4480
J. Am. Chem. Soc. 1992, 114, 4480-4486
Ligand Effects on the Blue Copper Site. Spectroscopic Studies of an Insulin-Stabilized Copper( 11) Chromophore Incorporating an Exogenous Thiolate Ligand Mark L. Brader,t Dan Borchardt,f and Michael F. Contribution from the Departments of Biochemistry and Chemistry, University of California at Riverside, Riverside, California 92521. Received October 16, 1991
Abstract: Insulin-stabilized type 1 Cu"N3L chromophores, in which L represents an exchangeable aromatic thiolate ligand, have been characterized by electron spin resonance (ESR), circular dicroism (CD), and UV-visible electronic absorption spectroscopy. The charge-transfer (CT) and ESR characteristics of these chromophores are strongly influenced by the nature of the thiolate ligand that coordinates to the copper. The complexes formed with pentafluorobenzenethiolate (PFBT) and tetrafluorobenzenethiolate (TFBT) possess axial ESR spectra (gll = 2.065, g, = 2.264, All= 87 X lo4 cm-I) that are analogous to those of the blue copper proteins plastocyanin and azurin. Furthermore, the resonance Raman spectrum of the PFBT complex is characterized by bands at 422, 393, 348, and 213 cm-I, features which indicate a strong resemblance to the resonance Raman spectrum of azurin. The complexes formed with benzenethiolate (BT) and 4-methylbenzenethiolate (4-MeBT) possess ESR spectra (g, = 2.025, g, = 2.090, g, = 2.230, A, = 60 X lo4 cm-I, A, = 33 X lo4 cm-I, A, = 38 X lo4 cm-l and g, = 2.020, g, = 2.090, g, = 2.210, A, = 60 X IO4 cm-I, A, = 32 X lo4 cm-I, A, = 42 X lo4 cm-I, respectively) that display substantial rhombic character and unusual hyperfine couplings, features which are strikingly similar to those that distinguish the ESR spectrum of the structurally uncharacterized blue copper protein, stellacyanin. The ligand 2-pyridinethiolate (2-PT) forms a complex possessing type 2 ESR (gll = 2.240, g, = 2.060, A,, = 152 X cm-l) and optical (A, = 500 nm, ,e, = 230 M-' cm-I;,,A = 720 nm, ,,e = 250 M-' cm-I) characteristics that are similar to those of the copper site in native bovine superoxide dismutase. This complex is proposed to incorporate 2-pyridinethiolate coordinated in a bidentate mode, thus forming a pentacoordinateCu(I1) center. Collectively, the ESR and CT data for the type 1 copper chromophores indicate that substitution in the aromatic ring of the coordinated benzenethiolate group affects the covalency of the Cu(I1)-S interaction, resulting in perturbations of the Cu(I1) site symmetry. It is concluded that an increased covalency of the Cu(I1)-S(thio1ate) bond in the pseudotetrahedral Cu"N3S unit can induce a change in the ESR spectrum from axially symmetric to one that possesses stellacyanin-like rhombic ESR parameters.
Introduction The exotic spectroscopic properties of blue (or type 1) copper proteins have stimulated considerable interest in the structural and electronic characteristics of the copper sites in these systems.' The blue copper spectral features comprise an unusually small ESR hyperfine coupling constant ( A , l< 70 X cm-I) and a very intense optical absorption envelope (t(60@630) = 3000-5000 M-' cm-l) with which distinctive resonance Raman bands are associated. The origins of these features have been elucidated via single-crystal X-ray crystallographic studies2 of several blue copper proteins in conjunction with detailed spectroscopic studies3+ of these proteins and relevant model systems.'O The blue copper protein crystal structures all identify highly distorted copper sites that incorporate two histidine imidazolyl nitrogens, a cysteine thiolate sulfur, and a methionine thioether sulfur as ligands to the copper. One impetus for extending chemical experience of the type 1 copper center beyond this now familiar Cu"N2(His)S(Cys)S(Met) structural motif stems from interest in atypical copper proteins, such as stellacyanin and cytochrome c oxidase, which possess exceptional spectroscopic properties but which remain structurally uncharacterized. Stellacyanin is a blue copper protein that contains a single type 1 copper center. The spectrochemical properties of stellacyanin'd." are distinctive in comparison to the other blue copper proteins; notably, the ESR spectrum is decidedly rhombic and displays peculiar hyperfine splittings. Spectroscopic studiesl2-I4indicate that two histidines and a cysteine residue ligate the copper; however, the copper site in stellacyanin cannot be entirely analogous to those of the structurally characterized blue copper proteins because stellacyanin does not contain methionine. The uncertainty regarding the stellacyanin active site exemplifies the difficulty associated with relating the spectroscopic nuances of pseudotetrahedral Cu(I1) sites to specific elements of biological structure and function. This difficulty highlights the need for a type 1 model system which *Author to whom correspondence should be addressed. 'Department of Biochemistry. *Department of Chemistry.
