J. Am. Chem. Soc. 1998, 120, 9621-9631
9621
Spectroscopic and Geometric Variations in Perturbed Blue Copper Centers: Electronic Structures of Stellacyanin and Cucumber Basic Protein Louis B. LaCroix,† David W. Randall,† Aram M. Nersissian,‡ Carla W. G. Hoitink,§ Gerard W. Canters,§ Joan S. Valentine,‡ and Edward I. Solomon*,† Contribution from the Department of Chemistry, Stanford UniVersity, Stanford, California 94305, Department of Chemistry and Biochemistry, UniVersity of California, Los Angeles, California 90095, and Department of Chemistry, Gorlaeus Laboratories, Leiden UniVersity, P.O. Box 9502, 2300 RA Leiden, The Netherlands ReceiVed February 23, 1998
Abstract: The electronic structures of the perturbed blue copper proteins stellacyanin (STC) and cucumber basic protein (CBP, also called plantacyanin, PNC) are defined relative to that of the well-understood “classic” site found in plastocyanin (PLC) by combining the results of low-temperature optical absorption, circular dichroism, and magnetic circular dichroism spectra with density functional calculations. Additionally, absorption and magnetic circular dichroism spectra of Alcaligenes denitrificans wild-type and M121Q azurin are presented and compared to PLC and STC, respectively. These studies show that the principal electronic structure changes in CBP/PNC, with respect to PLC, are a small shift of the ligand field transitions to higher energy and a rotation of the Cu dx2-y2 half-filled HOMO which increases the pseudo-σ and decreases the π interactions of the cysteine (Cys) sulfur with Cu dx2-y2 and, in addition, mixes some methionine (Met) sulfur character into the HOMO. The geometrical distortion responsible for the perturbed electronic structure, relative to PLC, involves a coupled angular movement of the Cys and Met residues toward a more flattened tetragonal structure. In contrast to CBP/PNC, STC (which has the axial Met substituted by Gln) has its ligand field transitions shifted to lower energy and undergoes much smaller degrees of HOMO rotation and Cys pseudo-σ/π mixing; no axial glutamine character is displayed in the HOMO. These changes indicate a tetrahedral distortion in STC. Therefore, perturbed spectral features are consistent with both tetragonal and tetrahedral geometric distortions relatiVe to PLC. These perturbations are discussed in terms of the increased axial ligand strength in these proteins (i.e., short Cu-S(Met) in CBP/PNC and O(Gln) in STC). This induces an ∼(u)-like distorting force which either results in a tetragonal distortion of the site (CBP/PNC) or is structurally restrained by the protein (STC and M121Q).
Introduction Blue (type 1) copper proteins exhibit rapid electron-transfer rates and high redox potentials compared with tetragonal “normal” copper complexes.1-4 Perhaps the most striking features of such proteins are an intense absorption at ∼600 nm and a small A| value in the EPR spectra.5,6 These features reflect novel electronic structures which contribute to reactivity.6-9 Classic blue copper proteins, such as plastocyanin and azurin, show intense ∼600 nm bands, a weak absorption envelope at * Author to whom correspondence should be addressed. † Stanford University. ‡ University of California, Los Angeles. § Leiden University. (1) Gray, H. B.; Solomon, E. I. In Copper Proteins; Spiro, T. G., Ed.; Wiley: New York, 1981; pp 1-39. (2) Adman, E. T. In Topics in Molecular and Structural Biology: Metalloproteins; Harrison, P., Ed.; MacMillan: New York, 1985; Vol. 1, pp 1-42. (3) Adman, E. T. In AdVances in Protein Chemistry; Anfinsen, C. B., Richards, F. M., Edsall, J. T., Eisenberg, D. S., Eds.; Academic Press: San Diego, CA 1991; Vol. 42, pp 145-198. (4) Sykes, A. G. AdV. Inorg. Chem. 1991, 36, 377-408. (5) Penfield, K. W.; Gewirth, A. A.; Solomon, E. I. J. Am. Chem. Soc. 1985, 107, 4519-4529. (6) Solomon, E. I.; Baldwin, M. J.; Lowery, M. D. Chem. ReV. 1992, 92, 521-542. (7) Solomon, E. I.; Lowery, M. D. Science 1993, 259, 1575-1581.
