Advertisement

EPR on Flavoproteins

  • Richard BrosiEmail author
  • Robert Bittl
  • Christopher EngelhardEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1146)

Abstract

Flavoproteins often employ radical mechanisms in their enzymatic reactions. This involves paramagnetic species, which can ideally be investigated with electron paramagnetic resonance (EPR) spectroscopy. In this chapter we focus on the example of flavin-based photoreceptors and discuss, how different EPR methods have been used to extract information about the flavin radical’s electronic state, its binding pocket, electron-transfer pathways, and about the protein’s tertiary and quaternary structure.

Key words

ENDOR spectroscopy Radical mechanisms Photoreceptors Transient EPR Protein structure 

Notes

Acknowledgements

We thank the UniCat Cluster of Excellence for funding.

References

  1. 1.
    Frey PA, Hegemann AD, Reed GH (2006) Free radical mechanisms in enzymology. Chem Rev 106:3302–3316PubMedCrossRefGoogle Scholar
  2. 2.
    Losi A (2007) Flavin-based blue-light photosensors: a photobiophysics update. Photochem Photobiol 83:1283–1300PubMedCrossRefGoogle Scholar
  3. 3.
    Demarsy E, Fankhauser C (2009) Higher plants use LOV to perceive blue light. Curr Opin Plant Biol 12:69–74PubMedCrossRefGoogle Scholar
  4. 4.
    Losi A, Gärtner W (2011) Old chromophores, new photoactivation paradigms, trendy applications: flavins in blue light-sensing photoreceptors. Photochem Photobiol 87: 491–510PubMedCrossRefGoogle Scholar
  5. 5.
    Weber S (2005) Light-driven enzymatic catalysis of DNA repair: a review of recent biophysical studies on photolyase. Biochim Biophys Acta 1707:1–23PubMedCrossRefGoogle Scholar
  6. 6.
    Sancar A (2003) Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors. Chem Rev 103:2203–2237PubMedCrossRefGoogle Scholar
  7. 7.
    Müller M, Carell T (2009) Structural biology of DNA photolyases and cryptochromes. Curr Opin Struct Biol 19:277–285PubMedCrossRefGoogle Scholar
  8. 8.
    Massey V (1994) Activation of molecular oxygen by flavins and flavoproteins. J Biol Chem 269:22459–22462PubMedGoogle Scholar
  9. 9.
    Massey V, Palmer G (1966) On the existence of spectrally distinct classes of flavoprotein semiquinones. A new method for quantitative production of flavoprotein semiquinones. Biochemistry 5:3181PubMedCrossRefGoogle Scholar
  10. 10.
    Weber S, Biskup T, Okafuji A, Marino AR, Berthold T, Link G, Hitomi K, Getzoff ED, Schleicher E, Norris JR (2010) Origin of light-induced spin-correlated radical pairs in cryptochrome. J Phys Chem B 114:14745–14754PubMedCrossRefGoogle Scholar
  11. 11.
    Mi QX, Ratner MA, Wasielewski MR (2010) Time-resolved EPR spectra of spin-correlated radical pairs: spectral and kinetic modulation resulting from electron-nuclear hyperfine interactions. J Phys Chem A 114:162–171PubMedCrossRefGoogle Scholar
  12. 12.
    Biskup T, Hitomi K, Getzoff ED, Krapf S, Koslowski T, Schleicher E, Weber S (2011) Unexpected electron transfer in cryptochrome identified by time-resolved EPR spectroscopy. Angew Chem Int Ed 50:12647–12651CrossRefGoogle Scholar
  13. 13.
