6934
J. Phys. Chem. 1993,97,6934-6940
Nonphotochemical Hole Burning of the Reaction Center of Rhodopseudomonas viridis N. Raja S. Reddy, Stephen V. Kolaczkowski, and Gerald J. Small' Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 5001 I Received: January 28, 1993; In Final Form: April 2, 1993
Reddy et al. (Science, accepted) have reported persistent, nonphotochemical hole-burned (NPHB) spectra for the Qystates of the reaction center of Rhodopseudomonas viridis. The photoinduced structural transformation was shown to be highly localized on the special pair. This transformation leads to a red shift of the special pair's lowest-energy absorption band, P960, of 150 cm-l and a comparable blue shift for a state at 850 nm, which, as a consequence, could be assigned as being most closely associated with the upper dimer component. Additional experimental results are presented here together with a theoretical analysis of the extent to which the NPHB spectra provide information on the contribution from the bacteriochlorophyll monomers of the special pair to the Qy states that absorb higher in energy than €960. Structured photochemical hole-burned (PHB) spectra of P960 are also presented that underscore the importance of strong electron-phonon coupling from a broad distribution of modes with a mean frequency of 30 cm-l for an understanding of the P960 absorption profile. These spectra also identify the zero-phonon hole of the strongly damped special pair marker mode (145 cm-l) and its associated phonon sideband structure. Calculated spectra are presented which are in good agreement with the experimental PHB spectra.
I. Introduction In the preceding paper,' referred to hereafter as paper 1, the results of a detailed study of the dependenceof the photochemical hole-burned (PHB) spectra of isolated reaction centers (RC) of Rhodobacter sphaeroides on burn frequency are reported. Attention was focused on P870, which is the lower-energy Q, absorption componentof the special pair (P) of bacteriochlorophyll a (BChl a) molecules. The associated excited state, P870*, is the primary electron-donor state of the RC. A number of new conclusions were drawn. For example, the lifetime of P870* or the electron-transfer rate from the total zero-point level is independent of the location of the zero-phonon line (ZPL) within the inhomogeneous distribution of ZPL frequencies. The independence of the lifetime is relevant to the question of dispersive kinetics for primary charge separation. In addition, the spectra allowed for a more precise analysis of the contribution from electron-phonon coupling to the P870 profile. As is the case for the Qy state of chlorophyllsin all other protein complexes studied,2J P870* couples to a broad distribution of low-frequency phonons. The mean frequency and Huang-Rhys factor are w, = 30 cm-1 and S 2 (strong coupling); see Table I1 of paper 1. The width of the one-phonon profile is -40 cm-I. The transition couples also to a special pair marker mode with a,, 120 cm-l and S,, = 1.5. We have assigned this mode as a pseudolocalized phonon and have referred to it as the special pair marker mode. Previous PHB studies of the RC of Rhodopseudomonas viridis have established that4 the underlying structure of P960 (absorption due to the special pair of BChl b molecules) is similar to that of P870; see Table I of paper 1. Perhaps the most significant difference is the us,of P960 is about 20 cm-1 higher. It was suggested that the increase may be to the fact that the average macrocycle separation in P870 and P960 is 3.5 and 3.3 A, respectively.5~~The similarity in the underlying structures of P870 and P960, as well as the entire Q, absorption spectrum, is not so surprising given that the basic structural features of the two RC are very similar.5-8 The two BChl monomers of P are closely juxtaposed with pyrrole rings I essentially perfectly overlapped. The Q, transition dipoles make an angle of 140°. In the literature the lowest-energy m*( Q y ) state of the pair, P*, is also designated as P- because P* is the antisymmetric linear combinationof thelocalized excitations, PMPL*and PM*PL,within the simple excitonic dimer model. The upper dimer component
-
-
-
