3278
J. Phys. Chem. 1996, 100, 3278-3287
Charge-Transfer Dynamics in Plastocyanin, a Blue Copper Protein, from Resonance Raman Intensities Ester Fraga, M. Adam Webb, and Glen R. Loppnow* Department of Chemistry, UniVersity of Alberta, Edmonton, Alberta, Canada T6G 2G2 ReceiVed: August 29, 1995; In Final Form: NoVember 8, 1995X
The resonance Raman intensities for parsley plastocyanin, a blue copper protein involved in electron transport in plant photosynthesis, have been measured at wavelengths throughout the S(Cys) f Cu charge-transfer absorption band centered at 597 nm in an effort to determine the structural and dynamic role of inner- and outer-sphere reorganization in the kinetics of charge transfer. Self-consistent analysis of the absorption band and the resulting resonance Raman excitation profiles demonstrates that the charge-transfer absorption band is primarily homogeneously broadened. The homogeneous line width is composed of population decay and solvent-induced dephasing. The excited-state lifetime of 20 ( 15 fs calculated here from the observed fluorescence suggests that the charge-transfer state decays rapidly via lower-lying ligand-field states. The spectral line shape dictates that this population decay be modeled as a Gaussian of line width 230 cm-1. The reorganization energy obtained from the resonance Raman intensities of specific vibrations is 0.19 eV. If the reorganization energy of the protein as measured from the solvent-induced dephasing component of the homogeneous line width is included, the observed reorganization energy is 0.25 eV, in quantitative agreement with a previous upper limit of 0.3 eV measured for the reorganization energy upon electron transport at the copper site in azurin, a similar blue copper protein. A crude comparison of the reorganization energies upon electron transport and charge transfer suggests that charge transfer may be a somewhat useful model for the geometry changes upon electron transfer. The resonance Raman spectrum indicates that reorganization occurs primarily along normal modes that involve the Cu-S(Cys) stretch, but significant reorganization also occurs along specific normal modes that involve internal cysteine stretches, Cu-N(His) stretches, and protein internal motions. An important result of this work is the two mechanisms by which the protein environment contributes to the reorganization energy: through coupling into specific resonance-enhanced normal modes and through a solvent-induced dephasing contribution as evidenced by the homogeneous line width. These results are compared to those of other methods for determining reorganization energies and are discussed in terms of the role of the environment in controlling electron- and charge-transfer processes.
Introduction The rate constant of electron transfer can be related to a number of factors by the Marcus equation:1 kET ∝ exp{(-∆G° + λ)/4λRT}, where ∆G° is the free-energy change upon electron transfer and λ is the reorganizational energy. The reorganization energy is usually written as the sum of the inner- and outer-sphere contributions (λ ) λi + λo), where λi is loosely defined as the structural reorganization of the donor and acceptor (nuclear) conformation and λo is defined as the structural reorganization of the solvent (electronic and nuclear) conformation to the new electronic distribution. Although measurements of the total reorganization energies in a number of systems have been made, the contributions of individual molecular motions have been difficult to determine, producing an inadequate molecular picture of the role of structure and dynamics in electron and charge transfer. Resonance Raman intensities provide detailed information about excited-state structure and dynamics on an extremely fast time scale.2 By tuning the exciting laser into an absorption band, resonant enhancement of those normal modes coupled to the electronic excitation occurs. The resonance Raman intensities directly reflect the conformational distortion of the molecule along each normal mode upon excitation to an electronic excited state. Measurement of the resonance Raman intensity of each * To whom correspondence should be addressed. E-Mail:
[email protected]. X Abstract published in AdVance ACS Abstracts, January 15, 1996.