0002-7863192115 14-4480$03.00/0
allows the relationship between structure and spectroscopic behavior to be probed by rational manipulation of the copper site. ( I ) For reviews, see: (a) Fee, J. A. Struct. Bonding (Berlin) 1975, 23, 1-60. (b) Gray, H. B.; Solomon, E. I. In Copper Proteins; Spiro, T. G., Ed.; Wiley: New York, 1981; Vol. 3, pp 1-39. (c) Solomon, E. I.; Penfield, K. W.; Wilcox, D. E. Struct. Bonding (Berlin) 1983, 53, 1-57. (d) Lappin, A. G In Metal Ions in Biological Systems. Copper Proteins; Sigel, H., Ed.; Marcel Dekker: New York, 1981; Vol. 13, pp 15-71. (e) Adman, E. T. In Advances in Protein Chemistry. Copper Protein Structures; Anfinsen, C. B.,
Edsall, J. T., Eisenberg, D., Richards, F. M., Eds.; Academic Press: New York, 1991; Vol. 42, pp 145-197. (2) (a) Norris, G. E.; Anderson, B. F.; Baker, E. N. J . Mol. Biol. 1983, 165, 501-521. (b) Norris, G. E.; Anderson, B. F.; Baker, E. N. J. Am. Chem. SOC.1986, 108, 2784-2785. (c) Baker, E. N. J . Mol. Biol. 1988, 203, 1071-1095. (d) Adman, E. T.; Jensen, L. H. Isr. J . Chem. 1981, 21, 8-12. (e) Korszun, Z. R. J . Mol. Biol. 1987, 196, 413-419. (f) Guss, J. M.; Freeman, H. C. J. Mol. Biol. 1983,169, 521-563. (9) Adman, E. T.; Stenkamp, R. E.; Sieker, L. C.; Jensen, L. H. J . Mol. Biol. 1978, 123, 35-47. (h) Guss, J. M.; Merritt, E. A.; Phizackerley, R. P.; Hedman, B.; Murata, M.; Hodgson, K. 0.;Freeman, H. C. Science 1988, 241, 806-811. (i) Adman, E. T.; Stewart, T.; Bramson, R.; Petratos, K.; Banner, D.; Tsernoglou, D.; Beppu, T.; Watanabe, H. J. Biol. Chem. 1989,264,87-99. (j) Messerschmidt, A.; Rossi, A.; Ladenstein, R.; Huber, R.; Bolognesi, M.; Gatti, G.;Marchesini, A.; Petruzelli, R.; Finazzi-Agro, A. J . Mol. Biol. 1989, 206, 513-529. (3) Solomon, E. I.; Hare, J. W.; Dooley, D. M.; Dawson, J. H.; Stephens, P. J.; Gray, H. B. J . Am. Chem. SOC.1980, 102, 168-178. (4) Penfield, K. W.; Gay, R. R.; Himmelwright, R. S.; Eickman, N. C.; Norris, V. A,; Freeman, H. C.; Solomon, E. I. J . Am. Chem. Soc. 1981,103, 4382-4388. (5) Penfield, K. W.; Gewirth, A. A.; Solomon, E. I. J . A m . Chem. SOC. 1985, 107, 4519-4529. (6) Dawson, J. H.; Dooley, D. M.; Clark, R.; Stephens, P. J.; Gray, H. B. J . Am. Chem. Soc. 1979, 101, 5046-5053. (7) Gewirth, A. A.; Solomon, E. I. J . Am. Chem. SOC. 1988, 110, 381 1-3819. (8) Woodruff, W. H.; Norton, K. A. J . Am. Chem. SOC. 1983, 105, 657-658. (9) Ainscough, E. W.; Bingham, A. G.; Brodie, A. M.; Ellis, W. R.; Gray,
H. B.; Loehr, T. M.; Plowman, J. E.; Norris, G. E.; Baker, E. N. Biochemistry 1987, 26, 71-82. (10) (a) Schugar, H. J. In Copper Coordination Chemistry: Biochemical and Inorganic Perspectiues; Karlin, K., Zubieta, J., Eds.; Adenine Press:
Guilderland, NY, 1983; pp 43-74. (b) Knapp, S.;Keenan, T. P.; Zhang, X.; Fikar, R.; Potenza, J. A,; Schugar, H. J. J . Am. Chem. SOC.1990, 112, 3452-3464.
