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Protein thiyl radicals directly observed by EPR spectroscopy
ABB Archives of Biochemistry and Biophysics 403 (2002) 141–144 www.academicpress.com
Research Report
Protein thiyl radicals directly observed by EPR spectroscopy €berg,c Matthias Kolberg,a G€ unther Bleifuss,a Astrid Gr€ aslund,b Britt-Marie Sjo d a a,d,* Wolfgang Lubitz, Friedhelm Lendzian, and G€ unter Lassmann a Max-Volmer-Laboratorium f€ur Biophysikalische Chemie, Technische Universit€at Berlin, D-10623 Berlin, Germany Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden Department of Molecular Biology and Functional Genomics, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden d Max-Planck-Institut f€ur Strahlenchemie, D-45470 M€ulheim/Ruhr, Germany b
c
Received 8 February 2002, and in revised form 8 March 2002
Thiyl radicals (R–S ) have been postulated to be directly involved in the turnover of all three known classes of ribonucleotide reductase (RNR)1 [1]. The evidence for their existence, however, is only indirect. With the exception of a thiyl radical that is strongly coupled to a cobalt complex in class II RNR [1], there is so far no spectroscopic evidence neither for thiyl radical appearance nor for their catalytic competence. In class Ia RNR, the thiyl radical is proposed to be generated at the active site in the R1 protein component via a tyrosyl radical in the R2 protein [1]. Because of the small chance to observe thiyl radicals spectroscopically under normal turnover conditions in RNR (R1/R2/substrate assay), model studies with artificially generated thiyl radicals in R1 (without R2) are necessary to analyze them in R1 and, finally, to assess their catalytic competence. This requires knowledge of the EPR spectroscopic properties of thiyl radicals in proteins in general which has so far been completely lacking. Recently, we reported on artificial generation of short-lived protein thiyl radicals in bovine serum albumin (BSA) and R1 of Escherichia coli RNR at room temperature by chemical oxidation of thiols using Ce(IV) and by photochemical release of NO from nitrosylated thiols and their indirect EPR detection using the spin trap phenyl-N-t-butylnitrone (PBN) [2]. Spin trapping, however, cannot provide information on spectroscopic properties like lineshape, g- and hfs-tensors, relaxation, and lifetime of the actual thiyl radical. So far, there are only few examples of EPR studies on thiyl radicals in low molecular weight thiol compounds, such as cysteine [3–5]. Here we report for the first time on the direct EPR detection of protein-based thiyl radicals and explore their unusual spectroscopic properties. In this work, thiyl radicals from proteins have been generated photolytically by UV irradiation at 77 K of frozen aqueous solutions of two different proteins, BSA as a protein model *
Corresponding author. Fax: +49-30-314-21122. E-mail address: [email protected] (G. Lassmann). 1 Abbreviations used: RNR, ribonucleotide reductase; EPR, electron paramagnetic resonance; BSA, bovine serum albumin; PBN, phenylN-t-butylnitrone.
(one single cysteine at C34 and several disulfides) and reduced R1 protein of class Ia RNR from E. coli (11 cysteines per monomeric unit of the R1 homodimer). Thiyl radicals from low-molecular-weight thiols in a frozen cysteine solution have also been studied for comparison.