is then P+. The subscripts M and L designate protein subunits. The RC possesses a pseudo-C* symmetry axis that extends from P to the non-heme Fe. The other cofactors are, in order of proximity to P, BChlM, and BChlL, then BPheoM (bacteriopheoPhytinM) and BPheoL, and, finally, the two quinones (QM/L). The relevance of recent resonance Raman datag to the absorption profile of P870 and its PHB spectra is considered in paper 1. It was concluded that until Raman excitation profiles, Huang-Rhys factors,and damping constantsof the low-frequency modes are obtained, it is premature to associate the Ramanactive modes with the Franck-Condon modes observed directly by hole burning. It was also pointed out that the Raman spectra do not speak to the important distribution of phonons with a mean frequency of 30 cm-I. In this paper the importance of this distribution is underscored by presenting PHB spectra of P96O in which both the real-phonon and pseudophonon sideband holes are discernible. New results are also presented on the marker mode of P960. However, the major purpose is to examine whether NPHB can shed further light on the nature of the states that absorb in the entire Qy absorption region which, for Rps. viridis, extends from 1000 to -780 nm at 4.2 K. Calculations based on the simple exciton modellOJ1 and semiempiricalquantum chemical methods'2-'4 have demonstrated that interactions between the six chromophores of the RC (quinones can be excluded) are required for an adequate description of the Qy states (vide infra). Photochemical holeburned spectra obtained through formation of P+Q- are, unfortunately, not very useful for assessing the chromophoric composition of the Qy absorbing states. Formation of P+Qnot only takes the Qvstate of one (on average) of the monomers of P out of the picture, the charges produce electrochromic shifting of zero-order state energies and, possibly, protein structural changes that also affect these energies. Nonphotochemical hole-burning offers more potential as has been demonstrated for the base-plate BChl a antenna complex of Prosthecochloris aestuarii's and the B80SB850and B875 antenna complexesofRb.~phaeroides.'~J~ There are two obvious reasons3why one would expect the rate of NPHB for P960 to be extremely slow relative, for example, to that for laser dyes in alcohol glasses and hydroxylated polymers.l* The first is the reduction in quantum yield of NPHB due to the short (- 1 ps) lifetime of P960*, and the second is the reduced absorption rate due to the large homogeneous broadening
0022-365419312097-6934%04.00/0 0 1993 American Chemical Society
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The Journal of Physical Chemistry, Vol. 97, No. 26, 1993 6935
Reaction Center of Rhodopseudomonas viridis
I
WAVELENGTH (nm)
WAVELENGTH (nm) 950 850
1050 I
750 I
I
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I
950
850
750
A
1
L
b h
I
100‘00
11000
12000
I 9000
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WAVENUMBERS (“1)
Figure 1. Polarized transient photochemical hole-burned spectra (a, b) and absorption spectra (c) in the Qvregion of the Rps. viridis RC. In (c), “sh”refers to the origin band (u,#)of the marker mode Franck-Condon progression. Features A-E correspond to the other Qu states of the RC (see text). Bum conditions are OB = 10 OOO cm-’ (a) and 10 210 cm-I (b) and burn intensity = 100 mW/cm2. Solid line and dashed line show hole spectra with read polarization parallel and perpendicular to the burn laser polarization. wlPI and u,? refer to the satellite holes due to the special pair marker mode progression.
of P960. Nevertheless, Reddy et al.19have observed NPHB for F960. The NPHB spectra and earlier PHB results show that the burning is due to a significant,persistent structural transformation that is highly localized on the special pair. The photoinduced structural transformation leads to a red shift of 150 cm-l for P960 (P-) and a comparable blue shift for a state at 850 nm, which, as a consequence,could be assigned as being most closely associated with P+, the upper dimer component. In addition, a new Q,, state at 818 nm was identified. Reddy et al. suggested that analysis of the NPHB spectra might lead to an improved understanding of the contributions from PL* and PM*to the Qy states that absorb higher in energy than P960. This paper reports on the results of such an analysis.