∼450 nm (R ) 450/600 < ∼0.15) and display approximately axial (gx ≈ gy) EPR signals. These spectral features derive from the Cu site’s distorted C3V tetrahedral ligand set in which two histidine (His) N’s with typical Cu-N bonds (∼2.0 Å) and a highly covalent cysteine (Cys) S with an unusually short Cu-S bond (∼2.1 Å) form an approximate trigonal plane which contains the half-occupied Cu dx2-y2 based HOMO. A weak axial ligand completes the site (usually a long methionine (Met) Cu-S bond at ∼2.8 Å).10 A strong π S(Cys)-Cu interaction orients the dx2-y2 orbital so that the Cu-S(Cys) bond bisects the lobes of this orbital and is responsible for the intense S(Cys) p f Cu 3dx2-y2 transition at 600 nm.8,11 Type 1 centers which exhibit spectral features12,13 substantially varied from those of the well-defined classic center are (8) Solomon, E. I.; Penfield, K. W.; Gewirth, A. A.; Lowery, M. D.; Shadle, S. S.; Guckert, J. A.; LaCroix, L. B. Inorg. Chim. Acta 1996, 243, 67-78. (9) Lowery, M. D.; Guckert, J. A.; Gebhard, M. S.; Solomon, E. I. J. Am. Chem. Soc. 1993, 115, 3012-3013. (10) Guss, J. M.; Bartunik, H. D.; Freeman, H. C. Acta Crystallogr. 1992, B48, 790-811. (11) Gewirth, A. A.; Solomon, E. I. J. Am. Chem. Soc. 1988, 110, 38113819. (12) Lu, Y.; LaCroix, L. B.; Lowery, M. D.; Solomon, E. I.; Bender, C. J.; Peisach, J.; Roe, J. A.; Gralla, E. B.; Valentine, J. S. J. Am. Chem. Soc. 1993, 115, 5907-5918.
S0002-7863(98)00606-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/05/1998
9622 J. Am. Chem. Soc., Vol. 120, No. 37, 1998 referred to as “perturbed” blue copper centers. Perturbed blue copper sites, such as those in stellacyanin (STC), cucumber basic protein (CBP), Achromobacter cycloclastes nitrite reductase (NiR), and pseudoazurin, exhibit rhombic EPR signals (i.e., ∆g⊥ ) gx - gy > 0.01) and increased 450 nm absorption intensities relative to those in classic blue copper proteins, usually accompanied by a decrease in the blue band intensity (R > ∼0.15). Such perturbed spectral features are observed in cases where the classic blue Cu ligands (CysHis2Met) are retained (i.e., cucumber basic protein, nitrite reductase, etc.) and when they are not (i.e., stellacyanin). The recently available structure14 of stellacyanin shows that the prototypical axial Sδ(Met) ligand is replaced by an amide oxygen from a glutamine side chain (O(Gln)). Our previous study15 on the perturbed site (i.e., green) in A. cycloclastes nitrite reductase (R ) 1.216) demonstrated this to be tetragonally distorted (toward square planar), in contrast to the tetrahedral distortion proposed for this site on the basis of resonance Raman measurements.17,18 To understand the electronic and geometric structure origins of the spectral features across the entire range of perturbed sites, blue centers with lesser degrees of perturbation and different axial ligation from nitrite reductase must be examined. Cucumber basic protein (also called plantacyanin, PNC)19,20 (R ≈ 0.6, ∆g⊥ ≈ 0.04) and Rhus Vernicifera stellacyanin21,22 (STC) (R ≈ 0.2, ∆g⊥ ≈ 0.05) both exhibit perturbed spectral features and allow for a comparison of Met and Gln axial ligation. The active site structures of CBP10 and STC14 are summarized in Figure 1. Compared with plastocyanin, in cucumber basic protein the Cu-S(Cys) bond has expanded by 0.1 Å and the Cu-S(Met) has contracted by 0.2 Å.10 The most prominent difference between Cucumis satiVa (cucumber) stellacyanin and plastocyanin is the replacement of the S(Met) axial ligand with O(Gln) at a distance of ∼2.2 Å; however, the Cu-S(Cys) bond also lengthens by 0.1 Å relative to plastocyanin.14 In this study, the electronic structure of the active sites in cucumber basic protein and stellacyanin are defined relative to that of the classic blue copper site in plastocyanin in order to describe the differences in bonding associated with the spectral changes and electronic effects on the geometry of the site. The energies and intensities of the excited-state spectral features are obtained from low-temperature absorption (Abs), circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopies.23 Density-functional calculations are used to probe further the electronic structure of these perturbed blue (13) Han, J.; Loehr, T. M.; Lu, Y.; Valentine, J. S.; Averill, B. A.; Sanders-Loehr, J. J. Am. Chem. Soc. 1993, 115, 4256-4263. (14) Hart, P. J.; Nesissian, A. M.; Herrmann, R. G.; Nalbandyan, R. M.; Valentine, J. S.; Eisenberg, D. Protein Sci. 1996, 5, 2175-2183. (15) LaCroix, L. B.; Shadle, S. E.; Wang, Y.; Averill, B. A.