Steven P. Berg

Chromium oxalate: A new spin label broadening agent for use with thylakoids

Potassium tris(oxalato)chromate(III) trihydrate (chromium ixalate) has been shown to be a more useful broadening agent than potassium ferricyanide for the spin label 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amine (Tempamine) in thylakoid suspensions. Our data show that chromium oxalate is less permeable than ferricyanide, does not inhibit thylakoid electron transport or photophosphorylation, and is not photoreduced by thylakoids.

Is functional manganese involved in hydrogen-peroxide-stimulated anomalous oxygen evolution in CACl2-washed photosystem II membranes?

When detergent-derived photosystem II (PSII) membranes are treated with CaCl2 to remove the three extrinsic proteins associated with the O2-evolving complex, the resulting membranes (CaPSII) can still catalyze water oxidation if sufficient Ca2+ and Cl- are present. When CaPSII membranes are exposed to single turnover flashes on an O2 rate electrode, anomalous O2 is produced by the first two flashes. The addition of catalase to the membrane suspension completely inhibits O2 produced by the first two flashes, but not by subsequent flashes. Exogenous H2O2 stimulates anomalous O2 production by the first few flashes in CaPSII membranes, but not in control PSII membranes. Diuron (DCMU) does not inhibit H2O2-stimulated O2 production by the first flash. However, it does inhibit the O2 yield of all subsequent flashes, indicating that all flash-induced O2 signals in CaPSII membranes are dependent on photosystem II electron transport. H2O2 stimulation of O2 yields is inhibited in Tris-, heat-, and EDTA-(ethylenediaminetetraacetic acid)-treated CaPSII. In the presence of high salt, H2O2 (but not EDTA) treatment of CaPSII, extracts Mn functional in normal photosynthetic O2 evolution. The addition of exogenous Mn2+ reconstitutes anomalous O2 production in Tris-and H2O2/EDTA-treated CaPSII preparations but only in the presence of H2O2. Anomalous H2O2-stimulated O2 production can be observed both with a Clark electrode (steady state) and an O2 rate electrode (flash sequence). The mechanism involves electron donation from H2O2, mediated by free Mn2+, to PSII, and the 33-kDa extrinsic protein under some conditions can block this process. Since H2O2 can remove functional Mn from CaPSII membranes, its presence can convert functional Mn to the Mn2+ mediator state required for anomalous O2 production. EDTA binds Mn in CaPSII disrupted by H2O2 and prevents anomalous O2 evolution.

The influence of spinach thylakoid lumen volume and membrane proximity on the rotational motion of the spin label Tempamine

Phosphorylating electron-transport reactions, using either Photosystem I or Photosystem II, were found to be competent in driving the light-induced increase in rotational hindrance of the spin label Tempamine. The uncoupler gramicidin prevented the change induced by either photosystem while the electron-transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) only prevented the light-induced change associated with Photosystem II. The internal lumen microviscosity is termed ‘η’ and was found to be inversely proportional to the lumen volume. Substances which should compete for Tempamine-binding sites on the thylakoid membrane were found to be largely ineffectual in changing η. These results further support the hypothesis that the magnitude of η is directly related to the proximity of Tempamine to a membrane surface.

Proton involvement with the light-induced hindrance of spin label motion in the lumen of spinach thylakoids

The light-induced hindrance of spin label motion increases linearly with light intensity. However, it has not been possible to unambiguously demonstrate light saturation due to the very high rates of spin label reduction at high light intensity. The light-induced hindrance of spin label motion may be mimicked in the dark by subjecting thylakoids to appropriately low pH regimes. Uncouplers such as gramicidin-D and methylamine reduce the light-induced hindrance to dark levels as does ethylenedinitrilotetraacetate (EDTA) treatment. Valinomycin plus KCl which destroys the electric potential is only partially effective in reducing the light-induced hindrance. These results indicate that protons in the aqueous lumen of the thylakoids are closely involved with the observed light-induced hindrance of spin label motion.

Electron acceptance at photosystem II in an oxygen-evolving photosystem II preparation: Clear discrimination of two sites of electron acceptance for quinones and quinonediimines associated with a photosystem II preparation

Oxygen-producing electron transport reactions of a photosystem II preparation are described. The preparation has six major peptides with apparent molecular weights of 36,000, 31,000, 28,000, 27,000, 25,000, and 21,000. Sucrose density gradient centrifugation indicates that the preparation is more homogeneous and more dense than control thylakoid membranes. The preparation photoreduces a number of known photosystem II oxidants including the Class I acceptor, ferricyanide; the Class II acceptor, 2,6-dichloroindophenol; and the Class III acceptor, 2,6-dichlorobenzoquinone. However, quinonediimines such as phenylenediimine are not reduced, suggesting that these substances are reduced at sites in the thylakoid membrane which are not found in the photosystem II preparation. All the oxygen-producing reactions are sensitive to inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea.

