Studies on the pathway of cyclic electron flow in mesophyll chloroplasts of a C4 plant

1. Cyclic photophosphorylation driven by white light, as followed by ¹⁴CO₂ fixation by mesophyll chloroplast preparations of the C₄ plant Digitaria sanguinalis, was specifically inhibited by disalicylidenepropanediamine (DSPD), antimycin A, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIb), 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDAC), and KCN suggesting that ferredoxin, cytochrome b₅₆₃, plastoquinone, cytochrome f, and plastocyanin are obligatory intermediates of cyclic electron flow. It was found that 0.2 μM DCMU and 40 μM o-phenanthroline blocked noncyclic electron flow, stimulated cyclic photophosphorylation, and caused a partial reversal (40–100%) of the inhibition by DBMIB and antimycin A, but not DSPD.

2. Cyclic photophosphorylation could also be activated using only far-red illumination. Under this condition, however, cyclic photophosphorylation was much less sensitive to the inhibitors DBMIB, EDAC and antimycin A, but remained completely sensitive to DSPD and KCN. Inhibition in far-red light was not increased by preincubating the chloroplasts with the various inhibitors for several minutes in white light.

3. The striking correspondence between the effects of photosystem II inhibitors, DCMU and o-phenanthroline, on cyclic photophosphorylation under white light and cyclic photophosphorylation under far-red light (in the absence of photosystem II inhibitors) suggests that electrons flowing from photosystem II may regulate the pathway of cyclic electron flow.     

Photosynthetic induction phenomena in spinach chloroplasts in relation to the nature of the isolating medium

1. Measurements were made of photosynthetic CO₂ fixation and O₂ evolution by spinach chloroplasts isolated in sorbitol media containing 2-(N-morpholino)ethanesulphonate (MES).

2. The chloroplasts isolated in MES-sorbitol media exhibited induction phenomena which were similar to those shown by chloroplasts isolated in orthophosphate-sugar mixtures. Added ribose 5-phosphate shortened the lags which preceded the attainment of maximal rates of CO₂ fixation and O₂ evolution. O₂ evolution reached its maximum rate almost immediately in the presence of 3-phosphoglycerate. Induction periods were shortened by pre-illumination of the parent tissue prior to separation of the chloroplasts.

3. In the absence of added substrate (other than CO₂) lags exhibited by chloroplasts isolated in MES-sorbitol were shorter than those observed with chloroplasts prepared in orthophosphate-sorbitol. These shorter lags could be extended by briefly exposing the chloroplasts to sugar media containing orthophosphate, malate or acetate or to Tris-NaCl.

4. The results are discussed in relation to photosynthetic induction phenomena and current methods of chloroplast isolation.     

A light- and cysteine-activated ATP-Pi exchange reaction in chloroplasts and its relationship to ATPase and photophosphorylation

1. ‘Broken’ chloroplasts from spinach if illuminated for a period in the presence of cysteine and phenazine methosulphate develop an ATP-P_i exchange activity which can be observed in the dark. The conditions giving rise to ATP-P_i exchange activity are similar to those giving rise to the thiol-activated light-triggered ATPase.

2. ATP is not necessary during illumination for development of ATP-P_i exchange activity, but the activity declines if a period of time elapses between illumination and addition of ATP. This is accompanied by a similar decline in the cysteine-activated light-triggered ATPase.

3. The ATP-P_i exchange and ATPase show the same dependence on ATP concentration and are both inhibited by added ADP.

4. Both reactions are inhibited by Dio-9.

5. Desaspidin, 4-octyl-2,6-dinitrophenol and carbonyl cyanide 4-trifluoromethoxyphenylhydrazone, added immediately after illumination, inhibit the ATP-P_i exchange. The ATPase is initially stimulated under these conditions and then inhibited. If present during illumination, desaspidin and octyldinitrophenol inhibit the ATPase.

6. It is concluded that the ATP-P_i exchange reaction and the ATPase are activities of the same enzyme complex in the chloroplast and that this is probably part of the terminal enzyme system of photophosphorylation.     

The measurement of cyclic photophosphorylation in isolated chloroplasts by determination of hydrogen ion consumption. An evaluation of the method using titration at constant pH

1. An improved method for the continuous measurement of phosphorylation is reported.

2. This method, in which the consumption of H⁺ during the reaction is followed by titration at constant pH using commercially available apparatus, has been evaluated for photophosphorylation by isolated chloroplasts.

3. By use of an independent assay for ATP it is shown that the method gives a valid and precise measurement of photophosphorylation.     