II. Experimental Section The samples used in the present experiments are from the same batch of samples used two years earlier. They were stored in the dark at liquid nitrogen temperature. Good glass samples were obtained by quick cooling of glycero1:water mixture (60:40 ratio) from 277 to 4.2 Kin a Janis liquid helium optical cryostat. Absorption and hole-burned spectra were obtained using a Bruker 120 HR Fourier transform spectrometer (resolving power = lo6). Optical density at P960 for the samples used in these experiments was about 0.4. High-resolution spectra were recorded at a resolution of 0.5 cm-l. A Coherent CR899 Ti:sapphire laser (line width = 0.06 cm-l), pumped by an argon ion laser (Coherent Innova 200),was used for hole burning. For polarization studies, a Glan-Thompson polarizer was placed in the probe beam path of the FTIR spectrometer. Required polarization of the burn laser beam was ensured by introducing an additional polarizer in its path. Using polarizers before and after the sample, the extent of depolarization of the probe beam on passing through the sample was found to be 40%. This was corrected for by placing the probe beam polarizer after the sample. Unfortunately, similar depolarization of the burn laser beam could not be corrected for. Typically, averaging over 100 scans was sufficient to obtain spectra with a good signal-to-noise ratio. 111. Results
The 4.2 K absorption of the Q, region for the Rps. viridis RC is shown as the lowest spectrum in Figure 1. The shoulder of
10600
11000
12000
13000
I
WAVENUMBERS (“1)
Figure 2. Nonphotochemicalhole-burned spectra obtained with the burn laser frequency at 9813 cm-I (a) and 983 1 cm-I (b). Burn fluences used to obtain the spectra are 250 mW/cm2for 15 min (a) and 250 mW/cmz for 30 min. The solid diamonds locate the ZPH at the burn frequency, and solid arrows identify the red-shifted antihole caused by burning in P960. Dashed arrows refer to the antihole generated by hole burning of the 850-nm band. Absorption spectrum is included as a guide. Asterisk locates the hole of the 818-nm band.
P960 is the origin band, , : s o of the special pair marker mode progression2s4(vide infra). The width of P96O is 430 cm-1, which is 10 cm-l broader than that measured about two years earlier for a sample from the same batch4 (cf. section 11). Aging in the dark and at 77 K appears to have only slightly increased the inhomogeneous broadening of the Qybands; the peak positions have not shifted. Linear and circular dichroism studies,2°J1 transient absorption measurements,22 and the observation of a positive correlation of site excitation energies by hole burninga had indicated that band A is a likely candidate for P+. The NPHB studies of Reddy et al.19establish that this is the case and that band C is an intrinsic state of the RC (vide infra). Band C is not resolved in low-temperature spectra of poorer quality, i.e., broader bandwidths. It has been generally ignored, perhaps because of an earlier suggestionthat it may be due to an impurityS20 Band B has often been assumed by experimentalists to be due to degenerate localized transitions of BChlL and BChlM. There is no basis for this assumption.10-l4 That bands D and E are more or less pure BPheoL and BPheoM transitions is supported by all theoretical calculations except those of Thompson and Zerner.14 The top and middle traces of Figure 1 are polarized PHB spectra for the “virgin” and aged samples, respectively. There are no significant differences between the two sets of linearly polarized spectra. The polarization anisotropy ratio, p = (41 - ZL)/(Z1l + ZI), for P960 and band A are of particular interest. The p value for P960 is 0.30. This is lower than the value of 0.45 determined by Breton et al.21J4 on the basis of photoselection experiments. Our lower p value is probably due to depolarization of the burn laser beam within the sample volume. While the depolarization of the probe beam of the FT spectrometer can be corrected for by inserting an additional polarizer after the sample, the depolarization of the burn beam cannot be. However, it is the polarization of the 850 nm (A) band relative to P960 that is most important. By measuring the absorbance changes from the AA = 0 base line, we obtained a value of p = -0.14. However, the hole of band A is interfered with by the red-shifted feature (2) of band B. Correcting for this led to a value of p = -0.22, which is in good agreement with the result of Paillotin et al.25 These workers concluded that the angle between the P960 and band A transition dipoles is R54O, consistent with the 850-nm state being closely associated with P+. The top and middle traces of Figure 2 are persistent NPHB spectra obtained with different burn fluences (cf. caption). The
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Reddy et al.