0 1992 American Chemical Society
J. Am. Chem. SOC.,Vol. 114, No. 12, 1992 4481
Insulin-Stabilized Cu(II)- Thiolate Complexes
Table I. Electronic Absorption Data' for the Cu(II)-R6-Thiolate Comdexes thiolateb 372 (1250), 406 (llOO), 626 (1700), 88Oe (520) PFBT 369 (1200), 416 (1250), 630 (1800), 88oC (550) TFBT BT 310 12700). 378 (900). 450 (700). 696 (2700), 89OC(1700) 4-MeBT 3lsd'(23O0), 390'(700), 448'(530), 702'(2500), 89oC (2200) 2-PT 500 (230), 720 (250) 432 (1000). 646 (1250). 900' (400) 4-PT oX,,, nm (e, M-I cm-I). emar = observed extinction coefficient calculated with respect to metal ion concentration. bBT = benzenethiolate, PFBT = pentafluorobenzenethiolate, TFBT = tetrafluorobenzenethiolate, 4-MeBT = 4-methylbenzenethiolate, 2-PT = 2-pyridinethiolate, 4-PT = 4-pyridinethiolate. Broad. dShoulder. ~~~
~~
(A)
(e)
Figure 1. View down the 3-fold symmetry axis of one half of the Zn(11)-& insulin hexamer showing the structural arrangement of the three subunits that form one zinc site (A). The locations of the protein-bound phenol molecules are shown also. Schematic representation of the copper environment postulated herein for the type 1 sites of the Cu(II)-R6thiolate complexes (B). The exogenous thiolate ligand is designated R-Sand is constrained by an -8-A long cylindrical tunnel that is formed by the B chain helices. A pseudotetrahedral array about the copper is completed by three HisBlO imidazolyl nitrogens. Coordinates for these drawings were kindly provided by Guy G. Dodson (University of York), and the computer graphics for the drawings were performed by Ole Hvilsted Olsen (Novo Research Institute).
Unfortunately, attempts to develop synthetic model analogues of the type 1 copper site have met with notorious difficulty due to the redox instability of the Cu(I1)-thiolate interaction15 and the marked preference of the Cu(I1) ion for tetragonal rather than pseudotetrahedral coordination geometries. The Cu(I1)-substituted R-state insulin hexamerI6-l8 is one system that has been postulated to stabilize Cu(I1)-thiolate ligation in a distorted tetrahedral Cu"N,S(thiolate) environment (Figure 1). X-ray crystallographic studieslFZzof Zn(I1)-insulin hexamers have identified a set of three structures designatedz3 as Tg, T3R3, and &. The & hexamer21*22 is stabilized by six phenol molecules which bind to hydrophobic pockets formed between adjacent subunits. This structure incorporates two identical Zn sites, each with a distorted tetrahedral arrangement of three B10 histidines and one chloride2I or phenolate ionz2(Figure 1A). The M(II)-&24 insulin hexamer provides, in effect, a constrained tridentate imidazolyl N 3 chelate in which a fourth coordination position is accessible to an exogenous ligand (Figure 1B). With copperinsulin, the spectrochemical versatility of this system is noteworthy because it permits the investigation of a type 1 Cu(I1) site in which the thiolate ligand may be varied. In this report we have characterized spectroscopically several Cu(II)-R6-thiolate adducts with the goal of demonstrating how perturbations of the Cu(1 1) Malmstrom, B. G.; Reinhammar, B.; Vanngard, T. Biochim. Biophys.
