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The CP43 chlorophyll a-core protein complex plays an important role in funneling excitation energy absorbed by more peripheral antenna complexes of photosystem II (PSII) to the reaction center (RC). Identification and characterization of the lowest energy Q y -states of CP43 is important for understanding the kinetics of excitation energy transfer (EET) from CP43 to the RC. We report the results of several types of spectroscopic experiments performed at liquid He temperatures on the isolated CP43 complex from spinach. Nonphotochemical hole burning (NPHB) and triplet bottleneck hole burning spectroscopies as well as zero-phonon hole (ZPH) action and Stark hole burning spectroscopies were employed. Two quasi-degenerate trap states at 682.9 nm (B state) and 683.3 nm (A state) are identified. The widths of their mainly inhomogeneously broadened Q y -absorption bands are 45 and 120 cm -1 , respectively. The uncorrelated site excitation distribution functions (SDF) of the two states are nearly the same as their absorption bands since the electron-phonon coupling is weak (optical reorganization energies of ∼6 cm -1 ). The NPHB spectra establish that the B state is the primary trap for EET from higher energy Q y -states. The permanent dipole moment change (∆µ) of the S 0 f Q y transition for both the B and A states is small, f‚∆µ ) 0.25 ( 0.05 and 0.47 ( 0.05, respectively, where f is the local field correction factor. These values, together with the weak electron-phonon coupling and other results, indicate that both states are highly localized on a single Chl a molecule. Holewidth measurements led to the remarkable finding that the rates of A f B and B f A EET processes are extremely slow, ∼(6 ns) -1 . This suggests that the Chl a molecules of the two states belong to different layers of Chl a molecules located at opposite sides of the membrane. The intriguing question of why CP43 possesses two quasi-degenerate trap states that are so weakly coupled is addressed. The possibility that they play a role in the photoinhibitory and photoregulatory processes is raised.

Introduction 4546 2. General Information on the Structure of Photosynthetic Complexes and StructureÀFunction Relationships 4548 2.1. Photosystem I (PSI) and Photosystem II (PSII) 4548 2.2. Basic Aspects of Bacterial Photosynthesis 4549 3. Interpigment Interactions, Excitation Energy Transfer (EET), and Charge Separation (CS) Rates-General Considerations 4550 4. Fundamentals of Spectral Hole-Burning (SHB) and Fluorescence Line-Narrowing Spectroscopy (FLNS) and Single Photosynthetic Complex Spectroscopy (SPCS) 4552 4.1. Zero-Phonon Lines, Homogeneous and Inhomogeneous Broadening 4553 4.1.1. ZPLs and Phonon Sidebands (PSBs) 4554 4.1.2. ElectronÀPhonon Coupling and Homogeneous Line Shapes 4555 4.2. Nonphotochemical, Photochemical, and Transient SHB Spectroscopy 4557 4.3. Mechanism of Nonphotochemical Hole-Burning (NPHB) 4558 4.4. Kinetics of NPHB 4559 4.5. Zero-Phonon Action (ZPA) Spectroscopy: Site Distribution Function (SDF) 4560 4.6. Hole Shapes and FLN Line Shapes-Electron Phonon Coupling and ΔFLNS 4561 4.7. Ground and Excited State Vibrational Frequencies 4567 4.8. SHB in Excitonically Coupled Systems 4567 4.9. Basic Principles of SPCS 4568 4.10. Basic Principles of Two-Dimensional Electronic Spectroscopy (2D ES) 4569 5. Examples of Applications of NPHB, FLNS, SPCS, and 2D ES to Photosynthesis 4570 5.1. Light-Harvesting and EET in Antenna Complexes 4570 5.1.1. Peripheral Antenna Systems of Photosystem II (