    Schleicher E, Wenzel R, Ahmad M, Batschauer A, Essen LO, Hitomi K, Getzoff ED, Bittl R, Weber S, Okafuji A (2010) The electronic state of flavoproteins: investigations with proton electron-nuclear double resonance. Appl Magn Reson 37:339–352CrossRefGoogle Scholar
  14. 14.
    Murataliev MB (1999) Applications of electron spin resonance (ESR) for detection and characterization of flavoprotein semiquinones. Methods Mol Biol 131:97–110PubMedGoogle Scholar
  15. 15.
    Müller F (1981) Spectroscopy and photochemistry of flavins and flavoproteins. Photochem Photobiol 34:753–758PubMedCrossRefGoogle Scholar
  16. 16.
    Heelis PF (1982) The photophysical and photochemical properties of flavins (isoalloxazines). Chem Soc Rev 11:15–39CrossRefGoogle Scholar
  17. 17.
    Weber S, Kay CWM, Bacher A, Richter G, Bittl R (2005) Probing the N(5)-H bond of the isoalloxazine moiety of flavin radicals by X- and W-band pulsed electron-nuclear double resonance. Chemphyschem 6:292–299PubMedCrossRefGoogle Scholar
  18. 18.
    Acocella A, Jones GA, Zerbetto F (2010) What is adenine doing in photolyase? J Phys Chem B 114:4101–4106PubMedCrossRefGoogle Scholar
  19. 19.
    Barquera B, Morgan JE, Lukoyanov D, Scholes CP, Gennis RB, Nilges MJ (2003) X- and W-band EPR and Q-band ENDOR studies of the flavin radical in the Na+-translocating NADH: quinone oxidoreductase from Vibrio cholerae. J Am Chem Soc 125:265–275PubMedCrossRefGoogle Scholar
  20. 20.
    Okafuji A, Schnegg A, Schleicher E, Möbius K, Weber S (2008) G-tensors of the flavin adenine dinucleotide radicals in glucose oxidase: a comparative multifrequency electron paramagnetic resonance and electron-nuclear double resonance study. J Phys Chem B 112:3568–3574PubMedCrossRefGoogle Scholar
  21. 21.
    Brosi R, Illarionov B, Mathes T, Fischer M, Joshi M, Bacher A, Hegemann P, Bittl R, Weber S, Schleicher E (2010) Hindered rotation of a cofactor methyl group as a probe for protein-cofactor interaction. J Am Chem Soc 132:8935–8944PubMedCrossRefGoogle Scholar
  22. 22.
    Schleicher E, Hitomi K, Kay CWM, Getzoff ED, Todo T, Weber S (2007) Electron nuclear double resonance differentiates complementary roles for active site histidines in (6-4) photolyase. J Biol Chem 282:4738–4747PubMedCrossRefGoogle Scholar
  23. 23.
    Kurreck H, Bock M, Bretz N, Elsner M, Kraus H, Lubitz W, Müller F, Geissler J, Kroneck PMH (1984) Fluid solutions and solid-state electron nuclear double resonance studies of flavin model compounds and flavoenzymes. J Am Chem Soc 106:737–746CrossRefGoogle Scholar
  24. 24.
    Cinkaya I, Buckel W, Medina M, Gomez-Moreno C, Cammack R (1997) Electron-nuclear double resonance spectroscopy investigation of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum: comparison with other flavin radical enzymes. Biol Chem 378:843–849PubMedCrossRefGoogle Scholar
  25. 25.
    Medina M, Lostao A, Sancho J, Gomez-Moreno C, Cammack R, Alonso PJ, Martinez JI (1999) Electron-nuclear double resonance and hyperfine sublevel correlation spectroscopic studies of flavodoxin mutants from Anabaena sp PCC 7119. Biophys J 77: 1712–1720PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Kay CWM, Feicht R, Schulz K, Sadewater P, Sancar A, Bacher A, Mobius K, Richter G, Weber S (1999) EPR, ENDOR, and TRIPLE resonance spectroscopy on the neutral flavin radical in Escherichia coli DNA photolyase. Biochemistry 38:16740–16748PubMedCrossRefGoogle Scholar