0022-3654/96/20100-3278$12.00/0
vibration as a function of excitation wavelength within an absorption band yields a set of resonance Raman excitation profiles. Self-consistent analysis of the resonance Raman excitation profiles and the absorption spectrum can yield such molecular parameters as the distortion along each normal mode upon photoexcitation, the excited-state lifetime, and transition moment.2 Recently, a number of studies have shown the ability of resonance Raman intensities to partition the reorganization energy among the normal coordinates of the donor, acceptor, and solvent for a charge-transfer system in solution by exciting at various wavelengths within a charge-transfer transition.3-11 In most of these studies, the reorganization energies are inferred from the absorption bandwidth and the relative Raman intensities obtained with a single pre- or postresonance excitation. In this model, it is assumed that the absorption bandwidth arises solely from Franck-Condon progressions in resonance Raman-active modes coupled to a single electronic transition. This assumption neglects broadening of the absorption band due to multiple electronic transitions and significant homogeneous and/or inhomogeneous broadening mechanisms. Use of this simple model cannot distinguish these effects on the calculated displacements and may lead to serious errors in quantitating the role of molecular reorganization energy contributions to electron- and charge-transfer kinetics. In the two most detailed of these studies,9-11 self-consistent analysis of the absolute resonance Raman cross sections and absorption spectrum suggested that the primary contributions to the total reorganization energy in the hexamethylbenzene/tetracyanoethylene © 1996 American Chemical Society
Resonance Raman Intensities of Plastocyanin SCHEME 1
charge-transfer complex were due to the solvent. For several reasons, however, the reorganization energy due to the solvent was not well-defined. Plastocyanin is a 10 500-D copper protein that acts as an electron-transport agent between the cytochrome bf complex and photosystem I in plant photosynthesis.12 The structure of poplar plastocyanin shows a single copper ion ligated to two histidine residues (His37 and His87), a cysteine (Cys84), and a methionine (Met92) in an irregular tetrahedral coordination geometry (Scheme 1).13 The copper ion is buried among hydrophobic amino acid residues in one end of the protein and does not show any solvent accessibility; both X-ray crystallography and NMR line shapes show no evidence of either water accessibility to or structured water at the copper binding site.13,14 The structure of the copper site appears to be imposed by the protein and is highly conserved between different plastocyanins.12,15 In fact, comparison of the resonance Raman spectra of 11 “cupredoxins” (blue copper proteins) has suggested that the structure of the copper site is highly conserved among all blue copper proteins.15 The electronic structure of the copper site in plastocyanin has been much more difficult to define. Plastocyanin exhibits three absorption bands in the visible spectrum centered at ca. 464, 597, and 790 nm. Substitution of the copper(II) ion with cobalt(II) led to assignment of the intense 597-nm absorption band as a S(Cys-σ) f Cu charge-transfer transition.16-18 More recent low-temperature absorption, CD, and MCD spectroscopy assigned this band as the S(Cys-π) f Cu charge-transfer and suggested the presence of at least seven transitions between 400 and 1000 nm.19,20 These transitions involve weak ligand-field, S(Met) f Cu charge-transfer, N(His) f Cu charge-transfer, and S(Cys-σ) f Cu charge-transfer transitions.19 Most recently, Solomon and Lowery have used EPR, X-ray absorption, and self-consistent-field XR scattered-wave bonding calculations to show that the large oscillator strength of the S(Cys-π) f Cu charge-transfer transition arises from the high covalency of the Cu-S(Cys) bond.21 The authors have argued that the highly covalent nature of the Cu-S(Cys) bond provides a possible superexchange pathway for electron transfer from the Cu, through the Cys84 residue, to the adjacent Tyr83 residue and subsequently to photosystem I.21 In this paper, we examine the partitioning of the reorganization energy among the donor, acceptor, and solvent in the blue copper protein plastocyanin. Because of the lack of solvent
