T.B. Melø

Quenching of chlorophyll a singlets and triplets by carotenoids in light-harvesting complex of photosystem II: comparison of aggregates with trimers

Abstract Laser-induced changes in the absorption spectra of isolated light-harvesting chlorophyll a/b complex (LHC II) associated with photosystem II of higher plants have been recorded under anaerobic conditions and at ambient temperature by using multichannel detection with sub-microsecond time resolution. Difference spectra (ΔA) of LHC II aggregates have been found to differ from the corresponding spectra of trimers on two counts: (i) in the aggregates, the carotenoid (Car) triplet–triplet absorption band (ΔA>0) is red-shifted and broader; and (ii) the features attributable to the perturbation of the Qy band of a chlorophyll a (Chla) by a nearby Car triplet are more pronounced, than in trimers. Aggregation, which is known to be accompanied by a reduction in the fluorescence yield of Chla, is shown to cause a parallel decline in the triplet formation yield of Chla; on the other hand, the efficiency (100%) of Chla-to-Car transfer of triplet energy and the lifetime (9.3 μs) of Car triplets are not affected by aggregation. These findings are rationalized by postulating that the antenna Cars transact, besides light-harvesting and photoprotection, a third process: energy dissipation within the antenna. The suggestion is advanced that luteins, which are buried inside the LHC II monomers, as well as the other, peripheral, xanthophylls (neoxanthin and violaxanthin) quench the excited singlet state of Chla by catalyzing internal conversion, a decay channel that competes with fluorescence and intersystem crossing; support for this explanation is presented by recalling reports of similar behaviour in bichromophoric model compounds in which one moiety is a Car and the other a porphyrin or a pyropheophorbide.

Does a leaf absorb radiation in the near infrared (780–900 nm) region? A new approach to quantifying optical reflection, absorption and transmission of leaves

The following question is addressed here: do healthy leaves absorb, as the spectra published over the last 50 years indicate, some 5–20% of incident radiation in the 780–900 nm region? The answer is found to be negative, and previous findings result from incomplete collection of the transmitted light by the detection system (even when the leaf is placed next to, but outside, the entrance port of an integrating sphere). A simple remedy for this inherent flaw in the experimental arrangement is applied successfully to leaves (of 10 unrelated species) differing in thickness, age and pigment content. The study has shown that, from an optical standpoint, a leaf tissue is a highly scattering material, and the infinite reflectance of a leaf is exceedingly sensitive to trace amounts of absorbing components. It is shown that water contributes, in a thick leaf (Kalanchoe blossfeldiana), an easily detectable signal even in the 780–900 nm region. The practical benefits resulting from improved measurements of leaf spectra are pointed out.

Electronic energy transfer involving carotenoid pigments in chlorosomes of two green bacteria: Chlorobium tepidum and Chloroflexus aurantiacus

Electronic energy transfer processes in chlorosomes isolated from the green sulphur bacterium Chlorobium tepidum and from the green filamentous bacterium Chloroflexus aurantiacus have been investigated. Steady-state fluorescence excitation spectra and time-resolved triplet-minus-singlet (TmS) spectra, recorded at ambient temperature and under non-reducing or reducing conditions, are reported. The carotenoid (Car) pigments in both species transfer their singlet excitation to bacteriochlorophyll c (BChlc) with an efficiency which is high (between 0.5 and 0.8) but smaller than unity; BChlc and bacteriochlorophyll a (BChla) transfer their triplet excitation to the Cars with nearly 100% efficiency. The lifetime of the Car triplet states is approximately 3 micros, appreciably shorter than that of the Car triplets in the light-harvesting complex II (LHCII) in green plants and in other antenna systems. In both types of chlorosomes the yield of BChlc triplets (as judged from the yield of the Car triplets) remains insensitive to the redox conditions. In notable contrast the yield of BChlc singlet emission falls, upon a change from reducing to non-reducing conditions, by factors of 4 and 35 in Cfx. aurantiacus and Cb. tepidum, respectively. It is possible to account for these observations if one postulates that the bulk of the BChlc triplets originate either from a large BChlc pool which is essentially non-fluorescent and non-responsive to changes in the redox conditions, or as a result of a process which quenches BChlc singlet excitation and becomes more efficient under non-reducing conditions. In chlorosomes from Cfx. aurantiacus whose Car content is lowered, by hexane extraction, to 10% of the original value, nearly one-third of the photogenerated BChlc triplets still end up on the residual Car pigments, which is taken as evidence of BChlc-to-BChlc migration of triplet excitation; the BChlc triplets which escape rapid static quenching contribute a depletion signal at the long-wavelength edge of the Qy absorption band, indicating the existence of at least two pools of BChlc.

Absorption and scattering of light by suspensions of cells and subcellular particles: an analysis in terms of Kramers–Kronig relations

An analysis of light scattering from suspensions of pigmented cells and particles is undertaken, and a practicable method, requiring only the experimentally measured extinction spectra, is documented. The analysis is based on two premises: Absorption and selective scattering from a single pool of pigments satisfy the Kramers-Kronig relations, which imply that one can be derived from the other; pigment-free domains contribute only nonselective scattering. This approach succeeds in simulating the spectra of many systems (human erythrocytes, chloroplasts and subchloroplast particles, algal cells) over a wide spectral range. Other, less favourable, cases are also examined, but even here the apparent discrepancy between theory adn experiment provides some clues that cannot be gleaned from absorption data alone.