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. J. Am. Chem. Soc. 1996, 118, 77557769. (16) Liu, M.-Y.; Liu, M.-C.; Payne, W. J.; LeGall, J. J. Bacteriol. 1986, 166, 604-608. (17) Andrew, C. R.; Yeom, H.; Valentine, J. S.; Karlsson, B. G.; Bonander, N.; van Pouderoyen, G.; Canters, G. W.; Loehr, T. M.; SandersLoehr, J. J. Am. Chem. Soc. 1994, 116, 11489-11498. (18) Andrew, C. R.; Sanders-Loehr, J. Acc. Chem. Res. 1996, 29, 365372. (19) Aikazyan, V. T.; Nalbandyan, R. M. FEBS Lett. 1975, 55, 272274. (20) Sakurai, T.; Okamoto, H.; Kawahara, K.; Nakahara, A. FEBS Lett. 1982, 147, 220-224. (21) Peisach, J.; Levine, W. G.; Blumberg, W. E. J. Biol. Chem. 1967, 242, 2847-2858. (22) Malmstro¨m, B. G.; Reinhammar, B.; Va¨nngård, T. Biochim. Biophys. Acta 1970, 205, 48-57. (23) Solomon, E. I.; Hanson, M. A. In Inorganic Electronic Spectroscopy; Solomon, E. I., Lever, A. P. B., Eds.; Wiley & Sons: New York, 1998, in press.
LaCroix et al.
Figure 1. Structure of the oxidized blue copper centers in (A) cucumber basic protein38 (B) and Cucumis satiVus stellacyanin14 viewed with the cysteine-histidine-histidine (Cys-His-His) NNS plane perpendicular (left) and parallel (right) to the plane of the paper. The angular orientation of the Cys, Met, and Gln ligands in relation to the His ligands is most apparent in the parallel view.
Cu proteins. The study presented here also provides an experimental and theoretical description of the electronic structure of a blue copper protein with an axial ligand other than methionine. Combined with previous results,15 these results allow for spectroscopic trends and their origins to be examined across the range of classic to perturbed proteins (both methionine and non-methionine containing). Finally, spectroscopic correlations between classic wild-type Alcaligenes denitrificans azurin (R ≈ 0.1, ∆g⊥ ≈ 0.01) and its perturbed axial mutant Gln M121Q24 (R ≈ 0.2, ∆g⊥ ≈ 0.05) provide further insight into possible protein contributions to active site electronic and geometric structure of perturbed relative to classic blue copper centers. Experimental Section Low-Temperature Optical Spectra. Low-temperature absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectra were obtained and fit as described in ref 15. Rhus Vernicifera stellacyanin,25 wild type and M121Q forms of A. denitrificans azurin,24 and cucumber basic protein26 were prepared as described elsewhere. Protein samples (∼0.5-1.0 mM) were prepared as glasses in 50% (v/ v) D2O/glycerol-d3 in either 50 mM phosphate (pD* ) 7.6) (cucumber basic protein), 10 mM phosphate (pD* ) 6.0) (stellacyanin), or 20 mM phosphate (pD* ) 7.0) (wild type and M121Q azurin). Electronic Structure Calculations. A. Active Site Geometry. In the C1(met)(his) approximation of the active site in cucumber basic protein used, the oxidized blue copper site is modeled by Cu[(S(CH3)2)(SCH3)(C3N2H4)2]+. The blue site in stellacyanin was modeled by the C1(gln)(his) approximation, where acetamide (CH3CONH2), occupies the axial position. The crystallographically determined coordinates were used for all atoms except hydrogens, which were added in appropriate geometries to complete the site. Each blue copper center was placed (24) Romero, A.; Hoitink, C. W. G.; Nar, H.; Huber, R.; Messerschmidt, A.; Canters, G. W. J. Mol. Biol. 1993, 229, 1007-1021. (25) Reinhammar, B. Biochim. Biophys. Acta 1970, 205, 35-47. (26) Nersissian, A. M.; Nalbandyan, R. M. Biochim. Biophys. Acta 1988, 957, 446.