J. Phys. Chem., Vol. 100, No. 8, 1996 3279 accessibility at the copper site in plastocyanin, the protein is the solvent. The primary goal of this work was to determine the influence of the solvent on charge-transfer kinetics in a more confident manner by probing a model system in which the charge-transfer complex has a well-defined structure and sits in a relatively isolated, well-characterized environment. Also, by choosing a system that is naturally involved in electron transfer, as well as exhibiting charge-transfer absorption, it was hoped that probing the charge-transfer absorption would help elucidate the mechanism of electron transport at the copper site. In this paper, we have used absorption, fluorescence, and resonance Raman spectroscopy to measure the excited-state photophysics in parsley plastocyanin and have calculated the mode-specific reorganization energies upon charge-transfer along each of the observed normal modes. The reorganization energies obtained here for charge-transfer in plastocyanin are compared to those measured in other systems from resonance Raman intensities. An important result of this paper is that the environmental contribution to the reorganization energy due to the protein arises from both specific motions of the protein and a solvent-induced dephasing contribution. Comparison of these results to an experimental determination of the total reorganization energy upon electron transport in a similar blue copper protein shows that, while the total reorganization energy is similar, the molecular changes in charge-transfer and electron transport are slightly different, reflecting the different nature of the orbitals involved in the two processes. Experimental Section Plastocyanin was isolated from parsley leaves according to literature procedures with slight modifications.22,23 Briefly, 5 kg of commercially-obtained parsley leaves, in 500-g batches, were homogenized with 450 g of crushed ice, 50 mL of 1 M TRIS-HCl (pH 7.6), and 500 mL of cold acetone. The homogenate was squeezed through four layers of cheesecloth and centrifuged. The supernatant was mixed with 1.16 vol of cold acetone to precipitate the proteins. The sediment was collected by centrifugation, mixed with the smallest possible volume (∼200 mL) of 0.06 M TRIS-HCl (pH 7.6), and dialyzed against 0.06 M TRIS-HCl (pH 7.6). After centrifugation, the supernatant was purified on DEAE-cellulose and Sephadex G-50 columns. A second DEAE-cellulose column was used to further purify the plastocyanin to an A278/A597 ratio of 1.6. A yield of 16-28 mg of plastocyanin/kg of parsley leaves was obtained with this method. Plastocyanin samples for the resonance Raman and fluorescence experiments were prepared by quantitative dilution of plastocyanin (50 mM TRIS-HCl, pH 7.6) with a cacodylate/ TRIS buffer solution (0.5 M cacodylic acid, 50 mM TRISHCl, pH 7.6). The addition of cacodylic acid/cacodylate buffer did not have a noticeable effect on the absorption or resonance Raman spectra of plastocyanin. Room-temperature resonance Raman spectra of plastocyanin were obtained with 450-750µL sample solutions having an absorbance of 4.6-6.0 OD/cm at 597 nm ( ) 4500 M-1 cm-1).12 Resonance Raman scattering was excited by spherically-focusing the laser onto a spinning 5-mm NMR tube containing the sample solution in a 135° backscattering geometry. Laser excitation was obtained with Kr and Ar ion lasers (Coherent, Santa Clara, CA) and HeNe lasers (PMS Electro-Optics, Boulder, CO). The wavelengths used were 514.5, 530.9, 568.2, 594, 612, and 647.1 nm. The laser power was 80-120 mW (Kr and Ar ion lasers) or 7 mW (HeNe lasers). Multichannel detection of the resonance Raman scattering was obtained with a liquid nitrogen-cooled CCD detector (Princeton Instruments, Trenton, NJ) connected to the
3280 J. Phys. Chem., Vol. 100, No. 8, 1996
Fraga et al.
