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1H NMR of oxidized blue copper proteins
www.elsevier.nl/locate/jinorgbio Journal of Inorganic Biochemistry 75 (1999) 153–157
1
H NMR of oxidized blue copper proteins
Gianantonio Battistuzzi, Lodovica Loschi, Marco Sola * Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, 41100 Modena, Italy Received 12 February 1999; received in revised form 27 April 1999; accepted 7 May 1999
Abstract The hyperfine-shifted 1H NMR resonances arising from the Cu(II) ligands of cucumber stellacyanin, plantacyanin (CBP) and plastocyanin, horseradish umecyanin, and of spinach plastocyanin and its L12G mutant are reported. It is shown that these broad paramagnetic signals are diagnostic of structural, electronic and solvation properties of the metal site and can be exploited to probe for differences and/or analogies among type I copper centers. q 1999 Elsevier Science Inc. All rights reserved. Keywords: Blue copper proteins; Cupredoxins; NMR spectroscopy; Phytocyanins; Plastocyanins
1. Introduction Type I or ‘blue’ copper centers are mononuclear metal sites found in low-molecular weight electron transport proteins, called cupredoxins, present in bacteria and green plants [1–6]. The metal is strongly bound by the thiolate sulfur of a cysteine and the nitrogen of two histidines, in an approximately trigonal arrangement, while the fourth (axial) coordination position is occupied either by the thioether sulfur of a methionine or the oxygen atom of a glutamine [1–6]. The structural and electronic properties of the metal sites, which are responsible for the peculiar spectroscopic and redox properties of these species, have been thoroughly investigated and are still the subject of extensive experimental and theoretical work [1–17]. The nucleus-unpaired electron coupling in paramagnetic metalloproteins can be profitably exploited to determine the protein structure in solution and the electronic properties of the metal center(s) through NMR [18–20]. The electron relaxation time of the Cu(II) ion (10y9 s) is relatively long and induces a large broadening (in most cases beyond detection) of the resonances of the protons neigh˚ [18–20]. Howboring the metal within approximately 5 A ever, broad contact-shifted resonances arising from protons outside the above limit belonging to the metal ligands could be recently detected in the 1H NMR spectra of oxidized plastocyanin, amicyanin, azurin and stellacyanin [21–25]. Estimation of the magnitude of the Fermi contact contribution to the hyperfine shift, which is related to the amount of spin * Corresponding author. Tel.: q39-059-378-421; fax: q39-059-373-543; e-mail: [email protected]
density delocalized over the ligands, provided further insight into the electronic properties of the Cu(II) site [21,22]. Here, we report the 400 MHz 1H NMR spectra of three oxidized phytocyanins, namely, umecyanin (UME) from horseradish, cucumber stellacyanin (CST) and plantacyanin (the latter also named cucumber basic protein, CBP), along with those of cucumber plastocyanin (CPL) and of recombinant wild-type plastocyanin from spinach (SPL) and of its L12G mutant [26,27]. Phytocyanins are a recently identified cupredoxin subfamily which includes a number of sequence and structurally related proteins from higher plants [28–31]. This group of proteins shows peculiar properties including, among others, a disulfide bridge at the Cu binding end of the polypeptide chain, the solvent accessibility of both Cu-binding histidines (instead of only one for azurins and plastocyanins), and a stronger interaction of the copper with the axial ligand (a methionine or a glutamine) as compared with the other cupredoxins [28–31]. The 1H NMR spectra of these species, which are unprecedented, have been compared with those recorded previously [21–25]. Since the overall architecture of the metal sites of these proteins obeys the same structural motif, the distribution of the spin density on the metal ligands, hence the pattern of the hyperfine-shifted resonances, is similar. However, these sites differ in the distortion of the coordination polyhedron and in the details of the electronic features of metal–ligand interactions, which result in moderate changes in position and linewidth of the signals. Here, the spectra of different cupredoxin subclasses are compared in search of typifying signal patterns and/or spectral signatures of certain metal site properties. This would allow exploitation of 1H NMR as an additional spectroscopic tool
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to recognize readily the nature of the metal site in oxidized cupredoxins. The utility of this work is related to the fact that independent signal assignment through one- or two-dimensional techniques for these species is hindered by the submillisecond T1 values, and it is almost hopeless unless signal assignments are available for the reduced form and favorable rates of electron self-exchange allow identification of the paramagnetic counterparts [21,22,24].
