tryptophan-heme covalent links in a single protein architecture. Biochemistry, 2007. 46(46): p. 13269-78.

Svistunenko, D.A., N. Davies, D. Brealey, M. Singer, and C.E. Cooper, Mitochondrial dysfunction in patients with severe sepsis: an EPR interrogation of individual respiratory chain components. Biochim. Biophys. Acta, 2006. 1757: p. 262-272.

Dunne, J., A. Caron, P. Menu, A.I. Alayash, P.W. Buehler, M.T. Wilson, R. Silaghi-Dumitrescu, B. Faivre, and C.E. Cooper, Ascorbate removes key precursors to oxidative damage by cell-free haemoglobin in vitro and in vivo. Biochem J, 2006. 399(3): p. 513-24.

Svistunenko, D.A., Reaction of haem containing proteins and enzymes with hydroperoxides: The radical view. Biochim. Biophys. Acta, 2005. 1707(1): p. 127-155.

Silkstone, G.G., C.E. Cooper, D. Svistunenko, and M.T. Wilson, EPR and optical spectroscopic studies of Met80X mutants of yeast ferricytochrome c. Models for intermediates in the alkaline transition. J Am Chem Soc, 2005. 127(1): p. 92-9.

Papadopoulou, N.D., M. Mewies, K.J. McLean, H.E. Seward, D.A. Svistunenko, A.W. Munro, and E.L. Raven, Redox and spectroscopic properties of human indoleamine 2,3-dioxygenase and a His303Ala variant: implications for catalysis. Biochemistry, 2005. 44(43): p. 14318-28.

Kagan, V.E., V.A. Tyurin, J. Jiang, Y.Y. Tyurina, V.B. Ritov, A.A. Amoscato, A.N. Osipov, N.A. Belikova, A.A. Kapralov, V. Kini, I.I. Vlasova, Q. Zhao, M.M. Zou, P. Di, D.A. Svistunenko, I.V. Kurnikov, and G.G. Borisenko, Cytochrome c acts as a cardiolipin oxygenase required for release of pro-apoptotic factors. Nature Chem. Biol., 2005. 1(4): p. 223-232.

Davies, N.A., D.A. Brealey, R. Stidwill, M. Singer, D.A. Svistunenko, and C.E. Cooper, Nitrosyl heme production compared in endotoxemic and hemorrhagic shock. Free Radic Biol Med, 2005. 38(1): p. 41-9.

Cooper, C.E., M. Jurd, P. Nicholls, M.M. Wankasi, D.A. Svistunenko, B.J. Reeder, and M.T. Wilson, On the formation, nature, stability and biological relevance of the primary reaction intermediates of myoglobins with hydrogen peroxide. Dalton Trans., 2005(21): p. 3483-3488.

Vanin, A.F., D.A. Svistunenko, V.D. Mikoyan, V.A. Serezhenkov, M.J. Fryer, N.R. Baker, and C.E. Cooper, Endogenous superoxide production and the nitrite/nitrate ratio control the concentration of bioavailable free nitric oxide in leaves. J. Biol. Chem., 2004. 279(23): p. 24100-24107.

Svistunenko, D.A., M.T. Wilson, and C.E. Cooper, Tryptophan or tyrosine? On the nature of the amino acid radical formed following hydrogen peroxide treatment of cytochrome c oxidase. Biochim. Biophys. Acta, 2004. 1655(1-3): p. 372-380.

Svistunenko, D.A. and C.E. Cooper, A new method of identifying the site of tyrosyl radicals in proteins. Biophys. J., 2004. 87(1): p. 582-595.

Reeder, B.J., D.A. Svistunenko, C.E. Cooper, and M.T. Wilson, The radical and redox chemistry of myoglobin and hemoglobin: from in vitro studies to human pathology. Antioxid. Redox Sign., 2004. 6(6): p. 954-966.

Malone, S.A., A. Lewin, M.A. Kilic, D.A. Svistunenko, C.E. Cooper, M.T. Wilson, N.E. Le Brun, S. Spiro, and G.R. Moore, Protein-template-driven formation of polynuclear iron species. J. Am. Chem. Soc., 2004. 126(2): p. 496-504.

Svistunenko, D.A., B.J. Reeder, M.T. Wilson, and C.E. Cooper, Radical formation and migration in myoglobins. Prog. React. Kinet. Mech., 2003. 28: p. 105-118.

McHugh, J.P., F. Rodriguez-Quinones, H. Abdul-Tehrani, D.A. Svistunenko, R.K. Poole, C.E. Cooper, and S.C. Andrews, Global iron-dependent gene regulation in Escherichia coli. A new mechanism for iron homeostasis. J. Biol. Chem., 2003. 278(32): p. 29478-29486.

