Probing the Structure–Function Relationship of Heme Proteins Using Multifrequency Pulse EPR Techniques

  • Sabine Van Doorslaer
Part of the Biological Magnetic Resonance book series (BIMR, volume 28)

Although EPR has been used regularly in the study of heme proteins for more than 40 years now, the use of multifrequency and/or pulse-EPR techniques is still quite scarce. Here, a review of the use of these methods is presented, highlighting the advantages, limitations, and challenges for the future.


Electron Paramagnetic Resonance Axial Ligand Heme Protein ENDOR Spectrum Electron Spin Echo Envelope Modulation 
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  1. 1.
    Kaim W, Schwederski B. 1994. Bioinorganic chemistry: Inorganic elements in the chemistry of life: an introduction and guide. Chichester: John Wiley & Sons.Google Scholar
  2. 2.
    Mathews CK, van Holde KE, Ahern KG. 2000. Biochemistry. San Francisco: Addison Wesley Longman.Google Scholar
  3. 3.
    Cowman JA. 1997. Inorganic biochemistry: an introduction. New York: Wiley-VCH.Google Scholar
  4. 4.
    Dickinson LC, Symons MCR. 1983. Electron Spin Resonance of Haemoglobin and Myoglobin. Chem Soc Rev 12(4):387–414.CrossRefGoogle Scholar
  5. 5.
    Smith DT, Pilbrow JR. 1980. ESR of iron proteins. In Biological Magnetic Resonance, Vol. 2, p. 85. Ed LJ Berliner, J Reuben. New York: Plenum.Google Scholar
  6. 6.
    Walker FA. 1999. Magnetic spectroscopy (EPR, ESEEM, Mössbauer, MCD and NMR) studies of low-spin ferriheme centers and their corresponding heme proteins. Coord Chem Rev 186:471–534.CrossRefGoogle Scholar
  7. 7.
    Schweiger A, Jeschke G. 2001. Principles of pulse electron paramagnetic resonance. Oxford: Oxford University Press.Google Scholar
  8. 8.
    Prisner T, Rohrer M, MacMillan F. 2001. Pulsed EPR spectroscopy: biological applications. Annu Rev Phys Chem 52:279–313.PubMedCrossRefGoogle Scholar
  9. 9.
    Brunori M, 2001. Nitric oxide moves myoglobin centre stage. Trends Biochem Sci 26 (4):209–210.PubMedCrossRefGoogle Scholar
  10. 10.
    Nistor SV, Goovaerts E, Van Doorslaer S, Dewilde S, Moens L. 2002. EPRspectroscopic evidence of a dominant His-FeIII-His coordination in ferric neuroglobin. Chem Phys Lett 361 (5–6):355–361.CrossRefGoogle Scholar
  11. 11.
    Walker FA, Huynh BH, Scheidt WR, Osvath SR. 1986. Models of the cytochromes b: effect of axial ligand plane orientation on the electron paramagnetic resonance and mossbauer spectra of low-spin ferrihemes. J Am Chem Soc 108 (17):5288–5297.CrossRefGoogle Scholar
  12. 12.
    Migita CT, Iwaizumi M. 1981. Low-temperature electron-paramagnetic resonance study of highly anisotropic low-spin (protoporphyrinato)iron(III) complexes. J Am Chem Soc 103 (15):4378–4381.CrossRefGoogle Scholar
  13. 13.
    Salerno JC. 1984. Cytochrome electron spin resonance line shapes, ligand fields and components stochiometry in ubiquinol-cytochrome-c oxidoreductase. J Biol Chem 259 (4):2331–2336.PubMedGoogle Scholar
  14. 14.
    Guzov VM, Houston HL, Murataliev MB, Walker FA, Feyereisen R. 1996. Molecular cloning, overexpression in Escherichia coli, structural and functional characterization of house fly cytochrome b5. J Biol Chem 271 (43):26637–26645.PubMedCrossRefGoogle Scholar
  15. 15.