Results The X-band EPR spectrum of the cysteine sample (Fig. 1C) exhibits features (marked with an asterisk) which result from the gx and gy components of the rhombic EPR spectrum of perthiyl radicals (R–S–S) [6] and features (marked with C ) from carbon-centered radicals of cysteine [7]. However, the dominant feature in the integrated EPR spectrum (Fig. 2C) is an axially symmetric signal with g? ¼ 2:008 and gk ¼ 2:29. In particular, the gk component of the powder spectrum is clearly separated from the region of overlap with the other radicals and its large value represents a fingerprint for a thiyl radical [4]. The EPR spectrum of the thiyl radical in the BSA sample shows a shift of the gk component of a thiyl radical to gk ¼ 2:18 (Fig. 1B) and a broadening (Fig. 2B). In the EPR spectrum of R1, the gk component is broadened even more and can be visualized only by integration of the recorded first-derivative EPR spectrum after careful subtraction of the background (see Figs. 1A and 2A, hatched area). The intensity of both the broad gk shoulder and the g? component in the absorption EPR spectrum (Fig. 2A) has been quantified to determine the temperature-dependent stability of thiyl radicals in R1 as well as the EPR power saturation behavior. The annealing curve in Fig. 3 shows that protein thiyl radicals in R1 are stable in the frozen matrix up to 170 K and 50% decayed irreversibly after 10 min at 210 K, and at 270 K the thiyl radicals almost disappeared. Protein thiyl radicals in BSA are slightly less stable as in R1 (50% decay at 190 K). Thiyl radicals in a frozen solution of cysteine, however, decay to 50% already at 140 K indicating a considerable longer lifetime of protein-based thiyl radicals. Line shape analysis of thiyl-PBN spin adducts generated artificially in R1 protein
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Fig. 1. First-derivative X-band EPR spectra of thiyl radicals in UVirradiated frozen aqueous solutions of (A) R1 protein of E. coli ribonucleotide reductase (RNR) ð500 lMÞ at 80 K, microwave power (P) 200 mW; (B) bovine serum albumine (BSA) (4 mM) at 80 K, P 200 mW; and (C) cysteine (300 mM) at 10 K, P 0.1 mW (Bruker ESP 300E; background from untreated sample subtracted). UV photolysis at 77 K was performed with argon-flashed anaerobic samples in a finger cryostat.
revealed that the majority of thiyl radicals is located in a folded protein environment [2] which may explain the longer lifetime of the protein thiyl radicals. The annealing curve for R1 (Fig. 3) also demonstrates that the decay of the narrow g? and the broad gk components show the same temperature dependence. This confirms that they belong to the same radical species. The EPR saturation curve obtained at 10 K (Fig. 4) shows that the relaxation of the thiyl radicals in R1 is fast and anisotropic. The required power for saturation of the gk and g? components of the protein thiyl radicals in R1 differs by two orders of magnitude. An anisotropic relaxation of thiyl radicals was also observed in BSA; at 80 K, for R1 as well as for BSA the half-saturation microwave power for the g? component is about 1 mW and for the gk component larger than 200 mW (data not shown).
Discussion Several of the basic spectroscopic properties of the investigated protein thiyl radicals are similar to those commonly found for low-molecular-weight thiyl radicals observed earlier in nonprotein systems, as e.g., cysteine [4–6]. The large spin-orbit coupling constant of sulfur (382 cm1 compared to 151 cm1 for oxygen) [8] gives rise to a short spin-lattice relaxation time T1 .
Fig. 2. EPR absorption (integrated first derivative) spectra of thiyl radicals as in Fig. 1: (A) R1; (B) BSA; and (C) cysteine. Experimental conditions as in Fig. 1. (Inset) Simplified scheme of orbitals and molecular axes of a cysteine thiyl radical. The unpaired electron occupies mainly a 3pz -orbital, d-orbital contributions from p-d hybridization are neglected; a H-bonding residue interacts with a lone pair orbital in the x–y plane.
This leads to increasing linewidth with higher temperature and consequently, thiyl radicals are directly observable by EPR only in the frozen state below 80 K and not in liquid solution [4,9]. Direct EPR observation of thiyl radicals therefore requires generation in a solid matrix at low temperature. Single crystal studies at low temperature on X-irradiated Nacetylcysteine [10] have revealed two thiyl radicals with gtensor components gx;y;z of 2.493, 1.923, 1.897, and 2.164, 2.012, and 2.003. Based on the determined 33 S-hyperfine tensors, significant d-orbital contributions of up to 50% for the former radical were concluded. The latter one with significantly less d-contributions was observed to transform into two further thiyl radicals at higher temperature with rather similar g-values of 2.239, 2.005, 1.986, 2.231, 1.976, and 1.962 for which d-orbital contributions of up to 20% were concluded. The large gx -value and the gz -value below the free electron value were attributed to the d-orbital admixture. All four radicals were reported to have similar structures, but different H-bonding situations to the sulfur [10]. In powder spectra of cysteine thiyl radicals a nearly axially symmetric spectrum with gk 2:24 and g? 2:00 was observed [4,5]. The large gk ðgx Þ value depends strongly on the Hbonds to the sulfur of the thiyl radical [3,4]. Neglecting d-orbital contributions, the molecular symmetry about the C–S bond direction usually leads to a degeneracy of the remaining sulfur sp3 hybrid orbitals in isolated thiyl radicals; H-bonds, however, may lift this degeneracy [3,9] (see Fig. 1, inset). In frozen solutions of low-molecular-weight thiols, the gk com-
M. Kolberg et al. / Archives of Biochemistry and Biophysics 403 (2002) 141–144
143
Fig. 3. Irreversible decay (relative EPR intensity I at 80 K of the g? component) of thiyl radicals in R1 ð Þ, BSA ðÞ, and cysteine ð}Þ after stepwise annealing the sample for 10 min at the respective temperature. For R1, both components, g? ð Þ and gk ( ) were plotted to show the same decay behavior. For Figs. 3 and 4, the intensities were quantified from the EPR absorption spectrum, for the broad gk component by scaling the dashed area, and for g? , via its amplitude. The relative error of spectral intensities within a set of spectra in case of unchanged line shape is given by error bars.