first half of a double monochromator (Spex Industries, Metuchen, NJ). Spectral slit widths were 5-7 cm-1. Frequency calibration was done by measuring Raman scattering of solvents of known frequencies (benzene, chloroform, and carbon tetrachloride). Reported frequencies are accurate to (2 cm-1. Single-channel detection was obtained by using a photon-counting system composed of a double monochromator (Spex) and a cooled photomultiplier tube. Absorption spectra were measured by using a diode array spectrophotometer (Hewlett-Packard, Sunnyvale, CA). The resonance Raman spectra were analyzed by using a 486DX2-66V computer (Gateway Computers, North Sioux City, SD). A buffer solution (50 mM TRIS-HCl, pH 7.6) background was subtracted from all spectra. The spectra were corrected for the wavelength dependence of the spectrometer efficiency by dividing the resonance Raman spectra by the spectrum of a standard lamp (Electro-Optics Associates, Palo Alto, CA) and multiplying the resulting spectrum by the standard lamp spectral output. Spectra were smoothed using a 5-point Savitsky-Golay function. The baselines were leveled by subtracting multiple joined line segments from the spectrum. Intensities of plastocyanin relative to the (605 + 638)-cm-1 lines of cacodylate were measured by integration of peak areas. Overlapping peaks were separated by fitting regions of the spectra to sums of Lorentzian/Gaussian peaks. To minimize errors in the fitting of overlapped peaks, the frequencies and bandwidths of the peaks were fixed to the same values for all spectra; only the heights of the peaks were allowed to vary. Bleaching of the sample was corrected by measuring the absorbance at 700 nm ( ) 1295 M-1 cm-1) before and after each scan, and the average absorbance was used to determine the plastocyanin concentration. This approximation assumes that the bulk photoalteration parameter24 is small. The observed bleaching in an 80-min scan was kT, it is assumed that only the V ) 0 vibrational level of the ground electronic state is significantly populated and that no molecules are in a
vibrationally excited initial state. The excited-state potential energy surface along each normal coordinate is modeled as a bound harmonic potential with a force constant identical to that of the ground state but displaced along the normal coordinate and in energy. This model is expected to be incorrect for long propagation times, due to the anharmonicity of the excited state potential surface and Duschinsky rotation2 in the excited state. However, it will be shown later that the resonance Raman process occurs on a very short time scale; the 〈i|i(t)〉 and 〈f|i(t)〉 overlaps decay rapidly and the wavepacket samples only a small portion of the excited-state potential surface near the Franck-Condon geometry. This approximation has been shown previously to be valid in the interpretation of the resonance Raman intensities of polyenes and other chargetransfer systems.2,31 Self-consistent analysis of the absorption spectrum and resonance Raman excitation profiles with eqs 6 and 7 permits the partitioning of the spectral breadth into inhomogeneous and homogeneous components. These two factors affect the observed absorption spectrum and resonance Raman excitation profile differently.2,25 The homogeneous (single-molecule) absorption spectrum arises from the Franck-Condon vibronic transitions in each normal mode, each of which is broadened by the homogeneous line width. As the homogeneous line width increases, the absorption spectrum and resonance Raman excitation profiles broaden and become spectrally diffuse. In addition, the magnitude of the resonance Raman excitation profiles decreases. Therefore, the overall homogeneous line width can be precisely determined from the resonance Raman excitation profiles. In plastocyanin, the absorption and resonance Raman excitation profile line shapes dictate that the homogeneous line width function G(t) be a Gaussian of the form e-Γ2t2/p2, where Γ is the homogeneous line width in cm-1. The inhomogeneous component arises from ensemble “site” effects and is usually modeled as a Gaussian distribution of purely electronic excitation energies with a standard deviation Θ. Inhomogeneous distributions of molecules in different sites affect the resonance Raman excitation profile and absorption spectrum similarly; as the inhomogeneous distribution gets larger, the resonance Raman excitation profile and absorption spectrum become more spectrally diffuse, but the integrated intensity of each remains constant. For the analysis, the initial guesses for the displacements along each normal coordinate (∆) were found from the relative resonance Raman vibrational intensities at 568.2 nm assuming the intensities were proportional to ∆2 and with I376 scaled arbitrarily to 1.0. All eight observed fundamental vibrational modes were used in the time-dependent calculations. Other parameters were selected to give the best calculated absorption spectrum and resonance Raman excitation profiles. The relative ∆’s were scaled to give the experimentally observed absorption and resonance Raman excitation profile bandwidths. The homogeneous line width (Γ) was determined primarily by the absolute magnitudes of the resonance Raman excitation profiles. The inhomogeneous line width (Θ) was determined by the absorption and resonance Raman excitation profile bandshapes. The transition moment (M) and the energy gap between potential surfaces (E0) were determined by the magnitude and position of the absorption spectrum. The parameters were optimized iteratively until the calculated and experimental absorption spectrum and resonance Raman excitation profiles are in agreement. Results The resonance Raman spectra of plastocyanin are shown in Figure 2, and the cross sections are summarized in Table 1.
