We present temperature-dependent fluorescence quantum yield and lifetime measurements on the LH-1 and LH-2 complexes of Rhodobacter sphaeroides and on the isolated B820 subunit of Rhodospirillum rubrum. From these measurements the superradiance is calculated, which is related to the delocalization of excitations in these complexes. In the B820 preparation we find a radiative rate that is 30% higher than that of monomeric bacteriochlorophyll, in agreement with a dimer model of this subunit. At room temperature both LH-1 and LH-2 are superradiant relative to monomeric Bchl-a with enhancement factors of 3.8 and 2.8, respectively. In LH-2 the radiative rate does not change significantly upon lowering the temperature to 4 K. LH-1 however exhibits a strong temperature dependence, giving rise to a 2.4 times higher radiative rate at 4 K relative to room temperature. From modeling of the superradiance using a Hamiltonian based on the LH-2 structure and including site inhomogeneity, we conclude that the ratio of inhomogeneity over the coupling between the pigments is around 1 for LH-1 and 2-3 for LH-2. From the Monte Carlo simulations we estimate the delocalization length in LH-1 and LH-2 to be on the order of 3-4 pigments at room temperature.

Polarized site-selected fluorescence spectroscopy of isolated Photosystem I particles P ol ari zed site-sel ected fluo resc ence sp ec tro sco py of iso lat ed P hot osy stem I pa rtic les Bas Gobets, Herbert van Amerongen, René Monshouwer, Jochen Kruip, Matthias Rögner, Rienk van Grondelle and Jan P. Dekker Polarized steady-state fluorescence spectra have been obtained from Photosystem I core complexes of the cyanobacterium Synechocystis PCC 6803 and from LHCI containing Photosystem I (PSI-200) complexes of spinach by selective laser excitation at 4 K. Excitation above 702 nm in Synechocystis and 720 nm in PSI-200 results in highly polarized emission, suggesting that pigments absorbing at these and longer wavelengths are not able to transfer excitation energy at 4 K. In both systems the peak wavelength of the emission (λ em ) depends strongly on the excitation wavelength (λ ex ). This indicates that in both systems the long-wavelength bands responsible for the steady-state emission are inhomogeneously broadened. The width of the inhomogeneous distribution is estimated to be about 215 cm -1 in Synechocystis and 400 cm -1 in PSI-200. We conclude that the peaks of the total absorption spectra of the long-wavelength pigments of Synechocystis and PSI-200 are at 708 and 716 nm, respectively, and therefore designate these pigments as C708 and C716. The results further show that C708 and C716 are strongly homogeneously broadened, i.e. carry broad phonon side-bands. The width of these bands is estimated to be about 170 and 200 cm -1 for C708 and C716, respectively. The Stokes' shifts appear to be large: about 200 cm -1 (10 nm) for C708 and about 325 cm -1 (17 nm) for C716. These values are much higher than usually observed for 'normal' antenna pigments, but are in the same order as found previously for a number of dimeric systems. Therefore, we propose that the long-wavelength pigments in Photosystem I are excitonically coupled dimers. Based on fitting with Gaussian bands the presence of one C708 dimer per P700 is suggested in the core antenna of Synechocystis. This chapter has been published in Biochimica etBiophysica Acta 1188, 75-85, reproduced with permission.

Isolated trimeric Photosystem I complexes of the cyanobacterium Synechococcus elongatus have been studied with absorption spectroscopy and site-selective polarized fluorescence spectroscopy at cryogenic temperatures. The 4 K absorption spectrum exhibits a clear and distinct peak at 710 nm and shoulders near 720, 698 and 692 nm apart from the strong absorption profile located at 680 nm. Deconvoluting the 4 K absorption spectrum with Gaussian components revealed that Synechococcus elongatus contains two types of long-wavelength pigments peaking at 708 nm and 719 nm, which we denoted C-708 and C-719, respectively. An estimate of the oscillator strengths revealed that Synechococcus elongatus contains about 4-5 C-708 pigments and 5-6 C-719 pigments. At 4 K and for excitation wavelengths shorter than 712 nm, the emission maximum appeared at 731 nm. For excitation wavelengths longer than 712 nm, the emission maximum shifted to the red, and for excitation in the far red edge of the absorption spectrum the emission maximum was observed 10-11 nm to the red with respect to the excitation wavelength, which indicates that the Stokes shift of C-719 is 10-11 nm. The fluorescence anisotropy, as calculated in the emission maximum, reached a maximal anisotropy of r=0.35 for excitation in the far red edge of the absorption spectrum (at and above 730 nm), and showed a complicated behavior for excitation at shorter wavelengths. The results suggest efficient energy transfer routes between C-708 and C-719 pigments and also among the C-719 pigments.

