JBIC Journal of Biological Inorganic Chemistry

, Volume 9, Issue 7, pp 828–838

SufA/IscA: reactivity studies of a class of scaffold proteins involved in [Fe-S] cluster assembly

  • S. Ollagnier-de-Choudens
  • Y. Sanakis
  • M. Fontecave
Original Article

Abstract

IscA/SufA proteins belong to complex protein machineries which are involved in iron-sulfur cluster biosynthesis. They are defined as scaffold proteins from which preassembled clusters are transferred to target apoproteins. The experiments described here demonstrate that the transfer reaction proceeds in two observable steps: a first fast one leading to a protein–protein complex between the cluster donor (SufA/IscA) and the acceptor (biotin synthase), and a slow one consisting of cluster transfer leading to the apoform of the scaffold protein and the holoform of the target protein. Mutation of cysteines in the acceptor protein specifically inhibits the second step of the reaction, showing that these cysteines are involved in the cluster transfer mechanism but not in complex formation. No cluster transfer from IscA to IscU, another scaffold of the isc operon, could be observed, whereas IscU was shown to be an efficient cluster source for cluster assembly in IscA. Implications of these results are discussed.

Keywords

Cluster assembly Iron-sulfur proteins IscA protein Scaffold proteins SufA protein 

Abbreviations

AdoMet

S-adenosylmethionine

APS

adenosine-5′-phosphosulfate

BioB

biotin synthase

DAF

deazaflavin

DTB

dethiobiotin

DTT

dithiothreitol

EDTA

ethylenediaminetetraacetic acid

hisIscU/A

six histidine residues at the N-terminus of IscU/A

PCR

polymerase chain reaction

PLP

pyridoxal 5-phosphate

SufAhis

six histidine residues at the C-terminus of SufA

References

  1. 1.
    Beinert H, Holm RH, Münck E (1997) Science 277:653–659CrossRefPubMedGoogle Scholar
  2. 2.
    Jacobson MR, Cash VL, Weiss MC, Laird NF, Newton WE, Dean DR (1989) Mol Gen Genet 219:49–57PubMedGoogle Scholar
  3. 3.
    Olson JW, Agar JN, Johnson MK, Maier RJ (2000) Biochemistry 39:16213–16219CrossRefPubMedGoogle Scholar
  4. 4.
    Ali V, Shigeta Y, Tokumoto U, Takahashi Y, Tomoyoshi N (2004) J Biol Chem 279:16863–16874CrossRefPubMedGoogle Scholar
  5. 5.
    Zheng L, Cash VL, Flint DH, Dean DR (1998) J Biol Chem 273:13264–13272CrossRefPubMedGoogle Scholar
  6. 6.
    Skovran E, Down DM (2000) J Bacteriol 182:3896–3903CrossRefPubMedGoogle Scholar
  7. 7.
    Kispal G, Csere P, Prohl C, Lill R (1999) EMBO J 18:3981–3989CrossRefPubMedGoogle Scholar
  8. 8.
    Strain J, Lorenz CR, Bode J, Garland S, Smolen GA, Ta DT, Vickery LE, Culotta VC (1998) J Biol Chem 273:31138–31144CrossRefPubMedGoogle Scholar
  9. 9.
    Pelzer W, Mühlenhoff U, Diekert K, Siegmund K, Kispal G, Lill R (2000) FEBS Lett 476:134–139CrossRefPubMedGoogle Scholar
  10. 10.
    Li J, Kogan M, Knight SAB, Pain D, Dancis A (1999) J Mol Chem 274:33025–33034CrossRefGoogle Scholar
  11. 11.
    Lange H, Kaut A, Kispal G, Lill R (2000) Proc Natl Acad Sci USA 97:1050–1055CrossRefPubMedGoogle Scholar
  12. 12.
    Jensen LT, Culotta VC (2000) Mol Cell Biol 20:3918–3927CrossRefPubMedGoogle Scholar
  13. 13.