Svistunenko, D.A., J. Dunne, M. Fryer, P. Nicholls, B.J. Reeder, M.T. Wilson, M.G. Bigotti, F. CutruzzolĂ , and C.E. Cooper, Comparative study of tyrosine radicals in hemoglobin and myoglobins treated with hydrogen peroxide. Biophys. J., 2002. 83(5): p. 2845-2855.

Reeder, B.J., D.A. Svistunenko, M.A. Sharpe, and M.T. Wilson, Characteristics and mechanism of formation of peroxide-induced heme to protein cross-linking in myoglobin. Biochemistry, 2002. 41(1): p. 367-375.

Svistunenko, D.A., An EPR study of the peroxyl radicals induced by hydrogen peroxide in the haem proteins. Biochim. Biophys. Acta, 2001. 1546(2): p. 365-378.

Svistunenko, D.A., M.A. Sharpe, P. Nicholls, M.T. Wilson, and C.E. Cooper, A new method for quantitation of spin concentration by EPR spectroscopy: application to methemoglobin and metmyoglobin. J. Magn. Reson., 2000. 142(2): p. 266-275.

Svistunenko, D.A., M.A. Sharpe, P. Nicholls, C. Blenkinsop, N.A. Davies, J. Dunne, M.T. Wilson, and C.E. Cooper, The pH dependence of naturally occurring low-spin forms of methaemoglobin and metmyoglobin: an EPR study. Biochem J, 2000. 351(Pt 3): p. 595-605.

Svistunenko, D.A., A. Rob, A. Ball, J. Torres, M.C.R. Symons, M.T. Wilson, and C.E. Cooper, The electron paramagnetic resonance characterisation of a copper-containing extracellular peroxidase from Thermomonospora fusca BD25. Biochim. Biophys. Acta, 1999. 1434: p. 74-85.

Dunne, J., D.A. Svistunenko, A.I. Alayash, M.T. Wilson, and C.E. Cooper, Reactions of cross-linked methaemoglobins with hydrogen peroxide. Adv Exp Med Biol, 1999. 471: p. 9-15.

Torres, J., D. Svistunenko, B. Karlsson, C.E. Cooper, and M.T. Wilson, Fast copper reduction in laccase by nitric oxide and formation of a stable peroxide intermediate. Biochem. J., 1998.

Moore, K.P., S.G. Holt, R.P. Patel, D.A. Svistunenko, W. Zackert, D. Goodier, B.J. Reeder, M. Clozel, R. Anand, C.E. Cooper, J.D. Morrow, M.T. Wilson, V. Darley-Usmar, and L.J. Roberts, 2nd, A causative role for redox cycling of myoglobin and its inhibition by alkalinization in the pathogenesis and treatment of rhabdomyolysis- induced renal failure. J. Biol. Chem., 1998. 273(48): p. 31731-31737.

Dunne, J., D.A. Svistunenko, M.T. Wilson, A.I. Alayash, and C.E. Cooper, Reactions of cross-linked ferric haemoglobins with hydrogen peroxide. Biochem. Soc. Trans., 1998. 26(4): p. S320.

Svistunenko, D.A., R.P. Patel, S.V. Voloshchenko, and M.T. Wilson, The globin-based free radical of ferryl hemoglobin is detected in normal human blood. Journal of Biological Chemistry, 1997. 272(11): p. 7114-7121.

Svistunenko, D.A., N.A. Davies, M.T. Wilson, R.P. Stidwill, M. Singer, and C.E. Cooper, Free radical in blood: a measure of haemoglobin autoxidation in vivo? J. Chem. Soc., Perkin Trans. 2, 1997(12): p. 2539-2543.

Svistunenko, D.A., R.P. Patel, and M.T. Wilson, An EPR investigation of human methaemoglobin oxidation by hydrogen peroxide: methods to quantify all paramagnetic species observed in the reaction. Free Rad. Res., 1996. 24(4): p. 269-280.

Shergill, J.K., R. Cammack, C.E. Cooper, J.M. Cooper, V.M. Mann, and A.H. Schapira, Detection of nitrosyl complexes in human substantia nigra, in relation to Parkinson's disease. Biochem. Biophys. Res. Commun., 1996. 228(2): p. 298-305.

Patel, R.P., D.A. Svistunenko, V.M. Darley-Usmar, M.C. Symons, and M.T. Wilson, Redox cycling of human methaemoglobin by H2O2 yields persistent ferryl iron and protein based radicals. Free Rad. Res., 1996. 25(2): p. 117-123.

Cooper, C.E., G.R. Lynagh, K.P. Hoyes, R.C. Hider, R. Cammack, and J.B. Porter, The Relationship of Intracellular Iron Chelation to the Inhibition and Regeneration of Human Ribonucleotide Reductase. J. Biol. Chem., 1996. 271(34): p. 20291-20299.

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