6936 The Journal of Physical Chemistry, Vol. 97, No. 26, 1993 WAVELENGTH (nm)
WAVELENGTH (nm) 1050
1000
opo
1opo
950
990
1
I
0 al C 0
g -.01 s 2
s -.02
0" -.03 I
9600
' 9500
10000
10500
Figure 3. Nonphotochemical hole-burned spectrum (b) and transient photochemicalhole-burnedspectrum (c) obtained with a bum frequency of 10 750 cm-1. Differencespectrum, (b) - (c),shows that the absorption of the phototransformed P960 is characterized by the marker mode progression.
diamonds locate the ZPH which are coincident with WB. For convenience, the absorption spectrum is included as the bottom trace. Comparison of Figures 1 and 2 shows that the NPHB spectra arevery different from thePHB spectra. The solid arrows in Figure 2 locate the red-shifted antihole of the P960 hole. As discussed in ref 19, the P960 NPHB profile is distinct from the PHB profile for the same WB because the marker mode progression that builds on the origin band of the antihole origin (solid arrow) interferes with the P960 nonphotochemical hole. It was shown that, by normalizing a NPHB and PHB profile (obtained with the same burn frequency) to the same intensity over a suitable frequency range on the high-energy side of P960 and subtracting the two, one obtains the entire antihole spectrum.19 A different example of this is shown in Figure 3 for OB = 10 750 cm-l. The middle and bottom traces are the NPHB and PHB spectra, respectively. The top trace is the P960 antihole spectrum which shows clearly the marker mode progression with os, 140 cm-l and S,, 1, equal, within experimental uncertainty, to the values determined earliere4 Thus, the phototransformed P960 is also characterized by the marker mode progression. The waPoorigin of the antihole spectrum is red-shifted from wSpOof P960 by 150 cm-1. Turning next to the band A-E region of Figure 2, one observes that the nonphotochemicalhole coincident with band A is second in intensity only to the P960 hole. As discussed in section IV, the meaningful quantity for comparison is the ratio of the integrated hole intensity to that of the absorption band. Cursory inspection of Figure 2 indicates that these ratios for P960 and band A are roughly equal. The antihole of the band A hole is only approximately located by the dashed arrow. The antihole is interfered with by a relatively weak hole from band B which is located just to the right of the dashed arrow;lg see spectrum b. On the basis of previous studies of antiholes free of interference,16J' Reddy et al. determined that the antihole of band A is blue-shifted by 150 cm-1 relative to its hole.19 This result, the large fractional intensity of the hole, and other results were used to assign band A as the state most closely associated with P+; cf. section IV. That the shift of the antihole of P+ is equal in magnitude but opposite in sign to that of P- was noted by Reddy et al. to be consistent with the simple excitonic dimer model when the photoinduced structural transformation of P does not produce a significant change in the dispersion energy term. Whereas bands D and E are relatively silent in the NPHB
-
-
-
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WAVENUMBERS
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(cml)
Figure 4. High-resolution transient photochemical hole-burned spectra of P960 for w g = 9837 cm-1 (a) and 9810 cm-1 (b). The burn intensity
WAVENUMBERS (cm-3
-
9800
is 50 mW/cmz, and read resolution is 0.5 cm-l. Features wgl and w.2 are described in the caption to Figure 1. Various arrows refer to the realand pseudo- PSBH building on the ZPH at w g and WB + 145 cm-l.
