Heterogeneity of the mechanism of water splitting in photosystem II.

We measured the temperature dependence of oxygen evolution in thylakoids from tobacco using mass spectrometry and high resolution polarography. We determined the initial S-state distribution and the efficiency of the transition between these states including the probability of the O(2) yield through a fast mode. We observed discontinuous changes of the parameters at the temperatures 11 degrees C, 15 degrees C and 21 degrees C. Due to the mass spectroscopy data we think that the irregularity observed at 11 degrees C is due to conformational changes within the water catalytic site. We show that the different contributions of the slow and fast modes of oxygen evolution and of the water molecule exchange are correlated and that their behavior can be explained in terms of the H(2)O accessibility to the water splitting enzyme.

An estimation of the size of the water cluster present at the cleavage site of the water splitting enzyme.

In time-dependent measurements of oxygen evolution in tobacco thylakoid membranes we varied the fraction of H(2)(18)O and the temperature and measured water splitting as (18)O(2), (16)O(18)O, and (16)O(2) by mass spectrometry. We show that the approach to the equilibrium of the system after H(2)(18)O addition can be very well understood in terms of the diffusion of water molecules. The equilibrium states of (16)O(2), (16)O(18)O, and (18)O(2) evolution differ from the theoretical binomial distributions, which are expected under the prerequisite of ideal mixing of the water molecules and that of the chemical equivalence of H(2)(18)O and H(2)(16)O for an infinite cluster. The presence of this deviation means that there is a typical size of water clusters having access to cleavage by the water splitting enzyme. We estimated that this cluster contains about 12+/-2 water molecules.

Capacity of enzymes of the euphorbiacea Aleurites montana involved in CO2-fixation, compared to plants having C3-, C4- and Crassulacean acid metabolism.

Capacities of phosphoenolpyruvate carboxylase (PEP-Co), ribulose bisphosphate carboxylase (Rubisco), NADP+ malic enzyme (ME) and of malate dehydrogenase (MDH) were measured in the Euphorbiacea Aleurites montana, grown under 700 ppm CO2 for four weeks prior to enzyme extraction. For comparison Bryophyllum daigremontiana (CAM). Saccharum officinarum (C4) and Capsicum frutescens (C3) were treated in the same way. PEP-Co capacity of Aleurites was in the range of 12-, that of Capsicum approx. 26 nmol x min(-1) x mg protein(-1), without significant influence of the light period or CO2-treatment. In contrast, the activity of the enzyme from Saccharum was, depending on the duration of light, 160- respectively 96 times higher than that of the tung-oil tree. In Bryophyllum a rather low activity in the morning was increased during the day to approx. 230 nmol x min(-1) x mg protein(-1) in plants grown in the greenhouse and to approx. 115 nmol x min(-1) x mg protein(-1) in those from the growth chamber. Malate was hardly detectable in extracts of Aleurites, whereas it was high in Bryophyllum, depending on the light period. The ratio of average PEP-Co to Rub-Co capacity was high for the CAM-plant (20:1), somewhat lower for sugar cane (10:1), but almost at equality for Aleurites (0.9:1) and chilli (0.8:1). For the NADP+ malic enzyme, low capacity (20 to 28 nmol x min(-1) x mg protein(-1)) was found for Aleurites and for Capsicum, whereas it was 10 to 17 times higher in Saccharum. In Bryophyllum, the activity was up to 80 nmol x min(-1) x mg protein, dependent on light period. MDH capacity was extremely high in all plants investigated. Highest rates (10-20 micromol x min(-1) x mg protein(-1)), were obtained for Bryophyllum, followed by sugar cane and Capsicum with 5-8 micromol x min(-1) x mg protein(-1). Again, the lowest capacity was found in extracts of Aleurites with approx. 1.3 to 1.6 micromol x min(-1) x m protein(-1). Thus, in Aleurites montana no indication for C4- or Crassulacean acid metabolism was obtained. Therefore, the earlier observed very efficient uptake of CO2 cannot be explained by a high expression of the PEP-Co protein, known to occur in CAM- and C4-plants.

Seeds of Trichosanthes kirilowii, an energy-rich diet.

