Probing Structural and Electronic Parameters in Randomly Oriented Metalloproteins by Orientation-Selective ENDOR Spectroscopy

  • Reinhard Kappl
  • Gerhard Bracic
  • Jürgen Hüttermann
Part of the Biological Magnetic Resonance book series (BIMR, volume 28)

Electron nuclear double resonance (ENDOR) spectroscopy in its continuous wave (CW) and pulsed operational modes is now widely used to characterize the functional, structural, and electronic properties particularly of paramagnetic centers found in metalloproteins. Its essential advantage is based on the intrinsic high resolution, which permits the analysis of small interactions of the metal nucleus or of nuclei in the vicinity of the metal center with the electron spin not observable by standard EPR techniques. Because most of the protein samples are available only as frozen solutions, the essential concept introduced for the analysis of such “powder”-type ENDOR spectra is a method for calculation of “orientation-selected” ENDOR spectra. In this approach the EPR resonance condition is solved for a set of g-, hyperfine-tensors of the paramagnetic center(s), yielding the orientational distribution of the subset of molecules contributing to the ENDOR spectrum recorded at a certain magnetic field value. For these orientations the ENDOR transitions can be calculated and the ENDOR powder spectrum composed. Typically, the resonance lines arise from cumulations (turning points) of the selected orientations in defined frequency ranges. By recording several ENDOR spectra across an EPR spectrum, the tensors of the interactions, not resolvable in EPR, are probed.


