New Insights on Persistent Nonphotochemical Hole Burning and Its

J. Phys. Chem. B 2001, 105, 5083-5098

5083

FEATURE ARTICLE New Insights on Persistent Nonphotochemical Hole Burning and Its Application to Photosynthetic Complexes Tonu Reinot, Valter Zazubovich, John M. Hayes, and Gerald J. Small* Department of Chemistry and Ames Laboratory-U.S. Department of Energy, Iowa State UniVersity, Ames, Iowa 50011 ReceiVed: December 31, 2000; In Final Form: March 12, 2001

Nonphotochemical hole burning (NPHB) at low temperatures of the electronic absorption bands of molecular chromophores imbedded in amorphous solids (glasses and polymers) and in proteins is a striking manifestation of configurational tunneling triggered by electronic excitation. The current mechanism of NPHB has it due to a hierarchy of relaxation events that begin in the outer shell and involve the intrinsic two-level systems (TLSint) of the glass and terminate in the inner shell where the rate determining step involving the extrinsic TLS (TLSext) occurs. The TLS correspond to asymmetric intermolecular double well potentials. The TLSext are associated with the chromophore and the inner shell of solvent molecules. The TLSint are intimately associated with the excess free volume of glasses. Their tunneling leads to diffusion of excess free volume. Results for Al-phthalocyanine tetrasulfonate (APT) in hyperquenched glassy water (HGW) and ethanol (HGE) films that provide strong support for the critical role of excess free volume are discussed. Hole spectra of APT/HGW obtained over eight decades of burn fluence reveal that the current mechanism needs to be modified to include multilevel extrinsic systems (MLSext) in order to explain why the antihole (“photoproduct” absorption) lies to the blue of the burn frequency for sufficiently high burn fluences, an intriguing up-conversion process. The spectra also reveal, for the first time, that the zero-phonon hole (ZPH) profile is non-Lorentzian. This is shown to be a natural consequence of the interplay between the three distributions that result in dispersive hole growth kinetics. They are associated with the tunnel parameter λ of the TLSext, the angle R between the laser polarization and transition dipole, and off-resonant absorption of the zero-phonon line (the ω distribution). Theoretical simulations of hole growth data for APT/HGW obtained over six decades of burn fluence show that the λ distribution is of primary importance, describing well the first 80% of the saturated burn. The paper ends with an application of NPHB combined with high pressure and external electric (Stark) fields to the critically important “red” antenna states of photosystem I. The addition of pressure and Stark fields enhances the already impressive selectivity of NPHB. The results show that the linear pressure shift, permanent dipole moment change, and linear electron-phonon coupling are correlated. Of particular importance is that these properties can be used to identify states which involve interacting chlorophyll molecules that possess significant charge transfer character because of electron-exchange coupling. The results also show that the site distribution functions of the antenna states are largely uncorrelated, consistent with the findings for previously studied complexes. This is important because the absence of correlation means that the electronic energy gaps of donor and acceptor states are distributed which, in turn, means that the kinetics can be dispersive under certain conditions.

1. Introduction Persistent nonphotochemical hole burning (NPHB) at low temperatures of inhomogeneously broadened electronic absorption bands of molecular chromophores in structurally disordered hosts such as glasses, polymers, and proteins is a consequence of tunneling between host-chromophore configurations. The tunneling is triggered by excitation of the chromophore and occurs while the chromophore is in its excited state (for reviews see refs 1 and 2). NPHB along with, for example, photochemical hole burning3 and fluorescence line narrowing4,5 are site * To whom correspondence should be addressed. E-mail: gsmall@ ameslab.gov.

excitation energy selective techniques (spectroscopies) designed to unearth the wealth of static and dynamical information hidden by severe site inhomogeneous broadening (∼100-300 cm-1). It was Personov and co-workers6 who first observed NPHB as it later came to be called. The systems studied at liquid He temperatures were perylene and 9-aminoacridine in alcohol glasses. Although it was clear that the mechanism for production of the zero-phonon holes (ZPH) was not photochemical, i.e., not due to photodecomposition of the perylene “isochromat” selected by the narrow line burn laser, a plausible mechanism was not proposed. [A ZPH is the result of burning out zerophonon line (ZPL) transitions. The ZPL transition associated with the origin absorption band is a pure electronic transition,

10.1021/jp010126y CCC: $20.00 © 2001 American Chemical Society Published on Web 04/26/2001

5084 J. Phys. Chem. B, Vol. 105, No. 22, 2001

Figure 1. Fluorescence excitation spectrum of APT in HGW at 5 K following burning of 10 000 holes. The burn direction is to lower energy to minimize laser-induced hole filling during subsequent burns. Holes with an average width of 0.49 GHz were burned 1.5 GHz apart over 15 000 GHz. The top inset shows an expanded frequency scale for the blue-colored segment of the burn. The bottom inset shows a further 10 times expansion of the blue-colored section of that segment. Regions of no burning were deliberately included in the experiment. Total holeburning time (t2 - t1) was 4 h; total read time (t4 - t3) was 16 h.

i.e., phononless, and analogous to a gas-phase electronic transition that involves no change in rotational quantum numbers.] It remained for Hayes and Small7 to make the connection between NPHB and the aforementioned phononassisted tunneling. Their mechanism is based on the standard tunneling model, vide infra. NPHB is a striking manifestation of configurational tunneling in amorphous solids. In simplest terms, it is a consequence of the postburn ground state configuration of the chromophoreglass system being different from and highly kinetically inaccessible to the preburn configuration. Thus it is that NPHB is generally restricted to amorphous hosts. Because of their structural disorder, amorphous solids exhibit a poor memory of their structure prior to excitation of the chromophore. Figure 1 shows over 10 000 ZPH burned into the fluorescence excitation spectrum of the Qx(S1) state8 of Al-phthalocyanine tetrasulfonate (APT) in a hyperquenched glassy water (HGW) film at 5.0 K. The spacing between the ZPH is ∼1.5 GHz. How this multihole “spectrum” that set a world record was obtained will be described later and discussed in the context of frequency domain optical data storage and other applications. Of course, a device that drips at room temperature and, moreover, is quite difficult to prepare is hardly practical.9 A solution to this problem is to use water-containing polymers such as poly(2hydroxyethyl methacrylate), known as poly-HEMA and used in soft contact lenses because of its high water content, ∼40%. Although the hole-burning properties of APT/poly-HEMA are not on a par with those of APT/HGW, they are still impressive.10 The fractional depths of the holes in Figure 1 are ∼0.1 and were obtained with a burn intensity and time of 280 µW cm-2 and 0.1 s, corresponding to a burn fluence of only 28 µJ cm-2. The very low “photon budget” is noteworthy.

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Figure 2. Preburn and postburn (burn fluence ) 17 J/cm2) fluorescence excitation spectra of APT/HGW, T ) 5 K. The left inset shows the difference spectrum. The right inset is a blow up of the ZPH region.

A signature of NPHB is that absorption intensity should be conserved.11 That is, the integrated intensity of the antihole should equal that of the hole structure. This is the case for APT/ HGW9,12 although it is not apparent in Figure 1. It is in Figure 2, where the postburn excitation spectrum is the result of a “hard” burn with a burn wavelength (λB) equal to 677.5 nm and fluence of 17 J cm-2. Note that the antihole (positive absorption) is located to higher energy of λB. The ZPH located at λB and the pseudophonon sideband hole (pseudo-PSBH) are close to being saturated. The pseudo-PSBH is due to sites whose ZPL transition frequencies lie lower than ωB, the burn frequency. They absorb via their phonon sideband. Hole burning occurs from the zero-point level of the excited electronic state following rapid relaxation of the excited phonons.13 As will be seen, the pseudo-PSBH, together with other data, can be used to characterize the linear electron-phonon coupling. Such coupling plays an important role, for example, in the dynamics of excitation energy transfer in photosynthetic antenna complexes. The electron-phonon coupling of APT/HGW is weak with a Huang-Rhys factor S ∼ 0.2.14 As a result, the displacement of the maximum of the pseudo-PSBH from the ZPH in Figure 2 is the peak energy of the coupling phonons, ∼36 cm-1(as determined more accurately from shallower holes). That the antihole in Figure 2 is almost entirely blue-shifted relative to ωB is food for thought because it represents an up-conversion process. Energy is conserved, however, because the groundstate energy of the chromophore-glass system in the postburn configuration is lower than that of the preburn configuration, vide infra. In this paper, recent results are presented that shed further light on the mechanism of NPHB. In this regard, the evolution of the antihole with increasing burn fluence turns out to be important. As mentioned, the Hayes-Small mechanism is based on the standard tunneling model. The standard tunneling model was originally proposed to explain respectively the near linear and quadratic dependencies on temperature (j2 K) of the specific

Feature Article

Figure 3. Extrinsic two-level system model (A) and multilevel system model (B) for NPHB. Indices e and g indicate the chromophore in its excited and ground state. ωB is the burn frequency of the laser, and k1, k2, and k3 are the tunneling rates. I, II, and III denote the chromophore host configurations where I corresponds to the preburn configuration and II and III correspond to postburn configurations. Note that configuration II in B can absorb at ωB via phonon sideband transitions. The inset defines the TLS parameters. W is the tunneling frequency.