Fig. 4. Microwave power (P) saturation curve of thiyl radicals in R1 at 10 K for gk and g? , respectively.
ponent of thiyl radicals is broadened probably as the result of a distribution of slightly different H-bonds to the individual thiyl radicals in the sample (g-strain due to structural heterogeneity) [4,9]. In proteins, a number of different sites for thiyl radicals obviously leads to a larger distribution of the gk value. The knowledge of these characteristic spectroscopic features of protein thiyl radicals, e.g., the line shape of the weak and extended gk component as described here for R1 thiyl radicals is important for their identification by EPR. Thiyl radicals (R–S ) exhibit spectroscopic properties similar to oxygen radicals (R–O ) [9]. For R–O , the largest g value ðgx Þ
is an indicator of the strength of H-bonding to the oxygen and the polarity of the environment. This has been shown by EPR at various frequency bands for nitroxide spin labels in bacteriorhodopsin [11], for OH radicals in glassy and crystalline ice [12],2 and for protein-based tyrosyl radicals. Here the gx values vary from 2.0090 for weak H-bonds to 2.0064 for strong
2
In this study a similar broadened shape of the gk component (ascending slope) of OH radicals was observed as for thiyl radicals in the present paper.
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H-bonds [13,14]. Thiyl radicals from low-molecular-weight thiols (e.g., cysteine) exhibit an analogous dependence of their gx values on H-bonding as tyrosyl radicals. But due to the larger spin orbit coupling of the sulfur, the gx component of thiyl radicals varies over a much larger range, from about 2.29 for weak H-bonds to 2.158 for strong H-bonds in polar solvents [3,4]. Recent DFT calculations (considering only p-orbitals) of g-values from thiyl radicals support the experimentally observed gk shifts upon binding of a water molecule [15]. In thiyl radicals, however, the H-bonding situation is more complicated as for R–O radicals since H-bonds may affect the p-d-hybridization [10,16] leading to different d-orbital admixtures which also influence gk . In the present case of protein thiyl radicals, the actual H-bonding situation is unknown. Nevertheless, in protein- based thiyl radicals gk may give information about the strength of H-bonds between the thiyl sulfur and the protein environment. The observed gk ¼ 2:18 value of thiyl radicals in BSA (Fig. 1B) indicates a strongly H-bonded thiyl radical. The broad distribution of the gk range observed for R1 (Fig. 2A), however, suggests different hydrogen bonds for the different (up to 11) cysteines in R1.3 Further studies on thiyl radicals in single-thiol proteins are required to discriminate between a g-strain due to a structural heterogeneity and superimposed different gk -values from different thiol sites. In summary, the present study provides first insights into gtensors, H- bonding situations, relaxation behavior, and temperature-dependent lifetimes of protein-based thiyl radicals deduced directly from EPR spectroscopy. The data are of general importance for recognizing trapped functional intermediate thiyl radicals in RNR and other proteins and may also be a prerequisite for future ENDOR studies.