spectrum, band C yields a significant fractional hole intensity (the hole is located by * in Figure 2), second only to that of P+ in the A-E region. On the basis of arguments stemming from perturbation theory, they concluded that PL* and PM*make a significant contribution to the intrinsic Qystate responsible for band C. However, details were not given. On the basis of the NPHB spectra, it was also concluded that the special pair marker mode, which couplesstrongly to P-, is, at most, only weakly coupled to P+. This is apparent also from the low-temperature difference spectrum obtained by Vermeglio and PaillotinZo from the absorption of the normal (dark adapted) and oxidized (P+)RC. The significance of this finding was considered in ref 19. An acceptable deconvolution must yield widths for bands A and B that are in reasonable agreement with their NPHB widths obtained under non-line-narrowingconditions,e.g., with OB located on the high-energy side of P960. Averaging over several NPHB profiles led to a width of 140 cm-l for both. Deconvolution is aided also by the fact that band B is resolved (width = 200 cm-1) and that the interference to band E from the relatively intense 750- and 920-cm-l BChl modes, which build on band B, can be accounted for since their Franck-Condon factors (1 0) are known.16 For both this factor equals 0.05. These vibronic bands were assigned a width equal to that of band B. Several approaches were used, all of which yielded similar results. The one that yielded the smallest residuals led to the deconvolution shown in Figure 5 . Lorentzian profiles were used for bands B, D, and E (after removal of the above vibronic interferences). Gaussian profiles led to larger residuals. Because time domain and holeburning studies haveshown that the states corresponding to bands B, D, and E relax in 5 100 fs,4.22there is a significant Lorentzian contribution to their profiles (in addition to a Gaussian contribution from inhomogeneous broadening). However, we decided to use Lorentzians rather than Voigt profiles because weakvibronic bands (5350 cm-1) must produce tailing on the high-energy sides of bands B, D, and E. Utilization of a Lorentzianservesto partially compensate for the tailing. The residual absorption obtained by subtracting bands B, D, and E form the experimental absorption spectrum yielded profiles for bands A and C that were described best as Voigt (as shown in Figure 5 ) . The widths and integrated intensities (relative to P960) of bands A-E are given in Table I. It should be noted that the widths of 170 and 130 cm-1 for bands A and C are in acceptable agreement with their aforementioned hole widths. We end this section by showing in Figure 4 two PHB spectra obtained with WB values that are in the near vicinity of the center of the inhomogeneous distribution of ZPL frequencies of the wSp1
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The Journal of Physical Chemistry, Vol. 97, No. 26, 1993 6937
Reaction Center of Rhodopseudomonas viridis WAVELENGTH (nm)
850
750
800
1
B
12000
13000
WAVENUMBERS (cm-1)
Figure5. Dtconvolution of various bands absorbing in the higher-energy Q region. Experimental spectrum is shown in (a) (solid line) and Lorentzian line shapes for bands B, D, and E (dashed lines, a). Voigt 1incshaptswereuscdtofitbandsAandC(sectextandTablc1). Spectrum (b) is the residual absorption not accounted for by any of the absorption bands.
TABLE I: Band Parameters and R Values band hole band band maximuma bandwidth areab intensit? A
B C
D E!
11 740 (852.0) 11 970 (835.2) 12 200 (819.2)e 12 380 (807.0) 12 670 (789.0)
170 200 130 260 260
0.12 1.30 0.11
0.57 0.42
0.110 0.039 0.039 0.028 0.037
R valueb 0.90 0.03 0.36
0.05 0.09
Units in cm-l. R value is defined as the ratio of hole intensity to that of the correspondingabsorption band, relative to unit value for P960. When measured directly from the absorption spectrum, the value is 818.2 nm. A value of 818 nm is used in the text. a
(zero point) level. They are the most highly structured PHB spectra of P960 reported to date. In the upper and lower spectra the distinct ZPH is coincident with OB = 9836 and 9810 cm-', respectively. The appearance of the real-phonon and pseudophonon sideband holes (PSBH) at OB f-30 cm-' (solid arrows) is particularly apparent in the lower spectrum. Although the pseudo-PSBH has been observed as a distinct "bulging" to lower energy of the ZPH (see paper 1 and references therein), the real PSBH at WB 30 cm-I had, until now, not been resolved. We attribute its observation to the higher signal/noise ratio of the spectra in Figure 4. Increased structure in the marker mode region is also apparent. In the lower spectrum the barely discernible feature labeled as osPlis displaced from the ZPH W B at by 145 cm-I. This feature is assigned as the satellite ZPH of the marker mode ( w , ~ ) ;see section 1V.D. To higher and lower energy of this ZPH one can observe the real- and pseudo-PSBH bulges (solid arrows). They are also discernible in the upper spectrum. Observation of these PSBH associated with the ZPH of q P l is expected within the Condon approximation since they build on the origin ZPH at WB. The broad shoulder, labeled as w8pZ in both spectra, is the overtone of the wSp1 hole profile.