heat and thermal conductivity of glasses.15,16 It approximates that subset of the configurations of the rugged potential energy surface capable of undergoing tunneling transitions at low temperatures by a static distribution of two-level systems (TLS). The TLS are asymmetric interatomic (molecular) double well potentials (see inset of Figure 3). The tunnel frequency is W, and λ ) d(2mV)1/2 is the tunnel parameter, where m is the effective mass of the tunneling entity and d is the displacement between the two potential energy minima. In the standard tunneling model, TLS-TLS coupling or connectivity is neglected. Anderson et al.15 and Phillips16 introduced simple, phenomenological distribution functions for W and the asymmetry parameter ∆ that led to an explanation for the above temperature dependencies under the assumption that the TLS density of states F(E) is constant over the relevant temperature interval. Here, E ) (W2 + ∆2)1/2 is the tunnel state splitting. As more detailed data emerged, other phenomenological distribution functions were introduced (see ref 17, and refs therein). In our studies, we have employed the distribution functions of Jankowiak et al.17 They are based on Gaussian distributions for λ and ∆ which are still assumed to be uncorrelated. They have been successfully applied to a number of problems including the temperature and time dependence of the specific heat of SiO2 glasses with different -OH (impurity) content.18 The Cv(t) data were explained in terms of intrinsic (int) and extrinsic (ext) TLS with the TLSext intimately associated with the impurity and its inner shell of “solvent” molecules and the TLSint associated with the “pure” glass. The time dependence of Cv is due to the relaxation rates, R, of the TLSint and TLSext being distributed, with the rates for the TLSext being the slower. The rates are proportional to W2. To a good approximation, the distributions, P(R), are dominated by the distributions for W2 which, with Gaussian distributions for the tunnel parameter λ, are log-normal.19 The same basic approach

J. Phys. Chem. B, Vol. 105, No. 22, 2001 5085 also explained the time-dependence of spectral diffusion of dye molecules in molecular glasses and polymers.20,21 The Hayes-Small mechanism7 for NPHB is based on pictures of the type shown in Figure 3A. It assumes that a static distribution of TLSext is sufficient to understand the phenomenon. The problem is more complicated than that encountered with specific heat because one needs to consider TLSext with the chromophore in its ground and excited state (TLSgext and TLSeext). At about the same time Hayes and Small proposed their mechanism, the connection between the anomalous magnitude and near linear dependence on temperature (j10 K) of the pure dephasing of optical transitions and TLS tunneling was made.22-26 What emerged was that NPHB is intimately associated with the TLSext, whereas pure dephasing/spectral diffusion is the result of “spin flips” of the TLSint, with the electron-TLSint coupling in the ground and excited states being different (for a review see ref 2). Importantly, hole-burning studies of dye molecules in hydrogen-bonding glasses such as ethanol and glycerol and polymers such as poly(vinyl alcohol) provided, early on, valuable insights on the TLSext and TLSint.27-29 Whereas deuteration of the hydroxyl proton of the host had no effect on the pure dephasing/spectral diffusion of the optical transition, it led to a marked decrease (≈ × 30) in hole-burning efficiency. In recent studies of APT in hyperquenched glassy films of H2O and D2O, deuteration led to a decrease of the average quantum yield of NPHB by a factor of 200-500, depending on whether the films were annealed.30 Such results establish that the TLSint are spatially extended hydrogenbonding networks with an effective coordinate that involves only small amplitude motion of the H atoms. In sharp contrast, the TLSext coordinate involves large amplitude motion of H atoms that signals a significant rearrangement of the hydrogen-bonding network in the inner shell. A number of observations made in the 80s led Shu and Small11,31 to conclude that NPHB cannot be understood in terms of a static distribution of TLSext. It is instructive to consider two of them. First, hole spectra of S0 f S1 (ππ*) transitions of several chromophore/glass (polymer) systems that featured a pronounced pseudo-PSBH exhibited an antihole that lies predominantly to higher energy of the burn frequency ωB. As in the case of the spectrum shown in Figure 2, the antihole stemmed mainly from the pseudo-PSBH whose maximum lies to lower energy of ωB. Because transitions of the above type typically exhibit a gas to condensed phase energy shift that is to the red, the blue-shifted antihole was attributed to hole burning leading to an increase in free volume around the chromophore. This would result in the intermolecular interactions of the chromophore with inner shell solvent molecules in antihole configurations being weaker than in preburn configurations. The second observation was that at liquid helium temperatures, ∼100% of the ZPL could be burned when the contribution from phononic transitions to the absorption was taken into account. The TLSext energy diagram (A) in Figure 3 depicts the situation where phonon-assisted tunneling in the excited state involves phonon emission and the antihole site absorbs to higher energy of ωB. There are seven other energy level schemes. Four of the eight lead to blue-shifted antihole sites, whereas the other four lead to red-shifted sites. Concerning phonon-assisted tunneling in the excited state, four of the schemes involve phonon absorption, the other four phonon emission. For a static distribution of TLSext, the former would not be expected to hole burn in the T f 0 K limit. That is, one would expect that only 50% of the ZPL could be burned. Shu and Small proposed that NPHB is the result of a hierarchy of

5086 J. Phys. Chem. B, Vol. 105, No. 22, 2001 tunneling events triggered by optical excitation that begin with the faster relaxing TLSint in the outer shell and terminate in the inner shell. This outside-in chain of events would result in diffusion of excess free volume to the inner shell, opening the way for the rate determining step of NPHB that involves the TLSeext. In this regard, it is widely accepted that the TLSint are a consequence of the excess free volume of glasses as first proposed by Cohen and Grest.32 With the Shu-Small mechanism, one can view the picture shown in Figure 3A as being the result of prior relaxation events; the system at some instant of time is poised to undergo downward or isoenergetic tunneling in the excited state that is operative in the T f 0 K limit. The Shu-Small mechanism explains the two aforementioned observations. An important prediction of their model is that NPHB should cease upon formation of the crystalline phase from the glass because the former should be devoid of excess free volume. Such was observed for APT in HGW upon formation of cubic ice at 150 K.9 It was shown later that the NPHB quantum yield of APT in hyperquenched glassy ethanol also undergoes a dramatic decrease upon formation of the crystalline phase.33 These observations provided convincing proof that excess free Volume plays a key role in NPHB and appears to be correlated with the density of TLSint. This article has two main objectives. The first is to present recent results for APT/HGW that provide new insights on the mechanism of NPHB and how the structural disorder of amorphous solids leads to highly dispersive kinetics for hole growth. Concerning the former, the burn fluence dependence of the hole spectrum, which encompasses the ZPH, PSBH, and antihole, indicates that the Shu-Small mechanism for NPHB is incomplete in that it does not adequately account for the effects of multiple excitations of preburn and antihole sites capable of absorbing the burn photons. Also, the Shu-Small mechanism assumes that all initially burned sites result in blueshifted antihole sites. The new results show that this is not generally the case. Analysis of the dispersive kinetics of ZPH growth show that it is due primarily to structural disorder, specifically, the distribution of λ values. The “intrinsic” distributions from the angle (R) between the burn laser polarization and transition dipole and off-resonant excitation of the ZPL (ω distribution) are of secondary importance, becoming noticeable for the last ∼20% of the burn that leads to near saturation of the ZPH. Non-Lorentzian ZPH profiles are reported and shown to be a natural consequence of the interplay of the λ, R, and ω distributions. The second objective is to show how NPHB has proven to be a valuable technique for studying the excited state electronic structure and excitation energy transfer dynamics of photosynthetic antenna complexes. Its attributes will be illustrated using recently obtained results on photosystem I (PS I) of cyanobacteria. 2. Experimental Section A block diagram of the apparatus used for preparation of hyperquenched films and hole burning of those films is shown in Figure 4. The laser system for fluorescence excitation and hole burning and the detection system have been described in detail in refs 9 and 34. Briefly, a Coherent 699-29 ring dye laser is used for burning and for hole reading (via fluorescence excitation). The laser line width is j10 MHz for burning. The laser power density for burning can be varied from 10 nW cm-2 to several hundred mW cm-2 by the use of neutral density filters in the beam. Use of a laser power stabilizer (Cambridge Research and Instrumentation, LS100) obviates the need for a reference beam as constant power may be maintained over the

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Figure 4. Block diagram of the experimental apparatus used for hole burning and reading by fluorescence excitation. The thermospray system used for deposition of amorphous solid films is depicted at left. For film deposition, the sample holder, S, is rotated to be perpendicular to the thermospray nozzle. The surrounding cold shroud is also rotated after the thermospray flow is established. L is an f/1.8, 50 mm camera lens. F is a long pass filter chosen to reject scattered excitation light and pass fluorescence.

scan range of the laser. For hole reading, laser power densities in the nW cm-2 range are used. Low power densities are obtained by using a set of neutral density filters and by expanding the laser beam to match the beam and sample diameters (∼ 1 cm) in order to maximize the fluorescence signal and minimize destructive hole burning at the hole reading time. The experiment control and data acquisition are performed by a dedicated computer using locally developed software. Simultaneously, the fluorescence signal from photon counter, sample temperature, laser power, and scanning properties can be monitored. The fluorescence signal is collected through a lowpass filter using a cooled GaAs photomultiplier tube. For hole burning of photosynthetic complexes, the same laser or a similarly configured Ti:sapphire ring laser (Coherent 899-29) is used, but the holes are measured either as above or by absorption spectroscopy with a Bruker IFS-120 Fourier transform spectrometer. This allows absorption changes over a broad spectral range to be detected simultaneously.35 In the lower left of Figure 4, the thermospray deposition source is shown. This is a Vestec VT-1499-13R vaporizer which is a resistively heated 150 µm i.d. capillary tube inserted into the vacuum system through a compression fitting. Thermocouples attached to the inlet and outlet of the capillary allow monitoring of these temperatures. The capillary is heated with a variable voltage along its length from a 0-5 V source. Liquid samples are delivered to the thermospray by an LDC-396-89 pump connected to the capillary through a four-way valve with 0.2 mm i.d. PEEK tubing. One port of the four-way valve is connected to a vacuum pump so that residual liquid in the capillary may be removed after film deposition. This prevents slow diffusion of the liquid into the sample chamber that can cause the film thickness to vary over time. By adjusting the voltage applied to the thermospray, it is possible to vary the size of clusters emerging from the source over a small range. The average cluster size is typically ∼2 µm in diameter as determined by microscopic viewing and by infrared spectroscopy. Clusters emanating from the thermospray impinge on a conduction-cooled copper plate attached to the coldfinger of a