Note added in proof. Based on some recent experiments in our laboratory, thiyl and perthiyl radicals are also formed by UV irradiation of frozen aqueous solution of methionine at 77 K. Thus, methionine residues in the proteins investigated in this paper (5 for BSA and 15 for R1) can also give rise to the observed thiyl radicals. However, we expect that the characteristic EPR spectra reported here for protein-associated thiyl radicals (R–S ) are the same irrespective whether the radicals are generated from cysteine (R–SH) or from methionine (R–SCH3 ).
Acknowledgments This work was supported by Deutsche Forschungsgemeinschaft (DFG) (La 751/3-1; Le 812/1-2), Fonds der
3
High-field EPR (e.g. at W-band) of protein thiyl radicals may resolve g? into rhombic g2 and g3 and separate the superimposed perthiyl and C-centered radical from the thiyl g? . However, it would not help to identify the thiyl-specific gk component which is already very broad at X-band.
Chemischen Industrie (to G.L. and W.L.), the Swedish Natural Science Council (to A.G.), the Swedish Cancer Society (to B.M.S.), and the EU ERBFMRXCT 98027 (to A.G. and B.M.S.).
References [1] J. Stubbe, W.A. van der Donk, Chem. Rev. 98 (1998) 705– 762. [2] M. Kolberg, G. Bleifuss, B.M. Sj€ oberg, A. Gr€aslund, W. Lubitz, F. Lendzian, G. Lassmann, Arch. Biochem. Biophys. 397 (2002) 57–68. [3] D.J. Nelson, M.C.R. Symons, Chem. Phys. Lett. 36 (1975) 340– 341. [4] D.J. Nelson, R.L. Petersen, M.C.R. Symons, J. Chem. Soc. Perkins Trans. II (1977) 2005–2014. [5] M.D. Sevilla, D. Becker, M. Yan, Int. J. Radiat. Biol. 57 (1990) 65–81. [6] D. Becker, S. Swarts, M. Champagne, M.D. Sevilla, Int. J. Radiat. Biol. 53 (1988) 767–786. [7] H.C. Box, H.G. Freund, E.E. Budzinski, J. Chem. Phys. 45 (1966) 809–811. [8] A. Carrington, D.M. McLachlan, Introduction to Magnetic Resonance, Harper & Row, New York, 1969. [9] M.C.R. Symons, J. Chem. Soc. Perkin II (1974) 1618–1620. [10] K. Matsuki, J.H. Hadley, W.H. Nelson, C. Yang, J. Magn. Res. 103 (1993) 196–202. [11] H.J. Steinhoff, A. Savitzki, C. Wegener, M. Pfeiffer, M. Plato, K. M€ obius, Biochim. Biophys. Acta 1457 (2000) 253–262. [12] H. Riederer, J. H€ uttermann, P. Boon, M.C.R. Symons, J. Magn. Res. 54 (1983) 54–66. [13] S. Un, X.S. Tang, B. Diner, Biochemistry 35 (1996) 679–684. [14] A. Ivancich, T.A. Mattioli, S. Un, J. Am. Chem. Soc. 121 (1999) 5743–5753. gren, Chem. Phys. Lett. 328 (2000) [15] M. Engstr€ om, O. Vahtras, H. A 483–491. [16] J.H. Hadley, W. Gordy, Proc. Natl. Acad. Sci. USA 74 (1977) 216–220.
