On the mechanism of photosynthetic sulfate reduction

Summary An enzyme has been purified from the green alga Chlorella which transfers sulfate from APS onto glutathion and dithiotreitol. The enzyme does not utilize PAPS unless a crude fraction containing a 3-phosphonucleotidse is used. From the data it is assumed that in photosynthetic sulfte reduction of plants APS rather than PAPS transfers its sulfte group onto a carrier-bound SH-group—replaceable in vitro by glutathion—to form an S-sulfocompound which then might be reduced to the level of sulfide.Abbreviations APS adenosine-phosphosulfate - PAPS 3 phospho-adenosine-5-phosphosulfate - GSH reduced glutathion - GS-SG oxidized glutathion - GS-SO₃H S-sulfoglutathion     

Silicomolybdate reduction by isolated pea chloroplasts

Conditions for the optimization of silicomolybdate reduction by isolated pea chloroplasts are described. Maximum rates of reduction are related to time of addition to the chloroplasts and the presence of an oxidizing cofactor, such as ferricyanide. Silicomolybdate or silicomolybdate plus ferricyanide reduction is only partially inhibited by a concentration of CMU which totally abolishes ferricyanide reduction. Evidence for a differing response of the two reduction sites to silicomolbydate is described.     

Superoxide reduction as a mechanism of ascorbate-stimulated oxygen uptake by isolated chloroplasts

Addition of 1m$\text{M}$ ascorbate to isolated chloroplasts with methyl viologen (MV) as electron acceptor trebled the rate of oxygen uptake and decreased the $\text{ADP}\text{O}$ ratio to a third of that with no ascorbate present. These effects of ascorbate were reversed by superoxide dismutase (SOD), which in the absence of ascorbate had little effect on O₂ uptake or $\text{ADP}\text{O}$ ratio. A chloroplast-associated SOD activity equivalent to 500 units/mg chlorophyll was detected. The effects of ascorbate and SOD on O₂ uptake were similar in both coupled and uncoupled chloroplasts. The results are consistent with the hypothesis that ascorbate stimulates O₂ uptake by reduction of superoxide, which is formed by autoxidation of the added electron acceptor (MV), and which dismutates in the absence of ascorbate. Ascorbate does not seem to stimulate O₂ uptake by replacing water as the photosystem II donor.     

Inhibition of the photosynthetic capacity of isolated chloroplasts by ozone

Isolated spinach chloroplasts have been used as a model system for studying the interaction of ozone, a component of photochemical smog, with plant membranes. Ozone bubbled into a suspension of isolated chloroplasts inhibits electron transport in both photosystems without uncoupling ATP production. Photosystem I (reduced 2,6-dichlorophenolindolphenol → NADP⁺) is a little more sensitive than photosystem II (H₂O → 2,6-dichlophenolindolphenol). Ozone does not act as an energy transfer inhibitor, since the drop in ATP production and high energy intermediate (measured by amine-induced swelling) is nearly parallel to the decline in electron transport. A reasonable hypothesis is that ozone disrupts the normal pathway of energy flow from light-excited chlorophyll into the photoacts by a disruption of the components of the membrane but not a general disintegration of the membrane. In addition, ozone does not seem to penetrate into the grana region through the outer membrane of intact plastids, since ozone lowers the bicarbonate-supported O₂ evolution but does not affect the rate of ferricyanide reduction in the same plastids after osmotic disruption. This would indicate that the effect of ozone on green plants, at low concentrations, may be due to the interaction of ozone with the first membrane it contacts and not directly with internal metabolic processes.     

Factors influencing photosynthetic enhancement in isolated chloroplasts

Photosynthetic enhancement of oxygen evolution (linked to CO₂ assimilation) in isolated chloroplasts was found to be governed by the supply of ATP. The addition of ATP (but not AMP) abolished enhancement that consistently occurred without added ATP. Enhancement in the H₂O → NADP reaction by chloroplasts was investigated in the light of one recent report that the phenomenon occurs when pure ferredoxin is replaced by a crude preparation (PPNR) and another report that the phenomenon is governed by Mg⁺⁺ concentration. Fractionation of PPNR led to the isolation of a protein factor which when added to pure ferredoxin induced enhancement. However, the rate of NADP reduction with pure ferredoxin and without enhancement was greater than the maximum rate of NADP reduction with enhancement induced by either the protein factor of PPNR. The report that Mg⁺⁺ concentration governs enhancement was not confirmed.     

