Establishment of the Qy Absorption Spectrum of Chlorophyll a Extending to Near-Infrared.

A weak absorption tail related to the Qy singlet electronic transition of solvated chlorophyll a is discovered using sensitive anti-Stokes fluorescence excitation spectroscopy. The quasi-exponentially decreasing tail was, at ambient temperature, readily observable as far as -2400 cm-1 from the absorption peak and at relative intensity of 10-7. The tail also weakened rapidly upon cooling the sample, implying its basic thermally activated nature. The shape of the spectrum as well as its temperature dependence were qualitatively well reproduced by quantum chemical calculations involving the pigment intramolecular vibrational modes, their overtones, and pairwise combination modes, but no intermolecular/solvent modes. A similar tail was observed earlier in the case of bacteriochlorophyll a, suggesting generality of this phenomenon. Long vibronic red tails are, thus, expected to exist in all pigments of light-harvesting relevance at physiological temperatures.

Controlling Photosynthetic Excitons by Selective Pigment Photooxidation.

As a basis of photosynthesis, photoinduced oxidation of (bacterio)chlorophyll molecules in the special reaction center complexes has been a subject of extensive research. In contrast, the generally harmful photooxidation of antenna chromoproteins has received much less attention. Here, we have established the permanent structural changes in the LH2 antenna bacteriochlorophyll-protein complex from a sulfur photosynthetic purple bacterium Ectothiorhodospira haloalkaliphila taking place at physiological conditions upon intense optical irradiation. To this end, a crystal structure of the LH2 complex from E. haloalkaliphila was first resolved by X-ray diffraction to 3.7 Å, verifying a great similarity with the earlier structure from Phaesporillum molischianum. Analysis of the various steady-state and picosecond time-resolved optical spectroscopy data and related model simulations then confirmed that the major spectral effects observed-bleaching and blue-shifting of the B850 exciton band and correlated emergence of a higher-energy C700 exciton band-are associated with photooxidation of increasing numbers of B850 bacteriochlorophylls into 3-acetyl-chlorophylls, with no noticeable damage to the pigment-binding protein scaffold. A prospective noninvasive method for an in situ optical control of excitons by selective photooxidation of pigment chromophores was thus revealed and demonstrated in a structurally well-defined native system.

Vibronic Origin of the Qy Absorption Tail of Bacteriochlorophyll a Verified by Fluorescence Excitation Spectroscopy and Quantum Chemical Simulations.

The long-wavelength tail of the lowest-energy Qy singlet absorption band of bacteriochlorophyll a in triethylamine peaking at 768.6 nm was examined by means of fluorescence excitation spectroscopy at ambient temperature of 22 ± 1 °C. The tail, usually considered a Gaussian, does in fact weaken quasi-exponentially, being clearly evident as far as 940 nm, nearly 2400 cm-1 (∼12 kBT) away from the absorption peak. Quantum chemical simulations identified vibronic transitions from the thermally populated normal modes and their overtones in the ground electronic state as the origin of this tail. Because energy transfer and relaxation processes generally depend on vibronic overlap integrals, these findings may have important implications on the interpretation of numerous photoinduced phenomena that involve bacteriochlorophyll and similar molecules, including photosynthesis.

Up-converted fluorescence from photosynthetic light-harvesting complexes linearly dependent on excitation intensity.

Weak up-converted fluorescence related to bacteriochlorophyll a was recorded from various detergent-isolated and membrane-embedded light-harvesting pigment-protein complexes as well as from the functional membranes of photosynthetic purple bacteria under continuous-wave infrared laser excitation at 1064 nm, far outside the optically allowed singlet absorption bands of the chromophore. The fluorescence increases linearly with the excitation power, distinguishing it from the previously observed two-photon excited fluorescence upon femtosecond pulse excitation. Possible mechanisms of this excitation are discussed.

Fluorescence micro-spectroscopy study of individual photosynthetic membrane vesicles and light-harvesting complexes.

Single-molecule spectroscopy, by getting rid of unwanted ensemble averaging effects, has proved to be a very valuable tool in the research of individual photosynthetic light-harvesting (LH) complexes. However, to learn about real photosynthetic processes the minimal unit to study is a single photosynthetic membrane complete with all elements of its machinery. In the present work, the fluorescence spectra of excitons in lone intracytoplasmic (IC) photosynthetic membrane vesicles of the wild type purple bacterium Rhodobacter sphaeroides that involve peripheral (LH2) and core (RC-LH1-PufX) antenna pigment-protein complexes were investigated at ambient temperature under continuous-wave laser excitation into the Q(x) absorption band of the bacteriochlorophyll-a (BChl) chromophores at 594 nm. In parallel, the spectra of mutant membrane vesicles occupied by just one type of complexes (either LH2 or RC-LH1-PufX) and the spectra of individual purified LH2 and RC-LH1-PufX complexes were measured. The fluorescence from full IC membranes shows a high sensitivity to excitation intensity, being varied over more than four orders of magnitude between 0.1 W/cm(2) and 2 kW/cm(2). At low to moderate excitation intensities, the spectra of IC membranes could be well reproduced by its component spectra, the ratio of the spectra related to peripheral and core complexes being the only adjustable parameter. The spectra of both intact chromatophores and individual membrane components recorded over 1-50 s experimental time frames are robust, strongly suggesting that large spectral fluctuations hardly play a role in the functional photosynthetic process. The significant, up to 14 times, variation of the LH2 and LH1 emission ratio observed in individual IC membranes could be related to variations in the stoichiometric ratio of the peripheral and core complexes. Evidence was found for the presence of LH2 parts that are detached from efficient energy transfer pathways. Upon strong and prolonged illumination, the membrane spectra reveal significant permanent modifications. These alterations, which mostly concern peripheral antenna complexes, were shown to be due to photo-oxidation of various numbers of BChl molecules in the B850 compartment of LH2.

Pressure-induced spectral changes for the special-pair radical cation of the bacterial photosynthetic reaction center.

The effect of pressure up to 6 kbars on the near to mid infrared absorption spectrum (7500-14,300 cm(-1) or 1333-700 nm) of the oxidized reaction center of Rhodobacter sphaeroides is measured and interpreted using density-functional B3LYP, INDO, and PM5 calculations. Two weak electronic transition origins at approximately 8010 and approximately 10,210 cm(-1) are unambiguously identified. The first transition is assigned to a Qy tripdoublet band that involves, in the localized description of the excitation, a triplet absorption on one of the bacteriochlorophyll molecules (PM) in the reaction center's special pair intensified by the presence of a radical cation on the other (PL). While most chlorophyll transition energies decrease significantly with increasing pressure, the tripdoublet band is found to be almost pressure insensitive. This difference is attributed to the additional increase in the tripdoublet-band energy accompanying compression of the pi-stacked special pair. The second band could either be the anticipated second Qy tripdoublet state, a Qx tripdoublet state, or a state involving excitation from a low-lying doubly occupied orbital to the half-occupied cationic orbital. A variety of absorption bands that are also resolved in the 8300-9600 cm(-1) region are assigned as vibrational structure associated with the first tripdoublet absorption. These sidebands are composites that are shown by the calculations to comprise many unresolved individual modes; while the calculated pressure sensitivity of each individual mode is small, the calculated pressure dependence of the combined sideband structure is qualitatively similar to the observed pressure dependence, preventing the positive identification of possible additional electronic transitions in this spectral region.