Abstract
Iron–sulfur clusters constitute a group of cofactors found in many proteins that play key roles in an exceptionally wide range of metabolic processes. The chemical reactivity of iron–sulfur clusters means that they can be particularly prone to damage when removed from the protective environment of the cell. In general, the key to obtaining an intact, biologically active iron–sulfur cluster-containing protein is to maintain a strictly anaerobic environment throughout the entire process of protein purification and analysis. For many proteins, particularly those with more labile clusters, it is essential.
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References
Balk J, Pilon M (2011) Ancient and essential: the assembly of iron-sulfur clusters in plants. Trends Plant Sci 16:218–226
Sheftel A, Stehling O, Lill R (2010) Iron-sulfur proteins in health and disease. Trends Endocrinol Metab 21:302–314
Beinert H (2000) Iron-sulfur proteins: ancient structures, still full of surprises. J Biol Inorg Chem 5:2–15
Johnson MK (1998) Iron-sulfur proteins: new roles for old clusters. Curr Opin Chem Biol 2:173–181
Beinert H, Holm RH, Munck E (1997) Iron-sulfur clusters: nature’s modular, multipurpose structures. Science 277:653–659
Fleischhacker AS, Kiley PJ (2011) Iron-containing transcription factors and their roles as sensors. Curr Opin Chem Biol 15:335–341
Crack JC, Green J, Thomson AJ, Le Brun NE (2012) Iron-sulfur cluster sensor-regulators. Curr Opin Chem Biol 16:35–44
Crack JC, Green J, Hutchings MI, Thomson AJ, Le Brun NE (2012) Bacterial iron-sulfur regulatory proteins as biological sensor-switches. Antioxid Redox Signal 17:1215–1231
Carter KR, Rawlings J, Orme-Johnson WH, Becker RR, Evans HJ (1980) Purification and characterization of a ferredoxin from Rhizobium japonicum bacteroids. J Biol Chem 255:4213–4223
Averill BA, Bale JR, Ormejohnson WH (1978) Displacement of iron-sulfur clusters from ferredoxins and other iron-sulfur proteins. J Am Chem Soc 100:3034–3043
Macomber L, Imlay JA (2009) The iron-sulfur clusters of dehydratases are primary intracellular targets of copper toxicity. Proc Natl Acad Sci U S A 106:8344–8349
Cruz-Ramos H, Crack J, Wu GG, Hughes MN, Scott C, Thomson AJ, Green J, Poole RK (2002) NO sensing by FNR: regulation of the Escherichia coli NO-detoxifying flavohaemoglobin, Hmp. EMBO J 21:3235–3244
Crack JC, den Hengst CD, Jakimowicz P, Subramanian S, Johnson MK, Buttner MJ, Thomson AJ, Le Brun NE (2009) Characterization of [4Fe-4S]-containing and cluster-free forms of Streptomyces WhiD. Biochemistry 48:12252–12264
Crack JC, Gaskell AA, Green J, Cheesman MR, Le Brun NE, Thomson AJ (2008) Influence of the environment on the [4Fe-4S]2+ to [2Fe-2S]2+ cluster switch in the transcriptional regulator FNR. J Am Chem Soc 130:1749–1758
Sutton VR, Kiley PJ (2003) Techniques for studying the oxygen-sensitive transcription factor FNR from Escherichia coli. Meth Enzymol 370:300–312
Yan A, Kiley PJ (2009) Techniques to isolate O2-sensitive proteins: [4Fe-4S]-FNR as an example. Meth Enzymol 463:787–805
Kuchenreuther JM, Grady-Smith CS, Bingham AS, George SJ, Cramer SP, Swartz JR (2010) High-yield expression of heterologous [FeFe] hydrogenases in Escherichia coli. PLoS One 5:e15491
Stehling O, Smith PM, Biederbick A, Balk J, Lill R, Muhlenhoff U (2007) Investigation of iron-sulfur protein maturation in eukaryotes. Meth Mol Biol 372:325–342
Orme-Johnson WH, Holm RH (1978) Identification of iron-sulfur clusters in proteins. Meth Enzymol 53:268–274
Lazazzera BA, Beinert H, Khoroshilova N, Kennedy MC, Kiley PJ (1996) DNA binding and dimerization of the Fe-S-containing FNR protein from Escherichia coli are regulated by oxygen. J Biol Chem 271:2762–2768
Winkler LW (1888) Die Bestimmung des im Wasser gelösten Sauerstoffes (the determination of oxygen dissolved in water). Berichte der deutschen chemischen Gesellschaft 21:2843–2854
Crack JC, Stapleton MR, Green J, Thomson AJ, Le Brun NE (2013) Mechanism of [4Fe-4S](Cys)4 cluster nitrosylation is conserved amongst NO-responsive regulators. J Biol Chem 288:11492–11502
Crack JC, Green J, Cheesman MR, Le Brun NE, Thomson AJ (2007) Superoxide-mediated amplification of the oxygen-induced switch from [4Fe-4S] to [2Fe-2S] clusters in the transcriptional regulator FNR. Proc Natl Acad Sci U S A 104:2092–2097
Crack JC, Jervis AJ, Gaskell AA, White GF, Green J, Thomson AJ, Le Brun NE (2008) Signal perception by FNR: the role of the iron-sulfur cluster. Biochem Soc Trans 36:1144–1148
Crack JC, Le Brun NE, Thomson AJ, Green J, Jervis AJ (2008) Reactions of nitric oxide and oxygen with the regulator of fumarate and nitrate reduction, a global transcriptional regulator, during anaerobic growth of Escherichia coli. Meth Enzymol 437:191–209
Stephens PJ, Thomson AJ, Dunn JB, Keiderling TA, Rawlings J, Rao KK, Hall DO (1978) Circular dichroism and magnetic circular dichroism of iron-sulfur proteins. Biochemistry 17:4770–4778
Crack JC, Smith LJ, Stapleton MR, Peck J, Watmough NJ, Buttner MJ, Buxton RS, Green J, Oganesyan VS, Thomson AJ, Le Brun NE (2011) Mechanistic insight into the nitrosylation of the [4Fe-4S] cluster of WhiB-like proteins. J Am Chem Soc 133:1112–1121
Pinske C, Bonn M, Kruger S, Lindenstrauss U, Sawers RG (2011) Metabolic deficiences revealed in the biotechnologically important model bacterium Escherichia coli BL21(DE3). PLoS One 6:e22830
Zheng L, White RH, Cash VL, Jack RF, Dean DR (1993) Cysteine desulfurase activity indicates a role for NIFS in metallocluster biosynthesis. Proc Natl Acad Sci U S A 90:2754–2758
Beinert H (1983) Semi-micro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins. Anal Biochem 131:373–378
Acknowledgements
This work has been supported by a series of grants from the BBSRC, held jointly between UEA and the University of Sheffield, over several years.
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Crack, J.C., Green, J., Thomson, A.J., Le Brun, N.E. (2014). Techniques for the Production, Isolation, and Analysis of Iron–Sulfur Proteins. In: Fontecilla-Camps, J., Nicolet, Y. (eds) Metalloproteins. Methods in Molecular Biology, vol 1122. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-794-5_4
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DOI: https://doi.org/10.1007/978-1-62703-794-5_4
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