    Garland SA, Hoff KG, Vickery LE, Culotta VC (1999) J Mol Biol 294:897–907CrossRefPubMedGoogle Scholar
  14. 14.
    Schilke B, Voisine C, Beinert H, Craig E (1999) Proc Natl Acad Sci USA 96:10206–10211CrossRefPubMedGoogle Scholar
  15. 15.
    Voisine C, Schilke B, Ohlson M, Beinert H, Marszalek K, Craig EA (2000) Mol Cell Biol 20:3677–3684CrossRefPubMedGoogle Scholar
  16. 16.
    Voisine C, Cheng YC, Ohlson M, Schilke B, Hoff K, Beinert H, Marszalek J, Craig E (2001) Proc Natl Acad Sci USA 98:1483–1488CrossRefPubMedGoogle Scholar
  17. 17.
    Wu G, Mansy SS, Hemann C, Hille R, Surerus KK, Cowan JA (2002) J Biol Inorg Chem 7:526–532CrossRefPubMedGoogle Scholar
  18. 18.
    Wu G, Mansy SS, Wu S, Surerus KK, Foster MW, Cowan JA (2002) Biochemistry 41:5024–5032CrossRefPubMedGoogle Scholar
  19. 19.
    Wu S, Wu G, Surerus KK, Cowan JA (2002) Biochemistry 41:8876–8885CrossRefPubMedGoogle Scholar
  20. 20.
    Takahashi Y, Tokumoto U (2002) J Biol Chem 277:28380–28383CrossRefPubMedGoogle Scholar
  21. 21.
    Patzer SI, Hantke K (1999) J Bacteriol 181:3307–3309PubMedGoogle Scholar
  22. 22.
    Ellis KES, Clough B, Saldanha JW, Wilson RJM (2001) Mol Microbiol 41:973–981CrossRefPubMedGoogle Scholar
  23. 23.
    Zheng M, Wang X, Templeton LJ, Smulski DR, laRossa RA, Storz G (2001) J Bacteriol 183:4562–4570CrossRefPubMedGoogle Scholar
  24. 24.
    Nachin L, El Hassouni M, Loiseau L, Expert D, Barras F (2001) Mol Microbiol 39:960–972CrossRefPubMedGoogle Scholar
  25. 25.
    Hantke K (2002) J Mol Microbiol Biotechnol 4:217–222PubMedGoogle Scholar
  26. 26.
    Nachin L, Loiseau L, Expert D, Barras D (2003) EMBO J 22:427–437CrossRefPubMedGoogle Scholar
  27. 27.
    Schwartz CJ, Djaman O, Imlay JA, Kiley P (2000) Proc Natl Acad Sci USA 97:9009–9014CrossRefPubMedGoogle Scholar
  28. 28.
    Loiseau L, Ollagnier-de Choudens S, Nachin L, Fontecave M, Barras F (2003) J Biol Chem 278:38352–38359CrossRefPubMedGoogle Scholar
  29. 29.
    Ollagnier-de Choudens S, Lascoux D, Loiseau L, Forest E, Barras F, Fontecave M (2003) FEBS Lett 555:263–267CrossRefPubMedGoogle Scholar
  30. 30.
    Wayne-Outten F, Wood MJ, Munoz FM, Stortz G (2003) J Biol Chem 278:45713–45719CrossRefPubMedGoogle Scholar
  31. 31.
    Hoff KG, Silberg JJ, Vickery LE (2000) Proc Natl Acad Sci USA 97:7790–7795CrossRefPubMedGoogle Scholar
  32. 32.
    Hoff KG, Ta DT, Tapley TL, Silberg JJ, Vickery LE (2002) J Biol Chem 277:27353–27359CrossRefPubMedGoogle Scholar
  33. 33.
    Silberg JJ, Hoff KG, Tapley TL, Vickery LE (2001) J Biol Chem 276:1696–1700CrossRefPubMedGoogle Scholar
  34. 34.
    Ollagnier-de Choudens S, Sanakis Y, Loiseau L, Nachin L, Barras F, Fontecave M (2003) J Biol Chem 278:17993–18001CrossRefPubMedGoogle Scholar
  35. 35.
    Ollagnier-de Choudens S, Mattioli T, Takahashi Y, Fontecave M (2001) J Biol Chem 276:22604–22607CrossRefPubMedGoogle Scholar
  36. 36.
    Agar JN, Krebs C, Frazzon J, Huynh BH, Dean DR, Johnson MK (2000) Biochemistry 39:7856–7862CrossRefPubMedGoogle Scholar
  37. 37.
    Agar JN, Zheng L, Cash VL, Dean DR, Johnson MK (2000) J Am Chem Soc 122:2136–2137CrossRefGoogle Scholar
  38. 38.
    Krebs C, Agar JN, Smith AD, Frazzon J, Dean DR, Huynh BH, Johnson MK (2001) Biochemistry 40:14069–14080CrossRefPubMedGoogle Scholar
