Abstract
Continuous-wave (CW) electron paramagnetic resonance (EPR) spectroscopy is a powerful ally in characterizing the multitude of redox-active iron-sulfur cluster-containing ([Fe-S]) species present in biological samples. The technique detects only those clusters that are paramagnetic—having a nonzero total electron spin (S > 0)—thus, it can discriminate between clusters in different oxidation states. The low-temperature CW-EPR spectrum of an [Fe-S] yields the three magnetic g-values that serve as a fingerprint of its electronic structure. This chapter briefly describes the underlying theory that defines this electronic structure and provides a recipe for the acquisition and analysis of EPR spectra of [Fe-S] proteins.
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References
Palmer G (1975) 1 iron—sulfur proteins. In: Boyer PD (ed) The enzymes. Academic Press, Cambridge, Massachusetts, pp 1–56
Beinert H (2003) Iron-sulfur proteins: properties and functions. Imperial College Press, London, pp 47–72
Beinert H, Kennedy MC, Stout CD (1996) Aconitase as iron-sulfur protein, enzyme, and iron regulatory protein. Chem Rev 96:2335–2373
Evans MCW (1982) Iron-sulfur centers in photosynthetic electron transport. Metal Ions Biol 4:249–284
Seefeldt LC, Yang Z-Y, Lukoyanov DA et al (2020) Reduction of substrates by Nitrogenases. Chem Rev 120:5082–5106
Jasniewski AJ, Lee CC, Ribbe MW et al (2020) Reactivity, mechanism, and assembly of the alternative Nitrogenases. Chem Rev 120:5107–5157
Cammack R (1992) Iron—sulfur clusters in enzymes: themes and variations. In: Cammack R (ed) Adv Inorg Chem. Academic Press, Cambridge, Massachusetts, pp 281–322
Hagen WR (2018) EPR spectroscopy of complex biological iron–sulfur systems. J Biol Inorg Chem 23:623–634
Abragam A, Bleaney B (1986) In electron paramagnetic resonance of transition ions. Dover Publications, New York
Weil JA, Bolton JR (2007) Electron paramagnetic resonance: elementary theory and practical applications. John Wiley & Sons, Hoboken, New Jersey
Hendrich MP, Debrunner PG (1989) Integer-spin electron-paramagnetic resonance of iron proteins. Biophys J 56:489–506
Hagen WR (1992) EPR spectroscopy of iron—sulfur proteins. In: Cammack R (ed) Adv Inorg Chem. Academic Press, Cambridge, Massachusetts, pp 165–222
Dunham WR, Palmer G, Sands RH et al (1971) On the structure of the iron-sulfur complex in the two-iron ferredoxins. Biochim Biophys Acta 253:373–384
Sands RH, Dunham WR (1974) Spectroscopic studies on two-iron ferredoxins. Q Rev Biophys 7:443–504
Agar JN, Krebs C, Frazzon J et al (2000) IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU. Biochemistry 39:7856–7862
Chandramouli K, Unciuleac M-C, Naik S et al (2007) Formation and properties of [4Fe-4S] clusters on the IscU scaffold protein. Biochemistry 46:6804–6811
Ribbe M, Tanifuji K, Jasniewski A et al (2020) Nitrogenase M-cluster assembly. American Chemical Society, Washington, p INOR-0100
Dicus MM, Conlan A, Nechushtai R et al (2010) Binding of histidine in the (Cys)3(his)1-coordinated [2Fe−2S] cluster of human mitoNEET. J Am Chem Soc 132:2037–2049
Fee JA, Findling KL, Yoshida T et al (1984) Purification and characterization of the Rieske iron-sulfur protein from Thermus Thermophilus - evidence for a 2Fe-2S cluster having non-cysteine ligands. J Biol Chem 259:124–133
Gurbiel RJ, Batie CJ, Sivaraja M et al (1989) Electron-nuclear double resonance spectroscopy of nitrogen-15-enriched phthalate dioxygenase from pseudomonas cepacia proves that two histidines are coordinated to the [2Fe-2S] Rieske-type clusters. Biochemistry 28:4861–4871
Iwasaki T, Kounosu A, Samoilova RI et al (2006) 15N HYSCORE characterization of the fully deprotonated, reduced form of the archaeal Rieske [2Fe-2S] center. J Am Chem Soc 128:2170–2171