The kernels of Trichosanthes kirilowii seeds contain a green oil which makes up for 62% of their dry matter. This oil consists up to 95% of triglycerides, 2% of glycolipids, 1.3% of phospholipids and 1.8% of chlorophylls. As fatty acid components the triglycerides, glycolipids and phospholipids contain the unsaturated fatty acids linoleic and oleic acid and the saturated palmitic acid. In the triglycerides 19% of the C18:3 acid occur with the configuration delta9 cis, delta11 trans, delta13 cis. This acid is called trichosanic acid and is absent in glycolipids and phospholipids which contain instead another C18:3 fatty acid, which has conjugated double bounds and occurs with an amount of 21% and 3%, respectively. Typically, these oil seeds contain in addition up to 30% of their dry matter proteins and up to 2.5% mono- and oligosaccharides. The monosaccharides consist of rhamnose, galactose and glucose and the oligosaccharides represent a mixture of tri- and tetrasaccharides.

Phosphatidylglycerol is involved in the dimerization of photosystem II.

Photosystem II core dimers (450 kDa) and monomers (230 kDa) consisting of CP47, CP43, the D1 and D2 proteins, the extrinsic 33-kDa subunit, and the low molecular weight polypeptides PsbE, PsbF, PsbH, PsbI, PsbK, PsbL, PsbTc, and PsbW were isolated by sucrose density gradient centrifugation. The photosystem II core dimers were treated with phospholipase A2 (PL-A2), which cuts phosphatidylglycerol (PG) and phosphatidylcholine molecules at the sn-2 position. The PL-A2-treated dimers dissociated into two core monomers and further, yielding a CP47-D1-D2 subcomplex and CP43. Thin layer chromatography showed that photosystem II dimers contained four times more PG than their monomeric counterparts but with similar levels of phosphatidylcholine. Consistent with this was the finding that, compared with monomers, the dimers contained a higher level of trans-hexadecanoic fatty acid (C16:1Delta3tr), which is specific to PG of the thylakoid membrane. Moreover, treatment of dimers with PL-A2 increased the free level of this fatty acid specific to PG compared with untreated dimers. Further evidence that PG is involved in stabilizing the dimeric state of photosystem II comes from reconstitution experiments. Using size exclusion chromatography, it was shown that PG containing C16:1Delta3tr, but not other lipid classes, induced significant dimerization of isolated photosystem II monomers. Moreover, this dimerization was observed by electron crystallography when monomers were reconstituted into thylakoid lipids containing PG. The unit cell parameters, p2 symmetry axis, and projection map of the reconstituted dimer was similar to that observed for two-dimensional crystals of the native dimer.

Cooperative binding of oxygen to the water-splitting enzyme in the filamentous cyanobacterium Oscillatoria chalybea.

In the filamentous cyanobacterium Oscillatoria chalybea photolysis of water does not take place in the complete absence of oxygen. A catalytic oxygen partial pressure of 15x10(-6) Torr has to be present for effective water splitting to occur. By means of mass spectrometry we measured the photosynthetic oxygen evolution in the presence of H(2)(18)O in dependence on the oxygen partial pressure of the atmosphere and analysed the liberations of (16)O(2), (16)O(18)O and (18)O(2) simultaneously. The observed dependences of the light-induced oxygen evolution on bound oxygen yield sigmoidal curves. Hill coefficient values of 3.0, 3.1 and 3.2, respectively, suggest that the binding is cooperative and that four molecules of oxygen have to be bound per chain to the oxygen evolving complex. Oxygen seems to prime the water-splitting reaction by redox steering of the S-state system, putting it in the dark into the condition from which water splitting can start. It appears that in O. chalybea an interaction of oxygen with S(0) and S(1) leads to S(2) and S(3), thus yielding the typical oxygen evolution pattern in which even after extensive dark adaptation substantial amounts of Y(1) and Y(2) are found. The interacting oxygen is apparently reduced to hydrogen peroxide. Mass spectrometry permits to distinguish this highly specific oxygen requirement from the interaction of bulk atmospheric oxygen with the oxygen evolving complex of the cyanobacterium. This interaction leads to the formation H(2)O(2) which is decomposed under O(2) evolution in the light. The dependence on oxygen-partial pressure and temperature is analysed. Structural peculiarities of the cyanobacterial reaction centre of photosystem II referring to the extrinsic peptides might play a role.

State of the lipid and fatty acid composition in chloroplasts of Nicotiana tabacum under the influence of an increased CO2 partial pressure of 700 p.p.m.