Polarographic study of oxaloacetate reduction by isolated pea chloroplasts

Suspensions of pea chloroplasts, prepared by differential centrifugation, catalyzed oxaloacetate-dependent O(2) evolution (mean rate of 29 determinations 10.9 micromoles per milligram of chlorophyll per hour, sd 3.2) with the concomitant production of malate. At optimum concentrations of oxaloacetate, both reactions were light-dependent, inhibited by 3-(3,4- dichlorophenyl)-1, 1-dimethylurea and oxalate, and enhanced 2.5- to 4-fold by 10 millimolar NH(4)Cl. At concentrations of oxaloacetate (<50 micromolar), 10 millimolar NH(4)Cl was inhibitory. The ratio of O(2) evolved to malate produced was 0.39 to 0.58. The ratio of O(2) evolved to oxaloacetate supplied was commensurate with the theoretical value of 0.5.Chloroplast suspensions contained both NAD- and NADP-malate dehydrogenase activities. It was concluded from oxalate inhibition studies and the promotion of oxaloacetate-dependent O(2) evolution by shocked chloroplasts by NADPH (but not NADH) that the reaction was mediated via the NADP enzyme.     

Inhibition of photosynthetic carbon metabolism in isolated chloroplasts by iodoacetol phosphate

Carbon dioxide-dependent and 3-phosphoglycerate (PGA)-dependent O₂ evolution by isolated chloroplasts of wheat is inhibited by micromolar levels of iodoacetol phosphate (IAP). Loss of the activity is time-dependent and a higher concentration of PGA increases the half-time for inhibition (e.g. at 40 micromolar IAP the half-time is about 0.5 minutes at 1 millimolar PGA compared to 1.5 minutes at 10 millimolar PGA). A marked inhibition of NADP glyceraldehyde-3-P dehydrogenase was observed when chloroplasts were pretreated with micromolar levels of IAP, osmotically shocked, and several stromal enzymes assayed.     

Level of photosynthetic intermediates in isolated spinach chloroplasts

The level of intermediates of the photosynthetic carbon cycle was measured in intact spinach chloroplasts in an attempt to determine the cause of the induction lag in CO₂ assimilation. In addition, transient changes in the level of the intermediates were determined as affected by a light-dark period and by the addition of an excess amount of bicarbonate during a period of steady photosynthesis. Assayed enzymically were: ribulose 1,5-diphosphate, pentose monophosphates (mixture of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, hexose monophosphates (mixture of glucose 6-phosphate, glucose 1-phosphate, and fructose 6-phosphate), glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, glycerate acid 3-phosphate, a mixture of fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP).     

Sulfide-induced oxygen uptake by isolated spinach chloroplasts catalyzed by photosynthetic electron transport

In addition to an inhibitory effect on the photoreduction of NADP⁺ by isolated spinach chloroplasts ( Spinacea oleracea L. cv. Melody Hybrid), sulfide initiated oxygen uptake by chloroplasts upon illumination, both in presence and absence of an electron acceptor. Sulfide-induced oxygen uptake was sensitive to DCMU demonstrating the involvement of photosynthetic electron transport. Addition of superoxide dismutase to the chloroplast suspension prevented the sulfide-induced oxygen uptake, which indicated that sulfide may be oxidized by the chloroplast, its oxidation being initiated by superoxide formed upon illumination (at the reducing side of PSI). Tris-induced inhibition of NADP⁺ photo-reduction could not be abolished by sulfide, which indicated that sulfide could not act as an electron donor for PSI.     