Electron acceptance at photosystem II in uncoupled spinach thylakoids: Resolution of two sites of electron acceptance prior to the DBMIB block with melittin, a new peptide inhibitor

Our understanding of photosystem II electron transport has been greatly fac&ated through the use of specific photosystem I inhibitors and the use of class III oxidants [ 11. However, all class III oxidants do not accept electrons at the same place near photosystem II. The sensitivity of the 2,5dibromo-3-methyl6-isopropyl-p-benzoquinone @BMIB)-mediated photosystem II reaction toward 3(3,4-dichlorophenyl)-1 ,I-dimethylurea (DCMU) was dependent on light intensity [2] while quinonediimines accepted electrons more directly from a photosystem II carrier, prior to the plastoquinone pool [2]. Based on steady. state kinetic analyses of photosystem II activities catalyzed by lipophilic electron acceptors, quinones and quinonediimines were concluded to accept electrons from two different sites on the reducing side of photosystem II. These conclusions are important because some properties of photosystem II electron transport depend on the type of lipophilic electron acceptor which is employed. For instance, photosystem II electron transport to various quinonediimines is inhibited by uncouplers [4-71, while the electron transport to the quinones is not [ 5,7,8]. An additional complexity indicated by kinetic studies [3] is the apparent presence of two photoreduction reactions in the quinonediimine-catalyzed reaction. Here we introduce a new, linear peptide inhibitor (melittin) which selectively inhibits between the sites of quinone and quinonediimine reduction. Melittin is a lytic peptide which contains 26 amino acids in a linear array [9]. It is isolated from the

Spin-label studies of the lipid regions of spinach thylakoids and a detergent-derived oxygen-evolving Photosystem II preparation

Abstract We have compared the fluidity of thylakoid membranes with the membrane present in a Triton X-100-derived, oxygen-evolving Photosystem II (PS II) preparation using two different spin labels. Data obtained with 2,2,6,6-tetramethylpipiridine- N -oxyl (TEMPO) shows that the PS II preparation contains less fluid membrane than the thylakoid. The TEMPO partition parameter ( f ) is about 2.5-times greater for the thylakoids at 6 mg chlorophyll/ml than for the PS II preparation at the same chlorophyll concentration. Similarly, the rotational correlation time, τ, of TEMPO residing in the membrane of the PS II preparation is about 2-times longer than the τ for TEMPO in the thylakoid membrane. A spin label which partitions more completely into the bilayer, 2-heptyl-2-hexyl-5,5-dimethyloxazolidine- N -oxyl (7N14), indicates a much greater fluidity in the thylakoid membrane than the membrane of the PS II preparation. The PS II preparation appears to have a hydrocarbon phase which approaches the rigid limit of EPR detectable motion. These results are discussed in terms of possible lipid depletion in the PS II preparation and in terms of lateral heterogeneity of hydrocarbon fluidity in the thylakoid membrane caused by the lateral heterogeneity in protein components.

The use of polyclonal antibodies to identify peptides exposed on the stroma side of the spinach thylakoid

We have raised polyclonal antibodies against an oxygen-evolving photosystem II preparation. Western Blot analysis of the whole serum revaals antibodies specific for at least 15 Coomassie visible bands ranging from 59 to 11 kDa. These antibodies are specific for proteins located on both sides of the membrane. Included are antibodies specific for Tris-removable peptides (33, 25 and 18 kda), which are thought to be exposed on the lumen surface of the PS II complex. Since the whole serum agglutinates thylakoids, antibodies specific for the stroma side of the PS II complex are also present. A sub-population of antibodies can be isolated by allowing the antibodies in whole serum to bind to EDTA-treated thylakoid membranes. The antibodies which specifically bind are cross-reactive with peptides with Mr of 59, 57, 34, 28, 27, 26, and 23 kDa. Our data indicate that these peptides have antigenic determinants exposed on the stroma side of the thylakoid membrane.

Transverse heterogeneity in lipid fluidity in spinach thylakoids, photosystem II preparations, and thylakoid galactolipid vesicles

We have used three doxyl stearic acid spin labels to study the transverse hetero-geneity in lipid fluidity in thylakoids, photosystem II (PS II) preparations, and thylakoid galactolipid vesicles. This comparative study shows that spin labels incorporated into the membrane of the PS II preparation experience far more immobilization than do the same spin labels incorporated into either thylakoids or vesicles prepared from the polar lipids extracted from thylakoids. The spin label immobilization found in the PS II preparation is manifest even near the center of the bilayer, where lipid mobility is normally at its maximum. Analysis of the lipid content of the PS II preparation, relative to chlorophyll, suggests that the PS II preparation may be lipid depleted. This lipid depletion could explain the results presented. However, electron microscopy [Dunahay et al. (1984) Biochim. Biophys. Acta 764:179–193] has not indicated that major delipidation has occurred, and so it remains possible that the immobilization found in the PS II preparation is due primarily to the normal (but close) juxtaposition of adjacent PS II complexes and the cooperative immobilization of their surrounding lipids. Based on the results presented, we conclude that highly mobile lipids are not required for oxygen evolution, the primary photochemistry or the secondary reduction of exogenously added quinones. Unfortunately, the relationship between the plastoquinone pool and the fluidity of the membrane in the PS II preparation remains ambiguous.

Light-induced hindrance of rotational motion of the spin label tempamine in the aqueous lumen of spinach thylakoids

Electron paramagnetic resonance (EPR) spectroscopy has been developed to measure internal viscosity, volume and pH in membrane-surrounded aqueous regions of cells and organelles [l-9]. In the viscosity studies, the motion of a spin label located in a membrane bound aqueous space is hindered relative to the motion of the spin label in bulk water. Thus, the rotational motion of 2,2,6,6-tetramethylpiperidine-N-oxyl4-amine (Tempamine) has been shown restricted by a factor of -7 when residing in the lumen of thyfakoids in the dark ]7,8]. However, experiments requiring light intensities which cansupport photophosphorylation are problematic since the thylakoids rapidly photoreduce nitroxyl radicals [7,8 ,l O-1 31 to the corresponding diamagnetic hydroxylamines 2131. Thus there are no reports of the motion of spin labels residing in the lumen of lightactivated thylakoids available to date. Here we report a light-induced change in the rotational motion of Tempamine located in the lumen of spinach thylakoids, and we discuss how this change may be related to light-induced merTlbrane conformational changes [14-215.