2. Experimental 2.1. Materials All chemicals were reagent grade and were used without further purification. Nanopure water was used throughout. 2.2. Protein purification Recombinant wild-type spinach plastocyanin and the L12G mutant were isolated from JM105 E. coli cells transformed with the expression system pTrc99A containing the construction of the peptide signal of P. aeruginosa azurin and the plastocyanin gene, following the procedure described elsewhere [26,32]. The transformed cells were a gift from Professors Klaus Bernauer and Peter Schurmann of the Univˆ ersite´ de Neuchatel, Switzerland. CBP, plastocyanin and stellacyanin from cucumber (Cucumis sativus) were isolated with slight modifications of the literature methods [33–35]. Umecyanin from horseradish (Armoracia laphatifolia) was from Sigma. 2.3. Spectroscopic measurements 1
H NMR spectra were recorded on a Bruker AMX-400 spectrometer, operating at 400.13 MHz, using 8K data points over a 100 kHz bandwidth. Suppression of the water peak was achieved by the super-WEFT pulse sequence (180-t-90AQqdelay) [36] with recycle time of 50 ms and delay times (t) of 30–50 ms. A typical spectrum run on a 0.1–0.3 mM protein sample required 100 000 scans. Spectra were recorded in water (100 mM phosphate buffer, pH 7), with 10% D2O for the lock, or in D2O (100 mM phosphate, pH 7) at 278C and are referenced to tetramethylsilane after calibration against the HDO peak, set at 4.78 ppm from TMS. The pH was adjusted by adding small amounts of concentrated NaOH and HCl under fast stirring.
Fig. 1. Paramagnetic region of the 400 MHz 1H NMR spectra of various oxidized cupredoxins in 0.1 M phosphate buffer, pH 7. CST, cucumber stellacyanin; UME, horseradish umecyanin; CBP, cucumber basic protein; CPL, cucumber plastocyanin; SPL, spinach plastocyanin; L12G-SPL, L12G mutant of spinach plastocyanin. Protein concentrations0.1–0.3 mM. Ts278C. Shaded signals correspond to exchangeable protons. Table 1 Chemical shift and tentative assignment of the 1H NMR resonances for oxidized phytocyanins at Ts278C Peak
UME
CST
a b (exch.) c
52 52 29
52 52 30
d e f (exch.) g
14.5 13 11 y5
17 14.2 11 y4
CBP
STC a
Assignment b
29.5
55 44 30
HisCdH GlnN´H HisCdH (MetCgH) c GlnCdH CysCaH CysNH HisCbH
17.5 12
14.6 12.1 y7
a
3. Results and discussion
From Ref. [25]. Assignments for STC are from Ref. [25]. Those for the other species are made upon spectral comparison with STC (this work). c Alternative assignment for CBP (this work).
The hyperfine-shifted resonances in the 400 MHz 1H NMR spectra of oxidized horseradish umecyanin, cucumber stellacyanin, cucumber basic protein, and plastocyanins from spinach and cucumber (SPL, CPL) at pH 7 are shown in Fig. 1. The chemical shifts are listed in Tables 1 and 2 for phy-
tocyanins and plastocyanins, respectively. Cucumber stellacyanin and horseradish umecyanin yield comparable spectra which are similar to that of Rhus vernicifera stellacyanin (STC) [25]. Two resonances arise from solvent exchangeable protons: signal ‘b’, which disappears in H2O at pH)8,
b
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G. Battistuzzi et al. / Journal of Inorganic Biochemistry 75 (1999) 153–157 Table 2 Chemical shift and assignment of the 1H NMR resonances of oxidized spinach (SPL) and cucumber (CPL) plastocyanins, L12G mutant of SPL, T. versutus amicyanin (AMI) and plastocyanin loop-containing amicyanin (Pc-AMI) at Ts278C Peak a b c (exch.) d e g
SPL 49 49 31.3 24.3 16.7
CPL
L12G AMI a
50 51 50 51 31.5 31 24.5 29 16.5 16.6
y5.1 y6.5
50 43 27.5 12–11.1 14.1 y9.5
Pc-AMI a 56 47.5 25.5 21.5 17.3 y9
Assignment b HisCdH a,c HisCdH a,c HisN´H a,c MetCgH a,c AsnCaH c (CysCaH) a CysCaH c (HisCbH) a
a
From Ref. [22]. Assignments for SPL and for AMI and Pc-AMI are from Ref. [21] and [22], respectively. Those for CPL and L12G (this work) must be considered as tentative, being based on the spectral similarities with SPL. c From Ref. [21]. b
as the corresponding peak in STC [25] and peak ‘f’, which disappears in D2O at pH 7. The similarity of these spectra is consistent with the presence of the same residues bound to the copper ion in these species [28–31], in which the oxygen atom of a glutamine residue acts as axial ligand. This allows extension of the tentative signal assignments obtained elsewhere for STC [25] to CST and UME. Thus, the broad signals ‘a’ and ‘c’ may be attributed to non-exchangeable CdH protons of the binding histidines, and peaks ‘b’ and ‘d’ to the N´H and the g-methylene protons of the axial glutamine, respectively. The greater shift of peak ‘d’ in CST as compared to UME and STC (17 versus 14.5 ppm, respectively) would indicate an increase in unpaired spin density over the axial ligand, possibly as a result of a somewhat stronger interaction with the copper ion and/or of a more effective spin delocalization mechanism. Peaks ‘e’ in CST and UME would correspond to the relatively slow-relaxing signal ‘e’ at 12.1 ppm observed for STC, which was assigned to the Ha proton of the binding cysteine [25]. The exchangeable signal ‘f’ detected for CST and UME at 11 ppm is not observed in STC. This resonance could arise from the peptide NH proton of the binding cysteine, which is only five bonds away from the metal and which could, in principle, experience a fraction of the relevant unpaired electron spin density delocalized over the S atom [7,22,23,25]. This assignment is supported by the small g anisotropy of the Cu(II) ion [21,22,25], which minimizes the pseudo-contact contribution to the hyperfine shift, virtually excluding any NH proton belonging to non-binding residues. The low-frequency signal ‘g’ at approximately y4 ppm in UME and CST should correspond to the analogous signal found at y7 ppm in STC and assigned to a CbH proton of a binding histidine. The absence of resonances attributable to solvent-exchangeable N´H protons of the binding histidines in these spectra is consistent with the pronounced solvent accessibility of the