    Rhynard D, Lang G., Spartalian K, Yonetani T. 1979. Mössbauer studies of lowsymmetry crystal fields in low-spin ferric heme complexes. J Chem Phys 71 (9):3715– 3721.CrossRefGoogle Scholar
  16. 16.
    Ioanitescu I, Dewilde S, Kiger L, Marden MC, Moens L, Van Doorslaer S. 2005. Characterization of nonsymbiotic tomato hemoglobin. Biophys J 89 (4):2628–2639.PubMedCrossRefGoogle Scholar
  17. 17.
    Simonneaux G, Schünemann V, Morice C, Carel L, Toupet L, Winkler H, Trautwein AX, Walker FA. 2000. Structural, magnetic, and dynamic characterization of the (dxz,dyz)4(dxy)1 ground-state low-spin iron(III) tetraphenylporphyrinate complex [(p- TTP)Fe(2,6-XylylNC)2]CF3SO3. J Am Chem Soc 122 (18):4366–4377.CrossRefGoogle Scholar
  18. 18.
    Blumberg WE, Peisach J. 1971. A unified theory for low spin forms of all ferric heme proteins as studied by EPR. In Probes of structure and function of macromolecules and membranes, Vol. 2, pp. 215 ff. Ed B Chance, T Yonetani, AS Mildvan. New York: Academic Press.Google Scholar
  19. 19.
    Siedow JN, Vickery LE, Palmer G. 1980. The nature of the axial ligands of spinach cytochrome f. Arch Biochem Biophys 203 (1):101–107.PubMedCrossRefGoogle Scholar
  20. 20.
    Rigby SEJ, Moore GR, Gray JC, Gadsby PMA, George SJ, Thomson AJ. 1988. Nmr, epr and magnetic-cd studies of cytochrome f: identity of the haem axial ligands. Biochem J 256 (2):571–577.PubMedGoogle Scholar
  21. 21.
    Scholes CP, Falkowski KM, Chen S, Bank J. 1986. Electron nuclear double resonance (ENDOR) of bis(imidazole)-ligated low-spin ferric heme systems. J Am Chem Soc 108 (7):1660–1671.CrossRefGoogle Scholar
  22. 22.
    Scholes CP, Van Camp HL. 1976. ENDOR from nitrogens and protons in low spin ferric heme and hemoprotein. Biochim Biophys Acta 434 (1):290–296.PubMedGoogle Scholar
  23. 23.
    Mulks CF, Scholes CP, Dickinson LC, Lapidot A. 1979. Electron nuclear double resonance from high- and low-spin ferric hemoglobins and myoglobins. J Am Chem Soc 101 (7):1645–1654.CrossRefGoogle Scholar
  24. 24.
    Fahnenschmidt M, Bittl R, Rau HK, Haehnel W, Lubitz W. 2000. Electron paramagnetic resonance and electron nuclear double resonance spectroscopy of a heme protein maquette. Chem Phys Lett 323 (3–4):329–339.CrossRefGoogle Scholar
  25. 25.
    Peisach J, Mims WB, Davis JL. 1979. Studies of the electron-nuclear coupling between Fe(III) and 14N in cytochrome P450 and in a series of low spin heme compounds. J Biol Chem 254 (24):12379–12389.PubMedGoogle Scholar
  26. 26.
    Magliozzo RS, Peisach J. 1992. Electron spin echo envelope modulation spectroscopic study of iron–nitrogen interactions in myoglobin hydroxide and Fe(III) tetraphenylporphyrin models. Biochemistry 31 (1):189–199.PubMedCrossRefGoogle Scholar
  27. 27.
    Magliozzo RS, Peisach J. 1993. Evaluation of nitrogen nuclear hyperfine and quadrupole coupling parameters for the proximal imidazole in myoglobin-azide, -cyanide and -mercaptoethanol complexes by electron spin echo envelope modulation. Spectroscopy 32 (33):8446–8456.Google Scholar
  28. 28.
    Theodorakis JL, Garber EAE, McCracken J, Peisach J, Schejter A, Margoliash E. 1995. A chemical modification of cytochrome-c lysines leading to changes in heme iron ligation. Biochim Biophys Acta 1252 (1):103–113.PubMedGoogle Scholar
  29. 29.