Feature Article continuous flow helium cryostat (Janis, ST-100). This results in a cooling rate of the clusters of ∼107 K s-1, sufficient to vitrify even hard to vitrify materials, e.g., water. Because the initial flow from the thermospray nozzle is atypical (the start of flow causes a slight drop in the nozzle exit temperature), a shutter between the nozzle and the copper plate is not opened until stable flow is established. Films of HGW up to ∼20 µm thick can easily be prepared with this system. Films deposited at 5.0 K are referred to as fresh. Films deposited at 5.0 K and annealed at ≈145 K are referred to as annealed. Although we have thus far studied in depth only HGW and hyperquenched glassy alcohols, the thermospray source should be applicable to vitrification of any room temperature liquid. Extension to higher melting materials is possible with easily envisioned modifications. The high-pressure apparatus used to study PS I of cyanobacteria has been described in detail in refs 36 and 37, including the procedure used to measure pressure. To ensure good optical quality, the sample was contained in a gelatin capsule (5 mm outside diameter) purchased from Torpac Inc. and then housed in a specially designed high pressure cell with four sapphire windows (thickness of 4 mm) providing optical access. The cell was connected to a three-stage hydraulic compressor (model U11, Unipress Equipment Division, Polish Academy of Sciences) through a flexible thick-walled capillary (o.d./i.d. ) 3.0 mm/0.3 mm). Helium gas was used as the pressure-transmitting medium. A specially designed Janis 11-DT cryostat was used for cooling of the high-pressure cell. High-pressure hole burning was performed at 12 K. At this temperature, liquid helium solidifies at ∼75 MPa. When the procedure given in ref 36 was followed, it was confirmed that pressure-induced structural changes are elastic. For studies at higher temperatures and pressures, a Lakeshore Cryotronic temperature controller (model 330) was used to stabilize and measure the temperature. The Stark hole-burning apparatus used was the same as that described in ref 37. Samples were contained in gelatin capsules. Prior to insertion into the Stark cell, the gelatin capsule filled with sample was allowed to soften for about 5 min at room temperature so that it could be mechanically squeezed by the two copper electrodes of the Stark cell. This procedure yielded an optical path length perpendicular to the applied field of ∼6 mm with a distance of ∼2 mm between the electrodes. Teflon spacers were used to set the distance between the electrodes ((0.05 mm). The Stark field could be applied parallel or perpendicular to the burn laser polarization by positioning a polarization rotator placed in front of the Stark cell. The probing light (unpolarized) was collinear with the burning beam. A Trek Inc. model 610 C dc high-voltage power supply (from 0 to ( 10 kV) was used. By changing the polarity of the power supply, we were able to acheive a maximum Stark field of ∼100 kV/ cm. Holes were initially burned at the highest field with a chosen polarity. All Stark measurements were performed at 1.8 K in a Janis 10 DT liquid helium cryostat. 3. Results and Discussion The basic NPHB properties and the spectral dynamics of APT in HGW9,34 and hyperquenched films of methanol33 and ethanol33 (HGM and HGE) have been determined. In all three systems, the lowest energy Q(ππ*) absorption band is split into components that are separated by ∼200 cm-1, as can be seen for APT/HGW in Figure 2. These components are designated as Qx and Qy, corresponding to the transition dipole moment along the molecular x and y axes. Molecular mechanics calculations have shown that the splitting for APT in HGW is

J. Phys. Chem. B, Vol. 105, No. 22, 2001 5087 due to breaking of the D4h molecular symmetry by ligation of water molecules to aluminum above and below the phthalocyanine plane.8 A similar explanation is no doubt relevant for the HGM and HGE films because the solvents used contained about 1% water. It is the band that lies lower in energy (arbitrarily assigned as Qx) that is routinely used in hole burning. Holes burned into the Qy band carried a width of 10 cm-1 which leads to a Qy f Qx internal conversion decay time of 1 ps.8 For annealed films of the three glasses, the low burn fluence ZPH widths at 5.0 K for the Qx band are ∼0.15, 1.5, and 2.0 GHz for HGW, HGE, and HGM, respectively.9,33 The hole spectra also differ in the peak energy, ωm, of the pseudo-PSBH apparent to the red of the ZPH. For HGW, ωm ) 36 cm-1, whereas for HGE and HGM, ωm is 26 and 17 cm-1, respectively. It is the high phonon frequency, narrow ZPH, and weak electronphonon coupling for APT in HGW that are responsible for the unique hole-burning properties of this system.39 We note that the temperature dependence of the intensity of the ZPL is governed by38 exp[-S(2nj + 1)] where S is the Huang-Rhys factor that defines the strength of the linear electron-phonon coupling and nj ) [exp(pωm/kT - 1]-1 is the phonon thermal occupation number. The weak coupling for APT/HGW (S ∼ 0.2) and high value of 36 cm-1 for ωm as well as the relatively weak dependence on temperature of the homogeneous width of the ZPL are responsible for its notable hole-burning properties at 77 K. At that burn temperature, ZPH with a fractional hole depth of 0.5 were burned.39 Before discussing hole growth kinetics and recent work on the hole and antihole profiles that shed further light on the mechanism of NPHB, a brief discussion of the temperature dependence of electronic dephasing of APT in hyperquenched films is appropriate. Electronic dephasing in HGW, HGM, and HGE could be studied over a broad temperature range (from 5 to >100 K).33,34 For all three films, the dephasing can be fit by the Jackson and Silbey expression40

Γ - Γ0 ) aTR + b1nj(ω1) + b2nj(ω2)

(1)

where Γ is half the measured holewidth and Γ0 is the lifetime limited line width of the ZPL (25 MHz). The homogeneous width of the ZPL is one-half that of the ZPH. The first term on the right-hand side of eq 1 describes dephasing from electronTLSint coupling. For a wide variety of probe molecules in amorphous hosts, R ≈ 1.3,2,41,42 as it is for APT in the three hyperquenched films.33,34 Above 10 K, the dephasing is dominated by exchange coupling of pseudolocalized modes. For HGW, two modes at ω1 ) 50 and ω2 ) 180 cm-1 are needed to obtain a good fit to the data. These mode frequencies correspond to the well-known transverse and longitudinal acoustic modes of water. The spatial extent of these modes in the liquid is temperature-dependent, and it is reasonable to assume that they are quite localized in the glass. Because it is associated with the tetrahedral arrangement of four water molecules about a central oxygen atom, the 180 cm-1 mode would not be expected to exist in either HGM or HGE because the H-bonding network in alcohols is not tetrahedral. Indeed, the dephasing of APT in HGE is well fit by the electron-TLSint coupling term and a single exchange coupling term with ω ) 47 cm-1.34 It is interesting that this frequency is essentially the same as ω1 for HGW. This may be due to the fact that the spectral densities of both liquids exhibit maxima near 50 cm-1.43,44 It should be noted that dephasing studies such as these provide a characterization of the quadratic electron-phonon coupling that gives rise to the exchange coupling mechanism, whereas


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Figure 5. Fits to the hole growth kinetics curve for APT in annealed HGW at 5 K (λB ) 676.5 nm) for the eight models described in the text.

measurement of the frequencies and intensities of the phonons that give rise to the PSBH characterize the linear electronphonon coupling. With this complete characterization of the electron-phonon coupling, it is possible to calculate the linear and nonlinear optical response functions of the chromophore as a function of temperature.45 As pointed out in refs 33 and 34, it would be illuminating to compare the pure electronic dephasing frequency as determined by photon echo measurements of optical coherence loss for a liquid just above its glass transition, with the predictions from hole-burning data. High frequency inertial modes such as those discussed above are likely to set the stage for larger amplitude nuclear motions associated with, for example, electron-transfer reactions in liquids. A. Dispersive Kinetics of Nonphotochemical Hole Growth. It has been known for some time that the kinetics for growth of the ZPH is highly dispersive.46 This is also the case for spontaneous hole filling (SPHF) which is a “dark” process that occurs after burning with the chromophore in its ground state.47 Given the immense structural disorder of amorphous solids, dispersive kinetics is expected for any physical process.48 It was recently shown that kinetics of NPHB in proteins are also dispersive.49 In the case of NPHB, however, the importance of the λ distribution to the dispersive kinetics relative to those of the R and ω distributions has been the subject of debate (see ref 14 and references therein). To determine the relative importance of the three distributions, it is essential to obtain kinetics data over many decades of burn fluence. For this, one needs a system like APT/HGW that exhibits very weak electron-phonon coupling, exhibits narrow ZPH, and, moreover, is homogeneous in the sense that all sites can eventually be burned. Thus, for example, polymer hosts that exhibit a degree of crystallinity should be avoided. Hole growth kinetic data for annealed APT/HGW obtained over an unprecedented six decades of burn fluence at 5.0 K are shown in Figure 5. The data were obtained by monitoring the decrease in fluorescence intensity that resulted from burning into the Qx band at 676.5 nm. The fits were obtained using14

D(Ω,t) ) 1.5

∫ dω L(Ω - ω)G(ω) ∫ dλ f(λ) ∫ dR sin R cos2 R e-Pσφ(λ)L(ω -ω)cos (R)t B

2

(2)

which is the fractional hole depth at frequency Ω following a burn for time t with photon flux P (number of photons cm-2

TABLE 1: Parameters Used for Fitting of Hole Growth Kinetics Data σ (cm2 s-1)

Γinh (cm-1)

S

γ (cm-1)

PSB widthsa (cm-1)

ωm (cm-1)

1.2 × 10-2

355

0.2

0.13

13,19

36

a

The PSB (phonon sideband) is modeled as a Gaussian on the low energy side and a Lorentzian on the high energy side with half-widths of 13 and 19 cm-1.