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metal site of STC [30], which would thus appear to be a common feature of this class of proteins, in agreement with electrochemical, spectroscopic and kinetic studies [15– 17,25,37–42]. Overall, this spectral comparison would indicate that the coordination features of the metal sites of CST, UME and STC are conserved, with only some minor differences in binding strength and/or residue orientation of the glutamine and cysteine ligands. The metal site of cucumber and spinach plastocyanins is characterized by a distorted trigonal pyramidal geometry and by a small solvent accessibility [1–14,43,44]. Their 1H NMR spectra (Fig. 1) are almost identical. The 600 and 800 MHz 1 H NMR spectra of SPL, which appeared recently [21], allowed a better resolution of the two large peaks ‘a’ and ‘b’, and detection of two additional overlapping broad resonances at approximately 35 ppm. Signal assignment was achieved through saturation transfer experiments with the reduced diamagnetic species. Interestingly, the spectra of SPL and CPL closely reproduce that of an amicyanin mutant (Pc-AMI) in which the loop separating the second metal-binding histidine and the axial methionine in the wild-type protein has been substituted with the corresponding loop of plastocyanin [22]. By referring to the spectral assignments for SPL [21] and for the amicyanin mutant [22], which are in reasonable agreement, the two-proton peak ‘a–b’ in CPL most likely arises from the CdH protons of the binding histidines, the exchangeable signal ‘c’ is due to the N´H proton of His37, which, in contrast to phytocyanins, exchanges more slowly with the bulk solvent [43,44], and the broad signal ‘d’ is due to one of the CgH protons of the axial methionine [21,22]. Signal ‘e’ has been assigned to the CaH of Asn38 in SPL [21], in contrast with a previous assignment to the CaH of the copper-binding cysteine in Pc-AMI [22]. The latter proton has been shown to fall in the upfield region, most likely corresponding to peak ‘g’ [21]. Signal ‘f’ is slow-relaxing and probably belongs to the reduced form [21]. The strong similarity of the 1H NMR spectra of the plastocyanins and Pc-AMI mutant parallels that of the UV–Vis and EPR spectra and demonstrates that the electronic properties of their metal sites are very similar. This finding is relevant for the understanding of the role of the polypeptide chain in modulating the electronic features of the Cu(II) center in these systems. In particular, it demonstrates that the length and flexibility of the aminoacidic loop between the second metal binding histidine and the axial methionine strongly influence the properties of the axial bond. The utility of the hyperfine-shifted resonances to probe for structural and electronic perturbations of the cupric site in blue copper protein is further testified by the spectrum of the L12G mutant of spinach plastocyanin (Fig. 1). Leu12 belongs to the ‘northern’ hydrophobic patch around the Cu ligand His87 which is suggested to be important in the electron transfer reaction of plastocyanin with redox partners [45]. Mutations of this residue resulted in significant changes in the kinetics of the reaction with photosystem I, as well as in the reduction potential and spectroscopic properties
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[45,46]. In particular, substitution of Leu12 with an alanine was found to induce a change of the chemical shift of the Cu binding His37 in the reduced protein [46], alteration of the EPR parameters of the cupric form, and a decrease in E8 [45] compared to the wild type, which were interpreted as indicative of a slight distortion of the coordination geometry and modification of the electronic structure of the Cu site. The 1H NMR spectrum of the L12G mutant of spinach plastocyanin (Fig. 1) clearly confirms that removal of the bulky leucine side chain perturbs, to a certain extent, the electronic structure of the cupric form. In particular, compared with the wild-type species, peak ‘d’ moves to higher frequencies and is almost overlapped with peak ‘c’. The minor change in chemical shift of the latter signal would indicate that removal of the above side chain, which is close to the metal site, does not noticeably affect the exposition of the site to the solvent. This information is of use for the interpretation of the mutation-induced changes in reduction potential and kinetics of electron transfer. The metal center of CBP, a species which is taken as the prototype of the phytocyanins [29], differs from that of stellacyanins and umecyanin due to the presence of a methionine as axial ligand [2,29]. It also differs from that of plastocyanins and azurins because of a shorter Cu–S(Met) bond [29,43,44], which results in a more tetrahedral copper coordination. These properties are reflected in the 1H NMR spectrum, which is rather peculiar. In particular, the resonances ‘a’ and ‘b’, which are present in all the other cupredoxins, are broadened beyond detection. The similarity in chemical shift and lineshape of the broad signal at 29.5 ppm with peak ‘c’ of the spectra of UME, CST and STC suggest its assignment to one of the CdH protons of one of the binding histidines. However, this peak could also correspond to a Cg proton of the axial methionine, since it has been shown that the contact shift of the methylene Cg protons increases with increasing strength of axial methionine ligation [22]. We tentatively assign the sharper signals ‘e’ (17 ppm) and ‘f’ (which disappears in D2O) (12 ppm) to the CaH and peptide NH protons of the binding cysteine, respectively. As in the case of stellacyanins and umecyanin, the absence of any solvent exchangeable resonance due to the N´H protons of the binding histidines can be attributed to the great solvent accessibility of the metal site [29].