Primary photosynthetic reactions in isolated chloroplasts fixed by glutaric aldehyde]

The primary photosynthetic reactions in isolated pea chloroplasts with the structures fixed by increasing concentrations of glutaric aldehyde were studied. It was shown that under chloroplast fixation by 5--25 mM of glutaric aldehyde, a significant inhibition of processes responsible for energy transformation in biological membranes was observed. The highest sensitivity was observed for the phosphorylation reactions, photo-induced changes in absorption at 520 nm, photo-induced quenching of atebrin fluorescence and slow component of delayed light emission. The photo-induced proton uptake was found to be less sensitive to fixation by glutaric aldehyde. It was also shown that on chloroplast fixation the extent of the steady-state P700 oxidation and the lifetime of the photosystem I and II chlorophyll fluorescence are both increased, a fact is indicating of loss in the effectiveness of light energy transfer from the antenna molecules to the reaction centres. Presumably the conformational changes play an essential role at the initial steps of light energy transformation.     

Inhibition of the photosynthetic activities of isolated spinach chloroplasts by phosphonate compounds

The effects of three closely related phosphonate compounds on several photosynthetic activities of isolated chloroplasts were investigated. Phosphonoformic and phosphonopropionic acid were found to inhibit both CO₂ fixation and the reduction of 3-phosphoglyceric acid, with CO₂ fixation being more sensitive. In contrast, phosphonoacetic acid was only slightly inhibitory. The lack of inhibition appeared to be due to its inability to enter the stroma via the phosphate translocator. Measurements of changes in stromal metabolite levels following the inhibition of CO₂ fixation by either phosphonoformic or phosphonopropionic acid indicated that the activity of ribulose bisphosphate carboxylase/oxygenase was reduced. Studies with the isolated enzyme confirmed that both of these compounds were effective competitive inhibitors of the carboxylase activity of the enzyme.     

Photoacoustic detection of photosynthetic activities in isolated broken chloroplasts

Methodology and demonstration how to utilize the photoacoustic technique in photosynthesis research are presented. Photoacoustic signals were obtained from suspensions of isolated broken chloroplasts. In the presence of strong, continuous (non-modulated) background light the signals were normally larger than without the background light. The effect of the background light was saturable and was absent when non-active (e.g. heat-treated) samples were used, showing that the normal smaller signal in the absence of background light is a genuine reflection of the loss of heat due to the competing photochemistry. The effect of the background light is to close the reaction-centers and hence to inhibit the photochemical process. The percent difference of the photoacoustic signal (± background light) is taken as a measure of the photochemical activity (‘photochemical loss’).

Initial results demonstrate the wavelength dependence of the ‘photochemical loss’. As expected there was a ‘red-drop’ decrease of the ‘photochemical loss’ for λ > 690 nm, when the cofactor methyl viologen was present. Surprisingly, however, there was a ‘red-rise’ increase for λ > 690 nm when no cofactor was present. These findings indicate that under the last conditions there is an unsuspected photoactivity of PS I which was not detected hitherto by the conventional techniques. The dependence on the background light intensity confirms this result. This photoactivity can be explained tentatively as a cyclic electron flow around PS I, present without any added cofactor.

Initial results on the modulation frequency dependence in the presence of electron acceptors are also demonstrated.     

The cupric ion as an inhibitor of photosynthetic electron transport in isolated chloroplasts

Strong inhibition of uncoupled photosynthetic electron transport by Cu²⁺ in isolated spinach chloroplasts was observed by measuring changes in O₂ concentration in the reaction medium. Inhibition was dependent not only on the concentration of the inhibitor, but also on the ratio of chlorophyll to inhibitor. Binding of Cu²⁺ to the chloroplast membranes resulted in removal of Cu²⁺ from solution. When chloroplasts were exposed to preincubation in light, there was increased inhibition as a result of Cu²⁺ binding to inhibitory sites. Preincubation in the dark resulted in Cu²⁺ binding to noninhibitory sites and decreased inhibition. The degree of inhibition was lower at low light intensities than at high light intensities.     

Effects of disalicylidenepropanediamines on photosynthetic electron transport of isolated spinach chloroplasts

The effects of disalicylidenepropanediamine (DSPD) and disulfo-disalicylidenepropanediamine (sulfo-DSPD) on the photosynthetic electron transport of isolated chloroplasts have been reexamined.