  27. 27.
    Kay CWM, El Mkami H, Molla G, Pollegioni L, Ramsay RR (2007) Characterization of the covalently bound anionic flavin radical in monoamine oxidase a by electron paramagnetic resonance. J Am Chem Soc 129: 16091–16097PubMedCrossRefGoogle Scholar
  28. 28.
    Weber S, Möbius K, Richter G, Kay CWM (2001) The electronic structure of the flavin cofactor in DNA photolyase. J Am Chem Soc 123:3790–3798PubMedCrossRefGoogle Scholar
  29. 29.
    Garcia JI, Medina M, Sancho J, Alonso PJ, Gomez-Moreno C, Mayoral JA, Martinez JI (2002) Theoretical analysis of the electron spin density distribution of the flavin semiquinone isoalloxazine ring within model protein environments. J Phys Chem B 106: 4729–4735CrossRefGoogle Scholar
  30. 30.
    Fuchs MR, Schleicher E, Schnegg A, Kay CWM, Törring JT, Bittl R, Bacher A, Richter G, Mobius K, Weber S (2002) g-Tensor of the neutral flavin radical cofactor of DNA photolyase revealed by 360-GHz electron paramagnetic resonance spectroscopy. J Phys Chem B 106:8885–8890CrossRefGoogle Scholar
  31. 31.
    Schnegg A, Okafuji A, Bacher A, Bittl R, Fischer M, Fuchs MR, Hegemann P, Joshi M, Kay CWM, Richter G, Schleicher E, Weber S (2007) Towards an identification of chemically different flavin radicals by means of their g-tensor. Appl Magn Reson 30:345–358CrossRefGoogle Scholar
  32. 32.
    Kay CWM, Schleicher E, Hitomi K, Todo T, Bittl R, Weber S (2005) Determination of the g-matrix orientation in flavin radicals by high-field/high-frequency electron-nuclear double resonance. Magn Reson Chem 43:S96–S102PubMedCrossRefGoogle Scholar
  33. 33.
    Kay CWM, Bittl R, Bacher A, Richter G, Weber S (2005) Unambiguous determination of the g-matrix orientation in a neutral flavin radical by pulsed electron-nuclear double resonance at 94 GHz. J Am Chem Soc 127:10780–10781PubMedCrossRefGoogle Scholar
  34. 34.
    Murphy DM, Farley RD (2006) Principles and applications of ENDOR spectroscopy for structure determination in solution and disordered matrices. Chem Soc Rev 35:249–268PubMedCrossRefGoogle Scholar
  35. 35.
    Van Doorslaer S, Vinck E (2007) The strength of EPR and ENDOR techniques in revealing structure-function relationships in metalloprotein. Phys Chem Chem Phys 9:4620–4638PubMedCrossRefGoogle Scholar
  36. 36.
    Kulik L, Lubitz W (2009) Electron-nuclear double resonance. Photosynth Res 102: 391–401PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Kim ST, Malhotra K, Smith CA, Taylor JS, Sancar A (1994) Characterization of (6-4)-photoproduct DNA photolyase. J Biol Chem 269:8535–8540PubMedGoogle Scholar
  38. 38.
    Zhao XD, Liu JQ, Hsu DS, Zhao SY, Taylor JS, Sancar A (1997) Reaction mechanism of (6-4) photolyase. J Biol Chem 272:32580–32590PubMedCrossRefGoogle Scholar
  39. 39.
    Hitomi K, Kim ST, Iwai S, Harima N, Otoshi E, Ikenaga M, Todo T (1997) Binding and catalytic properties of Xenopus (6-4) photolyase. J Biol Chem 272:32591–32598PubMedCrossRefGoogle Scholar
  40. 40.
    Park HW, Kim ST, Sancar A, Deisenhofer J (1995) Crystal-structure of DNA photolyase from Escherichia coli. Science 268:1866–1872PubMedCrossRefGoogle Scholar
  41. 41.