4. Conclusions The comparison of the 1H NMR properties of several oxidized cupredoxins carried out in this work indicates that the hyperfine-shifted resonances are sensitive to the structural and electronic properties of the metal center and to its exposure to the solvent. The strong similarities of the spectra of proteins sharing the same metal coordination, such as the stellacyanins and umecyanin, or the plastocyanins, suggest that 1H NMR can be profitably exploited to recognize readily
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the nature of the metal center in either newly discovered or still not structurally characterized oxidized cupredoxins.
5. Abbreviations
CBP CPL CST SPL STC UME
cucumber basic protein (also named plantacyanin) cucumber plastocyanin cucumber (Cucumis sativus) stellacyanin spinach plastocyanin Rhus vernicifera stellacyanin horseradish (Armoracia laphatifolia) umecyanin
Acknowledgements This work was performed with the financial support of the Ministero della Pubblica Istruzione of Italy (quota ex 40%). NMR measurements were carried out at the Centro Intedipartimentale Grandi Strumenti of the University of Modena and Reggio Emilia. Thanks are expressed to Professors Klaus ˆ Bernauer and Peter Schurmann of the Universite´ de Neuchatel, Switzerland, for the generous gift of the transformed E. coli cells.
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G. Battistuzzi et al. / Journal of Inorganic Biochemistry 75 (1999) 153–157 [18] I. Bertini, C. Luchinat, Coord. Chem. Rev. 150 (1996) 1. [19] I. Bertini, P. Turano, A.J. Vila, Chem. Rev. 93 (1993) 2833. [20] I. Bertini, C. Luchinat, NMR of Paramagnetic Molecules in Biological Systems, Benjamin and Cummings, Menlo Park, CA, 1986. [21] I. Bertini, S. Ciurli, A. Dikiy, R. Gasanov, C. Luchinat, G. Martini, N. Safarov, J. Am. Chem. Soc. 121 (1999) 2037. [22] A.P. Kalverda, J. Salgado, C. Dennison, G.W. Canters, Biochemistry 35 (1996) 3085. [23] A.J. Vila, B.E. Ramirez, A.J. Di Bilio, T.J. Mizoguchi, J.H. Richards, H.B. Gray, Inorg. Chem. 36 (1997) 4567. [24] J. Salgado, S.J. Kroes, A. Berg, J.M. Moratal, G.W. Canters, J. Biol. Chem. 273 (1998) 177. [25] C.O. Fernandez, A.I. Sannazzaro, A.J. Vila, Biochemistry 36 (1997) 10566. [26] M. Nordling, T. Olausson, L.G. Lundberg, FEBS Lett. 276 (1990) 98. [27] K. Bernauer, P. Schurmann, C. Nusbaumer, L. Verardo, S. Ghizdavu, Pure Appl. Chem. 70 (1998) 985. [28] A.M. Nersissian, C. Immoos, M.G. Hill, P.J. Hart, G. Williams, R.G. Herrman, J.S. Valentine, Prot. Sci. 7 (1998) 1915. [29] J.M. Guss, E.A. Merritt, R.P. Phizackerley, H.C. Freeman, J. Mol. Biol. 262 (1996) 686. [30] P.J. Hart, A.M. Nersissian, R.G. Herrman, R.M. Nalbandyan, J.S. Valentine, D. Eisenberg, Prot. Sci. 5 (1996) 2175. [31] A.M. Nersissian, Z.B. Mehrabian, R.M. Nalbandyan, P.J. Hart, G. Franczkiewicz, R.S. Czernuszewicz, C.J. Bender, J. Peisach, R.G. Herrmann, J.S. Valentine, Prot. Sci. 5 (1996) 2184.
