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Structural basis for VO2+ inhibition of nitrogenase activity (A): 31P and 23Na interactions with the metal at the nucleotide binding site of the nitrogenase Fe protein identified by ENDOR spectroscopy

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An Erratum to this article was published on 24 April 2008

Abstract

We previously reported the vanadyl hyperfine couplings of VO2+–ATP and VO2+–ADP complexes in the presence of the nitrogenase Fe protein from Klebsiella pneumoniae (Petersen et al. in Biochemistry 41:13253–13263, 2002). It was demonstrated that different VO2+–nucleotide coordination environments coexist and are distinguishable by electron paramagnetic resonance (EPR) spectroscopy. Here orientation-selective continuous-wave electron–nuclear double resonance (ENDOR) spectra have been investigated especially in the low-radio-frequency range in order to identify superhyperfine interactions with nuclei other than protons. Some of these resonances have been attributed to the presence of a strong interaction with a 31P nucleus although no resolvable superhyperfine structure due to 31P or other nuclei was detected in the EPR spectra. The superhyperfine coupling component is determined to be about 25 MHz. Such a 31P coupling is consistent with an interaction of the metal with phosphorus from a directly, equatorially coordinated nucleotide phosphate group(s). Additionally, novel more prominent 31P ENDOR signals are detected in the low-frequency region. Some of these correspond to a relatively weak 31P coupling. This coupling is present with ATP for all pH forms but is absent with ADP. The ENDOR resonances of these weakly coupled 31P are likely to originate from an interaction of the metal with a nucleotide phosphate group of the nucleoside triphosphate and are attributed to a phosphorus with axial characteristics. Another set of resonances, split about the nuclear Zeeman frequency of 23Na, was detected, suggesting that a monovalent Na+ ion is closely associated with the divalent metal–nucleotide binding site. Na+ replacement by K+ unambiguously confirmed that ENDORs at radio frequencies between 3.0 and 4.5 MHz arise from an interaction with Na+ ions. In contrast to the low-frequency 31P signal, these resonances are present in spectra with both ADP and ATP, and for both low- and neutral-pH forms, although slight differences are detected, showing that these are sensitive to the nucleotide and pH.

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Notes

  1. It is noteworthy that ENDOR spectra may not select purely parallel or perpendicular spin packets. ENDOR spectra recorded at perpendicular field settings also possess different amounts of parallel contributions, and some parallel field settings may have perpendicular contributions. However, these can be neglected for the outer parallel resonances, and parallel contributions at a perpendicular EPR field setting are often negligible owing to the much higher intensity of the contributions from the latter. In addition, spectra recorded at, for example, the M I  = −5/2 parallel field setting will have contributions from other parallel spin manifolds, in particular the −7/2 hyperfine line, and correspondingly inner perpendicular hyperfine lines will have contributions from the perpendicular manifolds with larger M I .

  2. Thus, instead of listing all three g i and A i tensor components with i = x, y, z, we refer to g || and A || or g ⊥ and A ⊥, for parallel or perpendicular orientation, respectively, with regard to the fixed V=O bond direction.

  3. Assuming that the phosphorus superhyperfine coupling is predominantly isotropic, and using an atomic isotropic coupling for 31P of 1.02 × 104 MHz [37], we can calculate from the observed coupling that it accounts for only about 0.25% of the unpaired spin density in the phosphorus 3s orbital.

  4. This superhyperfine coupling was measured by EPR spectroscopy, the others by continuous-wave ENDOR spectroscopy or pulsed techniques. The coupling determined by ENDOR yielded a somewhat larger value of 20.6 MHz [39] and another study for VO–ADP at pH 6 reported a value of 19.3 MHz [40]. Moreover couplings for the [VO(ATP)2]2+ complex determined by hyperfine sublevel correlation (HYSCORE) were 14.8 and 9 MHz for interactions with the β- and γ-phosphate atoms of ATP, respectively [38]. Phosphorus interactions for vanadyl pyrophosphate doped into a host material have also been determined by HYSCORE to be 18.2 MHz [41]. In addition 31P couplings have been measured for some biological systems, such as the two nucleotide binding proteins S-adenosyl methionine synthetase [42, 43] with bound pyrophosphate and a single coupling constant of 23.8 MHz from presumably two equivalent 31P groups, and F1-ATPase [38] in the presence of VO–ATP with couplings of 15.5 and 8.7 MHz for interactions with β- and γ-phosphorus atoms, respectively.