Spin Density ENDOR Spectrum Electronic Parameter Field Position Outer Line 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Feher G, Gere EA. 1956. Polarization of phosphorus nuclei in silicon. Phys Rev 103:501–503.CrossRefGoogle Scholar
  2. 2.
    Manikandan P, Carmieli R, Shane T, Kalb AJ, Goldfarb D. 2000. W-band ENDOR investigation of the manganese-binding site of concanavalin A: determination of proton hyperfine couplings and their signs. J Am Chem Soc 122:3488–3494.CrossRefGoogle Scholar
  3. 3.
    van Gastel M, Matthias S, Brecht M, Schroder O, Lendzian F, Bittl R, Ogata H, Higuchi Y, Lubitz W. 2006. A single-crystal ENDOR and density functional theory study of the oxidized states of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. J Biol Inorg Chem 11:41–51.PubMedCrossRefGoogle Scholar
  4. 4.
    Schonland DS. 1959. On the determination of the principal g-values in electron spin resonance. Proc Phys Soc (Lond) 73:788–792.CrossRefGoogle Scholar
  5. 5.
    Rist GH, Hyde JS. 1970. Ligand ENDOR of metal complexes in powders. J Chem Phys 52:4633–4643.CrossRefGoogle Scholar
  6. 6.
    Hurst GC, Henderson TA, Kreilick RW. 1985. Angle-selected ENDOR spectroscopy, 1: theoretical interpretation of ENDOR shifts from randomly orientated transition metal complexes. J Am Chem Soc 107:7294–7299.CrossRefGoogle Scholar
  7. 7.
    Henderson TA, Hurst GC, Kreilick RW. 1985. Angle-selected ENDOR spectroscopy, 2: determination of proton coordinates from a polycrystalline sample of bis(2,4- pentanedionato)copper(II). J Am Chem Soc 107:7299–7303.CrossRefGoogle Scholar
  8. 8.
    Hoffman BM, Martinsen J, Venters RA. 1984. General theory of polycrystalline ENDOR patterns: g and hyperfine tensors of arbitrary symmetry and relative orientation. J Magn Reson 59:110–123.Google Scholar
  9. 9.
    Hoffman BM, Venters RA, Martinsen J. 1985. General theory of polycrystalline ENDOR patterns: effects of finite EPR and ENDOR component linewidths. J Magn Reson 62:537–542.Google Scholar
  10. 10.
    Hüttermann J. 1993. ENDOR of randomly oriented mononuclear metalloproteins: toward structural determinations of the prosthetic group. Biol Magn Reson 13:219–252.Google Scholar
  11. 11.
    Hüttermann J, Däges GP, Reinhard H, Schmidt G. 1995. Metalloprotein-ENDORspectroscopy. In Nuclear magnetic resonance of paramagnetic macromolecules, pp. 165–192. Ed GN La Mar. New York: Kluwer Academic.Google Scholar
  12. 12.
    Hüttermann J, Kappl R. 1987. ENDOR: probing the coordination environment in metalloproteins. In Metal ions in biological systems, Vol 22, pp1–80. Ed H Sigel. New York: Marcel Dekker.Google Scholar
  13. 13.
    Kappl R, Ciurli S, Luchinat C, Hüttermann J. 1999. Probing structural and electronic properties of the oxidized [Fe4S4]3+ cluster of Ectothiorhodospira halophila iso-II highpotential iron–sulfur protein by ENDOR spectroscopy. J Am Chem Soc 121:1925–1935.CrossRefGoogle Scholar
  14. 14.
    Canne C, Lowe DJ, Fetzner S, Adams B, Smith AT, Kappl R, Bray RC, Hüttermann J. 1999. Kinetics and interactions of molybdenum and iron-sulfur centers in bacterial enzymes of the xanthine oxidase family: Mechanistic implications. Biochemistry 38:14077–14087.PubMedCrossRefGoogle Scholar
  15. 15.
    Canne C, Ebelshäuser M, Gay E, Shergill JK, Cammack R, Kappl R, Hüttermann J. 2000. Probing magnetic properties of the reduced [2Fe-2S] cluster of the ferredoxin from Arthrospira platensis by 1H ENDOR spectroscopy. J Biol Inorg Chem 5:514–526.PubMedCrossRefGoogle Scholar
  16. 16.
    Iwasaki M. 1974. Second-order perturbation treatment of general spin hamiltonian in an arbitrary coordinate system. J Magn Reson 16:417–423.Google Scholar
  17. 17.
    Weil JA, Bolton JR Wertz JE. 1994. Electron paramagnetic resonance. New York: John Wiley & Sons.Google Scholar
  18. 18.
    Wang DM, Hanson GR. 1995. A new method for simulating randomly oriented powder spectra in magnetic resonance: the Sydney Opera House (SOPHE) method. J Magn Reson Series A 117:1–8.CrossRefGoogle Scholar
  19. 19.
    Bertrand P, Guigliarelli B, Gayda JP, Beardwood P, Gibson JF. 1985. A ligand-field model to describe a new class of 2Fe–2S clusters in proteins and their synthetic analogs. Biochim Biophys Acta 831:261–266.Google Scholar
  20. 20.
    Dugad LB, LaMar GN, Banci L, Bertini I. 1990. Identification of localized redox states in plant-type 2-iron ferredoxins using the nuclear overhauser effect. Biochemistry 29:2263–2271.PubMedCrossRefGoogle Scholar
  21. 21.
    Kappl R, Ebelshäuser M, Hannemann F, Bernhardt R, Hüttermann J. 2006. Probing electronic and structural properties of the reduced [2Fe–2S] cluster by orientationselective 1H ENDOR spectroscopy: adrenodoxin vs. Rieske ISP. Appl Mag Reson 30:427–459.CrossRefGoogle Scholar
  22. 22.