Acta 1970, 205, 48-57.
(12) Rist, G . H.; Hyde, J. S.; Vanngard, T. Proc. Natl. Acad. Sci. U.S.A. 1970, 67, 79-86. (13) Mims, W. B.; Peisach, J. Biochemistry 1976, 15, 3863-3869. (14) Roberts, J. E.; Brown, T. G.; Hoffman, B. M.; Peisach, J. J . Am. Chem. SOC.1980, 102, 825-829. (15) Anderson, 0.P.; Perkins, C. M.; Brito, K. K. Inorg. Chem. 1983,22, 1267-1273. (16) Brader, M. L.; Dunn, M. F. J. A m . Chem. SOC. 1990, 112, 4585-4587. (17) Brader, M. L.; Borchardt, D.; Dunn, M. F. Biochemistry, in press. (18) Brader, M. L.; Dum, M. F. Trends Biochem. Sci. 1991,16,341-345. (19) Baker, E. N.; Blundell, T. L.; Cutfield, J. F.; Cutfield, S . M.; Dodson, E. J.; Dodson, G. G.; Crowfoot-Hodgkin, D. M.; Hubbard, R. E.; Isaacs, N. W.; Reynolds, C. D.; Sakabe, K.; Sakabe, N.; Vijayan, N. M. Philos. Trans. R. Soc. London 1988, 319, 369-456. (20) Smith, G. D.; Swenson, D. C.; Dodson, E. J.; Dcdson, G. G.; Reynolds, C. D. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 7093-7097. (21) Derewenda, U.; Derewenda, Z.; Dodson, E. J.; Dodson, G. G.; Reynolds, C. D.; Smith, G. D.; Sparks, C.; Swenson, D. Nature 1989, 338, 594-596. (22) Smith, G. D.; Dodson, G. G. Biopolymers 1992, 32, 441-445. (23) Kaarsholm, N. C.; KO,H.-C.; Dunn, M. F. Biochemistry 1989, 28, 4427-4435. (24) M(I1)-T6 and M(I1)-R6 denote the metal-substituted T6 and R6 insulin hexamers, respectively,where M may be Cu, Co, or Zn.Two chemically equivalent metal ions are bound per hexamer.
(11)-thiolate bond affect the spectral properties of the type 1 Cu(I1) site.
Experimental Section Preparation of the Insulin Complexes. Cu(I1)-T6 hexamer solutions were prepared by the stoichiometric addition of Cuz+ ions to solutions of metal-free human insulin in the ratio of two Cu2+ions per six insulin subunits (M = 5800). These solutions were prepared in 50 m M TrisC104 buffer, pH 7.5. Insulin concentrations were determined from the absorbance at 280 nm (c = 5.7 X lo3 M-l cm-I). Except where indicated, the CU(II)-R6-thiOlate complexes were obtained upon the addition of thiolate to the solution of Cu(I1)-substituted insulin hexamer in the presence of 100 m M resorcinol.25 Co(II)-R6-thiolate solutions were prepared as described previously.26 A slow bleaching of the Cu(I1)-&-thiolate complexes occurs at room temperature. This process proceeds at varying rates, depending upon the thiolate ligand. The E S R spectral signals of the Cu(I1)-&-thiolate complexes were typically 50% less intense than that of the original Cu(II)-T6 solution, indicating that an appreciable degree of reduction of Cu(1) had taken place. Reduction of Cu(II)-R6-thiolate complexes has been shown to yield the corresponding optically- and ESR-unobservable Cu(1)-&-thiolate com~lexes.~'Thus the extinction coefficients observed for the UV-visible spectra must be regarded as minimum values. Electronic Absorption and Circular Dichroism Spectroscopy. Spectra were recorded at 298 K immediately following the preparation of the Cu(I1)-&-thiolate complexes using a 1:l M/thiolate ratio. Electronic absorption spectra were recorded using a Hewlett-Packard HP8450A spectrophotometer for the 300-800 nm range and a Cary 2390 spectrophotometer for the 800-1100 nm range. Circular dichroism spectra obtained using a JASCO 5-600spectropolarimeter