On the functional significance of the polypeptide PsbY for photosynthetic water oxidation in the cyanobacterium Synechocystis sp. strain PCC 6803.

Recent investigations have revealed that the cyanobacterial photosystem II complex contains more than 26 polypeptides. The functions of most of the low-molecular-mass polypeptides, including PsbY, have remained elusive. Here we present a comparative characterization of the wild-type Synechocystis sp. strain PCC 6803 and a PsbY-free mutant derived from it. The results show that growth of the PsbY-free mutant was comparable to that of the wild-type when cells were cultivated in complete BG11 medium or under initial manganese or chloride limitation, and when illuminated at 20 or 200 microE m(-2) s(-1). However, while growth rates of both the wild-type and the PsbY-free mutant were reduced when cells were cultivated in BG11 medium in the absence of calcium, the reduction was significantly greater in the case of the PsbY-free mutant. This differential effect on growth of the mutant relative to the wild-type in CaCl(2) deficient medium was detected when the cells were illuminated with high-intensity light (200 microE m(-2) s(-1)) but not when light levels were lower (20 microE m(-2) s(-1)). The differential effect on growth was associated with lower O(2) evolving activity in the mutant compared to wild-type cells. The mutant was also found to be more sensitive to photoinhibition, and showed an altered pattern of fluorescence emission at 77 K. In addition, mass spectrometric analysis revealed that PsbY-free cells cultivated in CaCl(2) sufficient medium (in which no growth reduction was observed) had a significantly higher O(2) evolution from hydrogen peroxide and a lower O(2) evolution from water under flash light illumination than wild-type cells. These results imply that photosystem II is slightly impaired in the PsbY-free mutant, and that the mutant is less capable of coping with low levels of Ca(2+) than the wild-type.

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.

Interaction of quaternary ammonium and phosphonium salts with photosynthetic membranes.

Distinct concentration ranges of selected quaternary ammonium and phosphonium salts were elaborated to induce stimulatory or inhibitory effects, respectively, on photosynthetic reactions. By means of fluorescence induction measurements 3 different effects of alkylbenzyldimethylammonium chloride (ABDAC; zephirol) in chloroplast preparations from Pisum sativum were observed. 60 microM ABDAC produced a strong increase in Fmax with concurrently improved Kautsky kinetics. Increased ABDAC concentration (500 microM) led to a strong fluorescence quenching - virtually indistinguishable from the conditions following the addition of photosystem II electron acceptors like K3Fe[CN]6. Further increase of ABDAC to 5 mM provoked a drastic increase in the fluorescence yield together with the complete loss of any detectable kinetics. We suggest a 3-step interaction of ABDAC with the thylakoid membranes of photosynthetic organisms similar to our earlier discussion (Bader and Höper (1993), Z. Naturforsch. 49c, 87-94). We examined a series of derivatives with selectively modified side chains, central atoms and counter ions, respectively. Both an alkyl chain of the type ([-CH2-]n; n approximately > 10) and effective polar components are indispensable for the adsorption and intercalation of the molecule onto and into the thylakoid membranes. The benzyl group could be replaced by a methyl residue without any loss of effectiveness; replacement of the central nitrogen (N) by phosphorus (P) and the counter ion Cl by Br did not modify the effects and the results were indistinguishable from the ABDAC effect proper. Shortening of the alkyl chain to (-CH2-)6 resulted in a less effective interaction of e.g. tetraoctylammonium bromide with the photosynthetic membrane. Flash-induced oxygen evolution measurements with selected derivatives (15 microm) substantiated our interpretation of an improved OEC functioning by a substantial lowering of the miss parameter a and the exclusion of a chemical reduction as the standard S-state distribution was not affected. As evidenced by both SDS-PAGE and Western blot analyses the investigated molecules showed a direct interaction. The polypeptide patterns were characterized by a severe shift of the molecular weight components from high (20-67 kDa) to low (< 20 kDa) values.

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.

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.

The IdiA protein of Synechococcus sp. PCC 7942 functions in protecting the acceptor side of Photosystem II under oxidative stress.

Synechococcus sp. strains PCC 7942 and PCC 6301 contain a 35 kDa protein called IdiA (Iron deficiency induced protein A) that is expressed in elevated amounts under Fe deficiency and to a smaller extent also under Mn deficiency. Absence of this protein was shown to mainly damage Photosystem II. To decide whether IdiA has a function in optimizing and/or protecting preferentially either the donor or acceptor side reaction of Photosystem II, a comparative analysis was performed of Synechococcus sp. PCC 7942 wild-type, the IdiA-free mutant, the previously constructed PsbO-free Synechococcus PCC 7942 mutant and a newly constructed Synechococcus PCC 7942 double mutant lacking both PsbO and IdiA. Measurements of the chlorophyll fluorescence and determinations of Photosystem II activity using a variety of electron acceptors gave evidence that IdiA has its main function in protecting the acceptor side of Photosystem II. Especially, the use of dichlorobenzoquinone, preferentially accepting electrons from Q(A), gave a decreased O(2) evolving activity in the IdiA-free mutant. Investigations of the influence of hydrogen peroxide treatment on cells revealed that this treatment caused a significantly higher damage of Photosystem II in the IdiA-free mutant than in wild-type. These results suggest that although the IdiA protein is not absolutely required for Photosystem II activity in Synechococcus PCC 7942, it does play an important role in protecting the acceptor side against oxidative damage.

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.

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.

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.