We analysed chloroplast lipids of Nicotiana tabacum var. John William's Broadleaf, cultivated under an increased PCO(2) of 700 p.p.m. Glycolipids and phospholipids remain constant under these conditions, whereas the carotenoid content undergoes a quantitative change. The saturation degree of fatty acids increases due to an increase in palmitic acid and decreases in hexadecatrienoic acid and linolenic acid.

Localization of NADPH-protochlorophyllide reductase in plastids of barley at different greening stages.

The localization of protochorophyllide (Pchlide) and of NADPH-protochlorophyllide oxidoreductase (POR, EC 1.6.99.1) within (etio)chloroplasts has been investigated at selected stages of greening of barley seedlings. Pchlide pigment and POR protein contents were evaluated in different plastid membrane fractions by fluorescence spectroscopy and immunoblot analysis using a monospecific polyclonal antibody raised against the purified enzyme. Fluorescence analysis showed the presence of Pchlide in both the envelope and thylakoid membranes. During greening, the Pchlide content, expressed on a total protein basis, decreased in thylakoid membranes, whereas it increased in the envelope membranes. POR proteins were detected mainly in thylakoid membranes at early greening stages. In contrast, the weak amount of POR proteins was associated more specifically with envelope membranes of mature chloroplasts. Whatever the greening stage, thylakoid-bound Pchlide and POR proteins were more abundant in the thylakoid regions which remained unsolubilized after mild Triton treatment used as standard procedure to prepare PS II particles. This suggests the preferential association of Pchlide and POR to the appressed regions of thylakoids.

Comparative immunological and chemical analysis of lipids and carotenoids of the D1-peptide and of the light-harvesting-complex of photosystem II of Nicotiana tabacum.

The light-harvesting-complex (LHCP) was isolated from photosystem II of Nicotiana tabacum var. John William's Broadleaf by means of the detergent acetyl-beta-D-glucopyranoside and fractionating centrifugation. The D1-peptide of photosystem II was isolated as a dimer with the molecular mass of 66 kDa from the chlorophyll-deficient tobacco mutant N. tabacum Su/su by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Both preparations were characterized by means of the Western blot procedure using monospecific antisera to the proteins of photosystem II and monospecific antisera to lipids with which the lipids bound to peptides were determined. In parallel to this, lipids bound to the isolated LHCP-complex and to the isolated D1-peptide were determined by lipido-chemical methods. The extraction of the isolated core peptide D1 with a mixture of boiling methanol and chloroform and subsequent HPLC-chromatography showed that in the D1-peptide isolated via SDS-polyacrylamide gel electrophoresis, monogalactolipids, phosphatidylglycerol and sulfolipid molecules are bound in the molar ratio 1:3:17. By means of the immunological procedure of Western blotting we were able to show that the 66 kDa band of the isolated dimeric D1 reacts positively only with the antisera to monogalactolipid, sulfolipid, beta-carotene and violaxanthin. With the antiserum to digalactolipid and that to phosphatidylglycerol a positive reaction is only observed if the preparation used in the Western blot is not the isolated D1-peptide but a "total" photosystem II-preparation. The lipid extraction of the LHCP-complex and the subsequent analysis by thin-layer chromatography led to the result that the isolated LHCP complex contained in bound form 3 molecules monogalactolipid, 1 molecule of digalactolipid, 1 molecule of phosphatidylglycerol and 1 molecule of lutein. Less than 1 molecule of sulfolipid, beta-carotene, neoxanthin and violaxanthin are found. In the Western blot analysis only the antiserum to monogalactolipid and phosphatidylglycerol and among the carotenoid antisera only the antisera to beta-carotene, violaxanthin and to neoxanthin reacted. With the antisera to the digalactolipid, to the sulfolipid and the antisera to the xanthophylls, namely to lutein and neoxanthin, a positive reaction occurred only if the material used in the Western Blot was the "total" photosystem II-preparation. By gas chromatography of the fatty acids of the isolated peptide fractions it was shown that, compared to the lipids of photosystem II and of the thylakoid membrane, in lipids of the isolated D1-peptide and of the LHCP-complex the saturation degree of fatty acids is strongly increased. Whereas palmitic acid in chloroplast lipids makes up for only 11% of the fatty acids, this saturated fatty acid increases in the lipids of the LHCP to 20% and makes up for 74% of total fatty acids in the lipids of the D1-peptide. Linoleic and linolenic acids are completely absent and oleic acid makes up for 14% of total fatty acids. In contrast to the lipids of the thylakoid membrane, the lipids bound to proteins/peptides are characterized by a strongly saturated character.