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[32] C. Nusbaumer, Ph.D. Thesis, Universite´ de Neuchatel, ˆ Switzerland, 1996. [33] K.A. Markossian, V.Ts. Aikazyan, R.M. Nalbandyan, Biochim. Biophys. Acta 359 (1974) 47. [34] V.Ts. Aikazyan, R.M. Nalbandyan, FEBS Lett. 104 (1979) 127. [35] B. Reinhammar, Biochim. Biophys. Acta 205 (1970) 35. [36] T. Inubushi, E.D. Becker, J. Magn. Reson. 51 (1983) 128. [37] A.J. Vila, FEBS Lett. 355 (1994) 15. [38] A.J. Vila, C.O. Fernandez, J. Am. Chem. Soc. 118 (1996) 7291. [39] N. Sailasuta, F.C. Anson, H.B. Gray, J. Am. Chem. Soc. 101 (1979) 455. [40] A.G. Mauk, R.A. Scott, H.B. Gray, J. Am. Chem. Soc. 102 (1980) 4360. [41] S.K. Chapman, W.H. Orme-Johnson, J. McGinnis, J.D. Sinclair-Day, A.G. Sykes, P.-I. Ohlsonn, K.-G. Paul, J. Chem. Soc., Dalton Trans. (1986) 2063. [42] C. Dennison, G. Van Driessche, J. Van Beeumen, W. McFarlane, A.G. Sykes, Chem. Eur. J. 2 (1996) 104. [43] J.M. Guss, H.D. Bartunik, H.C. Freeman, Acta Crystallogr., Sect. B 48 (1992) 790. ¨ Hansson, S. Young, Prot. Sci. 7 (1998) 2099. [44] Y.F. Xue, M. Okvist, O. ¨ Hansson, Biochemistry 35 (1996) [45] K. Sigfridsson, S. Young, O. 1249. ¨ Hansson, D.S. Bendall, [46] S. Modi, M. Nordling, L.G. Lundberg, O. Biochim. Biophys. Acta 1102 (1992) 85.
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Article: 6184
1
H NMR of oxidized blue copper proteins
Gianantonio Battistuzzi, Lodovica Loschi, Marco Sola * Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, 41100 Modena, Italy Received 12 February 1999; received in revised form 27 April 1999; accepted 7 May 1999
Abstract The hyperfine-shifted 1H NMR resonances arising from the Cu(II) ligands of cucumber stellacyanin, plantacyanin (CBP) and plastocyanin, horseradish umecyanin, and of spinach plastocyanin and its L12G mutant are reported. It is shown that these broad paramagnetic signals are diagnostic of structural, electronic and solvation properties of the metal site and can be exploited to probe for differences and/or analogies among type I copper centers. q 1999 Elsevier Science Inc. All rights reserved. Keywords: Blue copper proteins; Cupredoxins; NMR spectroscopy; Phytocyanins; Plastocyanins
1. Introduction Type I or ‘blue’ copper centers are mononuclear metal sites found in low-molecular weight electron transport proteins, called cupredoxins, present in bacteria and green plants [1–6]. The metal is strongly bound by the thiolate sulfur of a cysteine and the nitrogen of two histidines, in an approximately trigonal arrangement, while the fourth (axial) coordination position is occupied either by the thioether sulfur of a methionine or the oxygen atom of a glutamine [1–6]. The structural and electronic properties of the metal sites, which are responsible for the peculiar spectroscopic and redox properties of these species, have been thoroughly investigated and are still the subject of extensive experimental and theoretical work [1–17]. The nucleus-unpaired electron coupling in paramagnetic metalloproteins can be profitably exploited to determine the protein structure in solution and the electronic properties of the metal center(s) through NMR [18–20]. The electron relaxation time of the Cu(II) ion (10y9 s) is relatively long and induces a large broadening (in most cases beyond detection) of the resonances of the protons neigh˚ [18–20]. Howboring the metal within approximately 5 A ever, broad contact-shifted resonances arising from protons outside the above limit belonging to the metal ligands could be recently detected in the 1H NMR spectra of oxidized plastocyanin, amicyanin, azurin and stellacyanin [21–25]. Estimation of the magnitude of the Fermi contact contribution to the hyperfine shift, which is related to the amount of spin * Corresponding author. Tel.: q39-059-378-421; fax: q39-059-373-543; e-mail: [email protected]