Crouse BR, Meyer J, Johnson MK (1995) Spectroscopic evidence for a reduced Fe2S2 cluster with a S = 9/2 ground state in mutant forms of clostridium pasteurianum 2Fe ferredoxin. J Am Chem Soc 117:9612–9613
Achim C, Golinelli MP, Bominaar EL et al (1996) Mossbauer study of Cys56Ser mutant 2Fe ferredoxin from clostridium pasteurianum: evidence for double exchange in an Fe2S2 (+) cluster. J Am Chem Soc 118:8168–8169
Priem AH, Klaassen AK, Reijerse EJ et al (2005) EPR analysis of multiple forms of 4Fe-4S (3+) clusters in HiPIPs. J Biol Inorg Chem 10:417–424
Watt GD, Reddy KRN (1994) Formation of an all ferrous Fe4S4 cluster in the iron protein component of Azotobacter vinelandii nitrogenase. J Inorg Biochem 53:281–294
Pandelia M-E, Lanz ND, Booker SJ et al (2015) Mössbauer spectroscopy of Fe/S proteins. Biochim Biophys Acta 1853:1395–1405
Garcia-Serres R, Clémancey M, Latour J-M et al (2018) Contribution of Mössbauer spectroscopy to the investigation of Fe/S biogenesis. J Biol Inorg Chem 23:635–644
Telser J, Emptage MH, Merkle H et al (1986) Oxygen-17 electron nuclear double resonance characterization of substrate binding to the 4-iron-4-sulfur ([4Fe-4S]1+) cluster of reduced active aconitase. J Biol Chem 261:4840–4846
Rao G, Tao L, Suess DLM et al (2018) A [4Fe–4S]-Fe(CO)(CN)-l-cysteine intermediate is the first organometallic precursor in [FeFe] hydrogenase H-cluster bioassembly. Nat Chem 10:555–560
Tao L, Zhu W, Klinman JP et al (2019) Electron paramagnetic resonance spectroscopic identification of the Fe-S clusters in the SPASM domain-containing radical SAM enzyme PqqE. Biochemistry 58:5173–5187
Hagen WR, van den Berg WAM, van Dongen WMAM et al (1998) EPR spectroscopy of biological iron-sulfur clusters with spin-admixed S=3-2 ground states. J Chem Soc Faraday Trans 94:2969–2973
Lindahl PA, Day EP, Kent TA et al (1985) Moessbauer, EPR, and magnetization studies of the Azotobacter vinelandii iron protein. Evidence for a [4-iron-4-sulfur]1+ cluster with spin S = 3/2. J Biol Chem 260:11160–11173
Conover RC, Kowal AT, Fu WG et al (1990) Spectroscopic characterization of the novel iron-sulfur cluster in Pyrococcus furiosus ferredoxin. J Biol Chem 265:8533–8541
Rothery RA, Bertero MG, Cammack R et al (2004) The catalytic subunit of Escherichia coli nitrate reductase a contains a novel [4Fe-4S] cluster with a high-spin ground state. Biochemistry 43:5324–5333
Angove HC, Yoo SJ, Burgess BK et al (1997) Mössbauer and EPR evidence for an all-ferrous Fe4S4 cluster with S = 4 in the Fe protein of Nitrogenase. J Am Chem Soc 119:8730–8731
Eisenstein RS, Kennedy MC, Beinert H (1998) The iron responsive element (IRE), the iron regulatory protein (IRP), and cytosolic aconitase: posttranscriptional regulation of mammalian iron metabolism. Chapman & Hall, London, pp 157–216
Crack JC, Green J, Thomson AJ et al (2014) Iron–sulfur clusters as biological sensors: the chemistry of reactions with molecular oxygen and nitric oxide. Acc Chem Res 47:3196–3205
Moura JJG, Moura I, Kent TA et al (1982) Interconversions of [3Fe-3S] and [4Fe-4S] clusters. Moessbauer and electron paramagnetic resonance studies of Desulfovibrio gigas ferredoxin II. J Biol Chem 257:6259–6267
Kent TA, Huynh BH, Munck E (1980) Iron-sulfur proteins—spin-coupling model for 3-iron clusters. Proc Natl Acad Sci U S A 77:6574–6576
Fan C, Houseman ALP, Doan P et al (1993) Conformational distribution in protein-bound [3Fe-4S]+ clusters: CW and pulsed EPR and iron-57 ENDOR of D. gigas hydrogenase. J Phys Chem 97:3017–3021
Duff JLC, Breton JLJ, Butt JN et al (1996) Novel redox chemistry of [3Fe−4S] clusters: electrochemical characterization of the all-Fe(II) form of the [3Fe−4S] cluster generated reversibly in various proteins and its spectroscopic investigation in Sulfolobus acidocaldarius ferredoxin. J Am Chem Soc 118:8593–8603