are reported in the range 300-800 nm. ESR Spectroscopy. X-band ESR spectra (9.21 GHz) were recorded on frozen (1 10 K) solutions in quartz tubes utilizing a Bruker ER2OOD ESR spectrometer equipped with a Hewlett-Packard 5350B microwave frequency counter and a Bruker ER035M gaussmeter. Spectra were recorded immediately following the preparation of the Cu(I1)-&-thiolate complexes using a 1:2 M/thiolate ratio. No color changes due to freezing and thawing the solutions for ESR spectroscopy were observed. ESR Simulations?' These were performed using the program QPOW.2s The simulations utilized a spin Hamiltonian of the form given bYeq1 H = 0H-g.S S.A*I (1)
+
where H is the magnetic field vector, S and I are the electron and nuclear spins respectively, g is the g tensor, A is the hyperfine tensor, and j3 is the Bohr magneton. Transitions where AM, # 0 were not included in the simulations. A and g were assumed to have the same principal ax=, and a Gaussian lineshape was used. The values of the g and A parameters giving the best agreement with the experimental data were found by trial and error. Resonance Raman Spectroscopy. Spectra were recorded at 298 K on samples in glass melting point capillary tubes using a Dilor X Y laser (25) Our previous studies (ref 26 and Choi et al., unpublished results) have shown that a wide variety of phenolic derivatives stabilize the b conformation of the M(I1)-substituted insulin hexamer. For Co(I1)- and Cu(I1)-insulin hexamers, resorcinol displaces the T6 = & equilibrium more strongly in favor of the R6 species than does phenol. (26) Brader, M. L.; Kaarsholm, N. C.; Lee, R. W.-K.; Dunn, M. F. Biochemistry 1991, 30, 6636-6645. (27) We note that simulations of observed ESR signals provide a useful means of estimating the spectral parameters but do not necessarily produce unique fits. (28) Nilges, M. J. Ph.D. Thesis, University of Illinois, Urbana, Illinois, 1979.
4482 J. Am. Chem. SOC.,Vol. 114, No. 12, 1992
Brader et al.
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Table 11. Circular Dichroism Spectral Data' for the Cu(IIbR,-Thiolate Comulexes thiolateb PFBT 322 (+0.092), 364 (-0.040),428 (-0.165), 607 (+1.19) TFBT 320 (+0.034), 362 (-0.026), 433 (-0.127), 611 (+1.25) BT 312 (-0.085), 375c (+0.116), 442 (+0.522), 664 (+0.937), 780 (-0.810) 4-MeBTd 320 (+j, 380' (+), 446 (+), 673 (+), 780 (-) aAmax, nm ([e1 X IO4). bBT = benzenethiolate, PFBT = pentafluorobenzdnethiolatd,TFBT = tetrafluorobenzenethiolate, 4-MeBT = 4-methylbenzenethiolate. Broad, unresolved shoulder. dRapid reduction of this complex precluded measurement of [b'] values.
c
5
c
2000
h
c.
w
I000
400
600 Wavelength (nm)
800
Figure 2. UV-visible electronic absorption spectra (A) and circular dichroism spectra (B) of the Cu(I1)-R6-thiolate complexes formed with tetrafluorobenzenethiolate(a) and benzenethiolate (b). Spectra were recorded on solutions of hexamer complexes prepared in 50 mM TrisCIO, buffer, p H 7.5, incorporating a 0.2 X lo-' M concentration of copper sites.
Raman spectrometer equipped with Ar and Kr ion lasers and a 1024 channel intensified diode array. Spectra were recorded immediately following the preparation of the Cu(I1)-R6-PFBT complex in the presence of 100 m M phenol at pH 8. The M/thiolate ratio was 1:l.