Variations in Perturbed Blue Copper Centers
J. Am. Chem. Soc., Vol. 120, No. 37, 1998 9623
in a coordinate system chosen to give a Cu dx2-y2 ground state wave function, which is experimentally observed from the g values in the EPR spectrum of each site (g| > g⊥ > 2.0):19-22 the Cu-S(Cys) bond is 45° from the x and y axes, while the bond closest to z is Cu-S(Met) or O(Gln). This axis system diagonalizes the g-tensor for plastocyanin.11 The Cartesian coordinates for calculations performed in this study on the cucumber basic protein and stellacyanin active sites are provided as Supporting Information. B. SCF-Xr-SW Calculations. The 1982 QCPE release of the SCFXR-SW package27-30 was used to evaluate the electronic structure of cucumber basic protein and stellacyain as described in ref 15. Atomic sphere radii adjusted to reproduce the experimental g values in plastocyanin were employed for all calculations.11 The oxygen sphere radius in the stellacyanin calculations was set to 1.70 Bohr on the basis of the Norman criteria.31 The parameters used for the SCF-XR-SW calculations are listed as Supporting Information. C. LCAO Density Functional Calculations. Spin restricted calculations were performed as described previously15 using version 1.1.3 of the Amsterdam Density Functional (ADF) programs of Baerends and co-workers.32 Basis functions, core expansions functions, core coefficients, and fit functions for all atoms were used as provided from database IV, which includes Slater-type orbital triple-ζ basis sets for all atoms and a single-ζ polarization function for all atoms except Cu.
Results and Analysis Electronic Structure of Cucumber Basic Protein. A. Optical Spectroscopic Parameters. Low-temperature absorption, MCD, and CD spectra between 5000 and 30000 cm-1 for cucumber basic protein are presented in Figure 2. Simultaneous Gaussian resolution of these spectra requires eight bands to adequately fit the spectra and are included in top panel of Figure 2. Three bands (1-3) with approximately equal intensity are required to fit the ∼450 nm absorption transition envelope. A single transition (4) accounts for the intensity under the ∼600 nm band. Two transitions (5-6) and a portion of a third (7) contribute to the ∼750 nm band. The positions and relative intensities of the individual bands are easier to envision by inspection of the MCD and CD spectra (Figure 2). While the position and intensity of band 2 cannot be determined from the MCD spectrum, the presence of this band is required by the intense negative CD feature found at ∼22500 cm-1 for which bands 1 and 3 cannot account. The lowest energy band (8) is required by the MCD spectrum. The transition energies and values, at the absorption band maxima, are summarized in Table 1. Experimental oscillator strengths, fexp, listed in Table 1 have been calculated through the fitted absorption maxima and full widths at half-maxima according to the approximation
fexp ≈ 4.61 × 10-9 max νj1/2
(1)
where the absorption maximum, max, is expressed in M-1 cm-1 and νj1/2, the full width at half-maximum of the absorption band, is in cm-1. All features in the MCD spectrum of cucumber basic protein consist of C-term intensity and have magnetization-saturation curves that can be fit to an isotropic g ≈ 2.1, (27) Johnson, K. H.; Norman, J. G., Jr.; Connolly, J. W. D. In Computational Methods for Large Molecules and Localized States in Solids; Herman, F., McLean, A. D., Nesbet, R. K., Eds.; Plenum: New York, 1973; pp 161-201. (28) Rosch, N. In Electrons in Finite and Infinite Structures; Phariseu, P., Scheire, L., Eds.; Wiley: New York, 1977. (29) Slater, J. C. The Calculation of Molecular Orbitals; John Wiley & Sons: New York, 1979. (30) Connolly, J. W. D. In Modern Theoretical Chemistry; Segal, G. A., Ed.; Plenum: New York, 1977; Vol. 7, pp 105-132. (31) Norman, J. G., Jr. Mol. Phys. 1976, 31, 1191-1198. (32) te Velde, G.; Baerends, E. J. J. Comput. Phys. 1992, 99, 84-98.