Comparison of the absorption spectra of trimers and aggregates of chlorophyll ab light-harvesting complex LHC II

Abstract The absorption spectrum of a suspension containing aggregates of LHC II, the light-harvesting chlorophyll a b - protein complex associated with photosystem II, when corrected for distortions introduced by scattering and mutual shadowing of trimers within a single aggregate, turns out to be almost superposable on the absorption spectrum recorded after disrupting the aggregates by the addition of a detergent at a concentration close to its critical micelle concentration (CMC). The correction for scattering is effected by implementing a strategy proposed in 1962 by Latimer and Eubanks; that for shadowing, by using a relation derived by Duysens in 1956, which also furnishes an estimate of the aggregate size. The standard procedure for bringing down scattering-related distortions, namely the use of an opal-glass plate, is found to be unsatisfactory for LHC II samples. Extinction spectra (i.e. scattering-contaminated experimental absorption spectra), recorded over a limited range of the detergent concentration (lying between zero and the CMC), are found to pass through two isosbestic points, which differ from their counterparts in true absorption spectra: being points at which total extinction stays constant, their locations depend on the instrumental geometry as well as on the size of the aggregates.

More on the catalysis of internal conversion in chlorophyll a by an adjacent carotenoid in light-harvesting complex (Chla/b LHCII) of higher plants: time-resolved triplet-minus-singlet spectra of detergent-perturbed complexes

Abstract Wavelength-selective photo-excitation of samples containing a detergent and LHCII (the main light-harvesting complex pertaining to photosystem II of green plants) is used for recording time-resolved triplet-minus-singlet (TmS) difference spectra, with a view to probing interactions between chlorophyll a (Chla) and chlorophyll b (Chlb), and between Chla and lutein (Lut). Once the detergent concentration (CD) exceeds a threshold, C©, the TmS spectrum becomes sensitive to λ⊗, the wavelength of excitation, and to t, the delay between excitation and observation. Each increment in CD brings about a diminution in the efficiency of a†→x† transfer (triplet–triplet transfer from Chla to Lut) and a rise in both the triplet formation yield and the fluorescence yield of Chla. What is more, b*→a* transfer (singlet–singlet transfer from Chlb to Chla) slackens to such an extent that Chlb*→Chlb† intersystem crossing, negligible when CD is below C©, begins to vie with transfer, for the deactivation of Chlb* (in the foregoing an asterisk/dagger denotes singlet/triplet excitation). The reduction in the efficiencies of the two transfers is easily understood by: (i) invoking the Kuhlbrandt–Wang–Fujiyoshi model of LHCII, which posits each Chlb in contact with a Chla and each Chla in contact with a Lut, and (ii) assuming that the detergent severs contact between adjacent chromophores. That a growth in the triplet yield of Chla* accompanies the detergent-induced decrease in the efficiency of a†→x† transfer becomes intelligible if one assumes, further, that internal conversion in Chla * is faster than that in Chl a * , where under or over lining betokens the presence or absence of a carotenoid neighbour. When CD is close to C©, most Chla molecules are adjacent to a Lut, internal conversion dominates, and the overall triplet yield is low. As CD is gradually raised the Chl a → Chl a transformation sets in, causing concomitant drops in the efficiencies of a†→x† transfer and internal conversion, and a consequent rise in the overall yields of Chla fluorescence and formation of Chla triplets.

Assaying the chromophore composition of photosynthetic systems by spectral reconstruction: application to the light-harvesting complex (LHC II) and the total pigment content of higher plants

Abstract A new strategy for ascertaining the pigment composition of photosynthetic specimens is proposed, and its advantages pointed out; it entails recording, on the same machine, the absorption spectrum of the pigment extract as well as the spectra of the cognate chromophores, and in using the latter for piecemeal reconstruction of the former.

Pigment–pigment interactions in thylakoids and LHCII of chlorophyll a/c containing alga Pleurochloris meiringensis: analysis of fluorescence-excitation and triplet-minus-singlet spectra