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The kinetics of primary electron transfer in membrane-bound Rhodobacter sphaeroides reaction centers (RCs) were measured on both wild-type (WT) and site-directed mutant RC's bearing mutations at the tyrosine M210 position. The tyrosine was replaced by histidine (H), phenylalanine (F), leucine (L), or tryptophan (W). A mutant with histidine at both the M210 and symmetry-related L181 positions (YM210H/FL181H) was also examined. Rates of primary charge separation were determined by both single and multiple wavelength pump−probe techniques. The time constants for the decay of stimulated emission in the membrane-bound mutant RC's were approximately 27 ps (F), 36 ps (L), 72 ps (W), 5.8 ps (H), and 4.2 ps (HH), compared with 4.6 ps in WT membrane-bound RC's. For all RC's, the decay of stimulated emission was found to be multiexponential, demonstrating that this phenomenon is not a consequence of the removal of the RC from the membrane. The source of the multiexponential decay of the primary donor excited state was examined, leading to the conclusion that a distribution in the driving force (ΔG) for electron transfer cannot be the sole parameter that determines the multiexponential character. Further measurements on membrane-bound mutant RC's showed that chemical prereduction of the acceptor quinones resulted in a significant slowing of the rate of primary charge separation. This was most marked in those mutants in which the rate of charge separation had already been slowed down as a result of mutagenesis at the M210 position. The phenomenon is discussed in terms of the Coulombic interaction between QA - and the other pigments involved in electron transfer and the influence of this interaction on the driving force for charge separation.

We report the results of relative difference absorption measurements for the LH2 antenna of Rhodobacter sphaeroides and the B820 subunit of Rhodospirillum rubrum at room temperature. It is shown that significant differences between shapes and amplitudes of photoinduced, absorption changes reflect a different degree of exciton delocalization in the intact antenna compared with the dimeric subunit. Using the exciton model in the presence of static disorder, we have obtained a consistent and quantitative fit of the amplitudes and shapes of the pump-probe spectra of the LH2 antenna and the B820 subunit. We estimate that the nearest-neighbors interaction energy in the antenna is about 400 cm -1 and the diagonal disorder is about 450 cm -1 . For these values the coherence length (FWHM) of the steady-state exciton wavepacket corresponds to 5 BChl molecules at room temperature. The amplitude of the difference absorption reflects a cooperative behavior within at least 12 BChls of the B850 antenna. The calculations suggest that the dimeric subunit is characterized by a decrease of the interaction energy to 300 cm -1 together with an increase of the disorder value to about 600 cm -1 .

The LH-1 antenna of the purple bacterium Rhodopseudomonas viridis has been investigated using femtosecond pump−probe spectroscopy. We find that qualitatively the relaxation processes are comparable to those in Rhodobacter sphaeroides and Rhodospirillum rubrum. Both the spectral relaxation and depolarization take place in approximately 150 fs, which suggests that the excitations in the LH-1 ring are delocalized over two to three pigments. Strong oscillations of the pump−probe signal are observed at all temperatures. We conclude that these must be of a vibrational origin and could be related to the observed fine structure seen in the low-temperature absorption spectrum of Rps. viridis membranes. The main frequencies of the oscillations are 65 and 105 cm-1, and the damping time is ∼700 fs. From the amplitude of the oscillations and the slow damping time, we conclude that excitations are transferred in a vibrationally coherent manner from site to site.

In this work we explain the spectral heterogeneity of the absorption band (. Biochim. Biophys. Acta. 1229:373-380), as well as the spectral evolution of pump-probe spectra for membranes of Rhodopseudomonas (Rps.) viridis. We propose an exciton model for the LH1 antenna of Rps. viridis and assume that LH1 consists of 24-32 strongly coupled BChl b molecules that form a ring-like structure with a 12- or 16-fold symmetry. The orientations and pigment-pigment distances of the BChls were taken to be the same as for the LH2 complexes of BChl a-containing bacteria. The model gave an excellent fit to the experimental results. The amount of energetic disorder necessary to explain the results could be precisely estimated and gave a value of 440-545 cm(-1) (full width at half-maximum) at low temperature and 550-620 cm(-1) at room temperature. Within the context of the model we calculated the coherence length of the steady-state exciton wavepacket to correspond to a delocalization over 5-10 BChl molecules at low temperature and over 4-6 molecules at room temperature. Possible origins of the fast electronic dephasing and the observed long-lived vibrational coherence are discussed.