Figure 2. Electronic absorption (top), magnetic circular dichroism (middle), and circular dichroism (bottom) spectra of cucumber basic protein. Abs, MCD, and CD spectra were obtained at 4.2 K on 0.05 M phosphate (pD* 7.0)/glycerol-d3 glasses (50:50 v/v). Gaussian resolution of bands in the absorption spectra is based on a simultaneous linear least-squares fit of the Abs, MCD, and CD data. The numbering scheme is chosen to be consistent with the assignments of bands in plastocyanin (see Table 1 for assignments). The position and intensity of band 2 cannot be resolved in the MCD spectrum; however, its presence is required by the CD data.
which is consistent with the Cu(II) ground-state EPR spectrum.19,20 To differentiate ligand field from charge-transfer transitions C0/D0 ratios are particularly valuable since this ratio is sensitive the amount of metal character in the particular excited state (vide infra). For complexes exhibiting only C-term MCD intensity, C0/D0 ratios can be determined from the ∆ and values obtained from the Gaussian fit of the MCD spectrum (Table 1) taken within the linear 1/T region and the absorption spectrum,33 respectively Via
C0 kT ∆ ) D0 µBB max
( )
(2)
where T is the temperature, B is the external magnetic field strength, k is Boltzmann’s constant, µB is the Bohr magneton, is the absorption maximum in M-1 cm-1, and ∆ is MCD intensity maximum measured in M-1 cm-1 (k/µB ≈ 1.489 T K-1). B. SCF-Xr-SW Calculations. The SCF-XR-SW calculated ground-state energies and one-electron wave functions for the (33) Piepho, S. B.; Schatz, P. N. Group Theory in Spectroscopy With Applications to Magnetic Circular Dichroism; John Wiley & Sons: New York, 1983.
9624 J. Am. Chem. Soc., Vol. 120, No. 37, 1998 Table 1.
LaCroix et al.
Experimental Spectroscopic Parameters for Cucumber Basic Protein (with Plastocyanin Parameters15 for Comparison)
band
assignments in PLC11
8 7 6 5 4 3 2 1
dz2 dxy dxz+yz dxz-yz Cys π pseudo-σ His π1 Metd
energy (cm-1) CBP PLC diffa 5800 10800 12900 14100 17100 21000 22500 24750
5000 10800 12800 13950 16700 18700 21390 23440
fexp oscillator strength CBP PLC
(M-1 cm-1) CBP PLC
800 0 100 150 400 2300 1110 1310
300 1640 1480 3410 1320 1610 1290
250 1425 500 5160 600 288 250
0.0030 0.0151 0.0121 0.0381 0.0150 0.0185 0.0148
0.0031 0.0114 0.0043 0.0496 0.0048 0.0035 0.0030
∆ (M-1 cm-1 T-1) at 4.2 K CBP PLC +2.6 -4.5 +15.3 -35.0 -15.2 +3.8 b -2.7
C0/D0
(+)c -8.5 +20.9 -41.4 -10.2 +1.2 -0.5 -0.5
CBP
PLC
(+)c -0.093 +0.058 -0.148 -0.028 +0.018 b -0.013
(+)c -0.213 +0.092 -0.518 -0.012 +0.013 -0.011 -0.013
a Difference in transition energies (defined as cucumber basic protein minus plastocyanin energy). b The position and intensity of band 2 cannot be determined from the MCD data; however, its presence is required by the CD spectrum. c Only signs can be determined from the data for these parameters;35 however, the C0/D0 ratios should be greater than 0.1 based on the relative magnitude of MCD to upper limit in absorption. d This likely involves the Met b1 orbital. See Ref 15 for details.
Table 2.
Results of SCF-XR-SW Calculations for the Highest Occupied Valence Orbitals of the C1(met)(his) Site in CBP % Cu d orbital breakdownc
%Cu
%Cys
%Met
%His
level
orbital label
energy (eV)
Cua
sb
pb
db
dz2
dxz
dyz
dx2-y2
dxy
S
Cysd
S
Metd
Ne
Hisd
48 a 47 a 46 a 45 a 44 a 43 a 42 a 41 a 40 a 39 a 38 a 37 a 36 a 35 a
Cu dx -y Cu dxy Cu dz2 Cu dxz-yz f Cu dxz+yz f Cys π His π1 His π1 Met b1 Cys pseudo-σ His π2 Met a1 Cys σ His π2
-2.75 -3.24 -3.73 -4.00 -4.12 -4.46 -4.68 -4.87 -5.03 -5.47 -6.50 -7.02 -7.26 -7.48
58 58 78 79 82 52 14 13 30 40 8 8 19 7
1 2 0 0 0 0 0 0 3 2 0 0 0 0
2 7 4 2 1 4 0 1 1 5 0 2 3 2
52 46 73 75 78 45 13 10 25 31 6 4 11 3
1.4 0.0 29.4 4.1 6.4 3.7 3.8 2.6 2.1 5.9 0.1 1.2 0.3 0.8
0.4 16.8 17.5 4.7 44.2 16.2 1.9 0.7 14.5 0.1 3.8 1.6 0.4 0.1
0.4 8.7 0.4 65.0 5.2 3.5 0.3 3.5 1.4 6.0 0.5 0.4 7.2 0.1
49.5 0.1 11.1 0.9 11.4 12.4 0.3 0.8 1.9 2.8 0.0 0.4 1.7 0.0
0.2 20.4 14.6 0.3 10.8 9.2 6.8 2.4 5.1 17.2 1.6 0.5 0.7 2.0
29 26 2 5 8 32 1 2 0 49 0 5 41 6
2 2 1 1 1 4 0 0 0 8 0 3 26 4
2 8 10 0 1 0 2 14 45 0 6 47 4 0
0 0 2 0 0 0 0 2 8 0 3 29 2 0
6 2 3 0 3 5 8 8 2 1 44 5 4 44
2 2 2 9 5 7 75 50 15 1 36 3 2 37
2
2
a Total charge on the copper ion. b l quantum breakdown for the copper ion. c Specific d orbital contributions to the total Cu d charge. d Total charge for all atoms of the ligand except the S or N coordinated to Cu. e Total charge for the coordinating N atoms. f These labels are only strictly valid for Cs symmetry; in the lower symmetry used here, variable amounts of mixing between these orbitals can occur.
