Flash-induced reduction of cytochrome b-559 by Q infB sup− in chloroplasts in the presence of protonophores

Flash-induced, fast (t _1/2 1 ms), reversible reduction of the high potential cytochrome b-559 (cyt b-559HP) was observed in chloroplasts in the presence of 2 M protonophore, FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), CCCP (carbonylcyanide 3-chlorophenylhydrazone) or SF 6847 (2,6-di-(t-butyl)-4-(2,2-dicyanovinyl)phenol). These protonophores promote autooxidation of cyt b-559HP in the dark (Arnon and Tang 1988, Proc Natl Acad Sci USA 85: 9524). No fast photoreduction could, however, be observed if the molecules were oxidized with ferricyanide in the absence of protonophores. This suggests that the molecules must be deprotonated to be capable for fast photoreduction.Photoreduction of cyt b-559HP was largely insensitive to DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), but was inhibited by DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). With a train of flashes, no oscillation could be observed in the amplitudes of photoreduction. These data strongly suggest that cyt b-559HP is reduced by the semireduced secondary quinone acceptor (Q_B ^–) of Photosystem 2.Abbreviations ADRY- acceleration of the deactivation reactions of the water-splitting enzyme system Y of photosynthesis - Ant 2p- 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene - cyt- cyto-chrome - CCCP- carbonylcyanide 3-chlorophenylhydrazone - DBMIB- 2,5-dibromo-3-methyl-6-iso-propyl-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimehtylurea - FCCP- carbonylcyanide p-trifluoromethoxyphenylhydrazone - FeCy- ferricyanide - HP- high potential form - HQ- hydroquinone - PQ- plastoquinone - PS 2- Photosystem 2 - SF 6847- 2,6-di-(t-butyl)-4-(2,2-dicyanovinyl)-phenol     

Effect of dibromothymoquinone (DBMIB) on reduction rates of Photosystem I donors in intact chloroplasts

Dual effect of dibromothymoquinone ( DBMIB ), inhibitor and reducing agent at the donor side of Photosystem I, was investigated in isolated intact chloroplasts by flash-induced absorbance changes at 820 and 515 nm. We show that in the absence of other electron donors, rereduction of P700+ by DBMIB proceeds at a very low rate (half-time of approximately 10 s) Dual effect of DBMIB explains that the initial rise of electrochromic absorbance change induced by repetitive flashes is usually not diminished while the slow rise is fully inhibited by this compound.     

Involvement of superoxide dismutase in heat-induced stimulation of photosystem I-mediated oxygen uptake

We have investigated the effect of heat-treatment of chloroplast thylakoid membranes on photosystem I-mediated electron transport. Spectroscopic techniques, oxidation of dichlorophenolindophenol (donor side) and reduction of NADP or methyl purple (acceptor side), showed no indication of an increased activity of photosystem I electron transport. Enhancement of oxygen uptake in the heat-treated (40 degrees C-48 degrees C) samples could largely be accounted for by decline in the activity of superoxide dismutase.     

Lipid‐polymorphism of plant thylakoid membranes. Enhanced non‐bilayer lipid phases associated with increased membrane permeability

Earlier experiments, using ³¹P‐NMR and time‐resolved merocyanine fluorescence spectroscopy, have shown that isolated intact, fully functional plant thylakoid membranes, in addition to the bilayer phase, contain three non‐bilayer (or non‐lamellar) lipid phases. It has also been shown that the lipid polymorphism of thylakoid membranes can be characterized by remarkable plasticity, i.e. by significant variations in ³¹P‐NMR signatures. However, changes in the lipid‐phase behaviour of thylakoids could not be assigned to changes in the overall membrane organization and the photosynthetic activity, as tested by circular dichroism and 77 K fluorescence emission spectroscopy and the magnitude of the variable fluorescence of photosystem II, which all showed only marginal variations. In this work, we investigated in more detail the temporal stability of the different lipid phases by recording ³¹P‐NMR spectra on isolated thylakoid membranes that were suspended in sorbitol‐ or NaCl‐based media. We observed, at 5°C during 8 h in the dark, substantial gradual enhancement of the isotropic lipid phases and diminishment of the bilayer phase in the sorbitol‐based medium. These changes compared well with the gradually increasing membrane permeability, as testified by the gradual acceleration of the decay of flash‐induced electrochromic absorption changes and characteristic changes in the kinetics of fast chlorophyll a‐fluorescence transients; all variations were much less pronounced in the NaCl‐based medium. These observations suggest that non‐bilayer lipids and non‐lamellar lipid phases play significant roles in the structural dynamics and functional plasticity of thylakoid membranes.     