s-1). The frequency ω is that of the ZPL, and G(ω) is the Gaussian distribution of ZPL frequencies, i.e., the site distribution function (SDF). With t ) 0, D(Ω,0) is the normalized absorption spectrum prior to burning. In eq 2, σ is the integrated absorption cross-section with units of cm-2 s-1. Its value and the width (fwhm) Γinh of G(ω), as determined in ref 14, are given in Table 1. f(λ) is the normalized Gaussian distribution function for the TLSext tunnel parameter centered at λ0 with a standard deviation σλ. The NPHB quantum yield, φ(λ), is given by Ω0 exp(-2λ)[Ω0 exp(-2λ) + τfl-1]-1. The numerator is the tunneling rate. The quantum mechanical expression for Ω0 is given in.29 To be consistent with earlier works (see ref 9 and reference therein) we set Ω0 ) 7.6 × 1012 s-1 (40 cm-1) which was argued to be a reasonable value. τfl is the fluorescence lifetime which for APT is 6.5 ns.34 We hasten to add that the uncertainties in Ω0, σ, and P do not affect σλ and, therefore, the relative contributions of the λ, R, and ω distributions. Finally, L(ωi, ω) [ωi ) Ω, ωB] is the single site absorption spectrum which for weak electron-phonon coupling is

L(ωi, ω) ) LZPL(ωi - ω)e-S + LPSB(ωi + ωm - ω)(1 - e-S) (3) where S is the Huang-Rhys factor for the phonons. LZPL is the Lorentzian line shape of the ZPL with a homogeneous width of γ (see Table 1). The shape of the experimentally determined one-phonon profile, LPSB, is defined in Table 1. In each panel of Figure 5, the labels on the right indicate which of the distributions that contribute to dispersion are included in the fit. The relevant distributions are the λ distribution, the R distribution, in which R is the angle between the laser polarization and the transition dipole, and the ω distribution that describes off-resonant absorption of the ZPL. Eight models which include the λ, R, and ω distributions were used to analyze the data. As shown in the figure, they are

Feature Article referred to as 0, λ, R, ω, λR, λω, Rω, and λRω, designations that reflect the distributions used in the model. The fits derived from each model are labeled in the figure. In the λRω model, eq 2 is used as written, whereas, e.g., the λ model neglects the R and ω distributions. In the 0 model, all three distributions are turned off so that the kinetics is treated as a single exponential. For all models, λ0 and S were adjustable parameters as was σλ for models that include the λ distribution. The results for the 0, R, ω, and λ models are shown in the left panel of Figure 5. For the 0, R, and ω models, the fits are poor beyond the first decade and a half of burn fluence. The S values obtained for these models are 1.1, 0.93, and 0.75, values that are far too high on the basis of hole spectra that place an upper limit of 0.4 for S.9 For the λ model, a good fit is obtained over the first three decades of burn fluence (80% of the saturated burn) with λ0 ) 8.3, σλ ) 0.95, and S ) 0.33. From careful measurements of the saturated hole depth of the ZPH when it is located sufficiently red of the absorption maximum and when it is not interfered with by the PSBH or antihole, S ) 0.2 is obtained for annealed HGW. Thus, the value of 0.33 obtained from the λ model is far more reasonable than the values obtained by the other single distribution models. The fits for combinations of distributions are shown in the right panel of Figure 5. The Rω model fits poorly, failing over the last four decades of burn fluence, consistent with the λ distribution being most important. The λω model gives a good fit over the first four decades of burn fluence, with λ0 ) 7.9, σλ ) 0.95, and S ) 0.21; this S value equals the estimate from hole depth given above. The λR model is somewhat better than λω, showing that the λ, ω, and R distributions are of primary, secondary, and tertiary importance, respectively. Finally, the λRω model gives a good fit over five decades of burn fluence with λ0 ) 8.1, σλ ) 0.95, and S ) 0.21. The extent of the dispersiveness is significant and leads to the hole-burning rate at the point where the fractional depth is 0.1 being about a 1000 times higher than at a fractional depth of 0.9. Interestingly, the σλ values of the λ and λRω models are identical and their λ0 values almost so. It appears, therefore, that reliable values of λ0 and σλ can be obtained using only the λ distribution at a considerable saving in computation time, although the resulting S value is overestimated. The conclusions reached for APT/ HGW should generally apply because the σλ values for several other systems are J1.14 In the course of obtaining the kinetic data discussed above, ZPH profiles as a function of burn fluence were measured over nearly 8 orders of magnitude of fluence. Figure 6 shows fits to the experimental ZPH for eight burn fluences from 0.8 to 4500 µJ cm-2. The fits were obtained using eq 2. (The hole spectrum is defined as D(Ω,t) - D(Ω,t ) 0).) The values of the parameters used were obtained by simultaneous fitting of the ZPH for the first six burn fluences. Importantly, they are very similar to those determined from the kinetics of ZPH growth.14 As can be seen in the figure, the fits for fluences from 0.8 to 350 µJ cm-2 are close to perfect. The fit to the ZPH profile with a burn fluence of 1000 J cm-2 fails at the wings of the hole. The fit for 4600 µJ cm-2 is considerably worse. These discrepancies are attributed to antihole formation which, at low burn fluences, is quite symmetrical about the burn frequency. This has implications for the NPHB mechanism (vide infra). For the lowest three fluences, the ZPH can also be fit as a Lorentzian line shape. However, for burn fluences of 35 µJ cm-2 or higher, the experimental profile is more sharply tipped than a Lorentzian, as shown in the inset of Figure 6. The fits shown in Figure 6 were calculated including the λωR distributions.

J. Phys. Chem. B, Vol. 105, No. 22, 2001 5089

Figure 6. Set of eight ZPH burned sequentially at 671.8 nm into the same sample of APT in fresh HGW at T ) 5 K. The respective cumulative burn fluences in µJ/cm2 are given in the figure. A laser intensity of 0.8 µW/cm2 was used for the top six burns; the bottom two burns were performed with 47 µW/cm2. After each burn, the zerophonon hole was scanned over 1 cm-1. The smooth curves are fits to the ZPH obtained using eq 2. The fits for the top six ZPH are essentially perfect. The poor fits for the two lowest ZPH are due to the absorption of antihole sites that are not accounted for in eq 1 (see the text for details). The inset compares a Lorentzian fit (L) with the fit using the λωR distribution. Comparison of these with the experimental hole (E) shows that the Lorentzian is too bluntly tipped.

These provide an excellent fit to the data, including the tips of the ZPH where the Lorentzian fit deviates. Reinot and Small also considered that an alternative explanation of the Lorentzian deviation might be that there is a distribution of ZPL widths, γ. Such has been observed in single molecule studies.50 However, as shown in ref 12, a distribution of γ has only a minor effect on the ZPH profile. B. New Insights on the Mechanism of NPHB. Returning to the fits of Figure 6 we can see that, for the highest fluences, the calculated ZPH are deeper than the experimental in the wings of the hole. This difference is attributed to antihole formation, which was found to be nearly symmetrical about ωB at low burn fluences.12 This has also been observed for APT in HGE and HGM.33 That the antihole for low burn fluence is nearly symmetrical about ωB has implications for the hole-burning mechanism. The formation of both blue- and red-shifted antiholes was shown to continue at intermediate burn fluences. In the fitting of the ZPH and pseudo-PSBH, parameter values determined for low fluence burns were used. The parameter values are very similar to those given in Table 1.12 It was observed that the experimental depth of the PSBH was significantly less than the calculated depth even though the ZPH depth was correct to within 10%. The discrepancy is shown in the left inset of Figure 7 for a burn fluence of 15 mJ/cm2. The calculated pseudo-PSBH is about 10 times more intense than the observed pseudo-PSBH. This was attributed to interference between the pseudo-PSBH and the antihole.12 Letters A and B in the inset locate the low and high energy tails of the antihole. The expression for the hole spectrum was modified to take into account the antihole, whose position and shape were determined by fitting the experimental spectrum in the left inset.12 The resulting antihole is shown in the upper inset of Figure 7 and was used to fit the 50, 150, 670, and 5900 mJ/cm2 spectra in that figure. Thus, it is assumed that the shape and position of the antihole is independent of burn fluence; only its intensity was an adjustable parameter in the fitting. The fits to the 50


Figure 7. Hole spectra of APT/HGW for four burn fluences, T ) 5 K. The fluorescence excitation spectrum is shown at the top. The left inset shows the fit (eq 1) to a spectrum obtained with a fluence of 16 mJ/cm2. The arrow to the right of the ZPH locates the real-PSBH in the calculated spectrum. A and B indicate antihole absorption. The right inset shows the antihole profile.

and 150 mJ/cm2 spectra are good and essentially indistinguishable from the experimental spectra. However, the fits to the 670 and 5900 mJ/cm2 spectra are poor. Thus, the assumption that the position and shape of the antihole is independent of burn fluence fails at sufficiently high burn fluences. It can be seen that the amplitude of the peak of the antihole located to the blue of the ZPH in the experimental spectra has increased relative to the broad underlying antihole that extends several hundred wavenumbers to higher energy of the ZPH. These results, together with others presented in ref 12, suggest that a “second channel” for hole burning becomes important at higher burn fluences and that it is responsible for the above increase in peak amplitude. The results can be qualitatively explained by postulating the existence of extrinsic multilevel systems (MLSext). One plausible scenario is shown in Figure 3B for a three-level system in which burning of configuration I leads to antihole site (II), whose ZPL is red-shifted relative to ωB. Following relaxation to the ground state, site II can absorb ωB photons via its phonon sideband and revert to site I with rate constant k2. This is an example of light-induced hole filling (LIHF). Site I can then be reexcited and converted to site II. This process could be repeated many times. However, site II can be converted to site III (with rate constant k3) that is blueshifted relative to ωB. Thus, LIHF is not possible for site III. For this reason, we refer to configuration III as a “terminal” site, one that cannot absorb at ωB. With k3 , k2 and the II f III conversion being essentially irreversible, one can understand how multiple excitations eventually lead to an antihole that is, for all intent and purposes, entirely blue-shifted relative to ωB, see Figure 2. We hasten to add that the results in Figure 6 show that blue-shifted antihole sites are also populated at low burn fluences. Those sites may also be referred to as terminal. In summary, the new results lead to two modifications of the Shu-Small NPHB mechanism. First, both red- and blue-