highest occupied valence orbitals obtained for the C1(met)(his) approximation of the cucumber basic protein active site are presented in Table 2. Reference 15 describes SCF-XR-SW calculations on the C1(met)(his) sites in plastocyanin and nitrite reductase. The properties of the redox active, half-occupied HOMO directly affect the electron-transfer reactivity of the site. The origins of many of the distinctive characteristics of blue sites as well as differences between classic and perturbed blue sites can be observed in the properties of the HOMO (vide infra). Similar to plastocyanin, the cucumber basic protein dx2-y2 based HOMO (level 48 a, Table 2) is highly covalent (52% Cu d character, the same value calculated for PLC15), with the predominant Cu-ligand interaction involving 29% S(Cys) and smaller contributions from the ligating S(Met) (2%) and N(His) (6% total) atoms. Contour diagrams for the cucumber basic protein HOMO, plotted perpendicular and parallel to the S(Met)-Cu-S(Cys) plane are shown in Figure 3. These plots reveal the S(Cys)-Cu and S(Met)-Cu interactions. A predominantly π S(Cys)-Cu interaction is clearly evident in the contour plot of the cucumber basic protein HOMO shown in Figure 3A. Several significant Cu-ligand interactions not present in plastocyanin11 are apparent in the ground-state wave function of cucumber basic protein. Increased pseudo-σ S(Cys)-Cu and S(Met) antibonding interactions in the HOMO can be seen in Figure 3B. Finally, the 1.4% dz2 character that is mixed into the HOMO (Table 2) has been shown by Gewirth et al.34 to be sufficient to generate the rhombic splitting of the EPR spectrum.
C. Ligand Field Transitions and Site Geometry. The low symmetry of the blue Cu site removes all orbital degeneracy. In such a situation,11 the two orthogonal dipole moments required for MCD C-term intensity33 can only be acquired through out of state spin-orbit coupling (SOC). Therefore, the MCD intensities (i.e., C0/D0 ratios) will depend on the magnitude of SOC occurring at the centers involved in the transitions. Since the SOC parameter for Cu is greater than that for S or N (ξ3d(Cu) ≈ 828 cm-1 > ξ3p(S) ≈ 382 cm-1 > ξ2p(N) ≈ 70 cm-1), the Cu-based d f d transitions will exhibit greater C0/D0 ratios than the ligand-based charge-transfer transitions.11 The four CD and MCD spectral features and signs in the low-energy, ligand field region (bands 5-8) for cucumber basic protein (Figure 2) are qualitatively similar to plastocyanin,11 exhibiting similar signs and |C0/D0| ratios of ∼0.1 (Table 1).35 Thus, the specific assignments for bands 5-8 can be made to parallel those in plastocyanin (Table 1). The energies of d f d transitions are very sensitive to the ligand field at the copper site: The d f d transitions in cucumber basic protein are observed to be ∼100 cm-1 higher energy than their counterparts in plastocyanin. This indicates that the cucumber basic protein type 1 center experiences a slightly greater ligand field strength than that of plastocyanin.11 Such an increased ligand field would result from a small tetragonal geometric distortion from pseudo(34) Gewirth, A. A.; Cohen, S. L.; Schugar, H. J.; Solomon, E. I. Inorg. Chem. 1987, 26, 1133-1146. (35) While C0/D0 for band 8 cannot be determined from the data, estimates for the lower limit of ∆ (>1.0 M-1 cm-1 T-1) and the upper limit for (19 000 cm-1 (MCD) or >25 000 cm-1 (CD) which were recorded at 4.2 K in 0.01 M phosphate (pD* 6.0)/glycerol-d3 glasses (50:50 v/v). Gaussian resolution of bands in the absorption spectra is based on a simultaneous linear least-squares fit of the Abs, MCD, and CD data. The numbering scheme is chosen to be consistent with the assignments of bands in plastocyanin (see Table 3 for assignments). A counterpart for band 1 is not observed.