Research Report
Protein thiyl radicals directly observed by EPR spectroscopy €berg,c Matthias Kolberg,a G€ unther Bleifuss,a Astrid Gr€ aslund,b Britt-Marie Sjo d a a,d,* Wolfgang Lubitz, Friedhelm Lendzian, and G€ unter Lassmann a Max-Volmer-Laboratorium f€ur Biophysikalische Chemie, Technische Universit€at Berlin, D-10623 Berlin, Germany Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden Department of Molecular Biology and Functional Genomics, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden d Max-Planck-Institut f€ur Strahlenchemie, D-45470 M€ulheim/Ruhr, Germany b
c
Received 8 February 2002, and in revised form 8 March 2002
Thiyl radicals (R–S ) have been postulated to be directly involved in the turnover of all three known classes of ribonucleotide reductase (RNR)1 [1]. The evidence for their existence, however, is only indirect. With the exception of a thiyl radical that is strongly coupled to a cobalt complex in class II RNR [1], there is so far no spectroscopic evidence neither for thiyl radical appearance nor for their catalytic competence. In class Ia RNR, the thiyl radical is proposed to be generated at the active site in the R1 protein component via a tyrosyl radical in the R2 protein [1]. Because of the small chance to observe thiyl radicals spectroscopically under normal turnover conditions in RNR (R1/R2/substrate assay), model studies with artificially generated thiyl radicals in R1 (without R2) are necessary to analyze them in R1 and, finally, to assess their catalytic competence. This requires knowledge of the EPR spectroscopic properties of thiyl radicals in proteins in general which has so far been completely lacking. Recently, we reported on artificial generation of short-lived protein thiyl radicals in bovine serum albumin (BSA) and R1 of Escherichia coli RNR at room temperature by chemical oxidation of thiols using Ce(IV) and by photochemical release of NO from nitrosylated thiols and their indirect EPR detection using the spin trap phenyl-N-t-butylnitrone (PBN) [2]. Spin trapping, however, cannot provide information on spectroscopic properties like lineshape, g- and hfs-tensors, relaxation, and lifetime of the actual thiyl radical. So far, there are only few examples of EPR studies on thiyl radicals in low molecular weight thiol compounds, such as cysteine [3–5]. Here we report for the first time on the direct EPR detection of protein-based thiyl radicals and explore their unusual spectroscopic properties. In this work, thiyl radicals from proteins have been generated photolytically by UV irradiation at 77 K of frozen aqueous solutions of two different proteins, BSA as a protein model *
Corresponding author. Fax: +49-30-314-21122. E-mail address: [email protected] (G. Lassmann). 1 Abbreviations used: RNR, ribonucleotide reductase; EPR, electron paramagnetic resonance; BSA, bovine serum albumin; PBN, phenylN-t-butylnitrone.
(one single cysteine at C34 and several disulfides) and reduced R1 protein of class Ia RNR from E. coli (11 cysteines per monomeric unit of the R1 homodimer). Thiyl radicals from low-molecular-weight thiols in a frozen cysteine solution have also been studied for comparison.
Results The X-band EPR spectrum of the cysteine sample (Fig. 1C) exhibits features (marked with an asterisk) which result from the gx and gy components of the rhombic EPR spectrum of perthiyl radicals (R–S–S) [6] and features (marked with C ) from carbon-centered radicals of cysteine [7]. However, the dominant feature in the integrated EPR spectrum (Fig. 2C) is an axially symmetric signal with g? ¼ 2:008 and gk ¼ 2:29. In particular, the gk component of the powder spectrum is clearly separated from the region of overlap with the other radicals and its large value represents a fingerprint for a thiyl radical [4]. The EPR spectrum of the thiyl radical in the BSA sample shows a shift of the gk component of a thiyl radical to gk ¼ 2:18 (Fig. 1B) and a broadening (Fig. 2B). In the EPR spectrum of R1, the gk component is broadened even more and can be visualized only by integration of the recorded first-derivative EPR spectrum after careful subtraction of the background (see Figs. 1A and 2A, hatched area). The intensity of both the broad gk shoulder and the g? component in the absorption EPR spectrum (Fig. 2A) has been quantified to determine the temperature-dependent stability of thiyl radicals in R1 as well as the EPR power saturation behavior. The annealing curve in Fig. 3 shows that protein thiyl radicals in R1 are stable in the frozen matrix up to 170 K and 50% decayed irreversibly after 10 min at 210 K, and at 270 K the thiyl radicals almost disappeared. Protein thiyl radicals in BSA are slightly less stable as in R1 (50% decay at 190 K). Thiyl radicals in a frozen solution of cysteine, however, decay to 50% already at 140 K indicating a considerable longer lifetime of protein-based thiyl radicals. Line shape analysis of thiyl-PBN spin adducts generated artificially in R1 protein
0003-9861/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 3 - 9 8 6 1 ( 0 2 ) 0 0 2 6 4 - 3
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Fig. 1. First-derivative X-band EPR spectra of thiyl radicals in UVirradiated frozen aqueous solutions of (A) R1 protein of E. coli ribonucleotide reductase (RNR) ð500 lMÞ at 80 K, microwave power (P) 200 mW; (B) bovine serum albumine (BSA) (4 mM) at 80 K, P 200 mW; and (C) cysteine (300 mM) at 10 K, P 0.1 mW (Bruker ESP 300E; background from untreated sample subtracted). UV photolysis at 77 K was performed with argon-flashed anaerobic samples in a finger cryostat.