    Jordan SP, Jorns MS (1988) Evidence for a singlet intermediate in catalysis by Escherichia coli DNA photolyase and evaluation of substrate binding determinants. Biochemistry 27:8915–8923PubMedCrossRefGoogle Scholar
  42. 42.
    Hitomi K, Nakamura H, Kim ST, Mizukoshi T, Ishikawa T, Iwai S, Todo T (2001) Role of two histidines in the (6-4) photolyase reaction. J Biol Chem 276:10103–10109PubMedCrossRefGoogle Scholar
  43. 43.
    Lv XY, Qiao DR, Xiong Y, Xu H, You FF, Cao Y, He X, Cao Y (2008) Photoreactivation of (6-4) photolyase in Dunaliella salina. FEMS Microbiol Lett 283:42–46CrossRefGoogle Scholar
  44. 44.
    Christie JM (2007) Phototropin blue-light receptors. Annu Rev Plant Biol 58:21–45PubMedCrossRefGoogle Scholar
  45. 45.
    Briggs WR (2007) The LOV domain: a chromophore module servicing multiple photoreceptors. J Biomed Sci 14:499–504PubMedCrossRefGoogle Scholar
  46. 46.
    Swartz TE, Corchnoy SB, Christie JM, Lewis JW, Szundi I, Briggs WR, Bogomolni RA (2001) The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin. J Biol Chem 276:36493–36500PubMedCrossRefGoogle Scholar
  47. 47.
    Salomon M, Christie JM, Knieb E, Lempert U, Briggs WR (2000) Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin. Biochemistry 31:9401–9410CrossRefGoogle Scholar
  48. 48.
    Salomon M, Eisenreich W, Durr H, Schleicher E, Knieb E, Massey V, Rudiger W, Müller F, Bacher A, Richter G (2001) An optomechanical transducer in the blue light receptor phototropin from Avena sativa. Proc Natl Acad Sci U S A 98:12357–12361PubMedCentralPubMedCrossRefGoogle Scholar
  49. 49.
    Crosson S, Moffat K (2001) Structure of a flavin-binding plant photoreceptor domain: insights into light-mediated signal transduction. Proc Natl Acad Sci U S A 98: 2995–3000PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    Crosson S, Moffat K (2002) Photoexcited structure of a plant photoreceptor domain reveals a light-driven molecular switch. Plant Cell 14:1067–1075PubMedCentralPubMedCrossRefGoogle Scholar
  51. 51.
    Fedorov R, Schlichting I, Hartmann E, Domratcheva T, Fuhrmann M, Hegemann P (2003) Crystal structures and molecular mechanism of a light-induced signaling switch: the Phot-LOV1 domain from Chlamydomonas reinhardtii. Biophys J 84:2474–2482PubMedCentralPubMedCrossRefGoogle Scholar
  52. 52.
    Kasahara M, Swartz TE, Olney MA, Onodera A, Mochizuki N, Fukuzawa H, Asamizu E, Tabata S, Kanegae H, Takano M, Christie JM, Nagatani A, Briggs WR (2002) Photochemical properties of the flavin mononucleotide-binding domains of the phototropins from Arabidopsis, rice, and Chlamydomonas reinhardtii. Plant Physiol 129:762–773PubMedCentralPubMedCrossRefGoogle Scholar
  53. 53.
    Swartz TE, Tseng TS, Frederickson MA, Paris G, Comerci DJ, Rajashekara G, Kim JG, Mudgett MB, Splitter GA, Ugalde RA, Goldbaum FA, Briggs WR, Bogomolni RA (2007) Blue-light-activated histidine kinases: two-component sensors in bacteria. Science 317:1090–1093PubMedCrossRefGoogle Scholar
  54. 54.
    Bittl R, Kay CWM, Weber S, Hegemann P (2003) Characterization of a flavin radical product in a C57M mutant of a LOV1 domain by electron paramagnetic resonance. Biochemistry 42:8506–8512PubMedCrossRefGoogle Scholar
  55. 55.