    Thomann H, Bernardo M, Goldfarb D, Kroneck PMH, Ullrich V. 1995. Evidence for water binding to the Fe center in cytochrome P450cam obtained by 17O electron spin echo envelope modulation spectroscopy. J Am Chem Soc 117 (31):8243–8251.CrossRefGoogle Scholar
  30. 30.
    Goldfarb D, Bernardo M, Thomann H, Kroneck PMH, Ullrich V. 1996. Study of water binding to low-spin Fe(III) in cytochrome P450 by pulsed ENDOR and four-pulse ESEEM spectroscopies. J Am Chem Soc 118 (11):2686–2693.CrossRefGoogle Scholar
  31. 31.
    Fann YC, Gerber NC, Osmulski PA, Hager LP, Sligar SG, Hoffman BM. 1994. ENDOR determination of heme ligation in chloroperoxidase and comparison with cytochrome P450cam. J Am Chem Soc 116 (13):5989–5990.CrossRefGoogle Scholar
  32. 32.
    Lee HI, Dexter AF, Fann YC, Lakner FJ, Hager LP, Hoffman BM. 1997. Structure of the modified heme in allylbenzene-inactivated chloroperoxidase determined by Q-band CW and pulsed ENDOR. J Am Chem Soc 119 (17):4059–4069.CrossRefGoogle Scholar
  33. 33.
    Astashkin AV, Raitsimring AM, Walker FA. 1999. Two- and four-pulse ESEEM studies of the heme binding center of a low-spin ferriheme protein: the importance of a multi-frequency approach. Chem Phys Lett 306 (1–2):9–17.CrossRefGoogle Scholar
  34. 34.
    Astashkin AV, Raitsimring AM, Walker FA. 2001. 1H pulsed ENDOR and ESEEM evidence that the bis-imidazole complexes of iron(III) tetraphenylchlorin and tetraphenylporphyrin have the same order of g values, and the same electronic ground state. J Am Chem Soc 123 (9):1905–1913.PubMedCrossRefGoogle Scholar
  35. 35.
    Raitsimring AM, Borbat P, Shokhireva TK, Walker FA. 1996. Magnetic field (g-value) dependence of proton hyperfine couplings obtained from ESEEM measurements: Determination of the orientation of the magnetic axes of model heme complexes in glassy media. J Am Chem Soc 100 (13):5235–5244.Google Scholar
  36. 36.
    Raitsimring AM, Walker FA. 1998. Porphyrin and ligand protons as internal labels for determination of ligand orientation in ESEEMS of low-spin d5 complexes in glassy media: ESEEM studies of the orientation of the g tensor with respect tot the planes of axial ligands and porphyrin nitrogens of low-spin ferriheme systems. J Am Chem Soc 120 (5):991–1002.CrossRefGoogle Scholar
  37. 37.
    García-Rubio I, Martínez JI, Picorel R, Yruela I, Alonso PJ. 2003. HYSCORE spectroscopy in the cytochrome b 559 of the photosystem II reaction center. J Am Chem Soc 125 (51):15846–15854.PubMedCrossRefGoogle Scholar
  38. 38.
    Vinck E, Van Doorslaer S. 2004. Analysing low-spin ferric complexes using pulse EPR techniques: a structure determination of bis (4-methylimidazole) (tetraphenylporphyrinato) iron(III). Phys Chem Chem Phys 6 (23):5324–5330.CrossRefGoogle Scholar
  39. 39.
    Jeschke G, Rakhmatullin R, Schweiger A. 1998. Sensitivity enhancement by matched microwave pulses in one- and two-dimensional electron spin echo envelope modulation spectroscopy. J Magn Reson 131 (2):261–271.PubMedCrossRefGoogle Scholar
  40. 40.
    Liesum L, Schweiger A. 2001. A multiple quantum coherence in HYSCORE spectra. J Chem Phys 114 (21):9478–9488.CrossRefGoogle Scholar
  41. 41.