revealed that the majority of thiyl radicals is located in a folded protein environment [2] which may explain the longer lifetime of the protein thiyl radicals. The annealing curve for R1 (Fig. 3) also demonstrates that the decay of the narrow g? and the broad gk components show the same temperature dependence. This confirms that they belong to the same radical species. The EPR saturation curve obtained at 10 K (Fig. 4) shows that the relaxation of the thiyl radicals in R1 is fast and anisotropic. The required power for saturation of the gk and g? components of the protein thiyl radicals in R1 differs by two orders of magnitude. An anisotropic relaxation of thiyl radicals was also observed in BSA; at 80 K, for R1 as well as for BSA the half-saturation microwave power for the g? component is about 1 mW and for the gk component larger than 200 mW (data not shown).
Discussion Several of the basic spectroscopic properties of the investigated protein thiyl radicals are similar to those commonly found for low-molecular-weight thiyl radicals observed earlier in nonprotein systems, as e.g., cysteine [4–6]. The large spin-orbit coupling constant of sulfur (382 cm1 compared to 151 cm1 for oxygen) [8] gives rise to a short spin-lattice relaxation time T1 .
Fig. 2. EPR absorption (integrated first derivative) spectra of thiyl radicals as in Fig. 1: (A) R1; (B) BSA; and (C) cysteine. Experimental conditions as in Fig. 1. (Inset) Simplified scheme of orbitals and molecular axes of a cysteine thiyl radical. The unpaired electron occupies mainly a 3pz -orbital, d-orbital contributions from p-d hybridization are neglected; a H-bonding residue interacts with a lone pair orbital in the x–y plane.
This leads to increasing linewidth with higher temperature and consequently, thiyl radicals are directly observable by EPR only in the frozen state below 80 K and not in liquid solution [4,9]. Direct EPR observation of thiyl radicals therefore requires generation in a solid matrix at low temperature. Single crystal studies at low temperature on X-irradiated Nacetylcysteine [10] have revealed two thiyl radicals with gtensor components gx;y;z of 2.493, 1.923, 1.897, and 2.164, 2.012, and 2.003. Based on the determined 33 S-hyperfine tensors, significant d-orbital contributions of up to 50% for the former radical were concluded. The latter one with significantly less d-contributions was observed to transform into two further thiyl radicals at higher temperature with rather similar g-values of 2.239, 2.005, 1.986, 2.231, 1.976, and 1.962 for which d-orbital contributions of up to 20% were concluded. The large gx -value and the gz -value below the free electron value were attributed to the d-orbital admixture. All four radicals were reported to have similar structures, but different H-bonding situations to the sulfur [10]. In powder spectra of cysteine thiyl radicals a nearly axially symmetric spectrum with gk 2:24 and g? 2:00 was observed [4,5]. The large gk ðgx Þ value depends strongly on the Hbonds to the sulfur of the thiyl radical [3,4]. Neglecting d-orbital contributions, the molecular symmetry about the C–S bond direction usually leads to a degeneracy of the remaining sulfur sp3 hybrid orbitals in isolated thiyl radicals; H-bonds, however, may lift this degeneracy [3,9] (see Fig. 1, inset). In frozen solutions of low-molecular-weight thiols, the gk com-
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143
Fig. 3. Irreversible decay (relative EPR intensity I at 80 K of the g? component) of thiyl radicals in R1 ð Þ, BSA ðÞ, and cysteine ð}Þ after stepwise annealing the sample for 10 min at the respective temperature. For R1, both components, g? ð Þ and gk ( ) were plotted to show the same decay behavior. For Figs. 3 and 4, the intensities were quantified from the EPR absorption spectrum, for the broad gk component by scaling the dashed area, and for g? , via its amplitude. The relative error of spectral intensities within a set of spectra in case of unchanged line shape is given by error bars.