Results Cu(II)-R,-Thiolate Electronic and CD Spectra. UV-visible electronic absorption spectral data for a series of CU(II)-R6thiolate complexes are presented in Table I. The spectra of the TFBT and BT complexes are presented in Figure 2A. The data of Table I show that the Cu(II)-R,-thiolate complexes all give electronic absorption spectra which display a single intense charge-transfer (CT) band in the region 600-700 nm. This band is flanked by bands in the 300-450 nm region and an additional band at approximately 900 nm. The data (Table I) establish that the energy of the prominent visible C T band is strongly influenced by the nature of the thiolate ligand. This band is analogous to the intense absorption that occurs in the 600-630 nm region of blue copper protein spectra and is assigned as an S(thio1ate)Cu(I1) ligand-to-metal C T (LMCT) transition. The low energy of the band at -900 nm supports its assignment as a ligand field (d-d) transition attributable to a near tetrahedrally coordinated Cu(I1) ion. The bands in the 300-450 nm region probably arise from s(ImH)-Cu(II) LMCT transitions. Comparable transitions have been identified in the spectra of blue copper proteins and in the spectra of various small molecule Cu(I1)-imidazole chromophores.I0 The C D spectra of Cu(I1)-R,-TFBT and Cu(II)-R6-BT are shown in Figure 2B. The 696-nm C T band in the optical spectrum of Cu(I1)-%-BT is resolved into positive (664 nm) and negative (780 nm) C D bands. These C D bands are attributable to s(S)-Cu(II) and u(S)-Cu(II) LMCT transitions. This interpretation is in accordance with theory, which predicts29that the CD bands associated with the T and u C T transitions will have opposite signs. The 630-nm optical CT band in Cu(II)-R6-TFBT appears as a large positive C D band at 611 nm and an incompletely distinguished minor negative band at >750 nm. A comparison of the Cu(II)-R6-TFBT and Cu(I1)-%-BT CD spectra indicates that the a and u components of the S(thio1ate)-Cu(I1) LMCT (29) Ibarra, C.; Soto, R.; Adan, L.; Decinti, A,; Bunei, S. Inorg. Chim. Acta 1972, 6, 601-606.
la
3;oo
35
Magnetic Fiold ( 0 )
Figure 3. X-band ESR spectra (1 10 K) (A) of the Cu(II)-R6-thiolate complexes formed with pentafluorobenzenethiolate (PFBT) (a) and 2pyridinethiolate (2-PT) (b). The spectra were recorded on a solution of hexamer complex prepared in 50 mM Tris-C104 buffer, pH 7.5, incorporating a 0.4 X lo-' M concentration of copper sites. X-band ESR spectra (110 K) (B) of the Cu(I1)-R6-thiolate complexes formed with benzenethiolate (BT) (a) and 4-methylbenzenethiolate (4-MeBT) (c). Spectra were recorded on solutions of hexamer complexes prepared in 50 m M Tris-CIO, buffer, pH 7.5, incorporating a 0.4 X 10-3 M concentration of copper sites. Simulations of spectra a and c are shown in b and d, respectively. The spectrum of stellacyanin, simulated using the parameters of Gewirth et al. (ref 30), is shown in e for comparison.
envelope are quite different in each complex, reflecting the different character of the respective Cu(I1)-S(thio1ate) bonds. The C D spectral data for the Cu(II)-R,-thiolate complexes formed with PFBT, TFBT, BT, and 4-MeBT are summarized in Table 11. ESR Spectra. The ESR spectra of the Cu(I1)-R,-thiolate complexes are presented in Figure 3. The spectrum of the PFBT complex (Figure 3A, spectrum a) is virtually identical to that of the TFBT complex (not shown). The spectra of the complexes formed with BT and 4-MeBT are shown in Figure 3B (spectra a and c, respectively). Spectra b and d of Figure 3B are the respective simulations of spectra a and c that are obtained using the parameters shown in Table 111. The spectrum of the complex formed with 2-pyridinethiolate (2-PT) is shown in Figure 3A (spectrum b). The ESR spectral parameters are summarized in Table I11 along with literature values for azurin, plastocyanin, stellacyanin, cucumber basic protein (CBP), and Cu(I1)-substituted liver alcohol dehydrogenase (Cu(I1)-LADH). The Cu(I1) ESR signal is affected dramatically by the nature of the thiolate group ligated to the Cu(I1) ion (Figure 3). No ligand hyperfine couplings are observed. The spectrum of Cu(11)-%-PFBT (Figure 3A, a) exhibits a resolved copper hyperfine
J. Am. Chem. SOC.,Vol. 114, No. 12, 1992 4483
Insulin-Stabilized Cu(II)- Thiolate Complexes Table 111. ESR Parameters suecies' Cu(II)-&-PFBT Cu(II)-R-TFBT Cu(IIj-