    Martínez JI, Alonso PJ, Gómez-Moreno C, Medina M (1997) One- and two-dimensional ESEEM spectroscopy of flavoproteins. Biochemistry 36:15526–15537PubMedCrossRefGoogle Scholar
  56. 56.
    Weber S, Richter G, Schleicher E, Bacher A, Mobius K, Kay CWM (2001) Substrate binding to DNA photolyase studied by electron paramagnetic resonance spectroscopy. Biophys J 81:1195–1204PubMedCentralPubMedCrossRefGoogle Scholar
  57. 57.
    Eriksson LE, Ehrenberg A, Hyde JS (1970) Comparative electron-nuclear double resonance study of two flavoproteins. Eur J Biochem 17:539–543PubMedCrossRefGoogle Scholar
  58. 58.
    Hyde JS, Rist GH, Eriksson LE (1968) Endor of methyl matrix and alpha protons in amorphous and polycrystalline matrices. J Phys Chem 72:4269CrossRefGoogle Scholar
  59. 59.
    Mees A, Klar T, Gnau P, Hennecke U, Eker APM, Carell T, Essen LO (2004) Crystal structure of a photolyase bound to a CPD-like DNA lesion after in situ repair. Science 306:1789–1793PubMedCrossRefGoogle Scholar
  60. 60.
    Stehlik D, Möbius K (1997) New EPR methods for investigating photoprocesses with paramagnetic intermediates. Annu Rev Phys Chem 48:745–784PubMedCrossRefGoogle Scholar
  61. 61.
    Bittl R, Weber S (2005) Transient radical pairs studied by time-resolved EPR. Biochim Biophys Acta 1707:117–126PubMedCrossRefGoogle Scholar
  62. 62.
    Furrer R, Thurnauer MC (1981) Nanosecond time resolution in electron-electron paramagnetic-res transient nutation spectroscopy of triplet-states. Chem Phys Lett 79:28–33CrossRefGoogle Scholar
  63. 63.
    van Tol J, Brunel LC, Angerhofer A (2001) Transient EPR at 240 GHz of the excited triplet state of free-base tetra-phenyl porphyrin. Appl Magn Reson 21:335–340CrossRefGoogle Scholar
  64. 64.
    van Tol J, Brunel LC, Wylde RJ (2005) A quasioptical transient electron spin resonance spectrometer operating at 120 and 240 GHz. Rev Sci Instrum 76:074101CrossRefGoogle Scholar
  65. 65.
    Lin CT, Todo T (2005) The cryptochromes. Genome Biol 6:220PubMedCentralPubMedCrossRefGoogle Scholar
  66. 66.
    Weber S, Kay CWM, Mogling H, Mobius K, Hitomi K, Todo T (2002) Photoactivation of the flavin cofactor in Xenopus laevis (6-4) photolyase: observation of a transient tyrosyl radical by time-resolved electron paramagnetic resonance. Proc Natl Acad Sci U S A 99: 1319–1322PubMedCentralPubMedCrossRefGoogle Scholar
  67. 67.
    Biskup T, Schleicher E, Okafuji A, Link G, Hitomi K, Getzoff ED, Weber S (2009) Direct observation of a photoinduced radical pair in a cryptochrome blue-light photoreceptor. Angew Chem Int Ed 48:404–407CrossRefGoogle Scholar
  68. 68.
    Cashmore AR (2003) Cryptochromes: enabling plants and animals to determine circadian time. Cell 114:537–543PubMedCrossRefGoogle Scholar
  69. 69.
    Brudler R, Hitomi K, Daiyasu H, Toh H, Kucho K, Ishiura M, Kanehisa M, Roberts VA, Todo T, Tainer JA, Getzoff ED (2003) Identification of a new cryptochrome class: structure, function, and evolution. Mol Cell 11:59–67PubMedCrossRefGoogle Scholar
  70. 70.
    Devlin PF, Kay SA (2001) Circadian photoperception. Annu Rev Physiol 63:677–694PubMedCrossRefGoogle Scholar
  71. 71.
    Canamero RC, Bakrim N, Bouly JP, Garay A, Dudkin EE, Habricot Y, Ahmad M (2006) Cryptochrome photoreceptors cry1 and cry2 antagonistically regulate primary root elongation in Arabidopsis thaliana. Planta 224:995–1003PubMedCrossRefGoogle Scholar