3282 J. Phys. Chem., Vol. 100, No. 8, 1996
Figure 2. Resonance Raman spectra of plastocyanin at excitation wavelengths throughout the 597-nm absorption band. Typical concentrations were 1 mM plastocyanin and 375 mM cacodylate. The spectra are the sum of three to nine scans and have been divided by a tungstenhalogen lamp spectrum (Eppley Laboratory, Inc.). The intensity standard (cacodylate) appears at 606 and 638 cm-1. The broad peak at 828 cm-1 is composed of the overtones and combinations of the plastocyanin vibrations between 350 and 500 cm-1 and a cacodylate peak at ca. 825 cm-1. All of the indicated vibrations between 250 and 500 cm-1 and the vibration at 759 cm-1 were used in the analysis.
The cacodylate vibrations, used as the intensity standard, are visible as a pair of bands at 606 and 638 cm-1. Approximately five intense bands are observed between 350 and 500 cm-1 and have been previously assigned to normal modes involving the Cu-S stretch mixed with other internal coordinates,15,32-34 primarily those of the cysteinate ligand, although the exact assignments are still controversial32 (Vide infra). The moderately intense bands at 266 and 759 cm-1 have previously been assigned to the symmetric Cu-N stretch from the histidine ligand(s) and the C-S stretch of the cysteinate ligand, respectively.32 Finally, the broad band centered at ca. 830 cm-1 arises from combination and overtone transitions of the vibrations between 350 and 500 cm-1. In our samples, the band at 830 cm-1 also contains a contribution from the 825-cm-1 As)O stretch of the cacodylate internal standard.35,36 Because of the imprecision in the intensity of this band, it was not used in the resonance Raman intensity analysis. No vibrations arising from the plastocyanin are observed at frequencies greater than 1000 cm-1. Also, no reproducible features attributable to resonance Raman scattering from plastocyanin are observed at frequencies below 250 cm-1. The poor stray light rejection of the single monochromator used to obtain these resonance Raman spectra precludes the separation of residual Rayleigh scattering from low-frequency resonance Raman scattering. Thus, no quantitation of the low-frequency contribution to the solvent reorganization energy was possible. The intensities of the plastocyanin vibrations relative to the intensities of the cacodylate vibrations vary as a function of the excitation wavelength, as expected from the resonance enhancement effect.2 Also, the relative intensities of the plastocyanin vibrations with respect to each other remain constant as the excitation wavelength is scanned through the absorption spectrum. These two facts provide strong evidence that the observed modes are coupled to the same, single electronic transition, namely the S(Cys)-π f Cu charge-transfer transition. The experimental and calculated resonance Raman cross
Fraga et al. sections are summarized in Table 1. Figures 3 and 4 illustrate the good agreement between the experimental and calculated resonance Raman excitation profiles and absorption spectrum of plastocyanin using the parameters summarized in Table 1. Similarities in the relative intensities at different excitation wavelengths indicate that the enhancement profiles of these modes should be similar, and this is borne out in the calculations. Attempts at modeling the absorption spectrum and resonance Raman excitation profile line shapes by a Lorentzian line shape were unsuccessful because they predicted large tails to high and low energies, inconsistent with the observed absorption spectrum and the resonance Raman excitation profiles. Thus, we were forced to use a Gaussian homogeneous line shape. Deviations of the experimental absorption spectrum from the calculated absorption spectrum at both higher and lower energies are due to charge-transfer and ligand-field transitions which were not modeled (Vide infra). Although the calculated resonance Raman excitation profiles are in fairly good agreement with experiment, the experimental excitation profiles are somewhat