    Brown TG, Hoffmann BM. 1980. 14N, 1H and metal ENDOR of single crystal Ag(II)(TPP) and Cu(II)(TPP). Mol Phys 39 (5):1073–1109.CrossRefGoogle Scholar
  42. 42.
    Shokhirev NV, Walker FA. 1998. Co- and counterrotation of magnetic axes and axial ligands in low-spin ferriheme systems. J Am Chem Soc 120 (5):981–990.CrossRefGoogle Scholar
  43. 43.
    Vinck E, Van Doorslaer S, Dewilde S, Mitrikas G, Schweiger A, Moens L. 2006. Analyzing heme proteins using EPR techniques: the heme-pocket structure of ferric mouse neuroglobin. J Biol Inorg Chem 11 (4):467–475.PubMedCrossRefGoogle Scholar
  44. 44.
    Vinck E, Van Doorslaer S, Dewilde S, Moens L. 2004. Structural change of the heme pocket due to disulfide bridge formation is significantly larger for neuroglobin than for cytoglobin. J Am Chem Soc 126 (14):4516–4517.PubMedCrossRefGoogle Scholar
  45. 45.
    Ohba Y. 2003. Application of two-dimensional pulsed EPR nutation spectroscopy to a disordered system with large g anisotropy. Appl Magn Reson 23 (3–4):539–556.Google Scholar
  46. 46.
    van Lenthe E, van der Avoird A, Hagen WR, Reijerse EJ. 2000. Density functional calculations of g tensors of low-spin iron(I) and iron(III) porphyrins. J Phys Chem A 104 (10):2070–2077.CrossRefGoogle Scholar
  47. 47.
    Johansson MP, Sundholm D, Gerfen G, Wilkström M. 2002. The spin distribution in low-spin iron porphyrins. J Am Chem Soc 124 (39):11771–11780.PubMedCrossRefGoogle Scholar
  48. 48.
    Abragam A, Bleaney B. 1970. Electron paramagnetic resonance of transition ions. Oxford: Oxford UP.Google Scholar
  49. 49.
    Peisach J, Blumberg WE, Ogawa S, Rachmilewitz EE, Oltzik R. 1971. The effects of protein conformation on the heme symmetry in high spin ferric heme proteins as studied by electron paramagnetic resonance. J Biol Chem 246 (10):3342–3355.PubMedGoogle Scholar
  50. 50.
    Munro OQ, de Wet M, Pollak H, van Wyk J, Marques HM. 1998. Haempeptide models for haemoproteins, part 3L: N-Acetylmicroperoxidase-8: EPR, Mössbauer and magnetic susceptibility studies on an iron(III) porphyrin in thermal equilibrium between S = 3/2, 5/2 and S = 1/2 states. J Chem Soc, Faraday Trans 94 (12):1743–1752.CrossRefGoogle Scholar
  51. 51.
    van Kan PJM, van der Horst E, Reijerse EJ, van Bentum PJM, Hagen WR. 1998. Multi-frequency EPR spectroscopy of myoglobin: spectral effects for high-spin iron(III) ion at high magnetic fields. J Chem Soc, Faraday Trans 94 (19):2975–2978.CrossRefGoogle Scholar
  52. 52.
    Scholes CP, Lapidot A, Mascarenhas R, Inubushi T, Isaacson RA, Feher G. 1982. Electron nuclear double resonance (ENDOR) from heme and histidine nitrogens in single crystals of aquometmyoglobin. J Am Chem Soc 104 (10):2724–2735.CrossRefGoogle Scholar
  53. 53.
    Fann Y, Ong J, Nocek JM, Hoffman BM. 1995. 19F and 1,2H ENDOR study of distalpocket N(∈)–H...F hydrogen bonding in fluorometmyoglobin. J Am Chem Soc 117 (22):6109–6116.CrossRefGoogle Scholar
  54. 54.
    Lee HI. 2002. 14 N Mims pulsed ENDOR of proximal histidine and heme of aquometmyoglobin and fluorometmyoglobin. Bull Korean Chem Soc 23 (12):1769–1772.CrossRefGoogle Scholar
  55. 55.