Quantitative method for studying orientation of transition dipoles in membrane vesicles of spherical symmetry

Pigmented vesicular membranes embedded in polyacrylamide gel exhibit linear dichroism when the gel sample is squeezed [Abdourakhmanov, I.A., Ganago, A.O., Erokhin, Yu.E., Solov'ev, A.A. and Chugunov, V.A. (1979) Biochim. Biophys. Acta 546, 183-186]. The orientation technique of gel-squeezing was modified to enhance polarization effects in membrane vesicles of spherical symmetry. Model calculations were carried out to provide a tool for the quantitative evaluation of the dichroism of squeezed gel samples. The orientation angles of the dipoles can be calculated with reasonable precision by measuring two quantities: (i) the macroscopic deformation parameter of the gel sample, and (ii) a parameter (e.g. the polarization ratio of the fluorescence emission) characterizing the orientation of the transition dipoles in the membranes embedded in the squeezed gel. The validity of the model was confirmed through a series of polarization measurements relating to the fluorescence of chlorophyll a in membranes of osmotically shocked chloroplasts, 'blebs'.     

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

The effects of glycerol, polyethylene glycol, fructose, glucose, sorbitol, and saccharose on the conformation and catalytic activity of alpha-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 alpha-chymotrypsin.     

Trapping of the quenched conformation associated with non-photochemical quenching of chlorophyll fluorescence at low temperature

The kinetics of non-photochemical quenching (NPQ) of chlorophyll fluorescence was studied in pea leaves at different temperatures between 5 and 25°C and during rapid jumps of the leaf temperature. At 5°C, NPQ relaxed very slowly in the dark and was sustained for up to 30 min. This was independent of the temperature at which quenching was induced. Upon raising the temperature to 25°C, the quenched state relaxed within 1 min, characteristic for qE, the energy-dependent component of NPQ. Measurements of the membrane permeability (ΔA₅₁₅) in dark-adapted and preilluminated leaves and NPQ in the presence of dithiothreitol strongly suggest that the effect of low temperature on NPQ was not because of limitation by the lumenal pH or the de-epoxidation state of the xanthophylls. These data are consistent with the notion that the transition from the quenched to the unquenched state and vice versa involves a structural reorganization in the photosynthetic apparatus. An eight-state reaction scheme for NPQ is proposed, extending the model of Horton and co-workers (FEBS Lett 579:4201–4206, 2005), and a hypothesis is put forward concerning the nature of conformational changes associated with qE. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Effect of chromium on photosystem 2 in the unicellular green alga, <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis>

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.     

Thermo-optically Induced Reorganizations in the Main Light Harvesting Antenna of Plants. II. Indications for the Role of LHCII-only Macrodomains in Thylakoids

We have investigated the circular dichroism spectral transients associated with the light-induced reversible reorganizations in chirally organized macrodomains of pea thylakoid membranes and loosely stacked lamellar aggregates of the main chlorophyll a/b light harvesting complexes (LHCII) isolated from the same membranes. These reorganizations have earlier been assigned to originate from a thermo-optic effect. According to the thermo-optic mechanism, fast local thermal transients due to dissipation of the excess excitation energy induce elementary structural changes in the close vicinity of the dissipation [Cseh et al. (2000) Biochemistry 39: 15250–15257]. Here we show that despite the markedly different CD spectra in the dark, the transient (light-minus-dark) CD spectra associated with the structural changes induced by high light in thylakoids and LHCII are virtually indistinguishable. This, together with other close similarities between the two systems, strongly suggests that the gross short-term, thermo-optically induced structural reorganizations in the membranes occur mainly, albeit probably not exclusively, in the LHCII-only domains [Boekema et al. (2000) J Mol Biol 301: 1123–1133]. Hence, LHCII-only domains might play an important role in light adaptation and photoprotection of plants.