Reinot et al. shifted antihole sites are produced in the low to moderate burn fluence stages, and second, introduction of a “second channel” for hole burning is required to understand the evolution of the hole spectrum with increasing fluence. A plausible explanation for this “second channel” is the involvement of MLSext. The importance of TLSint-TLSext coupling and excess free volume still remain. Further support for the importance of the latter is provided by our study of free base pthalocyanine in HGW. Although the mechanism for hole burning in this material is primarily photochemical involving phototautomerization, it has hole-burning properties similar to those of APT/HGW. At 5 K, the low fluence ZPH width is ∼180 MHz in annealed films and ∼450 MHz in fresh films. The hole-burning efficiency is also similar to that of APT/HGW, and ωm is ∼ 35 cm-1, nearly identical to that of APT/HGW. Unlike APT/HGW, however, is the observation that warming to 170 K (well above the transition to cubic ice) and recooling to 5 K had a negligible effect on the hole-burning efficiency (unpublished results). This observation, in combination with the earlier observation that NPHB of APT/HGW decreases by >1000 fold on crystallization, provides further strong evidence for the importance of excess free volume in NPHB but not for photochemical hole burning. C. Nonphotochemical Hole Burning: Optical Storage Potential. As discussed by Moerner et al.,51 the potential of persistent spectral hole burning for high density frequency and time domain optical memory, processing, and other applications was recognized early on. They provide a thorough discussion of material requirements for applications. Analysis is based, in part, on storage density Fs ) (Γinh/γ), acceptable write/read rates, and nondestructive readout (minimization of hole burning and light-induced hole filling). Storage densities of 103-107 have been projected (see refs 52-54 for a perusal of such systems). For annealed APT/HGW, Fs ) 125 and 105 at 77 and 5 K, respectively.39 At 77 K, ZPH with a fractional depth of 0.1 are obtainable with a burn fluence of only 1.5 mJ/cm2. The photon budget at 4 K is several orders of magnitude lower. Furthermore, at both temperatures the holes exhibit an impressive resilience against destructive readout from hole burning and LIHF; it was estimated that 108 readouts could be executed before refresh was necessary.39 Contrary to the conclusion reached in ref 51, our results show that amorphous molecular NPHB materials deserve closer attention for optical memory/processing, communications and other applications. In Figure 1, a fluorescence excitation spectrum for APT in annealed HGW is shown in which more than 10 000 holes have been burned. These holes were burned from high to low energy with a fluence of 28 µJ cm-2 for each hole. The holes were generated with the laser described in section II using locally developed software to position the laser at the correct burn frequency, then opening a shutter for 0.1 s to burn the hole. The laser was then scanned to the next burn position with the shutter closed. In this manner, a group of seven holes spaced at 1.5 GHz intervals was burned. The laser wavemeter was then used to position the laser for the next group of holes to be burned. The first hole in each new group was spaced 1.0 GHz from the last hole in the previous group. The exposure time of the sample to burn the holes shown was ∼20 min. However, because of the iterative nature of the scanning/wavelength determination program, the hole burning took ∼4 h. Holes were read out scanning from low to high energy with a fluence of 9 nJ cm-2 and a resolution of 0.5 GHz. With the time required for burning and scanning, the last holes were detected 20 h after burning. The inset of the figure shows a representative section of the scan on an expanded scale. For burns near the absorption

Feature Article maximum, the fluorescence detected is ∼50 000 counts s-1 with a signal-to-noise ratio of ∼15. In the tail of the absorption, the fluorescence signal is only ∼3000 counts s-1, the hole depth is ∼300 counts s-1, and the signal-to-noise ratio is ∼4. Thus, even relatively shallow holes are easily detected. We note that the storage density of APT in water-containing poly-HEMA films is only a factor of 10 lower than that of APT/HGW and that its photon budget is acceptably low.10 D. Applications of Hole Burning to Photosynthetic Complexes. Nonphotochemical, photochemical, and triplet bottleneck hole-burning spectroscopies have proven to be powerful, highresolution frequency domain techniques for studying the excitedstate Qy-electronic structure and excitation energy/electrontransfer processes of photosynthetic protein-chlorophyll complexes. The types of information they provide include (a) the static inhomogeneous broadening (Γinh) of S0 f S1 (Qy) chlorophyll (Chl) transitions via ZPH action spectra,55-63 (b) electron-phonon coupling parameters (S and ωm) and intramolecular Chl Franck-Condon factors of those transitions via vibronic satellite hole structure, (c) the extent of correlation between the site excitation distribution functions (SDF) of different Qy states, and (d) the excitation energy transfer (EET) and electron-transfer rates from the zero-point vibrational level. These attributes were already recognized in the early 90s (for reviews see refs 64 and 65). Results of types (a) and (b) are essential for calculation of EET rates because they determine the spectral density in the nonadiabatic rate expression. This is also the case for (c). In this regard, hole-burned spectra have shown that the SDF of different Qy states are generally uncorrelated, which means that the donor-acceptor electronic energy gap value is distributed.66 When the width of this distribution is broader than the homogeneous width of the spectral density, the kinetics can be expected to be dispersive. The reader interested in rate calculations that take this into account and use, as input, hole-burning data is referred to refs 66-68. Concerning (d), transfer rates from the zero-point level, as determined by hole-burning or photon echo spectroscopies, are especially important when femtosecond pump-probe spectra exhibit beats because of vibrational coherences. The observation of vibrational coherences raises the question of whether they play a role in the transfer dynamics. The first observation of vibrational coherences was made on the special pair donor state, P870*, of the reaction center (RC) of Rhodobacter sphaeroides.69 However, the rate for the initial phase of charge separation at liquid helium temperatures was found to equal that determined by hole burning. Thus, and as discussed in ref 70, the vibrations responsible for the coherences appear to be “spectator” modes. To the best of our knowledge, vibrational coherences have not yet been shown to play an important role in the EET dynamics of photosynthetic complexes. Two recent developments that have broadened the scope of NPHB are its interfacing with external electric (Stark) fields and high pressure. Stark hole-burning spectroscopy has been applied to the Fenna-Matthews-Olson bacteriochlorophyll a antenna complex of Chlorobium tepidum,71 the light harvesting 1 and 2 complexes of purple bacteria,71,72 the chlorosome antenna from C. tepidum,73 the CP43 core antenna complex of PS II,74 and PS I of the cyanobacterium Synechocystis PCC 6803.75 The broadening or splitting of the ZPH with applied field yields the value of f∆µ, where ∆µ is the permanent dipole moment change associated with the ground to excited-state transition and f is the local field correction. Importantly, f∆µ values can be used to identify excitonic states associated with closely spaced Chls whose strong coupling is significantly

J. Phys. Chem. B, Vol. 105, No. 22, 2001 5091 contributed to by electron exchange. Electron-exchange interactions lead to charge-transfer states whose mixing with neutral Qy states endow them with charge-transfer character.76 High pressure-hole burning has been applied to all of the above complexes except CP43,55,56,77-80 as well as the LHC II Chl a/Chl b complex of PS II.81 The linear pressure shift (to the red) rates of ZPH and absorption bands can also be used to identify excitonically coupled states with appreciable chargetransfer character.55,56,79 In what follows, we present recently published and unpublished results on PS I of two cyanobacteria which illustrate the positive correlation between the magnitudes of f∆µ, the linear pressure shift rate, and the linear electron-phonon coupling strength. Red Antenna States of PS I. An interesting feature of several PSs, including PS II of green plants,82,83 is that the lowest energy Qy state of at least one antenna complex in close proximity to the RC lies lower in energy than the primary electron donor state of the RC. PS I of green plants, algae, and cyanobacteria is another example (for a review of early works see ref 84). The only PS I complex for which an X-ray structure is available is that of the cyanobacterium Synechococcus elongatus.85,86 The 4 Å resolution structure clearly reveals the “special pair” of Chl a molecules (P700) of the RC whose lowest energy absorption is at ∼700 nm. P700*, the asterisk indicating the S1(Qy) excited state, is the primary electron donor state. The two Chl a molecules are strongly coupled as evidenced by the observation that the optical reorganization of the S0 f P700* transition because of low-frequency phonons is large, ∼200 cm-1,87 and comparable to those of the analogous special pair transitions in the RC of the purple bacteria Rhodobacter sphaeroides and Rhodopseudomas Viridis (for a detailed comparison see ref 70). At low temperatures, the red absorbing Chl a molecules of Synechococcus give rise to absorption bands at 708 and 719 nm. These Chls are often referred to as C-708 and C-719.88 It was shown in ref 88 that selective excitation at room temperature at 719 nm and longer wavelengths results in efficient charge separation in the RC. Thus, thermally activated EET from the red antenna states to P700 is efficient. Such red excitations at liquid helium temperatures do not lead to charge separation because kT , the relevant energy gaps. In the case of the cyanobacterium Synechocystis PCC 6803 (hereafter referred to as Synechocystis), the red antenna Chl a molecules (C-708) give rise to a broad absorption band at ∼708 nm.89 The 4 K Qy absorption spectra of the wild-type (WT) trimer of Synechocystis and Synechococcus are shown in Figure 8 (frame A). The existence of red antenna states raises two immediate questions, one being how they affect the efficiency of EET from the bulk antenna Chls to the RC. Trissl has argued that the deleterious effects of the red “trap” states is compensated by the enhanced absorption at longer wavelengths.90,91 They can also serve as reservoirs which function to funnel energy to the RC and to reduce the probability of back transfer to the bulk antenna network where nonproductive radiationless decay processes can occur. The second question, which we address here, pertains to the structure of the red antenna states that leads to their lying lower in energy than P700* which, again, is associated with a strongly coupled dimer. The results presented below point to their being due to strongly coupled dimers (or, possibly, a trimer) rather than monomers whose structures and/ or special interactions with the protein result in large red shifting. However, first, we briefly discuss the overall structure of PS I of cyanobacteria on the basis of the X-ray structure of Synechococcus. WT PS I complexes exist in trimeric and


Reinot et al.

Figure 9. Depiction of the protein subunits of cyanobacterial PS I. The schematic shows the top view of a monomer and is based on information from X-ray crystallography.