distortion toward a tetragonal geometry that causes a rotation of dx2-y2 such that a there is significant Cu-S(Cys) pseudo-σ and nonzero Cu-S(Met) overlap in the HOMO in addition to a weakened Cu-S(Cys) bond. Electronic Structure of Stellacyanin. A. Optical Spectroscopic Parameters. Low-temperature absorption,40,41 MCD, and CD spectra between 5000 and 30000 cm-1 for stellacyanin are presented in Figure 4. The Gaussian resolution of the absorption spectrum, obtained from a simultaneous fit of the absorption, MCD, and CD spectra is included in the top panel of Figure 4. The transition energies, oscillator strengths, , ∆, and C0/D0 values have been determined as presented above (Table 3). All features in the MCD spectrum of stellacyanin exhibit magnetization-saturation behavior consistent with a paramagnetic Cu(II) ground state, and at the low temperatures employed here they consist entirely of C-term intensity (as determined from their linear 1/T dependence). In contrast to both classic and perturbed proteins containing an axial S(Met) ligand, only seven bands are required to fit the (40) 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. (41) Gewirth, A. A., Ph.D. Dissertation, 1987, Stanford University.
Variations in Perturbed Blue Copper Centers Table 3.
J. Am. Chem. Soc., Vol. 120, No. 37, 1998 9627
Experimental Spectroscopic Parameters for Stellacyanin (with Plastocyanin Parameters15 Included for Comparison)
band
assignments in PLC11
8 7 6 5 4 3 2 1
dz2 dxy dxz+yz dxz-yz Cys π pseudo-σ His π1 Metd
energy (cm-1) Stc PLC diffa 5500 8750 11200 12800 16800 18600 22750 b
5000 10800 12800 13950 16700 18700 21390 23440
fexp oscillator strength Stc PLC
(M-1 cm-1) Stc PLC
500 -2050 -1600 -1150 100 -100 1350
100 580 690 4970 890 1090 b
250 1425 500 5160 600 288 250
0.0009 0.0048 0.0056 0.0439 0.0074 0.0120 b
0.0031 0.0114 0.0043 0.0496 0.0048 0.0035 0.0030
∆ (M-1 cm-1 T-1) at 4.2 K Stc PLC +1.8 -5.7 11.6 -19.1 -28.6 +1.8 -9.4 b
C0/D0
(+)c -8.5 +20.9 -41.4 -10.2 +1.2 -0.5 -0.5
Stc
PLC
(+)c -0.356 0.105 -0.173 -0.036 0.013 -0.054
(+)c -0.213 +0.092 -0.518 -0.012 +0.013 -0.011 -0.013
a Difference in transition energies (defined as stellacyanin minus plastocyanin energy). b This band is not observed in the stellacyanin data. Only signs can be determined from the data for these parameters;35 however, the C0/D0 ratios should be greater than 0.1 based on the relative magnitude of MCD to upper limit in absorption. d This likely involves the Met b1 orbital. See Ref 15 for details.
c
Table 4.