  39. 39.
    Mansy SS, Wu G, Surerus KK, Cowan JA (2002) J Biol Chem 277:21397–21404CrossRefPubMedGoogle Scholar
  40. 40.
    Wollenberg M, Berndt C, Bill E, Schwenn JD, Seidler A (2003) Eur J Biochem 270:1662–1671PubMedGoogle Scholar
  41. 41.
    Ollagnier-de Choudens S, Sanakis Y, Hewitson KS, Roach P, Baldwin JE, Münck E, Fontecave M (2000) Biochemistry 39:4165–4173CrossRefPubMedGoogle Scholar
  42. 42.
    Ollagnier-De-Choudens S, Mulliez E, Hewitson KS, Fontecave M (2002) Biochemistry 41:9145–9152CrossRefPubMedGoogle Scholar
  43. 43.
    Ugulava NB, Gibney BR, Jarrett JT (2001) Biochemistry 40:8343–8351CrossRefPubMedGoogle Scholar
  44. 44.
    Ugulava NB, Sacanell CJ, Jarrett JT (2001) Biochemistry 40:8352–8358CrossRefPubMedGoogle Scholar
  45. 45.
    Cosper MM, Jameson GLN, Hernandez HL, Krebs C, Huynh BN, Johnson MK (2004) Biochemistry 43:2007–2021CrossRefPubMedGoogle Scholar
  46. 46.
    Jameson GLN, Cosper MM, Hernandez HL, Johnson MK, Huynh BH (2004) Biochemistry 43:2022–2031CrossRefPubMedGoogle Scholar
  47. 47.
    Berkovitch F, Nicolet Y, Wan JT, Jarrett JT, Drennan CL (2004) Science 303:76–79CrossRefPubMedGoogle Scholar
  48. 48.
    Ashton WT, Brown R, Tolman RL (1978) J Heterocycl Chem 15:489–491Google Scholar
  49. 49.
    Tokumoto U, Nomura S, Minami Y, Mihara H, Kato S, Kurihara T, Esaki N, Kanazawa H, Matsubara H, Takahashi Y (2002) J Biochem (Tokyo) 131:713–719Google Scholar
  50. 50.
    Hewitson K, Baldwin JE, Shaw NM, Roach P (2000) FEBS Lett 466:372–376CrossRefPubMedGoogle Scholar
  51. 51.
    Hewitson KS, Ollagnier-de Choudens S, Sanakis Y, Shaw NM, Baldwin JE, Münck E, Roach P, Fontecave M (2002) J Biol Inorg Chem 7:83–93CrossRefPubMedGoogle Scholar
  52. 52.
    Bradford MM (1976) Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  53. 53.
    Fish WW (1988) Methods Enzymol 158:357–364CrossRefPubMedGoogle Scholar
  54. 54.
    Beinert H (1983) Anal Biochem 131:373–378PubMedGoogle Scholar
  55. 55.
    Wu G, Cowan JA (2003) Biochemistry 42:5784–5791CrossRefPubMedGoogle Scholar
  56. 56.
    Bilder PW, Ding H, Newcomer ME (2004) Biochemistry 43:133–139PubMedGoogle Scholar
  57. 57.
    Cupp-Vickery JR, Silberg JJ, Ta DT, Vickery LE (2004) J Mol Biol 338:127–137CrossRefPubMedGoogle Scholar
  58. 58.
    Urbina HD, Silberg JJ, hoff KG, Vickery LE (2001) J Biol Chem 276:44521–44526CrossRefPubMedGoogle Scholar
  59. 59.
    Kato S, Mihara H, Kurihara T, Takahashi Y, Tokumoto U, Yoshimura T, Esaki N (2002) Proc Natl Acad Sci USA 99:5948–5952CrossRefPubMedGoogle Scholar
  60. 60.
    Nishio K, Nakai M (2000) J Biol Chem 275:22615–22618CrossRefPubMedGoogle Scholar

Copyright information

© SBIC 2004

Authors and Affiliations

  • S. Ollagnier-de-Choudens
    • 1
  • Y. Sanakis
    • 2
    • 3
  • M. Fontecave
    • 1
  1. 1.Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques, DRDC-CBCEA/CNRS/Université Joseph Fourier, UMR 5047Grenoble France
  2. 2.NCSR, DemokritosInstitute of Materials Science Ag. ParaskeviGreece
  3. 3.Department of Biological Applications and TechnologiesUniversity of IoanninaIoanninaGreece

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