  72. 72.
    Ahmad M, Jarillo JA, Smirnova O, Cashmore AR (1998) Cryptochrome blue-light photoreceptors of Arabidopsis implicated in phototropism. Nature 392:720–723PubMedCrossRefGoogle Scholar
  73. 73.
    Li YF, Heelis PF, Sancar A (1991) Active-site of DNA photolyase—tryptophan-306 is the intrinsic hydrogen-atom donor essential for flavin radical photoreduction and DNA-repair in vitro. Biochemistry 30:6322–6329PubMedCrossRefGoogle Scholar
  74. 74.
    Lukacs A, Eker APM, Byrdin M, Brettel K, Vos MH (2008) Electron hopping through the 15 angstrom triple tryptophan molecular wire in DNA photolyase occurs within 30 ps. J Am Chem Soc 130:14394PubMedCrossRefGoogle Scholar
  75. 75.
    Byrdin M, Sartor V, Eker APM, Vos MH, Aubert C, Brettel K, Mathis P (2004) Intraprotein electron transfer and proton dynamics during photoactivation of DNA photolyase from E. coli: review and new insights from an “inverse” deuterium isotope effect. Biochim Biophys Acta 1655:64–70PubMedCrossRefGoogle Scholar
  76. 76.
    Zeugner A, Byrdin M, Bouly JP, Bakrim N, Giovani B, Brettel K, Ahmad M (2005) Light-induced electron transfer in Arabidopsis cryptochrome-1 correlates with in vivo function. J Biol Chem 280:19437–19440PubMedCrossRefGoogle Scholar
  77. 77.
    Milov AD, Salikhov KM, Shirov MD (1981) Application of ENDOR in electron-spin echo for paramagnetic center space distribution in solids. Fiz Tverd Tela 23:975–982Google Scholar
  78. 78.
    Jeschke G (2002) Distance measurements in the nanometer range by pulse EPR. Chemphyschem 3:927–932PubMedCrossRefGoogle Scholar
  79. 79.
    Martin RE, Pannier M, Diederich F (1998) Determination of end-to-end distances in a series of TEMPO diradicals of up to 2.8 nm length with a new four-pulse double electron electron resonance experiment. Angew Chem Int Ed 37:2834–2837CrossRefGoogle Scholar
  80. 80.
    Millhauser GL (1992) Selective placement of electron-spin-resonance spin labels—new structural methods for peptides and proteins. Trends Biochem Sci 17:448–452PubMedCrossRefGoogle Scholar
  81. 81.
    Hubbell WL, Altenbach C, Hubbell CM, Khorana HG (2003) Rhodopsin structure, dynamics, and activation: a perspective from crystallography, site-directed spin labeling, sulfhydryl reactivity, and disulfide cross-linking. Adv Protein Chem 63:243–290PubMedCrossRefGoogle Scholar
  82. 82.
    Jeschke G, Panek G, Godt A, Bender A, Paulsen H (2004) Data analysis procedures for pulse ELDOR measurements of broad distance distributions. Appl Magn Reson 26: 223–244CrossRefGoogle Scholar
  83. 83.
    Swanson MA, Kathirvelu V, Majtan T, Frerman FE, Eaton GR, Eaton SS (2009) DEER distance measurement between a spin label and a native FAD semiquinone in electron transfer flavoprotein. J Am Chem Soc 131: 15978–15979PubMedCentralPubMedCrossRefGoogle Scholar
  84. 84.
    Kay CWM, Elsässer C, Bittl R, Farrell SR, Thorpe C (2006) Determination of the distance between the two neutral flavin radicals in augmenter of liver regeneration by pulsed ELDOR. J Am Chem Soc 128:76–77PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Fachbereich Physik, Freie Universität BerlinBerlinGermany

Personalised recommendations