narrower than the absorption spectrum, suggesting that more than one allowed electronic transition is present under the 597-nm absorption band. The lack of resonance enhancement of specific modes by these other transitions, however, indicates that they have low oscillator strength and/or minimal excited-state distortions. In order to completely characterize the excited-state photophysics of plastocyanin, measurements of the fluorescence spectrum and fluorescence quantum yield were performed. Figure 5 shows the experimental fluorescence spectrum of plastocyanin excited at 568 nm. In Figure 5, curve A is the absorption spectrum and curve B is the experimentallydetermined fluorescence spectrum, corrected for detector sensitivity. Although we were unable to measure the fluorescence at energies smaller than ∼14 000 cm-1 due to a rapidly decreasing spectrometer sensitivity function, it is clear that very little fluorescence is observed; the sharp peaks superimposed on the fluorescence spectrum are the resonance Raman modes of plastocyanin (modes at energies > 16 400 cm-1) and the Raman modes of the TRIS buffer (modes at energies < 16 400 cm-1). Because we were unable to measure the complete fluorescence spectrum, we fit the observed spectrum to a Gaussian band (curve C, Figure 5) which had a frequency maximum at 14 050 cm-1 and a standard deviation of 900 cm-1. The integrated intensity of the fluorescence emission from this Gaussian band is approximately 98 times larger than the intensity of the (605 + 638) cm-1 of cacodylate. The integrated fluorescence cross section of 3.3 × 10-8 Å2/molecule is calculated using eq 4. This is divided by the absorption cross section at 568 nm (0.139 Å2/molecule) to give a total emission quantum yield of 2.3 × 10-7. The excited-state lifetime can be calculated from the radiative rate constant and the fluorescence quantum yield. The radiative rate constant was estimated to be 1.15 × 107 s-1 from eq 5, the absorption maximum at 16 750 cm-1, and the transition length of 0.581 Å (Table 1). Finally, these values of the fluorescence quantum yield and the radiative rate constant gave an excited-state lifetime due to population decay of 20 fs. It is clear that plastocyanin has a very short excited-state lifetime, although the exact value calculated here is a very crude estimate. Discussion Electronic Structure. Besides providing a structural probe of the excited-state dynamics, the resonance Raman excitation profiles provide a sensitive measure of the electronic structure of plastocyanin. As described above, the excitation profiles are narrower than the absorption band, suggesting that the absorption
Resonance Raman Intensities of Plastocyanin
J. Phys. Chem., Vol. 100, No. 8, 1996 3283
TABLE 1: Absolute Resonance Raman Cross Sections of Plastocyanina excitation wavelength, nm δν, cm-1
|∆|
514.5
530.9
568.2
594.0
612.0
647.1
266 376 390 403 421 436 473 759
0.98 1.35 1.20 0.49 1.44 0.98 0.24 0.35
0.26/0.23 1.50/0.95 1.02/0.81 0.17/0.15 2.18/1.40 0.98/0.70 0.04/0.05 0.27/0.34
1.07/0.53 2.76/2.13 2.38/1.82 0.33/0.33 4.10/3.11 2.22/1.56 0.11/0.11 1.25/0.71
2.03/1.78 6.00/6.85 5.49/5.83 0.82/1.04 8.67/9.81 4.21/4.88 0.17/0.35 1.73/1.89
1.95/2.20 8.84/8.16 7.18/6.91 1.26/1.23 11.90/11.50 5.74/5.69 0.44/0.40 1.73/1.94
1.05/1.90 6.97/6.82 5.97/5.75 0.94/1.02 9.85/9.49 4.00/4.67 0.22/0.32 1.58/1.46
0.95/0.76 2.03/2.58 1.83/2.16 0.37/0.38 3.02/3.52 1.41/1.72 0.14/0.12 1.23/0.47
a The cross sections are shown as experimental/calculated in units of Å2/molecule × 1010. Uncertainties in cross sections relative to cacodylic acid are 10% for strong lines and 20% for weak lines. ∆’s are in units of dimensionless normal coordinates. The calculations used E0 ) 15350 cm-1, transition length M ) 0.581 Å, temperature T ) 0 K, Gaussian homogeneous line width ) 385 cm-1, and no inhomogeneous broadening. The estimated errors in the parameters used in the calculation are as follows: zero-zero energy (E0) ) (1%, transition length (M) ) (1%, homogeneous line width ) (5%, displacements ) (5%.