  5. Note, that this is in contrast to the previous model of the Mg2+ coordination geometry in related guanosine binding proteins of the homologous family of switch proteins where the β- and γ-phosphate groups are believed to be both bound in equatorial positions of the metal ion [44]. The other difference is the purported axial coordination of Ser16 in the Fe protein; the equivalent serine/threonine in other nucleotide binding proteins presumably occupies an equatorial position.

  6. This small 31P coupling is observed at low and neutral pH. The trans-coordinated water molecule is, however, only present at low pH. If the phosphorus would coordinate VO2+ instead of the water molecule, it should only be observed at neutral pH, which is clearly not the case.

  7. This conformation should compensate the charges of the nucleotide phosphate moiety, especially at the hydrolytically important β- and γ-phosphate groups, very effectively. This may have relevance for the catalytic mechanism, since ATP hydrolysis appears to depend critically on the charge environment at the nucleotide binding site (for example, in the absence of metals no ATP hydrolysis occurs in nitrogenase, so they seem to be essential for the establishment of the appropriate steric and/or electrostatic environment).

Abbreviations

ADP:

Adenosine 5′-diphosphate

ATP:

Adenosine 5′-triphosphate

ENDOR:

Electron–nuclear double resonance

EPR:

Electron paramagnetic resonance

ESEEM:

Electron spin echo envelope modulation

HYSCORE:

Hyperfine sublevel correlation

Kp2:

Nitrogenase Fe protein from Klebsiella pneumoniae

Tris:

Tris(hydroxymethyl)aminomethane

References

  1. Thompson KH, McNeill JH, Orvig C (1999) Chem Rev 99:2561–2571

    Article  PubMed  CAS  Google Scholar 

  2. Shechter Y, Goldwaser I, Mironchik M, Fridkin M, Gefel D (2003) Coord Chem Rev 237:3–11

    Article  CAS  Google Scholar 

  3. Djordjevic C (1995) In: Sigel H, Sigel A (eds) Metal ions in biological systems, vanadium and its role in life. Marcel Dekker, New York, pp 595–616

  4. Evangelou AM (2002) Crit Rev Oncol Hematol 42:249–265

    Article  PubMed  Google Scholar 

  5. Davies DR, Hol GJ (2004) FEBS Lett 577:315–321

    Article  PubMed  CAS  Google Scholar 

  6. Simons TJB (1979) Nature 281:337–338

    Article  PubMed  CAS  Google Scholar 

  7. Stankiewicz PJ, Tracey AS, Crans DC (1995) In: Sigel H, Sigel A (eds) Metal ions in biological systems, vol 31. Marcel Dekker, Inc., New York, Basel, Hong Kong, pp 287–324

  8. Mortenson LE, Seefeldt LC, Morgan TV, Bolin JT (1993) In: Meister A (ed) Advances in enzymology, vol 67. Wiley, Chichester, pp 299–374

  9. Zumft WG, Mortenson LE, Palmer G (1974) Eur J Biochem 46:525–535

    Article  PubMed  CAS  Google Scholar 

  10. Yates MG (1972) Eur J Biochem 29:386–392

    Article  PubMed  CAS  Google Scholar 

  11. Walker GA, Mortenson LE (1974) Biochemistry 13:2382–2388

    Article  PubMed  CAS  Google Scholar 

  12. Stephens PJ, McKenna CE, Smith BE, Nguyen HT, McKenna MC, Thomson AJ, Devlin F, Jones JB (1979) Proc Natl Acad Sci USA 76:2585–2589