    Bowman MK, Berry EA, Roberts AG, Kramer DM. 2004. Orientation of the g-tensor axes of the Rieske subunit in the cytochrome bc(1) complex. Biochemistry 43:430–436.PubMedCrossRefGoogle Scholar
  23. 23.
    Gloux J, Gloux P, Lamotte B, Mouesca JM, Rius G. 1994. The different [Fe4S4]3+ and [Fe4S4]+ species created by gamma irradiation in single crystals of the (Et4N)2[Fe4S4(SBenz)4] model compound: their EPR description and their biological significance. J Am Chem Soc 116:1953–1961.CrossRefGoogle Scholar
  24. 24.
    Mouesca JM, Rius G, Lamotte B. 1993. Single-crystal proton ENDOR studies of the [Fe4S4]3+ cluster: determination of the spin population-distribution and proposal of a model to interpret the 1H-NMR paramagnetic shifts in high-potential ferredoxins. J Am Chem Soc 115:4714–4731.CrossRefGoogle Scholar
  25. 25.
    Breiter DR, Meyer TE, Rayment I, Holden HM. 1991. The molecular structure of the high-potential iron-sulfur protein isolated from Ectothiorhodospira halophila determined at 2.5 Å resolution. J Biol Chem 266:18660–18667.PubMedGoogle Scholar
  26. 26.
    Banci L, Bertini I, Capozzi F, Carloni P, Ciurli S, Luchinat C, Piccioli M. 1993. The iron–sulfur cluster in the oxidized high-potential iron protein from Ectothiorhodospira halophila. J Am Chem Soc 115:3431–3440.CrossRefGoogle Scholar
  27. 27.
    Dilg AWE, Capozzi F, Mentler M, Iakovleva O, Luchinat C, Bertini I, Parak FG. 2001. Comparison and characterization of the [Fe4S4]2+/3+ centre in the wild-type and C77S mutated HiPIPs from Chromatium vinosum monitored by Mössbauer, 57Fe ENDOR and EPR spectroscopies. J Biol Inorg Chem 6:232–246.PubMedCrossRefGoogle Scholar
  28. 28.
    Priem AH, Klaassen AAK, Reijerse EJ, Meyer TE, Luchinat C, Capozzi F, Dunham WR, Hagen WR. 2005. EPR analysis of multiple forms of [4Fe–4S]3+ clusters in HiPIPs. J Biol Inorg Chem 10:417–424.PubMedCrossRefGoogle Scholar
  29. 29.
    Bertini I, Briganti F, Luchinat C, Scozzafava A, Sola M. 1991. 1H-NMR spectroscopy and the electronic structure of the high-potential iron sulfur protein from Chromatium vinosum. J Am Chem Soc 113:1237–1245.CrossRefGoogle Scholar
  30. 30.
    Bertini I, Luchinat C. 1996. Electronic isomerism in oxidized Fe4S4 high-potential iron–sulfur proteins. ACS Symp Ser 653:57–73.CrossRefGoogle Scholar
  31. 31.
    Bertini I, Ciurli S, Luchinat C. 1995. The electronic structure of FeS centers in proteins and models, a contribution to the understanding of their electron-transfer properties. Struct Bonding 83:1–53.Google Scholar
  32. 32.
    Dunham WR, Sands RH. 2003. g-strain, ENDOR, and structure of active centers of two-iron ferredoxins. Biochem Biophys Res Comm 312:255–261.PubMedCrossRefGoogle Scholar
  33. 33.
    Guigliarelli B, Bertrand P. 1999. Application of EPR spectroscopy to the structural and functional study of iron–sulfur proteins. Adv Inorg Chem 47:421–497.CrossRefGoogle Scholar
  34. 34.
    Gibson JF, Hall DO, Thornley JH, Whatley FR. 1966. Iron complex in spinach ferredoxin. Proc Natl Acad Sci USA 56:987–990.PubMedCrossRefGoogle Scholar
  35. 35.
    Mouesca JM, Noodleman L, Case DA, Lamotte B. 1995. Spin-densities and spin coupling in iron–sulfur clusters: a new analysis of hyperfine coupling-constants. Inorg Chem 34:4347–4359.CrossRefGoogle Scholar
  36. 36.
    Fritz J, Anderson R, Fee J, Palmer G, Sands RH, Tsibris JCM, Gunsalus IC, Orme-Johnson WH, Beinert H. 1971. Iron electron-nuclear double resonance (ENDOR) of 2-iron ferredoxins from spinach, parsley, pig adrenal cortex and Pseudomonas putida. Biochim Biophys Acta 253:110–133.PubMedCrossRefGoogle Scholar
  37. 37.
    Rius G, Lamotte B. 1989. Single-crystal ENDOR study of a 57Fe-enriched iron sulfur [Fe4S4]3+ cluster. J Am Chem Soc 111:2464–2469.CrossRefGoogle Scholar
  38. 38.
    Middleton P, Dickson DPE, Johnson CE, Rush JD. 1980. Interpretation of the Mössbauer spectra of the high-potential iron protein from Chromatium. Eur J Biochem 104:289–296.PubMedCrossRefGoogle Scholar
  39. 39.
    Dilg AWE, Mincione G, Achterhold K, Iakovleva O, Mentler M, Luchinat C, Bertini I, Parak FG. 1999. Simultaneous interpretation of Mössbauer, EPR and 57Fe ENDOR spectra of the [Fe4S4] cluster in the high-potential iron protein I from Ectothiorhodospira halophila. J Biol Inorg Chem 4:727–741.PubMedCrossRefGoogle Scholar
  40. 40.
    Noodleman L, Chen JL, Case DA, Giori C, Rius G, Mouesca JM, Lamotte B. 1995. Isotropic hyperfine coupling in high-potential [Fe4S4]3+ models. In Nuclear magnetic resonance of paramagnetic macromolecules, pp. 339–367. Ed GN La Mar. New York: Kluwer Academic.Google Scholar

Copyright information

© Springer-Verlag New York 2009

Authors and Affiliations

  • Reinhard Kappl
    • 1
  • Gerhard Bracic
    • 1
  • Jürgen Hüttermann
    • 1
  1. 1.Fachrichtung BiophysikUniversität des Saarlandes66421 HomburgGermany

Personalised recommendations