Figure 3. Experimental (points) and calculated (solid line) resonance Raman excitation profiles of plastocyanin. The excitation profiles were calculated with eq 6 by using the parameters of Table 1. Error bars represent the uncertainties in the absolute resonance Raman cross sections. Note the difference in y-axis scales between the left and right panels.
Figure 4. Calculated (dashed line) and experimental (solid line) absorption spectrum of plastocyanin. The dashed line is generated from eq 7 by using the parameters of Table 1. Deviations of the calculated from the experimental absorption spectrum at higher and lower energies arise from other electronic transitions that were not modeled and that contribute no resonance enhancement to the observed resonance Raman spectra.
band at 597 nm is composed of more than one electronic transition. Previous studies have used EPR data, CD, MCD, and polarized single-crystal absorption spectroscopies to assign the electronic transitions within the 597-nm absorption band of plastocyanin to two S(Cys) f Cu charge-transfer transitions as well as a ligand-field transition.19-21 More specifically, absorption at energies 10 ps, whereas plastocyanin’s is ∼20 ( 15 fs, based on the fluorescence quantum yield measured here. This difference in excited-state lifetimes can have dramatic effects on the spectroscopic observables, primarily on the partitioning of the homogeneous line width between population decay and solvent reorganization energy effects. In HMB/TCNE, the population decay was ignored, whereas in plastocyanin, population decay accounts for almost 50% of the phenomenological Gaussian homogeneous line width. However, even if the total phenomenological Gaussian homogeneous line width in plastocyanin is attributed to solvent reorganization and the effects of population decay are neglected, a solvent reorganizational energy of 790 cm-1 (D ) 550 cm-1, Λ ) 28 cm-1) is obtained, still much less than the 2450 cm-1 reported for HMB/TCNE and in better agreement with previous measurements.42-44 The nature of the solvent is fundamentally different in the two studies as well; HMB/TCNE experiences random, fluctuating interactions with solvent which may significantly change the donor-acceptor bond length, geometry, and energetics, whereas the copper ion is covalently bound to the surrounding protein with a relatively well-characterized geometry and isolated from bulk solvent. A reasonable question to ask is whether the solvent should be treated qualitatively differently in the two cases. In plastocyanin, the protein seems to contribute to the reorganizational energy via two distinct mechanisms, directed motion along specific coordinates and nonspecific interactions similar to bulk solvent. Interpretation of the solvent’s effect on the reorganizational energy of HMB/ TCNE was examined by comparing the molecular parameters obtained from a resonance Raman intensity analysis9-11 in CH2Cl2 and CCl4. Unfortunately, the comparison seemed to indicate drastically different structures of the complex in the two solvents,11 complicating the determination of specific solvent effects on the excited-state dynamics but indicating that the solvent plays a significant role in at least the ground-state structure of the complex. A goal of this work was to see if the molecular geometry changes upon optical charge-transfer obtained from the resonance Raman intensities accurately reflect the molecular geometry changes upon electron transport during the natural photosynthetic process in plastocyanin. For this purpose, it is instructive to compare the molecular geometry changes deduced from the resonance Raman intensities with the observed changes in structure of the reduced and oxidized forms of plastocyanin from X-ray crystal structures. The X-ray structures13,50 show that the Cu-N(His87) bond increases 0.44 Å, the Cu-N(His37) bond increases 0.14 Å, the Cu-S(Cys84) bond increases 0.04 Å, and the Cu-S(Met92) bond increases 0.09 Å upon reduction. For a stretching vibration, the dimensionless displacements (∆) obtained in this work are related to a bond length change δR