Temperature dependence of the O2-oscillation pattern in the filamentous cyanobacterium Oscillatoria chalybea and in Chlorella kessleri.

Five characteristic discontinuities of the pattern of oxygen evolution have been detected for the filamentous cyanobacterium Oscillatoria chalybea in the temperature range of 0 degree C to 30 degrees C. The temperatures at which these discontinuities occur are: approximately 5 degrees C, approximately 11 degrees C, approximately 15 degrees C, approximately 21 degrees C and approximately 25 degrees C. The calculated initial 5-S state distribution, the miss parameter and the fraction of the fast transition S3-->So + O2 are affected. The discontinuities are observed at the same transition temperature also for Chlorella kessleri hence are not specific for the cyanobacterium. Based on these studies it is concluded that the not vanishing oxygen signal under the first flash of a flash train in Oscillatoria cannot have its origin in interactions between oxygen-evolving complexes. A decrease of temperature should slow down the expected charge exchanges, improve the oscillations thus reduce or lower the first two oxygen amplitudes of the oscillatoria pattern. Lowering of the temperature improves the oscillations but does not lower the first O2 signal of the pattern.

Action of an antiserum to alpha-tocoquinone on photosystem II-particle preparations of Nicotiana tabacum.

An antiserum to alpha-tocoquinone was prepared by immunization of rabbits. Immunization was obtained by injection of a conjugate consisting of the hapten alpha-tocoquinone attached to methylated ovalbumin into the rabbit. The antiserum recognizes the 3,4-dimethyl-p-benzoquinone group of the molecule as well as part of the immediate vicinity to the side chain. This is concluded from the fact that the antibody has some affinity also to plastoquinone. No reaction of the antibody is observed with alpha-tocopherol hydroquinone or alpha-tocopherol. Reaction of the antiserum to alpha-tocoquinone with photosystem II-particle preparations from tobacco affects the functionality of the preparation. Chlorophyll(a)-fluorescence emission is quenched without an alteration of the emission spectrum. Concomitant with this fluorescence quenching, the lifetime of two fluorescence components namely that of a fast and a slower component are shortened. By analogy with the literature the fast component is associated with chlorophyll(a) of the reaction center core and that of the slow component with the antenna system in which the lifetime parameter is shortened by the antibody from 3.42 ns to 1.795 ns. The action on the fast component is less and leads to a shortening of the lifetime parameter from 0.373 ns to only 0.249 ns. The effect is interpreted in terms of an enhancement of linear photosynthetic electron transport possibly due to an inhibition of the cyclic electron transport around PS II. discovered by Gruszecki et al. (1995), Z. Naturforsch. 50c, 61-68.

Comparative immunological detection of lipids and carotenoids on peptides of photosystem I from higher plants and cyanobacteria.