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Nonphotochemical hole burning and pressure-dependent absorption and hole-burning results are presented for the isolated (disaggregated) chlorophyll a/b light-harvesting II trimer antenna complex of green plants. Analysis of the 4.2 K burn-fluence dependent hole spectra and zero-phonon hole action spectra indicates that the three lowest energy states (Q y ) lie at 677.1, 678.4 and 679.8 nm. Their combined absorption intensity is equivalent to that of three Chl a molecules. The inhomogeneous broadening of their absorption bands is 70 cm-1. It is argued that these states, separated by 30 cm-1, are associated with the lowest energy state of the trimer subunit with the 30 cm-1 separations due to the indigenous structural heterogeneity of protein complexes. The linear electron−phonon coupling of the 679.8 nm state is weak and characterized, in part, by a mean phonon frequency of ωm = 18 cm-1 and Huang−Rhys factor of S m = 0.8, values which yield the correct Stokes shift for fluorescence from the 679.8 nm state at 4.2 K. The temperature dependence of the zero-phonon hole (ZPH) width for that state is consistent with optical dynamics due to coupling with glasslike two-level systems of the protein. The ZPH width at 1.9 K is 0.037 cm-1. Satellite hole structure produced by burning in the above three states as well as their low linear pressures shift rates (about − 0.08 cm-1/MPa) indicate that the Chl a molecule of the subunit associated with them is weakly coupled to other Chl molecules. The linear pressure shift rates for the main Q y -absorption bands are also low. The shift rates appear to be dictated by protein−Chl interactions rather than excitonic couplings. Holes burned into the 650 nm absorption band reveal energy transfer times of 1 ps and ∼100 fs which are discussed in terms of time domain measurements of the Chl b → Chl a transfer rates (Connelly et al. J. Phys. Chem. B 1997, 101, 1902). The holewidths associated with burning into the 676 nm absorption band lead to Chl a → Chl a transfer times in the 6−10 ps range, in good agreement with the time domain values (Savikhin et al. Biophys. J. 1994, 66, 1597).

We report low temperature (T) optical spectra of the isolated CP47 antenna complex from Photosystem II (PSII) with a low-T fluorescence emission maximum near 695 nm and not, as previously reported, at 690-693 nm. The latter emission is suggested to result from three distinct bands: a lowest-state emission band near 695 nm (labeled F1) originating from the lowest-energy excitonic state A1 of intact complexes (located near 693 nm and characterized by very weak oscillator strength) as well as emission peaks near 691 nm (FT1) and 685 nm (FT2) originating from subpopulations of partly destabilized complexes. The observation of the F1 emission is in excellent agreement with the 695 nm emission observed in intact PSII cores and thylakoid membranes. We argue that the band near 684 nm previously observed in singlet-minus-triplet spectra originates from a subpopulation of partially destabilized complexes with lowest-energy traps located near 684 nm in absorption (referred to as AT2) giving rise to FT2 emission. It is demonstrated that varying contributions from the F1, FT1, and FT2 emission bands led to different maxima of fluorescence spectra reported in the literature. The fluorescence spectra are consistent with the zero-phonon hole action spectra obtained in absorption mode, the profiles of the nonresonantly burned holes as a function of fluence, as well as the fluorescence line-narrowed spectra obtained for the Q(y) band. The lowest Q(y) state in absorption band (A1) is characterized by an electron-phonon coupling with the Huang-Rhys factor S of approximately 1 and an inhomogeneous width of approximately 180 cm(-1). The mean phonon frequency of the A1 band is 20 cm(-1). In contrast to previous observations, intact isolated CP47 reveals negligible contribution from the triplet-bottleneck hole, i.e., the AT2 trap. It has been shown that Chls in intact CP47 are connected via efficient excitation energy transfer to the A1 trap near 693 nm and that the position of the fluorescence maximum depends on the burn fluence. That is, the 695 nm fluorescence maximum shifts blue with increasing fluence, in agreement with nonresonant hole burned spectra. The above findings provide important constraints and parameters for future excitonic calculations, which in turn should offer new insight into the excitonic structure and composition of low-energy absorption traps.