    Bachmann R, Schweiger A, Aissaoui H, Woggon WD. 1998. On the origin of the low spin character of the resting state of cytochrome P450cam: investigation of enzyme models by 2D-HYSCORE spectroscopy. In Magnetic resonance and related phenomena, Vol. 2, pp. 822 ff. Ed D Ziessow, W Lubitz, F Lendzian. Berlin: Technische Universität Berlin.Google Scholar
  56. 56.
    Aissaoui H, Bachmann R, Schweiger A, Woggon WD 1998. On the origin of the lowspin character of cytochrome P450(cam) in the resting state: investigations of enzyme models with pulse EPR and ENDOR spectroscopy. Angew Chem, Int Ed 37 (21):2998–3002.CrossRefGoogle Scholar
  57. 57.
    Jeschke G, Spiess W. 1998. NMR-correlated high-field electron paramagnetic resonance spectroscopy. Chem Phys Lett 293 (1–2):9–18.CrossRefGoogle Scholar
  58. 58.
    Cooper CE. 1999. Nitric oxide and iron proteins. Biochim Biophys Acta 1411 (2–3):290–309.PubMedGoogle Scholar
  59. 59.
    Hori H, Ikeda-Saito M, Yonetani T. 1981. Single crystal EPR of myoglobin nitroxide: freezing induced reversible changes in the molecular orientation of the ligand. J Biol Chem 256 (15):7849–7855.PubMedGoogle Scholar
  60. 60.
    Morse RH, Chan SI. 1980. Electron paramagnetic resonance studies of nitrosyl ferrous heme complexes: determination of an equilibrium between two conformations. J Biol Chem 255 (16):7876–7882.PubMedGoogle Scholar
  61. 61.
    Bemski G. 1997. Contribution of electron paramagnetic resonance to the studies of hemoglobin: the nitrosylhemoglobin system. Mol Biol Rep 24 (4):263–269.PubMedCrossRefGoogle Scholar
  62. 62.
    Wajnberg E, Linhares MP, El-Jaick LJ, Bemski G. 1992. Nitrosyl hemoglobin: EPR components at low temperatures. Eur Biophys J 21 (1):57–61.PubMedCrossRefGoogle Scholar
  63. 63.
    Wajnberg E, Bemski G, El-Jaick J, Alves OC. 1996. Nitrosyl hemoglobins: EPR above 80 K. Int J Biol Macromol 18 (3):231–235.PubMedCrossRefGoogle Scholar
  64. 64.
    Trandafir F, Van Doorslaer S, Dewilde S, Moens L. 2004. Temperature dependence of NO binding modes in human neuroglobin. Biochim Biophys Acta 1702 (2):153–161.PubMedGoogle Scholar
  65. 65.
    Schmidt PP, Kappl R, Hüttermann. 2001. On the mode of hexacoordinated NO-binding to myo- and hemoglobin: Variable-temperature EPR studies at multiple microwave frequencies. Appl Magn Reson 21 (3–4):423–440.CrossRefGoogle Scholar
  66. 66.
    Flores M, Wajnberg E, Bemski G. 1997. Temperature dependence of Q-band electron paramagnetic resonance spectra of nitrosyl heme proteins. Biophys J 73 (6):3225–3229.PubMedCrossRefGoogle Scholar
  67. 67.
    Höhn M, Hüttermann J, Chien JCW, Dickinson LC. 1983. 14N and 1H ENDOR of nitrosylhemoglobin. J Am Chem Soc 105 (1):109–115.CrossRefGoogle Scholar
  68. 68.
    Kappl R, Hütterman. 1989. An ENDOR study of nitrosyl myoglobin single crystals. Isr J Chem 29 (1):73–84.Google Scholar
  69. 69.
    Hüttermann J, Burgard C, Kappl R. 1994. Proton ENDOR from randomly oriented NOligated hemoglobin: approaching the structural basis for the R-T transition. J Chem Soc, Faraday Trans 90 (20):3077–3087.CrossRefGoogle Scholar
  70. 70.