density delocalized over the ligands, provided further insight into the electronic properties of the Cu(II) site [21,22]. Here, we report the 400 MHz 1H NMR spectra of three oxidized phytocyanins, namely, umecyanin (UME) from horseradish, cucumber stellacyanin (CST) and plantacyanin (the latter also named cucumber basic protein, CBP), along with those of cucumber plastocyanin (CPL) and of recombinant wild-type plastocyanin from spinach (SPL) and of its L12G mutant [26,27]. Phytocyanins are a recently identified cupredoxin subfamily which includes a number of sequence and structurally related proteins from higher plants [28–31]. This group of proteins shows peculiar properties including, among others, a disulfide bridge at the Cu binding end of the polypeptide chain, the solvent accessibility of both Cu-binding histidines (instead of only one for azurins and plastocyanins), and a stronger interaction of the copper with the axial ligand (a methionine or a glutamine) as compared with the other cupredoxins [28–31]. The 1H NMR spectra of these species, which are unprecedented, have been compared with those recorded previously [21–25]. Since the overall architecture of the metal sites of these proteins obeys the same structural motif, the distribution of the spin density on the metal ligands, hence the pattern of the hyperfine-shifted resonances, is similar. However, these sites differ in the distortion of the coordination polyhedron and in the details of the electronic features of metal–ligand interactions, which result in moderate changes in position and linewidth of the signals. Here, the spectra of different cupredoxin subclasses are compared in search of typifying signal patterns and/or spectral signatures of certain metal site properties. This would allow exploitation of 1H NMR as an additional spectroscopic tool
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to recognize readily the nature of the metal site in oxidized cupredoxins. The utility of this work is related to the fact that independent signal assignment through one- or two-dimensional techniques for these species is hindered by the submillisecond T1 values, and it is almost hopeless unless signal assignments are available for the reduced form and favorable rates of electron self-exchange allow identification of the paramagnetic counterparts [21,22,24].
2. Experimental 2.1. Materials All chemicals were reagent grade and were used without further purification. Nanopure water was used throughout. 2.2. Protein purification Recombinant wild-type spinach plastocyanin and the L12G mutant were isolated from JM105 E. coli cells transformed with the expression system pTrc99A containing the construction of the peptide signal of P. aeruginosa azurin and the plastocyanin gene, following the procedure described elsewhere [26,32]. The transformed cells were a gift from Professors Klaus Bernauer and Peter Schurmann of the Univˆ ersite´ de Neuchatel, Switzerland. CBP, plastocyanin and stellacyanin from cucumber (Cucumis sativus) were isolated with slight modifications of the literature methods [33–35]. Umecyanin from horseradish (Armoracia laphatifolia) was from Sigma. 2.3. Spectroscopic measurements 1
H NMR spectra were recorded on a Bruker AMX-400 spectrometer, operating at 400.13 MHz, using 8K data points over a 100 kHz bandwidth. Suppression of the water peak was achieved by the super-WEFT pulse sequence (180-t-90AQqdelay) [36] with recycle time of 50 ms and delay times (t) of 30–50 ms. A typical spectrum run on a 0.1–0.3 mM protein sample required 100 000 scans. Spectra were recorded in water (100 mM phosphate buffer, pH 7), with 10% D2O for the lock, or in D2O (100 mM phosphate, pH 7) at 278C and are referenced to tetramethylsilane after calibration against the HDO peak, set at 4.78 ppm from TMS. The pH was adjusted by adding small amounts of concentrated NaOH and HCl under fast stirring.
Fig. 1. Paramagnetic region of the 400 MHz 1H NMR spectra of various oxidized cupredoxins in 0.1 M phosphate buffer, pH 7. CST, cucumber stellacyanin; UME, horseradish umecyanin; CBP, cucumber basic protein; CPL, cucumber plastocyanin; SPL, spinach plastocyanin; L12G-SPL, L12G mutant of spinach plastocyanin. Protein concentrations0.1–0.3 mM. Ts278C. Shaded signals correspond to exchangeable protons. Table 1 Chemical shift and tentative assignment of the 1H NMR resonances for oxidized phytocyanins at Ts278C Peak
UME
CST
a b (exch.) c
52 52 29
52 52 30
d e f (exch.) g
14.5 13 11 y5
17 14.2 11 y4
CBP
STC a
Assignment b
29.5
55 44 30
HisCdH GlnN´H HisCdH (MetCgH) c GlnCdH CysCaH CysNH HisCbH
17.5 12
14.6 12.1 y7
a
3. Results and discussion
From Ref. [25]. Assignments for STC are from Ref. [25]. Those for the other species are made upon spectral comparison with STC (this work). c Alternative assignment for CBP (this work).