Hearshen DO, Hagen WR, Sands RH et al (1986) An analysis of g strain in the EPR of two [2Fe-2S] ferredoxins. Evidence for a protein rigidity model. J Magn Reson 69:440–459
Lieder KW, Booker S, Ruzicka FJ et al (1998) S-Adenosylmethionine-dependent reduction of lysine 2,3-aminomutase and observation of the catalytically functional iron-sulfur centers by electron paramagnetic resonance. Biochemistry 37:2578–2585
Englander SW, Calhoun DB, Englander JJ (1987) Biochemistry without oxygen. Anal Biochem 161:300–306
Eaton GR, Eaton SS, Barr DP et al (2010) Quantitative EPR. Springer, New York
Petasis DT, Hendrich MP (2015) Chapter eight - quantitative interpretation of multifrequency multimode EPR spectra of metal containing proteins, enzymes, and biomimetic complexes. In: Qin PZ, Warncke K (eds) Methods enzymol. Academic Press, Cambridge, Massachusetts, pp 171–208
Hanson GR, Gates KE, Noble CJ et al (2004) XSophe-Sophe-XeprView®. A computer simulation software suite (v. 1.1.3) for the analysis of continuous wave EPR spectra. J Inorg Biochem 98:903–916
Stoll S (2015) Chapter six - CW-EPR spectral simulations: solid state. In: Qin PZ, Warncke K (eds) Methods enzymol. Academic Press, Cambridge, Massachusetts, pp 121–142
Stoll S, Schweiger A (2006) EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J Magn Reson 178:42–55
Rupp H, Cammack R, Rao KK et al (1978) Electron-spin relaxation of iron-sulfur proteins studied by microwave-power saturation. Biochim Biophys Acta 537:255–269
Beardwood P, Gibson JF, Bertrand P et al (1983) Temperature dependence of the electronic spin-lattice relaxation time in a 2-iron-2-Sulphur model complex. Biochim Biophys Acta 742:426–433
Gayda JP, Bertrand P, Deville A et al (1979) Temperature dependence of the electronic spin-lattice relaxation time in a 2-iron-2-sulfur protein. Biochim Biophys Acta 581:15–26
Yakovlev G, Reda T, Hirst J (2007) Reevaluating the relationship between EPR spectra and enzyme structure for the iron-sulfur clusters in NADH:quinone oxidoreductase. Proc Natl Acad Sci U S A 104:12720–12725
Dinis P, Suess DLM, Fox SJ et al (2015) X-ray crystallographic and EPR spectroscopic analysis of HydG, a maturase in FeFe -hydrogenase H-cluster assembly. Proc Natl Acad Sci U S A 112:1362–1367
Suess DLM, Britt RD (2015) EPR spectroscopic studies of FeFe -hydrogenase maturation. Top Catal 58:699–707
Lubitz W, Ogata H, Rudiger O et al (2014) Hydrogenases Chem Rev 114:4081–4148
Danyal K, Yang Z-Y, Seefeldt LC (2011) Electron paramagnetic resonance spectroscopy. Methods Mol Biol 766:191–205
Davis LC, Henzl MT, Burris RH et al (1979) Iron-sulfur clusters in the molybdenum-iron protein component of nitrogenase. Electron paramagnetic resonance of the carbon monoxide inhibited state. Biochemistry 18:4860–4869
Rupnik K, Hu Y, Lee CC et al (2012) P+ state of Nitrogenase P-cluster exhibits electronic structure of a [Fe4S4]+ cluster. J Am Chem Soc 134:13749–13754
Wiig JA, Hu Y, Ribbe MW (2011) NifEN-B complex of Azotobacter vinelandii is fully functional in nitrogenase FeMo cofactor assembly. Proc Natl Acad Sci U S A 108(8623–8627):S8623/8621–S8623/8622
Moebius K, Lubitz W, Cox N et al (2018) Biomolecular EPR meets NMR at high magnetic fields. Magnetochemistry 4:50
Beinert H, Holm RH, Munck E (1997) Iron-sulfur clusters: nature's modular, multipurpose structures. Science 277:653–659
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Stich, T.A. (2021). Characterization of Paramagnetic Iron-Sulfur Clusters Using Electron Paramagnetic Resonance Spectroscopy. In: Dos Santos, P.C. (eds) Fe-S Proteins. Methods in Molecular Biology, vol 2353. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1605-5_14
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DOI: https://doi.org/10.1007/978-1-0716-1605-5_14
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