Fig. 4. Microwave power (P) saturation curve of thiyl radicals in R1 at 10 K for gk and g? , respectively.
ponent of thiyl radicals is broadened probably as the result of a distribution of slightly different H-bonds to the individual thiyl radicals in the sample (g-strain due to structural heterogeneity) [4,9]. In proteins, a number of different sites for thiyl radicals obviously leads to a larger distribution of the gk value. The knowledge of these characteristic spectroscopic features of protein thiyl radicals, e.g., the line shape of the weak and extended gk component as described here for R1 thiyl radicals is important for their identification by EPR. Thiyl radicals (R–S ) exhibit spectroscopic properties similar to oxygen radicals (R–O ) [9]. For R–O , the largest g value ðgx Þ
is an indicator of the strength of H-bonding to the oxygen and the polarity of the environment. This has been shown by EPR at various frequency bands for nitroxide spin labels in bacteriorhodopsin [11], for OH radicals in glassy and crystalline ice [12],2 and for protein-based tyrosyl radicals. Here the gx values vary from 2.0090 for weak H-bonds to 2.0064 for strong
2
In this study a similar broadened shape of the gk component (ascending slope) of OH radicals was observed as for thiyl radicals in the present paper.
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H-bonds [13,14]. Thiyl radicals from low-molecular-weight thiols (e.g., cysteine) exhibit an analogous dependence of their gx values on H-bonding as tyrosyl radicals. But due to the larger spin orbit coupling of the sulfur, the gx component of thiyl radicals varies over a much larger range, from about 2.29 for weak H-bonds to 2.158 for strong H-bonds in polar solvents [3,4]. Recent DFT calculations (considering only p-orbitals) of g-values from thiyl radicals support the experimentally observed gk shifts upon binding of a water molecule [15]. In thiyl radicals, however, the H-bonding situation is more complicated as for R–O radicals since H-bonds may affect the p-d-hybridization [10,16] leading to different d-orbital admixtures which also influence gk . In the present case of protein thiyl radicals, the actual H-bonding situation is unknown. Nevertheless, in protein- based thiyl radicals gk may give information about the strength of H-bonds between the thiyl sulfur and the protein environment. The observed gk ¼ 2:18 value of thiyl radicals in BSA (Fig. 1B) indicates a strongly H-bonded thiyl radical. The broad distribution of the gk range observed for R1 (Fig. 2A), however, suggests different hydrogen bonds for the different (up to 11) cysteines in R1.3 Further studies on thiyl radicals in single-thiol proteins are required to discriminate between a g-strain due to a structural heterogeneity and superimposed different gk -values from different thiol sites. In summary, the present study provides first insights into gtensors, H- bonding situations, relaxation behavior, and temperature-dependent lifetimes of protein-based thiyl radicals deduced directly from EPR spectroscopy. The data are of general importance for recognizing trapped functional intermediate thiyl radicals in RNR and other proteins and may also be a prerequisite for future ENDOR studies.
Note added in proof. Based on some recent experiments in our laboratory, thiyl and perthiyl radicals are also formed by UV irradiation of frozen aqueous solution of methionine at 77 K. Thus, methionine residues in the proteins investigated in this paper (5 for BSA and 15 for R1) can also give rise to the observed thiyl radicals. However, we expect that the characteristic EPR spectra reported here for protein-associated thiyl radicals (R–S ) are the same irrespective whether the radicals are generated from cysteine (R–SH) or from methionine (R–SCH3 ).
Acknowledgments This work was supported by Deutsche Forschungsgemeinschaft (DFG) (La 751/3-1; Le 812/1-2), Fonds der
3
High-field EPR (e.g. at W-band) of protein thiyl radicals may resolve g? into rhombic g2 and g3 and separate the superimposed perthiyl and C-centered radical from the thiyl g? . However, it would not help to identify the thiyl-specific gk component which is already very broad at X-band.
Chemischen Industrie (to G.L. and W.L.), the Swedish Natural Science Council (to A.G.), the Swedish Cancer Society (to B.M.S.), and the EU ERBFMRXCT 98027 (to A.G. and B.M.S.).
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