+
Iv. Mscwclion A. State Compositions. The results of Table I and the experimental integrated nonphotochemical hole intensities (averaged over several spectra) were used to calculate the ratio ( R ) of each hole intensity relative to the integrated intensity of its associated absorption band. This ratio is a better measure of the
respbnse of each state to the photoinduced structural transformation of the special pair. The results are given in Table I with R(P960) = 1.0. Because of interferences between antiholes and holes, we estimate that the R values for bands R E are accurate to within only f30%. The estimated uncertainty of R for the 850-nm band (A) is *lo%. Table I shows that, while states A and C have a large response, the responses of states B, D, and E are weak. Within experimental uncertainty the R values for P960 and state A are equal while that for state C is about a factor of 2 lower. The identicalR value for the former two states together with the observation that the shifts of their antiholes are equal in magnitude but opposite in sign (negative correlation) and the aforementioned linear dichroism data led Reddy et al.19 to a definitive assignment of the 850-nm state as being most closely associated with P+. The negative correlation, together with the fact that earlier PHB studies23 had established that the ZPL frequency distributions for P960 (P-) and band A (P+) are positively correlated, led to the conclusion that the structural transformation associated with NPHB is highly localized on the special pair. The NPHB spectra presented in ref 19 and here lead to an adiabatic splitting between the zero-point levels of Pand P+ of 1930 cm-I at 4.2 K, which is increased to 2230 cm-l for the phototransformed special pair. Reddy et al. pointed out that the 300-cm-l increase is predicted from the simple excitondimer model for a ~OS-Adecreaseinthe 7.0-A M g M g distance of the special pair.' However, the structure of the "new special pair" was left as an open question. Reddy et al. suggested that the R ratios for the Qystates lying higher in energy than P+ can be used to gauge the contributions from PM,L*to their wave functions. For example, they asserted that the 8 18-nmstate (band C) also carries a quite significant contribution. Before pursuing this assertion, it is appropriate first to briefly review the results of electronic structure calculations. Calculationsbased on the simple, coupledchromophore-exciton modell0J and semiempiricalmethods,l"l4 which employ different and varying degrees of parametrization, predict that P960* is predominantly the lower-energy excitonic component of P, Le., P-. The semiempirical calculations reveal a relatively small (6s7and Thompson and Fajer,2*application of the exciton model would appear to be questionable. These workers find, as Parson and Warshell2 and Scherer and FischerI3had earlier, that the chargeresonance states PM+PLand P ~ P ~ - m a ka significant e contribution to P- (P960*). Thompson et al. conclude that charge resonance contributes as much to the red shift of P-of the BChl b dimer (relative to the Qy state of the monomer) as does the excitonic splitting. Furthermore, the blue shift of P870, relative to P960, is easily rationalized by an increase in the average interplanar spacing between