Photosystem I preparations were obtained from wild-type tobacco Nicotiana tabacum var. JWB, three chlorophyll-deficient tobacco mutants: Su/su, Su/su var. Aurea and yellow-green leaf patches of the variegated mutant NC 95, Spinacia oleracea and furthermore from the mesophilic cyanobacterium Synechococcus PCC 7942 and the thermophilic cyanobacterium Synechococcus sp.. Peptides from these preparations were analyzed by SDS polyacrylamide gel electrophoresis and transferred for detection of bound lipids and carotenoids according to the Western blot procedure to nitrocellulose membranes. The PS I preparations from the Nicotiana tabacum species and spinach consist of the core complex and the LHCP I complex, the latter containing, however, traces of the LHCP II polypeptides. The core complex consists of the two core peptides with the apparent molecular mass of 66 kDa each and peptides with molecular masses of 22, 20, 19, 17, 16, 10 and 9 kDa. The LHCP I complex contains 4 subunits with molecular masses of 28, 26, 25 and 24 kDa. The PS I preparations of the two mutants Su/su and Su/su var. Aurea contain as impurities traces of the core peptides (D1/D2) and the two chlorophyll-binding peptides (CP43/CP47) of photosystem II. The PS I preparation from the mesophilic and thermophilic cyanobacterium consists of the two core peptides with the apparent molecular mass of 66 kDa and peptides with molecular masses of 16, 14 and 10 kDa. The peptides of the PS I preparations were characterized by specific PS I, CP I and LHCP I antisera. The antiserum to the PS I complex reacts in the Western blot with the homologous peptides of PS I from higher plants, but only with the CP I complex from the two cyanobacteria. In comparative studies with PS II from higher plants the PS I antiserum reacts with the LHCP II complex as expected. The antiserum to the CP I complex reacts only with the 66 kDa peptides of PS I from all objects. There is no cross reaction with the 66 kDa peptides (heterodimer of the D1/D2 peptides) of PS II. The antiserum to the LHCP I complex reacts only with the four LHCP I peptides of PS I from higher plants and as expected with the LHCP II of PS II: Because cyanobacteria do not have LHCP complexes, there is no reaction with the LHCP I antiserum. By means of polyclonal monospecific antisera to lipids it was shown by Western blot procedure that only two lipid species are bound to PS I peptides. The galactolipid monogalactosyldiglyceride is bound to the CP I complex of the Nicotiana tabacum species, spinach and the two cyanobacteria as well as to the LHCP I complex of the higher plants. The phospholipid phosphatidylglycerol is only associated with the CP I complex of the analyzed higher plants and cyanobacteria. With polyclonal monospecific antisera to carotenoids it was demonstrated that beta-carotene, lutein, neoxanthin and zeaxanthin are associated with the CP I complex of the higher plants and the cyanobacteria analyzed. Violaxanthin is also bound to the CP I complex of the two cyanobacteria, whereas it is bound together with neoxanthin to the LHCP I complex of the higher plants.

Nucleotide sequence of the nar beta gene encoding assimilatory nitrate reductase from the cyanobacterium Oscillatoria chalybea.

The nucleotide sequence of the structural gene of nitrate reductase (n ar beta) has been determined from the filamentous, non-heterocystous cyanobacterium Oscillatoria chalybea. The nar beta gene encodes a protein of 737 amino acid residues, which shows 61% identity to nitrate reductase of the unicellular cyanobacterium Synechococcus sp. PCC 7942 and only weak homologies to different bacterial molybdoenzymes, such as nitrate reductases or formate dehydrogenases.

The role of phosphatidylglycerol as a functional effector and membrane anchor of the D1-core peptide from photosystem II-particles of the cyanobacterium Oscillatoria chalybea.

The intrinsic polypeptide D1, isolated from photosystem (PS) II-particles of the cyanobacterium Oscillatoria chalybea, was obtained by electroelution and fractionated extraction with organic solvents. Purification was demonstrated by Western blotting and amino acid sequencing. By carrying out D1-immunization in rabbits a polyclonal monospecific D1-antiserum was obtained. For the qualitative characterization of D1 as a lipid-binding peptide, the effect of the lipids phosphatidylglycerol (PG), monogalactosyldiacylglyceride (MGDG) and phosphatidylcholine (PC) on PSII-oxygen evolution was analysed in reconstitution experiments. In these experiments purified photosystem II (PSII)-particle preparations were treated with the enzyme phospholipase A2 and supplemented with lipid emulsions. We were able to show that the inhibition of electron transport, as the consequence of this lipase treatment, was only relieved, if phosphatidylglycerol was added to the preparation. A model was proposed, in which phosphatidylglycerol is a functional effector for the optimal conformation of D1 in the PSII core complex. Phosphatidylglycerol molecules are unusually tightly bound to the D1 peptide by hydrophobic interactions. A covalent binding seems not probable. The localisation of phosphatidylglycerol binding sites was found by trypsin treatment of D1 and analysis of the obtained oligopeptides with HPLC and immunoblotting. The binding sites could be confined to the hydrophobic amino acid section between arginine 27 and arginine 225, which is known to be the membrane anchor of D1. This has led us to the conclusion that the phospholipid phosphatidylglycerol plays an important role for anchoring the D1-peptide and for its orientation in the thylakoid membrane. Phosphatidylglycerol with its high amount of palmitic acid has in prokaryotic cyanobacteria apparently a role in stabilization and orientation. The high turn-over of D1 and the spatial separation of the synthesis- and incorporation-site in the membrane, developed during evolution in eukaryotic organisms, might have changed the requirement on the mobility and the orientation of D1 in photosynthetic membranes.