Results of SCF-XR-SW Calculations for the Highest Occupied Valence Orbitals of the C1(gln)(his) Site in Stellacyanin % Cu d orbital breakdownc
%Cu a
level
orbital label
energy (eV)
Cu
sb
50 a 49 a 48 a 47 a 46 a 45 a 44 a 43 a 42 a 41 a 40 a 39 a 38 a 37 a
Cu dx2-y2 Cu dxy Cu dz2 Cu dxz-yz f Cu dxz+yz f His π1 Cys π His π1 Cys pseudo-σ Gln Opy His π2 His π2 Gln Opx Cys σ
-2.99 -3.46 -3.94 -4.08 -4.26 -4.33 -4.63 -4.79 -5.70 -6.18 -6.42 -6.70 -6.75 -7.49
57 69 85 77 64 27 43 27 37 8 7 9 8 20
0 4 0 0 0 0 0 0 1 0 0 0 0 1
%Cys
% Gln
%His
pb
db
d z2
dxz
dyz
dx2-y2
dxy
S
Cysd
O
Glnd
Ne
Hisd
2 6 2 1 1 2 3 0 5 0 0 0 2 6
53 57 81 74 61 22 37 25 28 6 5 7 4 11
1.2 16.7 56 0.2 0.3 1.5 2.7 1.5 4.7 5.4 0.2 0.5 2.3 0.4
0.6 2.3 6.6 0.3 53.2 15.9 12.4 0.1 0.2 0.0 0.9 0.0 0.4 2.3
0.4 0.1 0.2 72.1 0.2 0.2 0.1 16.1 0.1 0.5 0.6 5.5 1.1 1.8
49.4 3.4 0.1 0.5 0.2 4.3 20.7 6.9 0.7 0.1 0.7 0.1 0.1 0.0
1.4 34.5 18.1 0.8 7.1 0.1 1.1 0.4 22.2 0.0 2.5 0.9 0.1 6.5
30 19 1 5 5 16 25 6 52 0 1 0 0 49
2 1 0. 1 1 1 2 0 7 0 0 0 0 29
0 2 4 1 0 0 0 1 1 54 2 16 39 0
0 1 1 0 0 0 0 0 0 22 0 15 35 0
7 4 5 6 6 10 5 7 2 6 47 32 9 2
2 4 3 10 23 45 25 58 1 8 42 29 6 0
a Total charge on the copper ion. b l quantum breakdown for the copper ion. c Specific d orbital contributions to the total Cu d charge. d Total charge for all atoms of the ligand except the S, O, or N coordinated to Cu. e Total charge for the coordinating N atoms. f These labels are only strictly valid for Cs symmetry; in the lower symmetry used here, variable amounts of mixing between these orbitals can occur.
spectra of stellacyanin. A single band (2) is required to fit the ∼450 nm absorption intensity, which is labeled band 2 (rather than 1, vide infra) due to its negative CD and MCD sign. Two transitions (3-4) contribute to the ∼600 nm absorption transition envelope. Two transitions of roughly equal intensity (56) and a third weaker band (7) at lower energy contribute to the ∼800 nm absorption envelope. A final low energy band (8) is required by the MCD spectrum. No spectral features can be attributed to a transition corresponding to band 1 in plastocyanin, which would exhibit a negative MCD and positive CD sign in the high-energy region of the spectrum. B. SCF-Xr-SW Calculations. The SCF-XR-SW calculated ground-state energies and wave functions for the highest occupied valence orbitals obtained with the C1(gln)(his) approximation of the stellacyanin active site are presented in Table 4. The SCF-XR-SW calculated half-occupied dx2-y2 based HOMO (level 50 a, Table 4) in stellacyanin is highly covalent (53% Cu d character), with the predominant Cu-ligand interaction involving 30% S(Cys), and minor contributions from the N(His) atoms (∼7% total). Contours for the half-filled HOMO of stellacyanin are plotted perpendicular and parallel to the O(Gln)-Cu-S(Cys) plane (Figure 5). In stellacyanins the dominant π S(Cys)-Cu antibonding interaction that is found for the HOMO of the classic blue copper plastocyanin11,15 is clearly evident perpendicular to this plane in Figure 5A. Only minor contributions from S(Cys)-Cu and O(Gln)-Cu σ antibonding interactions can be seen parallel to this plane in Figure 5B. The stellacyanin HOMO (level 50 a) contains no contribution from the axial ligand (i.e., glutamine-based orbitals).
Figure 5. Contours of the highest energy, half-occupied orbital (level 50 a in Table 4) for stellacyanin plotted (A) perpendicular and (B) parallel to the S(Cys)-Cu-O(Gln) plane. Contour lines are drawn at (0.64, (0.32, (0.16, (0.08, (0.04, (0.02, and (0.01 (e/bohr3)1/2.
9628 J. Am. Chem. Soc., Vol. 120, No. 37, 1998
LaCroix et al.
The two glutamine-based levels (41 a and 38 a in Table 4), which replace the methionine Met a1 and b1 levels, are labeled to indicate the specific O(Gln) p orbital which contributes to the wave function. As the Gln makes no contribution to the half-occupied HOMO, net Cu-Gln bonding is reflected in the amount of Cu 4s and 4p character mixed into the ligand based orbitals. The principal Gln-Cu bonding interaction is found to involve Gln Opx (level 38 a) which contains 2-3% Cu 4p (Table 4) (Gln Opy, level 41 a, contains