    Article  PubMed  CAS  Google Scholar 

  13. Chen L, Gavini N, Tsuruta H, Eliezer D, Burgess BK, Doniach S, Hodgson KO (1994) J Biol Chem 269:3290–3294

    PubMed  CAS  Google Scholar 

  14. Meyer J, Gaillard J, Moulis J-M (1988) Biochemistry 27:6150–6156

    Article  PubMed  CAS  Google Scholar 

  15. Orme-Johnson WH, Hamilton WD, Ljones T, Tso MYW, Burris RH, Shah VK, Brill WJ (1972) Proc Natl Acad Sci USA 69:3142–3145

    Article  PubMed  CAS  Google Scholar 

  16. Smith BE, Lowe DJ, Bray RC (1973) Biochem J 135:331–341

    PubMed  CAS  Google Scholar 

  17. Morgan TV, McCracken J, Orme-Johnson WH, Mims WB, Mortenson LE, Peisach J (1990) Biochemistry 29:3077–3082

    Article  PubMed  CAS  Google Scholar 

  18. Seefeldt LC, Mortenson LE (1993) Protein Sci 2:93–102

    Article  PubMed  CAS  Google Scholar 

  19. Bishop EO, Lambert MD, Orchard D, Smith BE (1977) Biochim Biophys Acta 482:286–300

    PubMed  CAS  Google Scholar 

  20. Petersen J, Gessner C, Fisher K, Mitchell CJ, Lowe DJ, Lubitz W (2005) Biochem J 391:527–539

    Article  PubMed  CAS  Google Scholar 

  21. Petersen J, Fisher K, Mitchell CJ, Lowe DJ (2002) Biochemistry 41:13253–13263

    Article  PubMed  CAS  Google Scholar 

  22. Chasteen ND (1993) Methods in enzymology. In: Riordan JF, Vallee BL (eds) Metallobiochemistry, vol 227. Academic Press, London, pp 232–244

    Google Scholar 

  23. Sen S, Krishnakumar A, McClead J, Johnson MK, Seefeldt MC, Szilagyi RK, Peters JW (2006) J Inorg Biochem 100:1041–1052

    Article  PubMed  CAS  Google Scholar 

  24. Eady RR, Smith BE, Cook KA, Postgate JR (1972) Biochem J 128:655–675

    PubMed  CAS  Google Scholar 

  25. Abragam A, Bleaney B (1970) Electron paramagnetic resonance of transition ions. Clarendon Press, Oxford

    Google Scholar 

  26. Tipton PA, McCracken J, Cornelius JB, Peisach J (1989) Biochemistry 28:5720–5728

    Article  PubMed  CAS  Google Scholar 

  27. Hoogstraten CG, Grant CV, Horton TE, DeRose VJ, Britt RD (2002) J Am Chem Soc 124:834–841

    Article  PubMed  CAS  Google Scholar 

  28. Hoffman BM, DeRose VJ, Doan PE, Gurbiel RJ, Houseman ALP, Telser J (1993) Biological magnetic resonance. In: Berliner LJ, Reuben J (eds) vol 13. Springer, Heidelberg, pp 151–214

  29. Mims WB, Peisach J (1989) In: Hoff AJ (ed) Advanced EPR, applications in biology and biochemistry. Elsevier, Amsterdam, pp 1–57

  30. van Willigen H, Chandrashekar TK (1983) J Am Chem Soc 105:4232–4235

    Article  Google Scholar 

  31. Pöppl A, Rudolf T, Manikandan P, Goldfarb D (2000) J Am Chem Soc 122:10194–10200

    Article  CAS  Google Scholar 

  32. Kirmse R, Böttcher R, Willems JP, Reijerse EJ, DeBoer E (1991) J Chem Soc Faraday Trans 87:3105–3111

    Article  CAS  Google Scholar 

  33. Sabbe K, Callens F, Boesman E (1998) Appl Magn Reson 15:539–555

    Article  CAS  Google Scholar 

  34. Luca V, MacLachlan DJ, Bramley R (1999) Phys Chem Chem Phys 1:2597–2606

    Article  CAS  Google Scholar 

  35. Boechat CB, Terra J, Eon J-G, Ellis DE, Rossi AM (2003) Phys Chem Chem Phys 5:44290–4298

    Article  CAS  Google Scholar 

  36. Gutjahr M, Hoentsch J, Böttcher R, Storcheva O, Köhler K, Pöppl A (2004) J Am Chem Soc 126:2905–2911

    Article  PubMed  CAS  Google Scholar 

  37. Kurreck H, Kirste B, Lubitz W (1988) Electron nuclear double resonance spectroscopy of radicals in solution; application to organic and biological chemistry. VCH Publishers, New York

