Gyözö Garab

The physiological role of ascorbate as photosystem II electron donor: protection against photoinactivation in heat-stressed leaves

Previously, we showed that ascorbate (Asc), by donating electrons to photosystem II (PSII), supports a sustained electron transport activity in leaves in which the oxygen-evolving complexes were inactivated with a heat pulse (49°C, 40 s). Here, by using wild-type, Asc-overproducing, and -deficient Arabidopsis (Arabidopsis thaliana) mutants (miox4 and vtc2-3, respectively), we investigated the physiological role of Asc as PSII electron donor in heat-stressed leaves (40°C, 15 min), lacking active oxygen-evolving complexes. Chlorophyll-a fluorescence transients show that in leaves excited with trains of saturating single-turnover flashes spaced 200 ms apart, allowing continual electron donation from Asc to PSII, the reaction centers remained functional even after thousands of turnovers. Higher flash frequencies or continuous illumination (300 μmol photons m−2 s−1) gradually inactivated them, a process that appeared to be initiated by a dramatic deceleration of the electron transfer from TyrZ to P680+, followed by the complete loss of charge separation activity. These processes occurred with half-times of 1.2 and 10 min, 2.8 and 23 min, and 4.1 and 51 min in vtc2-3, the wild type, and miox4, respectively, indicating that the rate of inactivation strongly depended on the Asc content of the leaves. The recovery of PSII activity, following the degradation of PSII proteins (D1, CP43, and PsbO), in moderate light (100 μmol photons m−2 s−1, comparable to growth light), was also retarded in the Asc-deficient mutant. These data show that high Asc content of leaves contributes significantly to the ability of plants to withstand heat-stress conditions.

Evidence for long-range excitation energy migration in macroaggregates of the chlorophyll ab light-harvesting antenna complexes

Abstract We investigated the picosecond transient absorbance kinetics under singlet-singlet annihilation conditions and the steady-state spectroscopic features, absorbance, circular dichroism and low-temperature fluorescence spectra, in large, three-dimensional, stacked lamellar aggregates of the purified light-harvesting chlorophyll a b complexes (LHCII) and its form of small aggregates. Our data strongly suggest that the macroorganizational parameters significantly influence the spectroscopic properties and strongly affect the energy migration pathways in the aggregates. In small aggregates ( d ≈ 100 nm) of LHCII trimers the excitation energy migration could be characterized with a percolation type of excitation migration in a small cluster of chromophores. In contrast, in chirally organized macroaggregates ( d ≈ 2–4 μ m) the annihilation kinetics were consistent with a model predicted for (infinitely) large three-dimensional aggregates, showing that LHCII macroaggregates can constitute a structural basis for long-range migration of the excitation energy.

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.

Effects of polyhydroxy compounds on the structure and activity of α-chymotrypsin

Abstract The effects of glycerol, polyethylene glycol, fructose, glucose, sorbitol, and saccharose on the conformation and catalytic activity of α-chymotrypsin were studied in 0.1 M sodium phosphate buffer and buffered aqueous 60% ethanol (pH 8.0). The enzyme activity was practically completely lost within 10 min in 60% ethanol, but in the presence of stabilizers the activity was retained. With the exception of polyethylene glycol, the stabilizing effect decreased with increase of the incubation time. The preservation of the catalytic activity was accompanied by changes in the secondary and tertiary structures of α-chymotrypsin.

Kinetic Analysis of the Light‐induced Fluorescence Quenching in Light‐harvesting Chlorophyll a/b Pigment‐Protein Complex of Photosystem II

We carried out a kinetic analysis of the light‐induced fluorescence quenching (AF) of the light‐harvesting chlorophyll a/b pigment‐protein complex of photosystem II (LHCII) that was first observed by Jennings et at (Pho‐tosynth. Res. 27, 57–64, 1991). We show that during a 2 min light, 2 min dark cycle, both the light and dark phases exhibit biexponential kinetics; this is tentatively explained by the presence of two types of light‐induced quenchers in different domains of aggregated LHCII. Quantitative analysis could be carried out on the faster kinetic component; the slower component that was not completed during the measurement was not amenable for quantitative analysis. Our analysis revealed that the rate of the light‐induced decrease of the fluorescence yield depended linearly on the light intensity, which shows that the generation of the quencher originates from a reaction that is first order with respect to the concentration of the excited domains. As shown by the estimated rate constant, pho‐togeneration of the quencher is a fast reaction that can compete with other excitation‐relaxation pathways. Both the decay and the recovery time constants of AF depended strongly on the temperature. Thermodynamic analysis showed that the fast light‐induced decline in the fluorescence was determined by a low fraction of the excited states. Recovery was associated with large decrease in the entropy of activation that indicated the involvement of large structural rearrangements. Macroaggregated LHCII exhibited larger ΔF than small aggregates, which is consistent with the proposed role of aggregated LHCII in thy‐lakoid membranes in nonphotochemical quenching.

