Klaus P. Bader

Study on the properties of the S3-state by mass spectrometry in the filamentous cyanobacterium Oscillatoria chalybea

Abstract In the present paper we analyzed the properties of the S 3 -state in the filamentous cyanobacterium Oscillatoria chalybea by mass spectrometry. In this organism a substantial O 2 -signal due to a single flash is observed even after extensive dark adaptation (20 min). This signal can be measured by mass spectrometry as well as amperometrically on an oxygen electrode and is not due to an interference of respiratory and photosynthetic electron transport in the prokaryotic membrane. The mass spectrometric analysis shows that, if S 3 is generated by two flashes in a medium containing only H 2 16 O, addition of H 2 18 O and subsequent firing of a third flash yields O 2 evolution labelled with 18 O. It appears that the isotopic composition of the O 2 evolved corresponds to the isotopic composition of the water in the suspension. This experiment shows that water oxidation does not proceed via an oxygen precursor or water derivatives bound to the S 3 -state. This conclusion has been reached shortly before ours by Radmer and Ollinger [15] in the reverse marker experiment. From our study with O. chalybea it appears that freshly generated S 3 can be distinguished from metastable S 3 by the mass spectrometric method. It looks as if in contrast to freshly generated S 3 metastable S 3 contained bound unexchangeable water or an oxidized water derivative.

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 H218O 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 H218O 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 H218O 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 18O-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.

A Study on Oxygen Evolution and on the S-State Distribution in Thylakoid Preparations of the Filamentous Blue-Green Alga Oscillatoria chalybea

When thylakoid preparations of the filamentous blue-green alga Oscillatoria chalybea are exposed to short (2 or 8 μs) saturating light flashes, the oxygen evolution pattern can be distinguished in several respects from the one usually observed in Chlorella. Thus, it appears that a substantial electrochemical signal is already seen under the first flash with maximal flash yield always occurring under the fourth flash. This refers to dark adapted preparations (up to 60 min). Fitting of such an experimental sequence in the 4-state Kok model yields an S-state population consisting of 36-41% S0, 40-49% S1, 1-10% S2 and up to 13% S3. No abnormality under the first flash is seen in such preparations. Characteristic for sequences with Oscillatoria preparations is a high level of misses which are in the region of 25 per cent, whereas double hits do not seem to play a substantial role in the damping of such sequences. The existence of metastable S3, anyway inconsistent with the coherent Kok model, is not confirmed by mass spectrometry. No 18O2 seems to be evolved under the first flash from Oscillatoria thylakoids suspended in 50% H218O. although, when judged from the absolute amperometric signal amplitude, mass spectrometric detection of O2 should have been possible. With the same method we are fully able to detect 18O2 under the second flash in Chlorella vulgaris. In Chlorella this is true for experimental conditions in which the amperometric signal amplitude under the second flash is even smaller than those under the first or second flash in Oscillatoria. The attempt to correlate the amperometrie signal observed under the first flash with a photoinhibition of respiration in our prokaryotic organism was not successful. However, the attempt to incorporate the phenomenon in the coherent Kok model shows that the Oscillatoria sequence fully resembles those with Chlorella, if the first flash signal and 40-50% of the signal observed under the second flash is simply removed from the sequence. The remaining sequence exhibits the usual properties known from Chlorella or higher plant chloroplasts. If one assumes contribution of the reduced state S-1 to the dark population of S-states, a fit in the five rank Kok model yields correct adjustments with a S-state distribution of 6-20% S-1, 31-40% S0, 49-54% S1, 0% S2 and 0% S3 which would be fully consistent with the Kok model and corresponds to the distribution observed with Chlorella or higher plant chloroplasts. The question what the first electrochemical signal is due to remains unanswered.