Figure 8. A. Absorption spectra of Synechococcus (a) and Synechocystis (b) at 4.2 K. To form a glass, sample solutions containing 10 mM MOPS-HCl (pH 7) and 0.05% dodecyl-2-maltoside were diluted with glycerol (2:1 v/v). B. Hole spectra of the two complexes following burning at 670 nm with a burn fluence of 900 J cm-2.

monomeric forms.92 A monomer contains eleven proteins (see Figure 9), about 90 Chl a molecules, 10-12 β-carotene molecules, two phylloquinones, and three [4Fe-4S] clusters. The phylloquinones and one of the [4Fe-4S] clusters belong to the RC which also houses six Chl a molecules, two of which are those of P700. These are located at the interface between the PsaA and PsaB proteins, which form a heterodimeric core. The reader interested in the spatial arrangement of the RC cofactors is referred to refs 85 and 86. The heterodimeric core contains about 80 Chl a antenna molecules (PS I of cyanobacteria are devoid of Chl b molecules unlike PS I of green plants). The PsaL protein is located at the trimer-forming side. Proteins PsaF, PsaI-M are integral membrane proteins. The PsaL and PsaK proteins are known to bind Chl a molecules; PsaL binds three. Because of the lack of Chl-coordinating residues, the PsaI protein is not thought to bind Chl a. In what follows, we refer to the proteins as A, B, etc. We recently reported the results of 4 K absorption and holeburning studies of the WT trimer, WT monomer, and mutants deficient in the L, K, F, and M proteins of Synechocystis.75 Comparison of the differences in absorption intensity of the 708 nm band led to the conclusion that the C-708 molecules belong to proteins A and B and, furthermore, that they have fragile structures which are sensitive to structural changes at the trimer forming side of PS I such as produced by formation of the monomer from the trimer (see also ref 89) and deletion of the M protein. Deletion of the F and K proteins, which are located

at the nontrimer forming side, did not affect the absorption intensity of the 708 nm band. Returning to frame A of Figure 8, the WT trimer Qyabsorption spectra of Synechococcus and Synechocystis are quite similar at wavelengths shorter than 700 nm. At longer wavelengths, the spectra are significantly different, with Synechococcus exhibiting two red bands at ∼708 and 719 nm and Synechocystis a single band at 708 nm. The integrated intensity of the red absorption for Synechococcus was estimated to be equivalent to that of 9-11 Chl a molecules88 (our estimate is 7, vide infra) and for Synechocystis ∼4 Chl a molecules.75 In frame B, persistent NPHB spectra obtained with λB ) 670 nm (coincident with the relatively sharp ZPH) are shown. The broad satellite holes at wavelengths longer than 670 nm are due to the burning of states populated by downward EET from the state(s) pumped at 670 nm. Note that the Synechococcus spectrum exhibits two broad holes at 708 and 717 nm, with the former appearing to correspond to the 708 nm absorption band. The 717 nm hole is most likely mainly associated with the 719 nm absorption band because, when the interference of the 717 nm hole by the 708 nm hole is taken into account, the adjusted position of the former hole is ∼719 nm. Synechocystis, however, exhibits only a single red hole at 714 nm, 6 nm longer than 708 nm of the absorption band. The 6 nm difference led Ra¨tsep et al.75 to conclude that more than one red antenna state contributes to the 708 nm absorption band. By way of digression, it was shown that sharp ZPH (fwhm ∼1 cm-1) coincident with λB could be burned throughout the entire Qyabsorption spectrum. (This is also the case for Synechococcus, results not shown.) This, and the observation of the broad (∼ 100-300 cm-1) satellite holes shown in frame B of Figure 8, establish that PS I is yet another example of a photosynthetic complex in which the SDF of different Qy states are uncorrelated. Figure 10 compares the 4.2 K hole spectra of Synechocystis reported in75 for six λB values (702-722 nm) with those of Synechococcus. The burn fluences are given in the caption. The sharpest hole in each spectrum is the ZPH coincident with λB. The solid vertical lines to the left and right of the ZPH locate the real and pseudo-PSBH, respectively, which are displaced by ∼20 cm-1 from the ZPH. Thus, in the simulations of the

Feature Article


TABLE 2: Parameters Used for Fitting of Hole Spectra of PS1 of Synechococcus and Synechocystisa

Synechococcus Synechocystis

Γinh (cm-1)

γ (cm-1)

ω1 (cm-1)

S1

Γb (cm-1)

ω2 (cm-1)

S2

Γc (cm-1)

λfmax (nm)

250 300

0.5 0.5

20 18

1.8 1.6

13, 15 15, 15

110 70

0.4 0.4

100 65

722 730

a The experimental maxima of the two complexes are also listed in the last column. b The ω phonon profile is modeled as a Gaussian on the low 1 energy side and a Lorentzian on the high energy side with half-widths of 13 and 15 cm-1, respectively. c The ω2 phonons are modeled as a Gaussian -1 with a fwhm of 100 and 68 cm for Synechococcus and Synechocystis, respectively.

Figure 10. Comparison of the hole spectra of Synechocystis (on the left) with those of Synechococcus (on the right) for the λB values shown. The burn fluences were 450 J cm-2. The vertical lines to the right and left of λB indicate the peaks of the real- and pseudo-PSBH. The vertical arrows at 692 and 699 nm for Synechocystis and at 694 and 699 nm for Synechococcus indicate high energy satellite holes which are discussed in ref 75 for Synechocystis.

hole spectra shown later, the peak frequency of the lowest energy phonons that couple to the optical transitions is fixed at ∼20 cm-1. Preliminary analysis of the Synechocystis spectra given in ref 75 indicated that modes at ∼100 cm-1 also contribute to the PSBH structure. The asterisks in Figure 10 locate the maximum of the broad, blue-shifted antihole associated with NPHB. Possible explanations for the high energy satellite holes at 692 and 699 nm for Synechocystis are given in ref 75. The corresponding features of Synechococcus are at 694 and 699 nm. For both species, the hole spectra of Figure 10 show that as λB is tuned to shorter wavelengths the ZPH at λB becomes more prominent relative to the broad underlying PSBH structure to higher and lower energy of the ZPH. On the basis of the theory of hole spectra, this is inconsistent with only a single state contributing to the red absorption. For Synechococcus, this is already obvious from its absorption spectrum. This is not the case, however, for Synechocystis. Theoretical simulations of the λB and burn fluence dependencies of the hole spectra for Synechocystis were presented in ref 93. They were obtained using eq 2 but with neglect of the R distribution which does not affect the spectral structure. A more exact form than eq 3 for the single site absorption profile was used that allows for participation of more than one type of phonon and includes multiphonon transitions (eq 3 of ref 93). Where the λ distribution in eq 2 is concerned, the experimental burn fluences were adjusted to compensate for dispersive kinetics and to provide good fits to the λB ) 722 nm spectra obtained with four burn fluences. This wavelength was chosen because it should be selective for the lowest energy red antenna state. The fits to the burn fluence dependence of the λB ) 722 nm spectra were obtained with the parameter values given in Table 2. These values, together with the same adjusted burn fluences, were then used to fit the hole spectra obtained with

Figure 11. Fits (using eq 2 and the parameters of Table 2) to the λB ) 714 nm holes of Synechocystis. For clarity, the calculated spectra are offset from the experimental spectra by 5 cm-1. The burn fluences for spectra a-d were 6, 30, 150, and 450 J, respectively. The asterisk marks the peak of the antihole. and the 699 nm satellite hole is also indicated.

λB ) 718, 714, 710, and 706 nm. Good fits for the λB ) 718 nm spectra were obtained. The fits for λB ) 714 nm, although not quite as good, are reasonable, Figure 11. The quality of the fits should be judged on the basis of the pseudo-PSBH, ZPH and region of the real-PSBH near its maximum (eq 2 does not account for the broad, blue-shifted antihole (see asterisk) that interferes with the real-PSBH on its high energy side.) For λB shorter than 714 nm, the fits worsened, as shown in Figure 12 for λB ) 706 nm which is close to the maximum of the 708 nm absorption band. As discussed in ref 93, the results establish that there are two states which contribute to the 708 nm absorption band of Synechocystis: the 714 nm state whose SDF lies at 717 nm (Table 2) and an ∼706 nm state with an SDF located at ∼708 nm. It was shown in ref 93 that the 706 and 714 nm states do not belong to the same aggregate of Chl molecules. Note that the electron-phonon coupling of the 714 nm state is strong, with a total S of 2.0, in sharp contrast with the weak coupling (S j 0.5) generally observed for antenna states highly localized on a single Chl molecule.1,81,65 The S value of the 706 nm state was estimated at 1.3,93 which explains why the ZPH in Figure 12 are more pronounced than in Figure 11. (In the low burn fluence limit, the Franck-Condon factor for the ZPH is exp(-2S).) That the S value of 2.0 is comparable to the value of ∼3 for the primary electron donor state of the RC of purple bacteria indicates that the 714 nm state is associated with a strongly coupled dimer (or possibly a trimer) and that it possesses significant charge-transfer character. Experiments and simulations of the above type were recently performed on PS I of Synechococcus. Simulation of the hole spectra obtained with λB ) 718, 722, and 725 nm resulted in


Figure 12. Fits (as in Figure 11) for λB ) 706 nm. In this figure, there is no offset between the calculated and experimental curves. The calculated curves are scaled so that the ZPH depths match those of the experimental curves. The burn fluences are as in Figure 11.