    Google Scholar 

  38. Buy C, Matsui T, Andrianambinintsoa S, Sigalat C, Girault G, Zimmermann J-L (1996) Biochemistry 35:14281–14293

    Article  PubMed  CAS  Google Scholar 

  39. Mustafi D, Telser J, Makinen MW (1992) J Am Chem Soc 114:6219–6226

    Article  CAS  Google Scholar 

  40. Alberico E, Dewaele D, Kiss T, Micera G (1995) J Chem Soc Dalton Trans 425–430

  41. Gromov I, Shane J, Forrer J, Rakhmatoullin R, Rozentzwaig Y, Schweiger A (2001) J Magn Reson 149:196–203

    Article  PubMed  CAS  Google Scholar 

  42. Markham GD, Leyh TS (1987) J Am Chem Soc 109:599–600

    Article  CAS  Google Scholar 

  43. Markham GD (1984) Biochemistry 23:470–478

    Article  PubMed  CAS  Google Scholar 

  44. Pai EF, Krengel U, Petsko GA, Goody RS, Kabsch W, Wittinghofer A (1990) EMBO J 9:2351–2359

    PubMed  CAS  Google Scholar 

  45. Dikanov SA, Liboiron BD, Orvig C (2002) J Am Chem Soc 124:2969–2978

    Article  PubMed  CAS  Google Scholar 

  46. Petersen J, Michell CJ, Fisher K, Lowe DJ (2008) J Biol Inorg Chem (in press). doi:10.1007/s00775-008-0364-9

  47. Wittinghofer A (1997) Curr Biol 7:R682–R685

    Article  PubMed  CAS  Google Scholar 

  48. Schindelin H, Kisker C, Schlessman JL, Howard JB, Rees DC (1997) Nature 387:370–376

    Article  PubMed  CAS  Google Scholar 

  49. Jang SB, Seefeldt LC, Peters JW (2000) Biochemistry 39:14745–14752

    Article  PubMed  CAS  Google Scholar 

  50. Suelter CH (1970) Science 168:789–795

    Article  PubMed  CAS  Google Scholar 

  51. Zhang C, Markham GD, LoBrutto R (1993) Biochemistry 32:9866–9873

    Article  PubMed  CAS  Google Scholar 

  52. Schneider B, Sigalat C, Amano T, Zimmermann J-L (2000) Biochemistry 39:15500–15512

    Article  PubMed  CAS  Google Scholar 

  53. Mildvan AS (1974) Ann Rev Biochem 43:357–399

    Article  PubMed  CAS  Google Scholar 

  54. Mildvan AS, Sloan DL, Fung CH, Gupta RK, Melamud E (1976) J Biol Chem 251:2431–2434

    PubMed  CAS  Google Scholar 

  55. Larsen TM, Benning MM, Wesenberg GE, Rayment I, Reed GH (1997) Arch Biochem Biophys 345:199–206

    Article  PubMed  CAS  Google Scholar 

  56. Larsen TM, Benning MM, Rayment I, Reed GH (1998) Biochemistry 37:6247–6255

    Article  PubMed  CAS  Google Scholar 

  57. Larsen TM, Loughlin LT, Holden HM, Rayment I, Reed GH (1994) Biochemistry 33:6301–6309

    Article  PubMed  CAS  Google Scholar 

  58. Jurica MS, Mesecar A, Heath PJ, Shi W, Nowak T, Stoddard BL (1998) Structure 6:195–210

    Article  PubMed  CAS  Google Scholar 

  59. Lord KA, Reed GH (1987) Inorg Chem 26:1464–1466

    Article  CAS  Google Scholar 

  60. Werneburg BG, Ash DE (1997) Biochemistry 36:14392–14402

    Article  PubMed  CAS  Google Scholar 

  61. Shimizu T, Mims WB, Peisach J, Davis JL (1979) J Chem Phys 70:2249–2254

    Article  CAS  Google Scholar 

  62. Buy C, Girault G, Zimmermann J-L (1996) Biochemistry 35:9880–9891

    Article  PubMed  CAS  Google Scholar 

  63. Deits TL, Howard JB (1990) J Biol Chem 265:3859–3867

    PubMed  CAS  Google Scholar 

  64. Zimmermann J-L, Schneider B, Morlet S, Amano T, Sigalat C (2000) Spectrochim Acta A56:285–299

    Google Scholar 