Physiological analyses of the hydrogen gas exchange in cyanobacteria

Abstract Mass spectrometric analysis of the light-induced hydrogen gas exchange in the cyanobacteria Oscillatoria chalybea, Synechocystis PCC 6803, Synechococcus PCC 6301 (Synechococcus leopoliensis; Anacystis nidulans) and Synechococcus elongatus has been carried out by direct detection of molecular hydrogen at m/e = 2 in the H/D collector of a ‘delta’ mass spectrometer. The time curves of the signals reveal an initial outburst of hydrogen at the onset of light, with a subsequent superimposition of a hydrogen uptake in the case of Oscillatoria chalybea. With in vivo cultures of Synechocystis PCC 6803, which have not been shown to photoevolve molecular hydrogen so far, we are able to measure very small but clearly detectable evolution and uptake signals. The principal qualitative features of the transition from hydrogen evolution to uptake during the illumination period in the cases of Oscillatoria chalybea and Synechocystis PCC 6803 are about identical. Upon addition of increasing concentrations of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), an increased stable hydrogen photoevolution shows up with the hydrogen uptake being completely suppressed. In the case of Oscillatoria chalybea this effect is obtained with 5 μM CCCP, whereas with Synechocystis PCC 6803 generally higher CCCP concentrations are required to stabilize the hydrogen evolution over time. However, in the presence of CCCP the hydrogen evolution from Synechocystis PCC 6803 (which is nearly negligible in the controls without additions) reaches values very similar to those observed with Oscillatoria chalybea. The stimulatory effect of CCCP is clearly distinct from an uncoupling of electron transport and photophosphorylation, as the addition of ammonium chloride inhibits hydrogen evolution but exerts the expected effect of stimulation of photosynthetic oxygen evolution. However, modification of the pH value of the reaction buffer results in a clear dependency of the hydrogen gas exchange on the external proton concentration. Basic pH values at about > 9 diminish the gas exchange signal as a whole. Acidic pH at about 4–5 substantially increases the evolution and decreases the uptake part of the gas exchange signals. Thus, at a pH of 4.4, the hydrogen gas exchange signal largely corresponds to the one observed with an assay at pH 7.5 but in the presence of CCCP. When the cyanobacterial reaction assays are flushed with pure nitrogen or with N2/CO2 (1%), the hydrogen evolution rates are by no means decreased; in some cases the hydrogen photoevolution appears rather to be stimulated by the presence of CO2 in the flushing gas. Concomitant recording of the light-induced carbon dioxide reveals a strong initial CO2, uptake which is substantially diminished within the first minute of illumination. Thus, mass spectrometric analysis of the light-induced carbon dioxide gas exchange favours the CO2 concentrating mechanism discussed for cyanobacteria rather than the light activation of a dark inactive Calvin cycle.

Physiological and evolutionary aspects of the O2/H2O2-cycle in cyanobacteria

Various cyanobacteria evolve oxygen upon illumination with the very first flash of a sequence and interact with the oxygen from the surrounding atmosphere. In the present paper I describe recent experiments allowing the discrimination between photosynthetic water splitting and the light induced peroxide decomposition, the latter showing a strong dependence on the oxygen partial pressure in the suspension. Under appropriate conditions a clear period-2 oscillation of a flash sequence is observed. This fits into the interpretation, as the decomposition of hydrogen peroxide requires only two light quanta and S2 has been shown to be the most reactive redox state within the S-state system. Comparison of the relaxation kinetics of the first two flashes of a sequence with the steady state signals as well as the detailed analysis of the mass spectrometric signals revealed completely different time constants for both water splitting and peroxide decomposition. These observations can be correlated to the suggestion that a peroxide might be involved in the higher oxidation states (Renger, G. (1987) Photosynthetica 21, 203–224). Moreover, the phenomena can be specifically and independently influenced and stimulated e.g. by the addition of various salts which strongly increases the water splitting reaction. In the present discussion on the participation of hydrogen peroxide the reaction sequences described for Oscillatoria in this and in previous papers might be the linking condition between oxygenic photosynthesis and the type of anoxygenic photooxidations such as the photooxidation of ferrous or sulfide ions which prevailed earlier. From the evolutionary point of view these two mechanisms might have been linked by the involvement of a quasi semireduced component like hydrogen peroxide.