Time-resolved triplet-minus-singlet (TmS) difference spectra, ΔA(λ; t), fluorescence excitation spectra, X(λ), and absorption spectra, A(λ), are used for probing pigment–pigment interactions in the thylakoids (Chla/c-Thyl) and isolated light-harvesting complexes associated with photosystem II (Chla/c-LHCII) of the alga Pleurochloris meiringensis, whose chromophores comprise chlorophyll a (Chla), chlorophyll c (Chlc), and several carotenoids. The data provide information about interactions between Car*-and-Chla0, Chla†-and-Car0, Car†-and-Chla0 (where the abbreviation Car stands for carotenoid, an asterisk and a dagger denote singlet and triplet excitation, respectively, and the superscript 0 denotes a molecule in the ground state). In Chla/c-Thyl, the efficiency of Car*→Chla* transfer (φLH), determined by comparing A(λ) and X(λ), is slightly less than unity (ca. 0.85), whereas the efficiency of Chla†→Car† transfer of triplet energy (φTT) must be much closer to unity, since no long-lived Chla† could be detected; an interaction between Car† and Chla0, already familiar from investigations concerning the TmS spectra of the trimers and aggregates of Chla/b-LHCII (the light-harvesting complex associated with the photosystem II of higher plants), which manifests itself through a depletion signal (in the Qy region of Chla) decaying at the same rate as the Car TmS signal, is observed, and explained likewise. In Chla/c-LHCII, both efficiencies are found to be much lower; the drastic reduction in the two yields is attributed to the perturbation of the native molecular architecture of the complex by the detergent used in the isolation procedure. The overall TmS signal from Chla/c-LHCII can be decomposed into two contributions, ΔA(λ; t)=Δ1A(λ; t)+Δ2A(λ; t), where Δ1A(λ; t) with a lifetime of about 8 μs; Δ2A(λ; t), which persists for several hundred microseconds, is contributed by those Chla† molecules which fail to transfer their excitation to a Car neighbour. A comparison of Δ1A(λ; t) with the TmS signal of thylakoids shows differences which parallel those previously reported for the TmS spectra of trimers and aggregates of Chla/b-LHCII: the carotenoid peak at 510 nm is broader, and the Qy depletion signal larger, in the difference spectrum of thylakoids. The absorption spectrum of Chla/c-LHCII show no signs of Chla–Chla excitonic interactions, since the Chla-contribution to the spectrum can be reproduced well by simply red-shifting (by about 200 cm−1) the Q bands and the Soret band in the absorption spectrum of an ethanolic solution of Chla, an observation consistent with the absence, reported in a recent study, of excitonic bands in the circular dichroism spectrum of Chla/c-LHCII.

Spectroscopic Characterization of Neutral and Cation Radicals of α-Tocopherol and Related Molecules: A Satisfactory Denouement

Notwithstanding the facile occurrence of one-electron oxidation in α-tocopherol and its acetate (TOH and TOAc, respectively), and despite the remarkable stability, under appropriate conditions, of the oxidation products (TOH(•+), TO(•), and TOAc(•+)), their spectroscopic characterization is in an unsatisfactory state, calling for a fresh attempt to acquire reliable data. A new, model-free method is developed for analyzing time-resolved spectra showing the progress of the reaction TOH + R(•) → TO(•) + RH, where R(•) is a stable free radical. The resulting absorption coefficients of TO(•) in dichloromethane and hexane are in severe disagreement with some recent values derived from stopped-flow spectrophotometry. The discrepancy is traced to the imposition of boundary conditions that do not take proper account of the dead time of the apparatus; when multiplied by a factor of two, the stopped-flow data fall mostly in the range ε = (7.5 ± 0.5) × 10(3) M(-1) cm(-1), conforming with the results of this study and the values found by Boguth and Niemann in 1969. Absorption spectra of the radical cations produced (electro)chemically are found to be reliable only in the visible region. Incomplete conversion of the parent compound to the radical cation, an obstacle to the determination of absorption coefficients from electrochemical studies, is circumvented by combining EPR and optical spectroscopy. The absorption coefficients of TOH(•+) and TOAc(•+), determined in this manner, are found to be, respectively, 1.6 × 10(4) and 1.3 × 10(4) M(-1) cm(-1), in accord with the values found first through similar means.

Comparison of the photochemical behaviors of α-tocopherol and its acetate in organic and aqueous micellar solutions.

Photoionization is known to take place when α-tocopherol (TOH) is excited to the S(1) state in a polar medium. It has been previously suggested that TO(•) is formed only as a result of proton release by TOH(•+), a process that is expected to occur, in a protic solvent, on the subnanosecond time scale. Recent redeterminations of the molar absorption coefficients of e(aq)(–) (Hare J. Phys. Chem. A 2010, 114, 1766) and of TOH(•+) and TO(•) (Naqvi J. Phys. Chem. A 2010, 114, 10795) have paved the way for testing the above suggestion, even if subnanosecond time resolution is not available, since it implies the equality of [e(aq)(–)](0) and [TO(•)](0), where [···](0) denotes the concentration of the enclosed species immediately after a nanosecond laser pulse. Nanosecond pump-probe spectroscopy of TOH in aqueous micellar solution (AMS) and two organic solvents with similar polarities (acetonitrile and methanol) has revealed that prompt formation of TO(•) through dissociation (TOH + hν → TO(•) + H(•)) is not negligible even in AMS. In acetonitrile, TOH(•+) and TO(•) are formed with comparable yields, and the former converts quantitatively into TO(•) within 15 μs. In methanol, TO(•) was observed, but no evidence was found for electron ejection from TOH. Only one photoproduct, namely TO(•), could be detected when α-tocopherol acetate (TOAc) was excited to the S(1) state in several polar and nonpolar solvents; TOAc has been found to be a more efficient energy degrader than TOH.