Abstract
The ferredoxin from the thermoacidophile Acidianus ambivalens is a representative of the archaeal family of di-cluster [3Fe-4S][4Fe-4S] ferredoxins. Previous studies have shown that these ferredoxins are intrinsically very stable and led to the suggestion that upon protein unfolding the iron-sulfur clusters degraded via linear three-iron sulfur center species, with 610 and 520 nm absorption bands, resembling those observed in purple aconitase. In this work, a kinetic and spectroscopic investigation on the alkaline chemical denaturation of the protein was performed in an attempt to elucidate the degradation pathway of the iron-sulfur centers in respect to protein unfolding events. For this purpose we investigated cluster dissociation, iron release and protein unfolding by complementary biophysical techniques. We found that shortly after initial protein unfolding, iron release proceeds monophasically at a rate comparable to that of cluster degradation, and that no typical EPR features of linear three-iron sulfur centers are observed. Further, it was observed that EDTA prevents formation of the transient bands and that sulfide significantly enhances its intensity and lifetime, even after protein unfolding. Altogether, our data suggest that iron sulfides, which are formed from the release of iron and sulfide resulting from cluster degradation during protein unfolding in alkaline conditions, are in fact responsible for the observed intermediate spectral species, thus disproving the hypothesis suggesting the presence of a linear three-iron center intermediate. Kinetic studies monitored by visible, fluorescence and UV second-derivative spectroscopies have elicited that upon initial perturbation of the tertiary structure the iron-sulfur centers start decomposing and that the presence of EDTA accelerates the process. Also, the presence of EDTA lowers the observed melting temperature in thermal ramp experiments and the midpoint denaturant concentration in equilibrium chemical unfolding experiments, further suggesting that the clusters also play a structural role in the maintenance of the conformation of the folded state.
Similar content being viewed by others
References
Wittung-Stafshede P (2002) Acc Chem Res 35:201–208
Fraústo da Silva JJR, Williams RJP (1991) The biological chemistry of the elements. Clarendon Press, Oxford
Sandberg A, Leckner J, Shi Y, Schwarz FP, Karlsson BG (2002) Biochemistry 41:1060–1069
Leckner J, Bonander N, Wittung-Stafshede P, Malmstrom BG, Karlsson BG (1997) Biochim Biophys Acta 1342:19–27
Gross EL, Draheim JE, Curtiss AS, Crombie B, Scheffer A, Pan B, Chiang C, Lopez A (1992) Arch Biochem Biophys 298:413–419
Moczygemba C, Guidry J, Jones KL, Gomes CM, Teixeira M, Wittung-Stafshede P (2001) Protein Sci 10:1539–1548
Foster MW, Bian S, Surerus KK, Cowan JA (2001) J Biol Inorg Chem 6:266–274
Bentrop D, Bertini I, Iacoviello R, Luchinat C, Niikura Y, Piccioli M, Presenti C, Rosato A (1999) Biochemistry 38:4669–4680
Lange C, Hervas M, De la Rosa MA (2003) Biochem Biophys Res Commun 310:215–221
Apiyo D, Jones K, Guidry J, Wittung-Stafshede P (2001) Biochemistry 40:4940–4948
Burova TV, Bernhardt R, Pfeil W (1995) Protein Sci 4:909–916
Bera AK, Grinberg A, Bernhardt R (1999) Arch Biochem Biophys 361:315–322
Russell MJ, Martin W (2004) Trends Biochem Sci 29:358–363
Frazzon J, Dean DR (2003) Curr Opin Chem Biol 7:166–173
Gomes C, Faria A, Carita J, Mendes J, Regalla M, Chicau P, Huber H, Stetter K, Teixeira M (1998) J Biol Inorg Chem 3:499–507
Fujii T, Hata Y, Oozeki M, Moriyama H, Wakagi T, Tanaka N, Oshima T (1997) Biochemistry 36:1505–1513
Wittung-Stafshede P, Gomes CM, Teixeira M (2000) J Inorg Biochem 78:35–41
Jones K, Gomes CM, Huber H, Teixeira M, Wittung-Stafshede P (2002) J Biol Inorg Chem 7:357–362
Griffin S, Higgins CL, Soulimane T, Wittung-Stafshede P (2003) Eur J Biochem 270:4736–4743
Pereira MM, Jones KL, Campos MG, Melo AM, Saraiva LM, Louro RO, Wittung-Stafshede P, Teixeira M (2002) Biochim Biophys Acta 1601:1–8
Higgins CL, Meyer J, Wittung-Stafshede P (2002) Biochim Biophys Acta 1599:82–89
Higgins CL, Wittung-Stafshede P (2004) Arch Biochem Biophys 427:154–163
Kennedy MC, Kent TA, Emptage M, Merkle H, Beinert H, Munck E (1984) J Biol Chem 259:14463–14471
Teixeira M, Batista R, Campos AP, Gomes C, Mendes J, Pacheco I, Anemuller S, Hagen WR (1995) Eur J Biochem 227:322–327
Mach H, Middaugh CR (1994) Anal Biochem 222:323–331
Ragone R, Colonna G, Balestrieri C, Servillo L, Irace G (1984) Biochemistry 23:1871–1875
Cowart RE, Singleton FL, Hind JS (1993) Anal Biochem 211:151–155
Shirley BA (1995) In: Shirley BA (ed) Methods in molecular biology. Humana, Totowa, NJ, USA, p 377
Lakowicz JR (1999) Principles of fluorescence spectroscopy, 2nd edn. Kluwer/Plenum, New York
Dorovska-Taran V, van Hoek A, Link TA, Visser AJ, Hagen WR (1994) FEBS Lett 348:305–310
Iametti S, Uhlmann H, Sala N, Bernhardt R, Ragg E, Bonomi F (1996) Eur J Biochem 239:818–826
Kojoh K, Matsuzawa H, Wakagi T (1999) Eur J Biochem 264:85–91
Cotton FA, Wilkinson G (1972) Advanced inorganic chemistry: a comprehensive text, 3rd edn. Interscience, New York
Russell JB (1980) General chemistry. McGraw-Hill, New York
Kennedy MC, Beinert H (1988) J Biol Chem 263:8194–8198
Burova TV, Beckert V, Uhlmann H, Ristau O, Bernhardt R, Pfeil W (1996) Protein Sci 5:1890–1897
Acknowledgements
Rita Delgado (ITQB/UNL) is acknowledged for a kind gift of an accurately titrated Fe(NO3)3 solution. ITQB analytical services are gratefully acknowledged for cell mass processing (N. Carita), N-terminal sequencing (M. Regalla) and preliminary mass spectrometry experiments (A. Coelho). This work was supported by research grants POCTI/QUI/37521 and POCTI/QUI/45758 (to C.M.G.) from the Fundação Ciência e Tecnologia (FCT/MCES, Portugal). S.S.L. was a Bolsa Investigação Científica (BIC) grantee from Fundação para a Ciência e Tecnologia (FCT/MCES, Portugal).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Leal, S.S., Teixeira, M. & Gomes, C.M. Studies on the degradation pathway of iron-sulfur centers during unfolding of a hyperstable ferredoxin: cluster dissociation, iron release and protein stability. J Biol Inorg Chem 9, 987–996 (2004). https://doi.org/10.1007/s00775-004-0599-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00775-004-0599-z