Effect of chromium on photosystem 2 in the unicellular green alga, Chlorella pyrenoidosa

We investigated the effect of chromium (20–40 g m−3, 8–72 h) on the photosystem 2 (PS2) activities of Chlorella pyrenoidosa cells. By using chlorophyll fluorescence transients, thermoluminescence, oxygen polarography, and Western blot analysis for D1 protein we found that inhibition of PS2 can be accounted for by the enhanced photodestruction of the reaction centres in the cells cultivated in the presence of Cr(VI) at 25 °C in “white light” (18 W m−2). Hence photodestruction of D1 is caused by an enhanced oxidative stress and lipid peroxidation, as indicated by the appearance of a high-temperature thermoluminescence band.

Characterization of the light induced reversible changes in the chiral macroorganization of the chromophores in chloroplast thylakoid membranes. Temperature dependence and effect of inhibitors

We investigated the temperature dependence and inhibitor sensitivity of the light-induced reversible changes in the circular dichroism (ΔCD) of chloroplast thylakoid membranes. Earlier, these changes, which originate from structural changes affecting the chiral macroorganization of the chromophores, were thought to be driven by photochemically generated proton and/or ion gradients in the thylakoids [Garab et al. (1988) Biochemistry 27: 2430]. However, more recently, these changes have been shown to be largely independent of the photochemical activity of thylakoids, and ΔCD has been observed in lamellar aggregates of the light harvesting chlorophyll a/b complex (LHC II) of Photosystem II [Barzda et al. (1996) Biochemistry 35: 8981]. Here, we show that in thylakoids (i) ΔCD is gradually and substantially decelerated upon gradually decreasing the temperature from 33 °C to 2 °C, and abruptly disappears above 35–37 °C; (ii) ΔCD is inhibited with nigericin with I50≈ 1 μM, which is about 10 times higher than the I50for the transmembrane ΔpH; (iii) ΔCD can be inhibited with dicyclohexylcarbodiimide that blocks proton binding at the lumenal side of LHC II; (iv) quinone antagonists, such as antimycin-A and myxothiazol, inhibit ΔCD without noticeably affecting the electron and proton transport, and the chiral macroorganization of the chromophores in the dark. We conclude that ΔCD is conditioned but not driven by the photochemical activity of the membranes, and the structural changes are given rise by a physical mechanism previously unrecognized in thylakoids, thermooptic effect described for liquid crystals. We discuss the possible link between the deactivation(s) of the excess excitation energy and ΔCD, the light-induced changes in the chiral macroorganization of the chromophores of the photophysical apparatus in thylakoids.

Organization of the pigment molecules in the chlorophyll a/c light-harvesting complex of Pleurochloris meiringensis (xanthophyceae). Characterization with circular dichroism and absorbance spectroscopy

Abstract By the aid of circular dichroism (CD), absorbance and fluorescence spectroscopy, we studied the molecular organization of the pigment molecules in cells, isolated chloroplasts and the chlorophyll a / c light-harvesting complex (LHC) associated with photosystem II of the chlorphyll c -containing alga, Pleurochloris meiringensis . In cells and chloroplasts, similarly to higher plant chloroplasts, a (+) 693 nm CD band accompanied by a tail outside the absorbance indicated a long-range chiral organization of the chlorophyll molecules. The LHCII of these algae exhibited an intense negative CD band at 679 nm. However, in contrast to the chlorophyll a / b LHCII of higher plants, where the intense, non-conservative (−)684 nm band has been shown to be associated with long-range chiral organization of the macro-aggregates, the intense, non-conservative (−)679 nm band in the chlorophyl a / c LHC originated from the non-aggregated form of the complexes. In sharp contrast to the trimers or monomers of the chlorophyll a / b LHCII, in the chlorophyll a / c LHC no split excitonic CD bands could be detected in the red spectral region, thus CD provided no indication for the occurence of excitonic interactions among the Q Y transition dipoles of the chlorophyll molecules. Gaussian analysis of the absorbance and CD bands showed that the (−)679 nm CD signal is given rise by a small number of long-wavelength absorbing chlorophyll a molecules. Data obtained with LHC treated with low concentrations of acetone or digitonin strongly suggest a specific binding site of chlorophyll a to the protein, which upon binding a chlorophyll a molecule induces a bathochromic shift and asymmetry in the electronic structure of the molecule. These results and literature data strongly suggest that the organization of the pigment molecules in chlorophyll a / c antenna complexes is significantly different from the organization of the chlorophyll a / b complexes of green algae and higher plants.

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.

Irreversible disassembly of chiral macrodomains in thylakoids due to photoinhibition

We investigated the effect of photoinhibitory illumination on the chiral macroorganization of the chromophores in spinach thylakoid membranes. By measuring circular dichroism (CD), we found that prolonged (15 min) illumination of membranes with intense white light led to irreversible diminishment of the main CD bands originating from the chiral macroorganization of the chromophores. The irreversible decrease of the main CD bands showed a nearly linear correlation with the extent of photoinhibition which was determined by chlorophyll fluorescence induction. CD measurements also revealed that the excitonic CD bands, which are given rise by short-range interactions between the chromophores inside the complexes or particles, were largely insensitive to the photoinhibitory illumination of the membranes. These data show that, whereas photoinhibitory treatment has no perceptible effect on the molecular architecture of the bulk of the pigment–protein complexes, it leads to a disorganization of their macroarray, and an irreversible disassembly of the chirally organized macrodomains.