  65. Bogumil R, Hüttermann J, Kappl R, Stabler R, Sudfeldt C, Witzel H (1991) Eur J Biochem 196:305–312

    Article  PubMed  CAS  Google Scholar 

  66. Mulks CF, Kirste B, vanWilligen H (1982) J Am Chem Soc 104:5906–5911

    Article  CAS  Google Scholar 

  67. Hanna PM, Chasteen ND, Rottman GA, Aisen P (1991) Biochemistry 30:9210–9216

    Article  PubMed  CAS  Google Scholar 

  68. Petersen J, Hawkes TR, Lowe DJ (1997) J Biol Inorg Chem 2:308–319

    Article  CAS  Google Scholar 

  69. Petersen J, Hawkes TR, Lowe DJ (1998) J Am Chem Soc 120:10978–10979

    Article  CAS  Google Scholar 

  70. Houseman ALP, LoBrutto R, Frasch WD (1994) Biochemistry 33:10000–10006

    Article  PubMed  CAS  Google Scholar 

  71. Zimmermann J-L, Amano T, Sigalat C (1999) Biochemistry 38:15343–15351

    Article  PubMed  CAS  Google Scholar 

  72. Crampton D J, LoBrutto R, Frasch WD (2001) Biochemistry 40:3710–3716

    Article  PubMed  CAS  Google Scholar 

  73. Houseman ALP, LoBrutto R, Frasch WD (1995) Biochemistry 34:3277–3285

    Article  PubMed  CAS  Google Scholar 

  74. Sakurai H, Goda T, Yoshimura T (1982) Biochem Biophys Res Commun 108:474–478

    Article  PubMed  CAS  Google Scholar 

  75. Sakurai H, Goda T, Shimomura S, Yoshimura T (1982) Biochem Biophys Res Commun 104:1421–1426

    Article  PubMed  CAS  Google Scholar 

  76. Baran EJ (1995) In: Sigel H, Sigel A (eds) Metal ions in biological systems, vanadium and its role in life. Marcel Dekker, New York, pp 129–146

  77. Sigel H (1987) Eur J Biochem 165:65–72

    Article  PubMed  CAS  Google Scholar 

  78. Dikanov SA, Liboiron BD, Thompson KH, Vera E, Yuen VG, McNeill JH, Orvig C (1999) J Am Chem Soc 121:11004–11005

    Article  CAS  Google Scholar 

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Acknowledgments

We gratefully acknowledge the help of C.J. Mitchell for preparing some of the samples. EPR simulations of the VO spectra were conducted using the program LSIM written by D. Collison and kindly provided by S. Fairhurst. D.J.L. thanks BBSRC for financial support through the Core Strategic Grant to the John Innes Centre.

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  1. Karl Fisher

    Present address: Manchester Interdisciplinary Biocentre, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK

Authors and Affiliations

  1. Department of Biological Chemistry, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK

    Jan Petersen, Karl Fisher & David J. Lowe

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  1. Jan Petersen
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Correspondence to Jan Petersen.

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An erratum to this article can be found at http://dx.doi.org/10.1007/s00775-008-0373-8

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Petersen, J., Fisher, K. & Lowe, D.J. Structural basis for VO2+ inhibition of nitrogenase activity (A): 31P and 23Na interactions with the metal at the nucleotide binding site of the nitrogenase Fe protein identified by ENDOR spectroscopy. J Biol Inorg Chem 13, 623–635 (2008). https://doi.org/10.1007/s00775-008-0360-0

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