    LoBrutto R, Wei YH, Mascarenhas R, Scholes CP, King TE. 1983. Electron nuclear double resonance and electron paramagnetic resonance study on the structure of the NO-ligated heme a3 in cytochrome c oxidase. J Biol Chem 258 (12):7437–7448.PubMedGoogle Scholar
  71. 71.
    Flores M, Wajnberg E, Bemski G. 2000. Proton electron nuclear double resonance from nitrosyl horse heart myoglobin: the role of His-E7 and Val-E11. Biophys J 78 (4):2107–2115.PubMedCrossRefGoogle Scholar
  72. 72.
    Tyryshkin AM, Dikanov SA, Reijerse EJ, Burgard C, Hüttermann. 1999. Characterization of bimodal coordination structure in nitrosyl heme complexes through hyperfine couplings with pyrrole and protein nitrogens. J Am Chem Soc 121 (14):3396–3406.CrossRefGoogle Scholar
  73. 73.
    Gilbert DC, Dikanov SA, Doetschman DC, Smeija JA. 1999. A study of pyridyl nitrosyl iron(II) tetraphenyl 15N4-porphyrin: NO geometry and spin coupling to the pyrrole nitrogens. Chem Phys Lett 315 (1–2):43–48.CrossRefGoogle Scholar
  74. 74.
    Gilbert DC, Doetschman DC. 2001. Five-coordinate nitrosyl iron(II) tetraphenylporphyrin exhibits porphyrin ring 14N symmetry about the Fe-NO plane: a hyperfine sublevel correlation spectroscopy study. Chem Phys Lett 269 (1–3):125–135.Google Scholar
  75. 75.
    Patchkovskii S, Ziegler T. 2000. Structural origin of two paramagnetic species in sixcoordinated nitrosoiron(II) porphyrins revealed by density functional theory analysis of the g tensors. Inorg Chem 39 (23):5354–5364.PubMedCrossRefGoogle Scholar
  76. 76.
    Hori H, Masuya F, Dou Y, Ikeda-Saito M. 2000. EPR studies on the photoinduced intermediates of NO complexes in recombinant ferric Mb trapped at low temperature. J Inorg Biochem 82 (1–4):181–187.PubMedCrossRefGoogle Scholar
  77. 77.
    Hubbell WL, Cafiso DS, Altenbach C. 2000. Identifying conformational changes with site-directed spin labeling. Nature Struct Biol 7 (9):735–739.PubMedCrossRefGoogle Scholar
  78. 78.
    Hubbell WL, Gross A, Langen R, Lietzow MA. 1998. Recent advances in site-directed spin labeling of proteins. Curr Opin Struct Biol 8 (5):649–656.PubMedCrossRefGoogle Scholar
  79. 79.
    Eaton SS, Eaton GG. 2000. Distance measurements by CW and Pulsed EPR. In Biological magnetic resonance, Vol. 19: Distance measurements in biological systems by EPR, pp. 1 ff. Ed LJ Berliner, SS Eaton, GR Eaton. New York: Kluwer Academic/Plenum Publishers.Google Scholar
  80. 80.
    Budker V, Du JL, Seiter M, Eaton GR, Eaton SS. 1995. Electron–electron spin–spin interaction in spin-labeled methemoglobin. Biophys J 68 (6):2531–2542.PubMedCrossRefGoogle Scholar
  81. 81.
    Rakowsky MH, More K, Kulikov AV, Eaton GR, Eaton SS. 1995. Time-domain electron paramagnetic resonance as a probe of electron–electron spin–spin interaction in spin-labeled low spin iron porphyrins. J Am Chem Soc 117 (7):2049–2057.CrossRefGoogle Scholar
  82. 82.
    Rakowsky MH, Zecevic A, Eaton GR, Eaton SS. 1998. Determination of high-spin iron(III)-nitroxyl distances in spin-labeled porphyrins by time-domain EPR. J Magn Reson 131 (1):97–110.PubMedCrossRefGoogle Scholar
  83. 83.