the parameter values for the red-most 719 nm antenna state given in Table 2. For the sake of brevity, we do not show the spectra and fits which are good for the ZPH and pseudo-PSBH. Given the similarity between the parameter values for Synechococcus and Synechocystis, it is reasonable to conclude that the dimers of the two species associated with the red-most antenna state have similar structures. That the absorption (also SDF) maxima differ by ∼5 nm may be due to differences in interactions with the protein. The fluorescence origin band is predicted to lie ∼ΣiSiωi (i ) 1 and 2) lower in energy than the maximum of the SDF. On the basis of the parameter values in Table 2, the static fluorescence origin bands of Synechocystis and Synechococcus are predicted to lie at 720 and 727 nm which are in reasonable agreement with the experimental values given in that table. The ∼2 nm discrepancy may be due to protein conformational changes that occur prior to fluorescence which occurs on a nanosecond time scale. Returning to the theoretical fits of the Synechococcus hole spectra, we found that the onset of poor fits occurred at burn wavelengths considerably longer than those for Synechocystis. For example, the poorness of the fits for λB ) 714 nm (not shown) is comparable to that seen in Figure 12 for Synechocystis (λB ) 706 nm). Thus, there is a higher energy state in Synechococcus whose absorption at 714 nm is appreciable and whose electron-phonon coupling is weaker than that of the 719 nm state. It is unlikely that it is the state associated with the 708 nm absorption band (Figure 8A) because Synechocystis exhibits a broader band near 708 nm and the fits to its λB ) 714 nm hole spectrum in Figure 11 are quite good. That is, the 714 nm state is the major contributor to the hole spectrum. We propose that Synechococcus possesses a third red state that lies between the 719 nm state and the state responsible for the 708 nm absorption band. In what follows, we will refer to this state as R2. As mentioned, the results in Table 2 indicate that the 714 and 719 nm states of Synechocystis and Synechococcus are analogous. The Stark results provide additional support for this, vide infra. Concerning the 708 nm state of Synechococcus, it may correspond to the 706 nm state of Synechocystis, although the absorption band of the former state is considerably sharper, Figure 8A. With that assignment, the R2 state would be unique

Reinot et al. to Synechococcus. On the other hand, the R2 state could be the 706 nm state of Synechocystis, leaving the 708 nm state as the new state of Synechococcus. Clearly, an X-ray structure of PS I of Synechocystis is required for a detailed comparison of the red antenna states of the two species. Very recently the resolution of the X-ray structure of PS I of Synechococcus was improved to 2.5 Å.94,95 There are 90 antenna Chl a molecules; the RC contains six. With these numbers, deconvolution of the low energy region of the absorption spectrum of Synechococcus led to the intensity of its red absorption being equivalent to that of seven Chl a monomers. The calculation took into account that under the conditions of our experiments about 50% of the P700 dimers are oxidized.75 (The Qy-absorption spectrum was cut at 650 nm (see Figure 8A).) We confirmed that75 the red absorption intensity of Synechocystis is equivalent to that of approximately four Chl a monomers. These results are consistent with Synechococcus possessing an additional red dimer (or trimer) state. It was concluded in ref 75 that the 706 and 714 nm states of Synechocystis are due to different dimers. The 2.5 Å resolution structure reveals three dimers and one trimer that are candidates for the red Chl a molecules.95 Mg‚‚‚Mg distances are in the 7.6-8.9 Å range; interplanar separations are ∼3.5 Å. Interestingly, Synechocystis is not expected to bind the trimer (N. Krauβ, private communication) which suggests that the trimer, which has a ladder-like structure, may be responsible for the additional red absorption of Synechococcus. It was suggested in ref 75 that both dimers in Synechocystis are coordinated to PsaA or B and located close to PsaL at the trimer forming side. This is the situation for the dimer with the short 7.6 Å Mg‚‚‚Mg distance and interplanar separation of 3.5 Å. This dimer would seem to be a plausible candidate for the redmost state of the two species. Unfortunately, the coordinates have not yet been released, and thus, we are unable to perform excitonic calculations that might lead to definite assignments. Stark hole-burning experiments were performed on PS I of Synechococcus using 12 λB values between 692 and 720 nm. Stark splitting of the ZPH was not observed with a read resolution of 0.5 cm-1, only Stark broadening. This is consistent with the results for Synechocystis.75 The absence of splitting may be a consequence of the direction of the matrix (protein)induced contribution to the permanent dipole moment change, ∆µ, varying somewhat from complex to complex. Stark broadening data for λB ) 706, 714, and 718 nm at T ) 1.8 K are shown in Figure 13. The solid lines are fits obtained using the theory of Kador et al.96 They define the parameter

F)

2f∆µES pγh

(4)

where ∆µ is the magnitude of the dipole moment change, Es is the applied field strength, f is the local field correction and γh is the width of the ZPH at zero applied field. When F j 3.5, which is the case in our experiments, their general expression for Stark broadening reduces to

Γ(F) ) γh(1 + F2)1/2

(5)

In the high field limit, Γ(F) ∝ Es. The f∆µ values for the above burn wavelengths are 0.60, 1.3, and 2.2 D. They are given in Table 3 along with the values for the other λB’s. For comparison, the results for Synechocystis from ref 75 are also given. Because the local field correction is not known, we prefer to report f∆µ values but note that f ∼ 1.5 (based on the Lorentz local field correction) for proteins has often been used.

Feature Article


Figure 13. Dependence of the 1.8 K widths of the zero-phonon holes of Synechococcus burned at λB ) 706, 714, and 718 nm on an electric (Stark) field. The solid curves are fits using eq 5. The f∆µ values are 0.6, 1.3, and 2.2 D, respectively.

TABLE 3: Values of f∆µ for Synechocystis and Synechococcus as a Function of λBa

a

system

λB, nm

f∆µ (D)

WT trimer PS I of Synechocystis

690.0 692.0 695.0 698.5 702.0 706.5 708.0 710.0 712.0 714.0 716.0

0.5 ( 0.20 0.6 ( 0.10 0.5 ( 0.10 0.7 ( 0.10 0.6 ( 0.10 0.8 ( 0.15 1.0 ( 0.40 2.0 ( 0.40 1.8 ( 0.20 2.3 ( 0.20 2.4 ( 0.20

WT trimer PS I of Synechococcus

692.0 694.0 696.0 698.0 701.0 704.0 706.0 708.0 710.0 712.0 714.0 716.0 718.0 720.0

0.5 ( 0.20 0.7 ( 0.20 0.8 ( 0.30 0.5 ( 0.20 0.6 ( 0.20 0.8 ( 0.20 0.6 ( 0.20 0.7 ( 0.30 1.0 ( 0.20 1.0 ( 0.20 1.3 ( 0.20 2.2 ( 0.20 2.2 ( 0.20 2.3 ( 0.30

Ch a:PVB polymer films

666.0 674.0

0.52 ( 0.05 0.52 ( 0.05

Chl a:LHC II

680.0

0.63 ( 0.10

Values for Chl a in a PVB film and in LHC II are also included.

As discussed in detail in ref 75, the f∆µ values for monomer Chl a in polymers or a Chl a molecule in a protein that is not strongly coupled to others is ∼0.5-0.6 D. The values for Chl a in a poly(vinylbutyral) film and in the LHC II antenna complex of PS II determined by hole burning are 0.52 and 0.63 D, Table 3. The latter value corresponds to the lowest energy Qy state of LHC II, which is highly localized on a single Chl a molecule.81 For the five or six shortest burn wavelengths, the f∆µ values for Synechococcus and Synechocystis are monomer-like (Table 3), consistent with the weak electron-phonon coupling observed at those wavelengths (unpublished results). Thus, the states excited are not associated with Chl a molecules whose

intermolecular spacings are short enough to result in electronexchange coupling of a magnitude that leads to appreciable charge-transfer (CT) character for the excited states. Substantial charge-transfer character can be expected to lead to a large dipole moment change which, in turn, leads to strong linear electron-phonon coupling. The reader is referred to ref 97 for an excellent discussion of the physics involved. In sharp contrast, the three or four longest burn wavelengths result in the largest f∆µ values yet observed for Chl a, ∼2.2 D. This value is only about a factor of 2 lower than that of the primary electron donor state (P870*) of Rb. sphaeroides98 which is known to possess significant charge-transfer character as reviewed in ref 70. A detailed discussion of the correlation between charge-transfer character and the dipole moment change is also given in ref 98. It is reasonable to conclude that the lowest energy red antenna state of Synechocystis and Synechococcus at 714 and 719 nm, respectively, is associated with a strongly coupled dimer (or, possibly, a trimer). The high-pressure hole-burning results are consistent with this, vide infra. This lowest energy state could be referred to as a “special pair” antenna state. The results in Table 3 provide support for our assertion that Synechococcus possesses a third red antenna state (R2) that lies between the 708 and 719 nm states. As discussed, it contributes significantly to absorption at 714 nm. The key result is that for λB ) 714 nm f∆µ equals 1.3 and 2.3 D for Synechococcus and Synechocystis, respectively. The significantly lower value for Synechococcus cannot be understood in terms of a two-state model. Finally, we point out that for Synechocystis the 706 nm state does not appear to contribute significantly to the 714 nm ZPH because its f∆µ value is ∼0.8 D (see 706.5 nm entry in Table 3). To conclude this subsection, we briefly consider the highpressure hole-burning results for Synechocystis reported in ref 75. (Such experiments are planned for Synechococcus.) The property of interest is the linear pressure shift rate, δν/δP ≡ Rp, of the ZPH. For 1ππ* states of isolated chromophores in polymers and glasses99 and Qy states of photosynthetic complexes that are highly localized on a single Chl molecule,55,56,76,80 Rp ∼ -0.05 to -0.15 cm-1/MPa (0.1 Mpa ) 1 atm). We mention in passing that in this case the theory of Laird and Skinner100 can be used to determine a “local” isotropic compressibility (κ) which has often been found to be in reasonable agreement with the bulk compressibility. For polymers, organic glasses, and proteins, κ ) ∼0.1 GPa-1. The above theory is not applicable to an excitonically coupled system. In a series of papers on the effects of pressure on the Qy states of photosynthetic complexes,55,56,79 it was concluded that |Rp| values > ∼0.2 cm-1/MPa are indicative of a state involving coupled Chl molecules. Values J0.4 cm-1/MPa signal that electron-exchange interactions contribute significantly to Rp. For example, the Rp values for the lowest exciton level of the strongly coupled B850 and B875 BChl a rings of the LH2 and LH1 complexes of purple bacteria are close to -0.6 cm-1/ MPa.55 A simple theoretical model was introduced that allows one to estimate the contribution from electron-exchange interactions to Rp when a structure of the coupled Chl molecules is available.55 A structure allows one to estimate the contribution from Coulombic interactions using a reasonable value for κ (∼0.1 GPa-1) and reasonable value of -0.1 cm-1/MPa for the contribution from protein-Chl interactions. For example, the contribution to Rp from electron-exchange coupling to the shift rate of -0.67 cm-1/MPa for the lowest exciton level of LH1 was estimated at -0.45 cm-1/MPa.56 Such estimates can serve