The hyperfine-shifted resonances in the 400 MHz 1H NMR spectra of oxidized horseradish umecyanin, cucumber stellacyanin, cucumber basic protein, and plastocyanins from spinach and cucumber (SPL, CPL) at pH 7 are shown in Fig. 1. The chemical shifts are listed in Tables 1 and 2 for phy-
tocyanins and plastocyanins, respectively. Cucumber stellacyanin and horseradish umecyanin yield comparable spectra which are similar to that of Rhus vernicifera stellacyanin (STC) [25]. Two resonances arise from solvent exchangeable protons: signal ‘b’, which disappears in H2O at pH)8,
b
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G. Battistuzzi et al. / Journal of Inorganic Biochemistry 75 (1999) 153–157 Table 2 Chemical shift and assignment of the 1H NMR resonances of oxidized spinach (SPL) and cucumber (CPL) plastocyanins, L12G mutant of SPL, T. versutus amicyanin (AMI) and plastocyanin loop-containing amicyanin (Pc-AMI) at Ts278C Peak a b c (exch.) d e g
SPL 49 49 31.3 24.3 16.7
CPL
L12G AMI a
50 51 50 51 31.5 31 24.5 29 16.5 16.6
y5.1 y6.5
50 43 27.5 12–11.1 14.1 y9.5
Pc-AMI a 56 47.5 25.5 21.5 17.3 y9
Assignment b HisCdH a,c HisCdH a,c HisN´H a,c MetCgH a,c AsnCaH c (CysCaH) a CysCaH c (HisCbH) a
a
From Ref. [22]. Assignments for SPL and for AMI and Pc-AMI are from Ref. [21] and [22], respectively. Those for CPL and L12G (this work) must be considered as tentative, being based on the spectral similarities with SPL. c From Ref. [21]. b
as the corresponding peak in STC [25] and peak ‘f’, which disappears in D2O at pH 7. The similarity of these spectra is consistent with the presence of the same residues bound to the copper ion in these species [28–31], in which the oxygen atom of a glutamine residue acts as axial ligand. This allows extension of the tentative signal assignments obtained elsewhere for STC [25] to CST and UME. Thus, the broad signals ‘a’ and ‘c’ may be attributed to non-exchangeable CdH protons of the binding histidines, and peaks ‘b’ and ‘d’ to the N´H and the g-methylene protons of the axial glutamine, respectively. The greater shift of peak ‘d’ in CST as compared to UME and STC (17 versus 14.5 ppm, respectively) would indicate an increase in unpaired spin density over the axial ligand, possibly as a result of a somewhat stronger interaction with the copper ion and/or of a more effective spin delocalization mechanism. Peaks ‘e’ in CST and UME would correspond to the relatively slow-relaxing signal ‘e’ at 12.1 ppm observed for STC, which was assigned to the Ha proton of the binding cysteine [25]. The exchangeable signal ‘f’ detected for CST and UME at 11 ppm is not observed in STC. This resonance could arise from the peptide NH proton of the binding cysteine, which is only five bonds away from the metal and which could, in principle, experience a fraction of the relevant unpaired electron spin density delocalized over the S atom [7,22,23,25]. This assignment is supported by the small g anisotropy of the Cu(II) ion [21,22,25], which minimizes the pseudo-contact contribution to the hyperfine shift, virtually excluding any NH proton belonging to non-binding residues. The low-frequency signal ‘g’ at approximately y4 ppm in UME and CST should correspond to the analogous signal found at y7 ppm in STC and assigned to a CbH proton of a binding histidine. The absence of resonances attributable to solvent-exchangeable N´H protons of the binding histidines in these spectra is consistent with the pronounced solvent accessibility of the
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metal site of STC [30], which would thus appear to be a common feature of this class of proteins, in agreement with electrochemical, spectroscopic and kinetic studies [15– 17,25,37–42]. Overall, this spectral comparison would indicate that the coordination features of the metal sites of CST, UME and STC are conserved, with only some minor differences in binding strength and/or residue orientation of the glutamine and cysteine ligands. The metal site of cucumber and spinach plastocyanins is characterized by a distorted trigonal pyramidal geometry and by a small solvent accessibility [1–14,43,44]. Their 1H NMR spectra (Fig. 1) are almost identical. The 600 and 800 MHz 1 H NMR spectra of SPL, which appeared recently [21], allowed a better resolution of the two large peaks ‘a’ and ‘b’, and detection of two additional overlapping broad resonances at approximately 35 ppm. Signal assignment was achieved through saturation transfer experiments with the reduced diamagnetic species. Interestingly, the spectra of SPL and CPL closely reproduce that of an amicyanin mutant (Pc-AMI) in which the loop separating the second metal-binding histidine and the axial methionine in the wild-type protein has been substituted with the corresponding loop of plastocyanin [22]. By referring to the spectral assignments for SPL [21] and for the amicyanin mutant [22], which are in reasonable agreement, the two-proton peak ‘a–b’ in CPL most likely arises from the CdH protons of the binding histidines, the exchangeable signal ‘c’ is due to the N´H proton of His37, which, in contrast to phytocyanins, exchanges more slowly with the bulk solvent [43,44], and the broad signal ‘d’ is due to one of the CgH protons of the axial methionine [21,22]. Signal ‘e’ has been assigned to the CaH of Asn38 in SPL [21], in contrast with a previous assignment to