the macrocycles of 0.2-0.3 A. The increase reduces the charge-resonance stabilization of P-. With the same parametrization, Thompson and Fajer28could account for the 2000-cm-l blue shift of P- of the BChl g dimer relative to P- of the BChl b dimer. (BChl gis obtained from BChl b by replacing the acetyl group of ring I by a vinyl group.) One may ask then whether the structural change of the special pair of Rps. viridis detected by NPHB is simply a slight reduction in the average interplanar spacing between the macrocycles. The results of Thompson and Fajer indicate that the answer is no since such a reduction leads also to a red shift for P+. It will be interesting to see whether calculations at this or a higher level will identify a structure(s) consistent with the NPHB results. The search for such a structure may have to include consideration of ruffling of the macrocycles and orientation of the ring I acetyl group29 (Fajer, J., Gudowska-Nowak,E., private communication). C. Additional Aspects of the Nonphotochemical Hole-Burned Spectra. With reference to Figure 2, the observed fractional b l changes at the peak of P960 are C0.04. In contrast with PHB, for which a fractional change of 0.6 is readily observed at 4.2 K, we have not succeeded in producing a change greater than -0.04 by NPHB. Although we cannot exclude the possibility that NPHB is operative for only a small subset of the ensemble of RC, we favor an interpretation based on laser (light)-induced hole filling or LIHF. The most detailed study of LIHF is to be found in ref 30, where it is proven that excitation of antihole sites causes them to revert efficiently to, more or less, their original preburn configurations. That is, the antihole sites retain, at the very least, a partial memory of their origin in configuration space. For 1 m * states of molecular monomers in amorphous solids, LIHF does not significantly limit the depth of the hole because their antihole is predominantly shifted to the blue of the laser burn frequency (ZPH), WB. Indeed, 100%nonphotochemical burning of the zero-phonon linescoincident with OB has been achieved.18 The situation for P960 is different because its antihole lies to the red of WB. Therefore, the very laser frequency that burns also excites the antihole sites. (Here the marker mode progression would be instrumental.) With this interpretation the NPHB spectra of Figure 2 are quite likely due to the RC that are most difficult to burn (the kinetics of NPHB are highly dispersive’*) and, therefore, most unlikelyto undergo LIHF.’O This is supported
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The Journal of Physical Chemistry, Vol. 97, No. 26, I993 6939
I
0 200 400 CENTER SITE OFFSET (cm-’) Figure 6. Simulated PHB spectra obtained with the following parameter values: the parameters wm = 30 cm-I, S = 2.2, w P = 145 cm-’, Ssp= 1.1, and = 140cm-I. The one-phonon profile is described by a function (fwhm = 42.5 an-’)which is Gaussian on the low-energy side (hwhm = 15 cm-l) and Lorentzian on the high-energy side (hwhm = 27.5 cm-I). The spectra were calculated for OB = +0, 20, and +40 cm-I from the center of the inhomogeneous ZPL frequency distribution. The arrows locate the real-phonon and pseudophonon sideband holes at uapoi 30 cm-I and wapl 30 cm-l.