5096 J. Phys. Chem. B, Vol. 105, No. 22, 2001 as benchmarks for electronic structure calculations that take into account charge transfer states. Because the coordinates of the 2.5 Å resolution X-ray structure of Synechococcus have not been released, we are constrained to make some general remarks concerning the f∆µ values for Synechocystis whose structure is expected to be very similar. The values for λB ) 688, 690, 692, 694, 696, 710, 713, and 718 nm are -0.17, -0.19, -0.19, -0.20, -0.20, -0.42, -0.45, and -0.49 cm-1/MPa with an uncertainty of e0.02. With reference to the f∆µ values in Table 3, one observes that the large ∼ -0.4 cm-1/MPa Rp values correlate with the large f∆µ values for the lowest energy red antenna state at 714 nm. Importantly, there is also a correlation with strong linear electron-phonon coupling. Thus, PS I serves as an excellent example of a complex that exhibits positive correlation between these three important properties. 4. Concluding Remarks and Future Prospects A Holy Grail for many interested in structural disorder and configurational tunneling in glasses at low temperatures has been to obtain data that signal a breakdown of the standard tunneling model. Studies of the pure dephasing and spectral diffusion of the ZPL of imbedded chromophores have failed to do so. Spectral diffusion data obtained over times ranging from nanoseconds to hours can be explained using distribution functions for the tunnel parameter (λ) for the TLSint and more slowly relaxing TLSext (see ref 21 and references therein). Inclusion of distribution functions for the asymmetry parameter (∆) would lead to greater flexibility in fitting, especially if correlation between ∆ and λ was included. Recently, Boiron et al.101 reported on the spectral diffusion of single terrylene molecules in polyisobutylene films at 2 K because of tunneling with terrylene in its ground state. The frequency range covered was 20 GHz. The “spectral trails” were followed for 1000 s with a time resolution of 1 s. About 70% of the trails of single molecules were consistent with the standard tunneling model. The results for the other molecules indicated, in part, that multilevel systems need to be considered. This could be viewed as a breakdown of the standard tunneling model, although Boiron et al. urge caution in drawing such a conclusion because photoinduced spectral jumps, which they attempted to avoid, could not be excluded as being responsible for the nonstandard tunneling model molecules. We note that single molecule spectroscopy is not a practical approach to studying photoinduced spectral jumps (NPHB) because this would require tracking over hundreds of reciprocal centimeters. The results on NPHB presented point convincingly to a breakdown of the standard tunneling model. Our mechanism for NPHB has it being due to a hierarchy of configurational relaxation events triggered by electronic excitation that begin with the TLSint and terminate with the TLSext. TLS-TLS coupling and diffusion of excess free volume are key ingredients. The importance of excess free volume was established using a novel hyperquenching apparatus that allowed for formation of the crystalline phases of water and ethanol from the hyperquenched glassy films. A detailed study of the hole spectra of APT/HGW led to the conclusion that the Shu-Small mechanism requires some modification, specifically, the inclusion of extrinsic multilevel systems in which tunneling eventually leads to a terminal configuration that absorbs to higher energy of the burn frequency, ωB. This model explains why the antihole for 1ππ* states is essentially entirely blue-shifted relative to ω B for sufficiently high burn fluences. Data of unprecedented detail on the dispersive kinetics of ZPH growth in annealed APT/

Reinot et al. HGW were presented which prove that the λ distribution is of primary importance, with the intrinsic distributions being due to laser polarization and off-resonant absorption of the zerophonon to the dispersive kinetics becoming important only for the last ∼20% of the burn. These conclusions should be generally applicable to other NPHB systems. The most detailed NPHB experiments have been performed on 1ππ* states of dye molecules in H-bonding glasses (water, methanol, ethanol, and glycerol) and polymers, e.g., poly(vinyl alcohol). Although the NPHB quantum yield undergoes a marked decrease upon deuteration of the hydroxyl group of the host, the pure dephasing/spectral diffusion is unaffected. Thus, tunneling of the TLSext involves significant H-bond rearrangement in the inner shell, whereas the effective coordinate of the TLSint involves only small amplitude motion of H atoms. In the case of low density, vapor deposited glassy water, the major source of disorder is a dispersion (∼8°) in the O-O-O angle from 109° for Ih, which is correlated with a departure from the linearity of the O-H‚‚‚O bond (up to 5°).102 (The HGW films we have studied are of low density.) It is conceivable that the TLSint coordinate is associated with hindered rotation of OH4 moieties. The excess free volume in HGW may be associated with the interface between higher and lower density regions, as suggested in ref 103. Molecular dynamics simulations coupled with statistical analysis and quantum mechanical calculations on a Lennard-Jones binary glass have indicated that the TLSint are linked to voids.104 Unfortunately, such calculations on APT/ HGW are not yet feasible. Concerning future studies of NPHB, we have several in mind. In the case of APT and free base phthalocyanine tetrasulfonate (H2-PT) in HGW and HGE, it would be interesting to determine the permanent dipole moment changes for fresh and annealed films by Stark hole-burning spectroscopy because annealing leads to a significant reduction (∼ × 5) in the density of the TLSint. The matrix-induced contribution to ∆µ may be sensitive to that reduction. We recently constructed a liquid-helium cryostat for the study of pressure effects (e15 MPa) on the hole burned spectra of hyperquenched glassy films.105 We anticipate that the linear pressure shift rates of APT and free base phthalocyanine tetrasulfonate in fresh and annealed films will be different because the rates are proportional to the local compressibility. We also plan to study the NPHB efficiency, spectral dynamics, and electron-phonon coupling of APT in high density (1.3 g cm-3 at 80 K) amorphous (HDA) water which can be formed from cubic ice with a pressure of ∼1 GPa at liquid-nitrogen temperatures.106 One might expect that the higher density of HDA water relative to HGW (∼0.95 g cm-3) will lead to a significant decrease in the hole-burning quantum yield and dephasing because of a decrease in the density of TLSint in HDA water, but this remains to be seen. Further experiments on APT in the high water content polymer polyHEMA are planned, with the objective being to determine and characterize the spectral dynamics from 5 to ∼80 K. Photon echo experiments would then be performed at temperatures between ∼80 and 300 K in order to determine whether the theoretical predictions (based on hole-burning data) of the T dependence of dephasing due to electron-phonon coupling associated with inertial modes are met. As a final example, we intend to use our novel hyperquenching apparatus to study the mechanism of NPHB in non-hydrogen-bonding glasses such as benzene and cyclohexane. Terrylene would be a good first choice as the probe molecule. We anticipate that the modified Shu-Small mechanism based on H-bonding glasses will still hold, but this also remains to be seen.

Feature Article We hope that we have convinced the reader that persistent NPHB and triplet bottleneck hole-burning spectroscopies are powerful high resolution techniques for the study of the excited state electronic structure and excitation energy/electron transfer processes of photosynthetic complexes. They can be routinely used to determine the homogeneous and static inhomogeneous broadening contributions to S0 f S1 (Qy) transitions, the extent of correlation between site distribution functions of different excited states, electron-phonon coupling parameters, and Franck-Condon factors and frequencies of intramolecular Chl modes. Hole-burning spectroscopies also yield the decay times of Qy states from their total zero-point vibrational level which is particularly important in assessing whether vibrational coherences observed in femtosecond pump-probe experiments play a role in transport dynamics. In this paper, we have emphasized the additional information that is obtained when NPHB is combined with pressure and external electric (Stark) fields. The results presented for the functionally important “red” antenna states of PS I of cyanobacteria further establish the positive correlation between electron-phonon coupling, linear pressure shift rates, and the permanent dipole moment change associated with Qy states. Noteworthy is that these properties can be used to identify states of Chl aggregates with significant charge transfer character due to electron-exchange coupling. Utilization of the Stark effect and pressure also results in higher selectivity, which is important for resolving and characterizing closely spaced states. Finally, we note, with some gratification, that our NPHB studies of the light harvesting 2 complex of purple bacteria and PS I of cyanobacteria have proven to be important for interpretation of spectroscopic data obtained on single complexes of these systems.107,108 Given the insights gained by combining NPHB with pressure and external electric fields, it would be interesting to employ pressure and the Stark effect in future studies of single photosynthetic complexes. Acknowledgment. Primary support for this research was provided by the Solid State Chemistry and Polymers Program of the National Science Foundation under Grant No. DMR9908714. Support from the Division of Chemical Sciences, Office of Basic Energy Sciences of USDOE is also acknowledged. We are indebted to Professor P. Chitnis and Dr. T. W. Johnson for providing the Synechococcus samples and Professor Norbert Krauβ for providing us with a preprint of ref 95 and a useful discussion. Useful discussions with Dr. Ryszard Jankowiak are also acknowledged. Finally, we would like to thank present and past graduate students and postdoctoral fellows who have contributed to our research on NPHB. Abbreviations and Acronyms APT: aluminum phthalocyanine tetrasulfonate. Chl: chlorophyll. EET: excitation energy transfer. fwhm: full width at half-maximum. HGE: hyperquenched glassy ethanol. HGM: hyperquenched glassy methanol. HGW: hyperquenched glassy water. NPHB: nonphotochemical hole burning. PS: photosystem. PSBH: phonon sideband hole. RC: reaction center. SDF: site distribution function. TLS: two level system. WT: wild type.

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