Fraga et al. by the relation δR ) (p/µω)1/2∆, where µ is the reduced mass and ω the vibrational frequency. Assuming the 421-cm-1 mode is a localized Cu-S stretch, a fair assumption since this mode shifts the most upon 34S substitution in poplar plastocyanin,46 a δR of 0.09 Å upon charge-transfer is obtained. Thus, the calculated value of 0.09 Å from the resonance Raman intensities is the same order of magnitude as the X-ray results. A further comparison can be made by assuming all the intensity between 250 and 500 cm-1 arises from a pure Cu-N stretch. Using the combined ∆ of 6.68 for all modes between 250 and 500 cm-1 and an average frequency of 400 cm-1 yields a bond length change of 0.57 Å, again of the same order of magnitude as the total bond length changes, at the copper site of 0.71 Å. Clearly, these are crude comparisons of bond length changes, and a quantitative comparison of the charge-transfer and electrontransfer reorganization energies will require a more detailed normal coordinate analysis of plastocyanin. The normal coordinate analysis is needed to determine the coefficients in the linear combinations of internal coordinates (bond stretch, angle bend, angle torsion, etc.) that make up the observed normal modes. For example, the bond stretch coordinate for an isolated bond is usually distributed among several normal modes of vibration of the molecule, sometimes with different phases in different normal modes. The total bond length change then is calculated from the ∆ along that normal mode weighted by the contribution that the bond stretch coordinate makes to the normal mode. Also, the resonance Raman intensities are proportional to ∆2, not ∆, making it difficult to determine the direction of the geometry change along the normal mode. Although these are crude comparisons, the similarities in the δR calculated from the resonance Raman intensities and the experimental δR from the X-ray crystal structures provide encouragement that the resonance Raman intensities of the charge-transfer transition may provide a useful model of the reorganization energies upon electron transport. Conclusions There are two main results of this work. The first is that the charge-transfer excited state of plastocyanin has a very low fluorescence quantum yield, indicating the state is very short lived (20 ( 15 fs), probably due to nonradiative internal conversion to lower-lying charge-transfer and ligand-field states. Second, the reorganization upon charge-transfer occurs primarily along the Cu-S(Cys) stretch with smaller but significant changes along the Cu-N(His) and protein modes. The protein component is composed of reorganization along specific motions coupled to the resonance-enhanced Raman vibrations and a bulk solvent-like contribution as determined from the phenomenological Gaussian homogeneous line width. The protein contribution is significant and indicates that the structure of the protein is important to the charge-transfer and to its function. Acknowledgment. We thank J. Lee and S. Mwaniki for help in the purification of plastocyanin and in the initial resonance Raman experiments, and Prof. M. Palcic and Prof. H. B. Dunford for equipment support. We also thank Dr. L. Kirkpatrick, Prof. A. English, and Prof. H. B. Gray for helpful discussions and Prof. J. E. Bertie for reading the manuscript. Financial support was provided by the Department of Chemistry at the University of Alberta, NSERC, and the Alberta Heritage Fund for Medical Research. References and Notes (1) Marcus, R. A.; Sutin, N. Biochim. Biophys. Acta 1985, 811, 265.
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