Europium- and Dysprosium-Ions as Probes for the Study of Calcium Binding Sites in Photosystem II.

Trivalent lanthanide cations are suitable probes for Ca2+-binding sites in photosystem II (PS II). PS II membranes prepared from Nicoticinci tabacum, intact and depleted of the extrinsic polypeptides were exposed to lanthanide ions (Dy3+ and Eu3+). Small concentrations of dysprosium and europium ions enhance oxygen evolution under short saturating flashes. Higher concentrations of the rare earth cations cause the release of the three extrinsic peptides (17, 23 and 33 kDa) and reduce O2 yield. The reactivation of the PS II membranes, thus depleted of the 33 kDa subunit, by Ca2+ ions is not possible. Comparing Eu3+ with Dy3+ in this effect shows that Eu3+ is more effective than Dy3+, because a lower Eu3+-concentration in comparison to that of Dy3+ inactivates O2-evolution. The differences between europium and dysprosium can be explained by their different ionic radius. Our results suggest the existence of two Ca-binding regions: one with a low affinity for calcium would be located on the contact surface of the 23 and 33 kDa proteins and the second one with a high affinity, should be located close to the Mn-cluster and to tyrosine-161 (Z ). The more tightly-bound calcium would be responsible for the activity of the PS II system.

Antioxidant properties of plastoquinol and other biological prenylquinols in liposomes and solution.

Oxidation of biological prenylquinols, like plastoquinol-9 (PQH2-9), ubiquinol-10 (UQH2-10), reduced vitamins K1 (VK1H2) and K2 (VK2H2), alpha-tocopherol quinol (alpha-TQH2) and alpha-tocopherol (alpha-T) was followed by their fluorescence during sonication of egg yolk lecithin/prenylquinol liposomes. The order of magnitude of oxidation of the prenylquinols by free radicals generated during sonication was UQH2-10 > VK2H2 > VK1H2 > alpha-TQH2 > PQH2-9 > alpha-T. It was shown that egg yolk lecithin undergoes degradation even when sonicated briefly under atmosphere of nitrogen and at 0 degree C. A kinetic study of free radical scavenging action of the prenylquinols in solvents of different polarity was performed. The pseudo-first-order rate constants, k, for the reaction of the prenylquinols with 1,1-diphenyl-2-picrylhydrazyl (DPPH) in hexane showed that their scavenging activity changes in the order VK2H2 > VK1H2 > alpha-TQH2 > PQH2-9 > alpha-T > UQH2-10, being the highest in hexane and methanol, whereas in acetone and ethyl acetate the scavenging activity appeared much lower. The reaction rate constants, k, were apparently not dependent on the solvent polarity. The antioxidant activity of the prenylquinols in natural membranes is discussed.

Inactivation of the water-oxidizing enzyme in manganese stabilizing protein-free mutant cells of the cyanobacteria Synechococcus PCC7942 and Synechocystic PCC6803 during dark incubation and conditions leading to photoactivation.

The previously constructed MSP (manganese stabilizing protein-psbO gene product)-free mutant of Synechococcus PCC7942 (Bockholt R, Masepohl B and Pistorius E K (1991) FEBS Lett 294: 59-63) and a newly constructed MSP-free mutant of Synechocystis PCC6803 were investigated with respect to the inactivation of the water-oxidizing enzyme during dark incubation. O2 evolution in the MSP-free mutant cells, when measured with a sequence of short saturating light flashes, was practically zero after an extended dark adaptation, while O2 evolution in the corresponding wild type cells remained nearly constant. It could be shown that this inactivation could be reversed by photoactivation. With isolated thylakoid membranes from the MSP-free mutant of PCC7942, it could be demonstrated that photoactivation required illumination in the presence of Mn(2+) and Ca(2+), while Cl(-) addition was not required under our experimental conditions. Moreover, an extended analysis of the kinetic properties of the water-oxidizing enzyme (kinetics of the S3→(S4)→S0 transition, S-state distribution, deactivation kinetics) in wild type and mutant cells of Synechococcus PCC7942 and Synechocystis PCC6803 was performed, and the events possibly leading to the reversible inactivation of the water-oxidizing enzyme in the mutant cells are discussed. We could also show that the water-oxidizing enzyme in the MSP-free mutant cells is more sensitive to inhibition by added NH4Cl-suggesting that NH3 might be a physiological inhibitor of the water oxidizing enzyme in the absence of MSP.

