Current Microbiology

, 59:559 | Cite as

Cys92, Cys101, Cys197, and Cys203 Are Crucial Residues for Coordinating the Iron–Sulfur Cluster of RhdA from Acidithiobacillus ferrooxidans

  • Yunjie Dai
  • Jianshe Liu
  • Chunli Zheng
  • Anna Wu
  • Jia Zeng
  • Guanzhou Qiu
Article
  • 102 Downloads

Abstract

By proteomic analysis, we found a rhodanese-like protein(RhdA) from Acidithiobacillus ferrooxidans ATCC 23270 whose C-terminal contained a cysteine motif (Cys-XX-Trp-XX-Cys), known to bind iron–sulfur clusters. But so far, there were no articles to confirm the existence of iron–sulfur cluster in RhdA. In this study, RhdA gene from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The UV–Vis scanning and EPR spectra results indicated that the wild-type proteins contained an iron–sulfur cluster. Site-directed mutagenesis results revealed that the four cysteines Cys92, Cys101, Cys197, and Cys203 were crucial residues for iron–sulfur cluster binding.

Notes

Acknowledgments

This work was supported by the National Basic Research Program of P.R. China (2004CB619204), National Natural Science Foundation of P.R.China (50621063, 50874032) and Foundation for the Author of National Excellent Doctoral Dissertation of P.R. China (200549).