-200
by our inability to generate polarized NPHB spectra that exhibit high linear dichroism ratios for P960 (P-) and P+ (results not shown). In paper 1, PHB spectra of the RC of protonated (R-26) and deuterated (wild type) Rb. sphaeroides are presented. With the burn intensities and fluences used to produce NPHB of P960, we were unable to observe NPHB of P870. This might be due to the structural differencesbetween the special pairs of Rps. viridis and Rb. sphaeroides. However, it might also be due to the fact that the Rps. uiridis RC samples used were “matured” in the dark, at 77 K for about two years. (We intend to check this possibility when “fresh” RC samples are made available to us.) Even if the latter possibility turns out to be correct, it is of no consequence to the results of this work. D. Structure of the Photochemical Hole-Burned Spectra of P960. With reference to the last paragraph of section 111, we consider further the structure of the PHB spectra in Figure 4. Figure 6 presents the simulated PHB spectra obtained with OB = Y, ,Y 20 cm-1, and Y, + 40 cm-1 and the values of the theoreticalparameters given in the caption. (The reader is referred to paper 1 and references therein for the hole profile theory used.) The frequency Y, is the center of the inhomogeneous ZPL frequency distribution of wap0. Comparison of these parameters values with those used in previous simulations (Table I of paper 1) for the “virgin”sample (see section 11) reveals only two notable changes: the homogeneouswidth of os; has been decreased from 50 to 25 cm-1 (without this decrease the satellite marker mode ZPH, wSpl in Figure 5, would not be observed, see Figure 3 of paper 1); and oSp has been increased from 135 to 145 cm-I. In our earlier ~ o r k ~we - * stated ~ that the value of wSp has an uncertainty of about 10%. The simulated spectra of Figure 5 obviously capture the main features of the experimental spectra of Figure 4. The discrepancy in the near vicinity of the ZPH at W B is similar to that observed for P810 of Rb. sphaeroides and is the result of an inadequate description of the low-energy side of the one-phonon profile (see paper 1). The two-mode (w,,~,,,) model was also found to provide an adequatedescriptionofthePHBspectraof P870 for the protonated RC (see paper 1) although the wSplsatellite ZPH was nor ooserved for P870. This suggests that the wspi ievel oi F d / u , kith a frequency of 120 cm-1, has a larger damping constant tnau the corresponding level of P960, which has a frequency of 145 cm-l.
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The Journal of Physical Chemistry, Vol. 97, No. 26, I993
Reddy et al.
The explanation for this may be that the protein phonon density of states at 145 cm-1 is significantly lower than at 120 cm-l (see paper 1).
us with the samples of the Rps. uiridis RC and Jack Fajer, Eva Gudowska-Nowak and Mark Thompson for illuminating discussions on electronic structure calculations.
V. Conclusions
References and Notes
On the basis of nonphotochemical hole-burned spectra and theoretical analysis the following conclusions are drawn: the 850nm state associated with absorption band A is predominantly P+ in character. The in-phase contributions from BChlL* and BChlM* are small relative to those from PL*and PM*;the weakly absorbing 814-nm state (band C) is an intrinsicstate of the Rps. uiridis RC. Next to P- and P+, it carries the largest contribution from PL*and PM*and; the contributions to the 835-, 807-, and 789-nm states (bands B, D, E) from PL* and PM*are relatively small. The hole-burned spectra provide support for certain predictions common to several theoretical investigati~ns,~O-l~ for example: the second most intense absorption band at 835 nm is due to a Qustate that contains a large out-of-phasecontribution from BChlL* and BChlM* (again, with little PL*, PM*contribution) and the fourth Qystate (818 nm) is characterized by a large in-phase contribution from BChlL* and BChlM* and a significant in-phasecontributionfrom PL*and PM*. With regard to the latter, it is to be appreciated that NPHB has not only identified the sixth Q, state, it has led to its assignment as state 4 of the above theoretical works. We hasten to add that the NPHB spectra shed no light on the extent to which the BPheoL* and BPheoM* states contribute, for example, to the 835- and 8 18-nm states. Experimentalistshave tended to assume that the 835-nm state is due to degenerate BChlL and BChlM monomer transitions. Although the electronic structure calculationshad indicated that there is no basis for this, the NPHB spectra, which identify the state at 8 18 nm, prove that the above assumption is invalid. This should be recognized in the interpretation of ultrafast transient absorption spectra. The conclusion that the light-induced, persistent structural transformation of the RC associated with NPHB is highly localized on the special pair had been drawn earlier.19 This transformation produces a quite remarkablered- and blue-shifting of P- and P+ equal to 150 cm-l. This effect is more profound than any produced by site-directed mutagenesis in which the special pair is left chemically intact. As such, it is highly deserving of theoretical investigation.
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Acknowledgment. Research at the Ames Laboratory was supported by the Division of Chemical Sciences, Office of Basic Energy Science, and the U S . Department of Energy. Ames Laboratory is operated for the U S . Department of Energy by Iowa State University under Contract W-7405-Eng-82. We thank David M. T i d e of the Argonne National Laboratory for providing