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 H2 18O and the temperature and measured water splitting as 18O2, 16O18O, and 16O2 by mass spectrometry. We show that the approach to the equilibrium of the system after H2 18O addition can be very well understood in terms of the diffusion of water molecules. The equilibrium states of 16O2, 16O18O, and 18O2 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 H2 18O and H2 16O 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.

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 QA, gave a decreased O2 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.

Light-induced oxygen uptake in tobacco chloroplasts explained in terms of chlororespiratory activity

Abstract Chloroplasts from higher plants (Nicotiana tabacum var. John Williams Broadleaf) exhibit a substantial oxygen uptake upon illumination with one or more short saturating light flashes. This uptake was detected and analyzed by means of mass spectrometry as 18O2-gas exchange following the addition of 5 ml 18O2 to the gas phase over the buffered reaction assay. Along with the light-induced oxygen uptake we measured photosynthetic water oxidation as oxygen evolution at m e = 32 . The oxygen uptake of 18O2 measured at m e = 36 can be completely inhibited by various electron acceptors and by silicomolybdate in particular. The same holds true for PS II-inhibitors like DBMIB. The effects of DCMU and of different light qualities (blue, red and far-red) on the light-induced oxygen uptake are discussed. We conclude from our results that the chlororespiratory activity within the thylakoid membranes is responsible for the observed oxygen uptake and that the plastoquinone pool is the component shared between both, photosynthetic and respiratory electron transport chains.

Mass spectrometric analysis of a photosystem-II-mediated oxygen uptake phenomenon in the filamentous cyanobacterium, Oscillatoria chalybea

Abstract Flash-induced oxygen production was studied by mass spectrometry in thylakoid particle preparations of the filamentous cyanobacterium Oscillatoria chalybea . Essentially, two oxygen uptake phenomena related to Photosystem II were observed. First, photosynthetic oxygen evolution requires the presence of a minimal threshold quantity of oxygen. Under completely anaerobic conditions the photosynthetic water-splitting reaction does not occur. In nitrogen-flushed assays, a small oxygen uptake precedes oxygen evolution induced by a train of short saturating flashes. Second, flash-induced photosynthetic oxygen evolution was measured in the presence of the oxygen isotope, 18 O 2 , in the ambient atmosphere of the assay. The oxygen evolved was labelled with 18 O 2 , which showed that the evolution reaction included an 18 O 2 -uptake phenomenon. The labelling density completely excludes 18 O 2 exchange via H 2 18 O (by respiration or other processes) and subsequent photosynthetic water splitting, since too little mixed ( 16 O 18 O) oxygen label was found. Since most of the label was found to be 18 O 2 , the label could come from hydrogen peroxide (or an equivalent) produced in the immediate vicinity of the S-state system. The decomposition of this hydrogen peroxide appears to be managed by the S-state system (S 2 ) itself. Addition of exogenous hydrogen peroxide or the addition of high amounts of catalase does not affect the behaviour of our preparation. The phenomenon seems to be an inherent property of our cyanobacterium and does not take place in tobacco chloroplasts under identical conditions. The phenomenon seems to play a role under natural conditions, also, and might be the consequence of the absence of two of the extrinsic polypeptides in cyanobacteria. It is enhanced by high concentrations of oxygen in the ambient atmosphere and is diminished by low oxygen tension.

18O isotope effect in the photosynthetic water splitting process

In mass spectroscopic experiments of oxygen evolution in Photosystem II at 50% enrichment of H(2)18O, one expects equal signals of 18O(2) and 16O(2) unless one of the isotopes is favored by the oxygen evolving complex (OEC). We have observed a deviation from this expectation, being a clear indication of an isotope effect. We have measured the effect to be 1.14-1.30, which is higher than the theoretically predicted value of 1.014-1.06. This together with the strong temperature variation of the measured effect with a discontinuity at 11 degrees C observed for wild-type tobacco and at 9 degrees C for a yellow-green tobacco mutant suggest that an additional mechanism is responsible for the observed high isotope effect. The entry of a finite size of water clusters to the cleavage site of the OEC can explain the observation.