References

  1. 1.
    Acosta M, Beard S, Ponce J, Vera M, Mobarec JC, Jerez CA (2005) Identification of putative sulfurtransferase genes in the extremophilic Acidithiobacillus ferrooxidans ATCC 23270 genome: structural and functional characterization of the proteins. OMICS J Integr Biol 9:13–29CrossRefGoogle Scholar
  2. 2.
    Agar JN, Krebs C, Frazzon J, Huynh BH, Dean DR, Johnson MK (2000) IscU as a scaffold for iron–sulfur cluster biosynthesis: sequential assembly of [2Fe–2S] and [4Fe–4S] clusters in IscU. Biochemistry 39:7856–7862PubMedCrossRefGoogle Scholar
  3. 3.
    Beinert H, Holm RH, Munck E (1997) Iron–sulfur clusters: nature’s modular, multipurpose structures. Science 277:653–659PubMedCrossRefGoogle Scholar
  4. 4.
    Berks BC, Ferguson SJ, Moir JWB, Richardson DJ (1995) Enzymes and associated electron transport systems that catalyze the respiratory reduction of nitrogen oxides and oxyanions. Biochim Biophys Acta 1232:97–173PubMedCrossRefGoogle Scholar
  5. 5.
    Bian S, Cowan JA (1999) Protein-bound iron–sulfur centers. Form, function, and assembly. Coord Chem Rev 190:1049–1066CrossRefGoogle Scholar
  6. 6.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  7. 7.
    Colnaghi R, Pagani S, Kennedy C, Drummond M (1996) Cloning, sequence analysis and overexpression of the rhodanese gene of Azotobacter vinelandii. FEBS Eur J Biochem 236:240–248CrossRefGoogle Scholar
  8. 8.
    Cupp-Vickery JR, Silberg JJ, Ta DT, Vickery LE (2004) Crystal structure of IscA, an iron–sulfur cluster assembly protein from Escherichia coli. J Mol Biol 338:127–137PubMedCrossRefGoogle Scholar
  9. 9.
    Davidson E, Ohnishi T, Atta-Asafo-Adjei E, Daldal F (1992) Potential ligands to the [2Fe–2S] Rieske cluster of the cytochrome bc1 complex of Rhodobacter capsulatus probed by site-directed mutagenesis. Biochemistry 31:3342–3351PubMedCrossRefGoogle Scholar
  10. 10.
    Fauman EB, Cogswell JP, Lovejoy B (1998) Crystal structure of the catalytic domain of the human cell cycle control phosphatase, Cdc25A. Cell 93:617–625PubMedCrossRefGoogle Scholar
  11. 11.
    Friedrich CG (1998) Physiology and genetics of sulfur-oxidizing bacteria. Adv Microb Physiol 39:235–289PubMedCrossRefGoogle Scholar
  12. 12.
    Fukumori Y, Yano T, Sato A, Yamanaka T (1988) Fe(II)-oxidizing enzyme purified from Thiobacillus ferrooxidans. FEMS Microbiol Lett 50:169–172CrossRefGoogle Scholar
  13. 13.
    Johnson DC, Dean DR, Smith AD, Johnson MK (2005) Structure, function, and formation of biological iron–sulfur clusters. Annu Rev Biochem 74:247–281PubMedCrossRefGoogle Scholar
  14. 14.
    Kakuta Y, Horio T, Takahashi Y, Fukuyama K (2001) Crystal structure of Escherichia coli Fdx, an adrenodoxin-type ferredoxin involved in the assembly of iron–sulfur clusters. Biochemistry 40:11007–11012PubMedCrossRefGoogle Scholar
  15. 15.
    Kerfeld CA, Salmeen AE, Yeates TO (1998) Crystal structure and possible dimerization of the high-potential iron–sulfur protein from Chromatium purpuratum. Biochemistry 37:13911–13917PubMedCrossRefGoogle Scholar
  16. 16.
    Krepinsky K, LeimkÜhler S (2007) Site-directed mutagenesis of the active site loop of the rhodanese-like domain of the human molybdopterin synthase sulfurase MOCS3. FEBS J 274:2778–2787PubMedCrossRefGoogle Scholar
  17. 17.
    Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  18. 18.
    Nogi T, Fathir I, Kobayashi M, Nozawa T, Miki K (2000) Crystal structures of photosynthetic reaction center and high-potential iron–sulfur protein from Thermochromatium tepidum: thermostability and electron transfer. Proc Natl Acad Sci USA 97:13561–13566PubMedCrossRefGoogle Scholar
  19. 19.
    Ramĺrez P, Toledo H, Guiliani N, Jerez CA (2002) An exported rhodanese-like protein is induced during growth of Acidithiobacillus ferrooxidans in metal sulfides and different sulfur compounds. Appl Environ Microbiol 68:1837–1845CrossRefGoogle Scholar
  20. 20.
    Rees DC, Howard JB (2003) The interface between the biological and inorganic worlds: iron–sulfur metalloclusters. Science 300:929–931PubMedCrossRefGoogle Scholar
  21. 21.
    Rogers A, Ding H (2001) l-Cysteine-mediated destabilization of dinitrosyl iron complexes in proteins. J Biol Chem 276:30980–30986PubMedCrossRefGoogle Scholar
  22. 22.
    Smíd O, Horáková E, Vilímová V, Hrdý I, Cammack R, Horváth A, Lukeš J, Tachezy J (2006) Knock-downs of iron–sulfur cluster assembly proteins IscS and IscU down-regulate the active mitochondrion of procyclic Trypanosoma Brucei. JBC 281:28679–28686 (in press)Google Scholar
  23. 23.
    Sodeoka M, Larson C, Chen L, Land W, Verdine G (1993) A multifunctional plasmid for protein expression by ECPCR: overproduction of the p50 subunit of NF-KB. Bioorg Med Chem Lett 3:1095–1100CrossRefGoogle Scholar
  24. 24.
    Watanabe S, Kita A, Kobayashi K, Miki K (2008) Crystal structure of the [2Fe–2S] oxidative-stress sensor SoxR bound to DNA. Proc Natl Acad Sci USA 105:4121–4126PubMedCrossRefGoogle Scholar
  25. 25.
    Yang J, Bitoun JP, Ding J (2006) Interplay of IscA and IscU in biogenesis of iron–sulfur cluster. J Biol Chem 281:27956–27963PubMedCrossRefGoogle Scholar
  26. 26.
    Zeng J, Geng M, Liu Y, Zhao W, Xia L, Liu J, Oiu G (2006) Expression, purification and molecular modelling of the Iro protein from Acidithiobacillus ferrooxidans Fe-1. Protein Expr Purif 52:146–152PubMedCrossRefGoogle Scholar
  27. 27.
    Zeng J, Geng M, Jiang H, Liu J, Qiu G (2007) The IscA from Acidithiobacillus ferrooxidans is an iron–sulfur protein which assemble the [Fe4S4] cluster with intracellular iron and sulfur. Arch Biochem Biophys 463:237–244PubMedCrossRefGoogle Scholar
  28. 28.
    Zeng J, Zhang X, Wang Y, Ai C, Liu Q, Qiu G (2008) Glu43 is an essential residue for coordinating the [Fe2S2] cluster of IscR from Acidithiobacillus ferrooxidans. FEBS Lett 582:3889–3892PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Yunjie Dai
    • 1
  • Jianshe Liu
    • 2
  • Chunli Zheng
    • 1
  • Anna Wu
    • 1
  • Jia Zeng
    • 1
  • Guanzhou Qiu
    • 1
  1. 1.Department of Bioengineering, School of Resources Processing and BioengineeringCentral South UniversityChangshaPeople’s Republic of China
  2. 2.Department of Environmental Science & EngineeringDonghua UniversityShanghaiPeople’s Republic of China

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