One-and two-color pump/probe femtosecond and hole-burning data are reported for the isolated B800-850 (LH2) antenna complex of Rhodopseudomonas acidophila (strain 10050). The two-color profiles are interpretable in terms of essentially monophasic B800fB850 energy transfer with kinetics ranging from 1.6 to 1.1 ps between 19 and 130 K for excitation at or to the red of the B800 absorption maximum. The B800 zero-phonon hole profiles obtained at 4.2 K with burn frequencies located near or to the red of this maximum yielded a transfer time of 1.8 ps. B800 hole-burning data (4.2 K) are also reported for chromatophores at ambient pressure and pressures of 270 and 375 MPa. At ambient pressure the B800-B850 energy gap is 950 cm -1 , while at 270 and 375 MPa it is close to 1000 and 1050 cm -1 , respectively. However, no dependence of the B800fB850 transfer time on pressure was observed, consistent with data for the B800-850 complex of Rhodobacter sphaeroides. The resilience of the transfer rate to pressure-induced changes in the energy gap and the weak temperature dependence of the rate are consistent with the model that has the spectral overlap (of Förster theory) provided by the B800 fluorescence origin band and weak vibronic absorption bands of B850. However, both the time domain and hole-burning data establish that there is an additional relaxation channel for B800, which is observed when excitation is located to the blue of the B800 absorption maximum. Several explanations for this faster channel are considered, including that it is due to intra-B800 energy transfer or a manifestation of coupling of B800 with quasi-degenerate upper exciton levels of the B850 molecules. The data indicate that it is not due to vibrational relaxation.

The cyclic (C n ) light harvesting 2 (LH2 or B800−850) complexes of Rps. acidophila (strain 10 050) and Rb. sphaeroides, isolated under identical conditions, are compared using the title spectroscopies. Thermal broadening and shifting data for the B850 absorption band reveal a structural change near 150 K for both species in the glycerol:water solvent used. The linear regions of thermal broadening above and below this temperature are shown to be consistent with dephasing via phonon-assisted relaxation between the B850 ring's exciton levels, which contribute to the B850 absorption band. The theoretical model used predicts, for both species, that the nearest neighbor coupling(s) between bacteriochlorophyll a (BChl a) molecules of the B850 ring is (are) significantly stronger, ca. 35%, for the low-temperature structures. Moreover, the linear thermal broadening rates of Rb. sphaeroides are significantly lower than those of Rps. acidophila for both the low- and high-temperature regions. Analysis of the difference in rates with the above model indicates that the nearest neighbor BChl a−BChl a coupling(s) is ca. 20% weaker for Rb. sphaeroides at all temperatures. The observation that the thermal shift rate for the B850 band of Rb. sphaeroides is 2.2 times smaller than that of Rps. acidophila is consistent with this weaker coupling. Pressure shift data for the B800 band indicate that the compressibility (κ) for Rb. sphaeroides is significantly larger than for Rps. acidophila, suggesting that the weaker excitonic coupling between B850 molecules of Rb. sphaeroides stems, at least in part, from looser packing of its α,β-polypeptide pairs. A higher κ value for Rb. sphaeroides provides an explanation for the observation that the linear rates for pressure broadening and shifting of the B850 band for the two species are similar. Although the pressure- and temperature-dependent data for the B800 band of both species are consistent with weak excitonic coupling between nearest neighbor B800 molecules, the data for the B850 band (including pressure shifting of zero-phonon holes burned into the lowest exciton level of the B850 ring (B870)) require interpretation in terms of strong coupling. Although large, the pressure-shift rate for B870 holes burned on the high-energy side of the B870 band (−0.46 cm-1/MPa) is a factor of 1.3 lower than on the low-energy side. An interpretation for this variation in terms of energy disorder is given. Zero-phonon hole action spectra (4.2 K) for the B870 exciton level are presented that yield similar inhomogeneous widths for the B870 band of both species, ∼120 cm-1. For both species the apparent displacement of this band below the maximum of the B850 band is close to 200 cm-1. A theoretical discussion of the relationship between the apparent displacement and excitonic level structure in the absence of energy disorder is given in the accompanying paper.