A mass spectrometric analysis of the water-splitting reaction.

Earlier mass spectrometric measurements, in which oxygen evolution was measured following short saturating light flashes, indicated that with a time resolution of about 30 s no form of bound water and/or an oxidation product exists up to the redox state S3 of the oxygen evolving center (R. Radmer and O. Ollinger, 1986, FEBS Lett 195: 285-289; K.P. Bader, P. Thibault and G.H. Schmid, 1987, Biochim Biophys Acta 893: 564-571). In the present study, isotope exchange experiments with H2 (18)O were performed under different experimental conditions. We found: a) the isotope exchange pattern is virtually the same at both pH 6.0 and 7.8, although marked structural changes of the PS II donor side are inferred to take place within this pH-range (Renger G., Messinger J. and Wacker U., 1992, Research in Photosynthesis, II: 329-332); b) injection of H2 (18)O at about 0°C gives rise to mass ratios of the evolved oxygen which markedly deviate from the theoretically expected values of complete isotope scrambling; and c) rapid injection of H2 (18)O into samples with high population of S1 and S2 and subsequent illumination with three and two flashes, respectively, spaced by a dark time of only 1.5 ms lead to similar (18)O-labeling of the evolved oxygen. Based on the published data on the interaction with redox active amines, possible pathways of substrate exchange in the water oxidase are discussed.

Comparison of photosystem II complexes isolated from tobacco and two chlorophyll deficient tobacco mutants.

A comparative study of photosystem II complexes isolated from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contains normal stacked thylakoid membranes, and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked grana or essentially unstacked thylakoids with occasional membrane doublings, has been carried out. The corresponding photosystem II complexes had an O2 evolving activity ranging from 290 (for the wild type) to 1100 µmol O2 x mg chlorophyll(-1) x h(-1) (for the mutant Su/su var. Aurea). The reduced photosynthetic unit size was also obvious in the mangenese and cytochromeb559 content. The photosystem II complex from the wild type contained 4 Mn and 1 cytochromeb559 per 200 to 280 chlorophylls, while the corresponding value for the mutant Su/su var. Aurea was 4 Mn and 1 cytochromeb559 per 35 to 60 chlorophylls. We have also examined the polypeptide composition and show that the photosystem II complex from the wild type consisted of polypeptides of 48, 42, 33, 32, 30, 28, 23, 21, 18, 16 and 10 kDa, while the mutant complex mainly contained the polypeptides of 48, 42, 33, 32, 30, 28 and 10 kDa. In the mutant photosystem II complex the light-harvesting chlorophyll protein (peptide of 28 kDa) was reduced by a factor of 5 to 6 as compared to the wild type. With respect to the peptide composition and the photosynthetic unit size, the Triton-solubilized photosystem II complex from the mutant Su/su var. Aurea was very similar to O2 evolving photosystem II reaction center core complexes.

Inhibition of photosynthetic electron transport in tobacco chloroplasts and thylakoids of the blue green alga Oscillatoria chalybea by an antiserum to synthetic zeaxanthin.

An antiserum to synthetic Zeaxanthin inhibits photosynthetic electron transport on the oxygen-evolving side of photosystem II in tobacco chloroplasts and thylakoids of the filamentous blue-green alga Oscillatoria chalybea. The inhibition site lies for both species between the site of electron donation of water or tetramethyl benzidine and that of diphenyl carbazide or manganese II ions. Typical photosystem I reactions are not impaired by the antiserum. The effect of the antiserum concerning the inhibition site is practically identical to that of the earlier described antiserum to violaxanthin. However, the degree of inhibition seems to be generally somewhat lower with the antiserum to Zeaxanthin, than with that to violaxanthin which hints at a lesser accessibility of zeaxanthin, in the tylakoid membrane in comparison to violaxanthin. In the course of these investigations new evidence was obtained that the oxygen-evolving side of the electron transport scheme is differently organized in Oscillatoria chalybea when compared to tobacco chloroplasts. Thus, the silicomolybdate reduction with water as the electron donor is sensitive to DCMU in these algae.