    Seiter M, Budker V, Du JL, Eaton GR, Eaton SS. 1998. Interspin distances determined by time domain EPR of spin-labeled high-spin methemoglobin. Inorg Chim Acta 273 (1–2):354–356.CrossRefGoogle Scholar
  84. 84.
    Klug CS, Eaton SS, Eaton GR, Feix JB. 1998. Ligand-induced conformational change in the ferric enterobactin receptor FepA as studied by site-directed spin labeling and time-domain ESR. Biochemistry 37 (25):9016–9023.PubMedCrossRefGoogle Scholar
  85. 85.
    Eaton SS, Eaton GR. 2000. Determination of distances based on T1 and Tm effects. In Biological magnetic resonance, Vol. 19: Distance measurements in biological systems by EPR, pp. 347 ff. Ed LJ Berliner, SS Eaton, GR Eaton. New York: Kluwer Academic/Plenum Publishers.Google Scholar
  86. 86.
    Zhou Y, Bowler BE, Lynch K, Eaton GR, Eaton SS. 2000. Interspin distances in spinlabeled metmyoglobin variants determined by saturation recovery EPR. Biophys J 79 (2):1039–1052.PubMedCrossRefGoogle Scholar
  87. 87.
    Hirsh DJ, Beck WF, Innes JB, Brudvig GW. 1992. Using saturation recovery EPR to measure distance in proteins. Biochemistry 31 (2):523–541.CrossRefGoogle Scholar
  88. 88.
    Hirsh DJ, Brudvig GW. 1993. Long-range electron spin–spin interactions in the bacterial photosynthetic reaction center. J Phys Chem 97 (50):13216–13222.CrossRefGoogle Scholar
  89. 89.
    Davydov R, Perera R, Jin SX, Yang TC, Bryson TA, Sono M, Dawson JH, Hoffman BM. 2005. Substrate modulation of the properties and reactivity of the oxy-ferrous and hydroperoxo-ferric intermediates of cytochrome P450cam as shown by cryoreduction EPR/ENDOR spectroscopy. J Am Chem Soc 127 (5):1403–1413.PubMedCrossRefGoogle Scholar
  90. 90.
    Kim SH, Yang TC, Perera R, Jin SX, Bryson TA, Sono M, Davydov R, Dawson JH, Hoffman BM. 2005. Cryoreduction EPR and 13C, 19F ENDOR study of substrate-bound substates and solvent kinetic isotope effects in the catalytic cycle of cytochrome P450cam and its T252A mutant. Dalton Trans 21:3464–3469.PubMedCrossRefGoogle Scholar
  91. 91.
    van Amsterdam IMC, Ubbink M, Canters GW, Huber W. 2003. Measurement of a Cu– Cu distance of 26 Å by a pulsed EPR method. Angew Chem, Int Ed 43 (1):62–64.CrossRefGoogle Scholar
  92. 92.
    Siddigui MK, Lyubenovab S, de vries MJMP, Prisner TF, Ludwig B. 2004. Distance and orientation studies of the CuA fragment of cytochrome c oxidase with different cytochrome c by pulsed EPR spectroscopy. Biochim Biophys Acta Bioenerg 1658:154–154.Google Scholar
  93. 93.
    Trandafir F, ter Heerdt P, Fittipaldi M, Vinck E, Dewilde S, Moens L, van Doorslaer S. 2007. Studying high-spin ferric heme proteins using pulsed EPR spectroscopy: analysis of the ferric form of the E7Q mutant of human neuroglobin. Appl Magn Reson 31:553–572CrossRefGoogle Scholar
  94. 94.
    Fittipaldi M, García-Rubio I, Trandafir F, Gromov I, Schweiger A, Bouwen A, Van Doorslaer S. 2008. A multi-frequency pulse EPR and ENDOR approach to study strongly coupled nuclei in frozen solutions of high-spin ferric heme proteins. J Phys Chem B 112:3859–3870.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag New York 2009

Authors and Affiliations

  • Sabine Van Doorslaer
    • 1
  1. 1.Department of PhysicsUniversity of Antwerp, SIBAC LaboratoryAntwerpBelgium

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