the CaH of the copper-binding cysteine in Pc-AMI [22]. The latter proton has been shown to fall in the upfield region, most likely corresponding to peak ‘g’ [21]. Signal ‘f’ is slow-relaxing and probably belongs to the reduced form [21]. The strong similarity of the 1H NMR spectra of the plastocyanins and Pc-AMI mutant parallels that of the UV–Vis and EPR spectra and demonstrates that the electronic properties of their metal sites are very similar. This finding is relevant for the understanding of the role of the polypeptide chain in modulating the electronic features of the Cu(II) center in these systems. In particular, it demonstrates that the length and flexibility of the aminoacidic loop between the second metal binding histidine and the axial methionine strongly influence the properties of the axial bond. The utility of the hyperfine-shifted resonances to probe for structural and electronic perturbations of the cupric site in blue copper protein is further testified by the spectrum of the L12G mutant of spinach plastocyanin (Fig. 1). Leu12 belongs to the ‘northern’ hydrophobic patch around the Cu ligand His87 which is suggested to be important in the electron transfer reaction of plastocyanin with redox partners [45]. Mutations of this residue resulted in significant changes in the kinetics of the reaction with photosystem I, as well as in the reduction potential and spectroscopic properties
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[45,46]. In particular, substitution of Leu12 with an alanine was found to induce a change of the chemical shift of the Cu binding His37 in the reduced protein [46], alteration of the EPR parameters of the cupric form, and a decrease in E8 [45] compared to the wild type, which were interpreted as indicative of a slight distortion of the coordination geometry and modification of the electronic structure of the Cu site. The 1H NMR spectrum of the L12G mutant of spinach plastocyanin (Fig. 1) clearly confirms that removal of the bulky leucine side chain perturbs, to a certain extent, the electronic structure of the cupric form. In particular, compared with the wild-type species, peak ‘d’ moves to higher frequencies and is almost overlapped with peak ‘c’. The minor change in chemical shift of the latter signal would indicate that removal of the above side chain, which is close to the metal site, does not noticeably affect the exposition of the site to the solvent. This information is of use for the interpretation of the mutation-induced changes in reduction potential and kinetics of electron transfer. The metal center of CBP, a species which is taken as the prototype of the phytocyanins [29], differs from that of stellacyanins and umecyanin due to the presence of a methionine as axial ligand [2,29]. It also differs from that of plastocyanins and azurins because of a shorter Cu–S(Met) bond [29,43,44], which results in a more tetrahedral copper coordination. These properties are reflected in the 1H NMR spectrum, which is rather peculiar. In particular, the resonances ‘a’ and ‘b’, which are present in all the other cupredoxins, are broadened beyond detection. The similarity in chemical shift and lineshape of the broad signal at 29.5 ppm with peak ‘c’ of the spectra of UME, CST and STC suggest its assignment to one of the CdH protons of one of the binding histidines. However, this peak could also correspond to a Cg proton of the axial methionine, since it has been shown that the contact shift of the methylene Cg protons increases with increasing strength of axial methionine ligation [22]. We tentatively assign the sharper signals ‘e’ (17 ppm) and ‘f’ (which disappears in D2O) (12 ppm) to the CaH and peptide NH protons of the binding cysteine, respectively. As in the case of stellacyanins and umecyanin, the absence of any solvent exchangeable resonance due to the N´H protons of the binding histidines can be attributed to the great solvent accessibility of the metal site [29].
4. Conclusions The comparison of the 1H NMR properties of several oxidized cupredoxins carried out in this work indicates that the hyperfine-shifted resonances are sensitive to the structural and electronic properties of the metal center and to its exposure to the solvent. The strong similarities of the spectra of proteins sharing the same metal coordination, such as the stellacyanins and umecyanin, or the plastocyanins, suggest that 1H NMR can be profitably exploited to recognize readily
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the nature of the metal center in either newly discovered or still not structurally characterized oxidized cupredoxins.
5. Abbreviations
CBP CPL CST SPL STC UME
cucumber basic protein (also named plantacyanin) cucumber plastocyanin cucumber (Cucumis sativus) stellacyanin spinach plastocyanin Rhus vernicifera stellacyanin horseradish (Armoracia laphatifolia) umecyanin
Acknowledgements This work was performed with the financial support of the Ministero della Pubblica Istruzione of Italy (quota ex 40%). NMR measurements were carried out at the Centro Intedipartimentale Grandi Strumenti of the University of Modena and Reggio Emilia. Thanks are expressed to Professors Klaus ˆ Bernauer and Peter Schurmann of the Universite´ de Neuchatel, Switzerland, for the generous gift of the transformed E. coli cells.
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