Studies on the enzyme systems involved in electron and energy transfer in isolated chloroplasts. I. Effect of endogenous phosphate on the photophosphorylation coupled with noncyclic electron transport in intact chloroplasts

1. It was found that the P/2e ratio was independent of the degree of electron transport stimulation in the presence of ADP and P_i and exceeded 1.0 in the preparations with slight (30 %) as well as with high (80 %) stimulation.

2. Chloroplast preparations having a low content of endogenous P_i showed higher stimulation than those with higher contents.

3. Illumination of the chloroplasts in the presence of ADP and electron acceptor led to a decrease of endogenous P_i content that resulted in an increase of electron transport stimulation in the presence of exogenous P_i.

4. Electron transport in the absence of exogenous P_i was inhibited by both exogenous ADP and ATP.

5. It appears that the electron transport in the absence of exogenous P_i is coupled to phosphorylation, which occurs because isolated chloroplasts contain endogenous P_i. Stimulation of the electron transport by the addition of ADP and P_i seems to be caused by acceleration of the existing electron transport pathway, and not from the initiation of a new one.     

The reduction of 3-phosphoglycerate by reconstituted chloroplasts and by chloroplast extracts

1. The rate of 3-phosphoglycerate reduction in extracts from spinach chloroplasts, assayed by spectrophotometric measurement of 3-phosphoglycerate-dependent NADPH oxidation, was strongly inhibited by ADP. AMP was much less inhibitory.

2. Oxygen evolution by reconstituted chloroplasts with 3-phosphoglycerate as substrate was also inhibited by the addition of ADP or following uncoupling by added NH₄Cl.

3. In all cases the inhibitory effects of ADP were reversed by addition of phosphocreatine and creatine phosphokinase activity.

4. The stoichiometry of 3-phosphoglycerate reduction to NADPH oxidation in chloroplast extracts was 1:1 and there was negligible turnover of the Benson-Calvin cycle in either chloroplast extracts or in reconstituted chloroplasts under the particular conditions employed.

5. The maximum rate of 3-phosphoglycerate-dependent O₂ evolution by reconstituted chloroplasts was ultimately limited by NADP reduction and photo-phosphorylation, and was similar to the maximum rate of oxygen evolution under optimal conditions by intact chloroplasts. In the presence of sufficient ADP phosphorylating activity, the rate of enzymic 3-phosphoglycerate reduction was relatively high. The inhibition of this reaction by ADP may represent a control mechanism in photosynthesis.     

Photochemical systems in mesophyll and bundle sheath chloroplasts of C4 plants

1. The agranal bundle sheath chloroplasts of Sorghum bicolor possess very low Photosystem II activity compared with the grana-containing mesophyll chloroplasts.

2. Sorghum mesophyll chloroplasts have a chlorophyll (chl) and carotenoid composition similar to that of spinach chloroplasts. In contrast, the sorghum bundle sheath chloroplasts have a higher chl a/chl b ratio; they are enriched in β-carotene and contain relatively less xanthophylls as compared to sorghum mesophyll or spinach chloroplasts.

3. Sorghum mesophyll chloroplasts with 1 cytochrome f, 2 cytochrome b₆ and 2 cytochrome b-559 per 430 chlorophylls have a cytochrome composition similar to spinach chloroplasts. Sorghum bundle sheath chloroplasts contain cytochrome f and cytochrome b₆ in the same molar ratios as for the mesophyll chloroplasts, but cytochrome b-559 is barely detectable.

4. The chl/P700 ratios of mesophyll chloroplasts of S. bicolor and mesophyll and bundle sheath chloroplasts of Atriplex spongiosa are similar to that of spinach chloroplasts suggesting that these chloroplasts possess an identical photosynthetic unit size to that of spinach. The agranal bundle sheath chloroplasts of S. bicolor possess a photosynthetic unit which contains only about half as many chlorophyll molecules per P700 as found in the grana-containing chloroplasts.

5. The similarity of the composition of the bundle sheath chloroplasts of S. bicolor with that of the Photosystem I subchloroplast fragments, together with their smaller photosynthetic unit and low Photosystem II activities suggests that these chloroplasts are highly deficient in the pigment assemblies of Photosystem II.     

Fluorescence properties of chloroplasts from manganese deficient and mutant tobacco

1. The kinetics of the fluorescence induction are described for chloroplasts from normal green tobacco, from the aurea tobacco mutant Su/su, from the photosynthetically inactive yellow patches of a variegated tobacco, and from tobacco plants grown in absence of manganese. The first two types display the well-known biphasic induction, but the Su/su chloroplasts have a distinctly slower rise time. Manganese deficient chloroplasts show a significantly higher fluorescence yield than any other type of chloroplasts studied. The kinetics of their fluorescence, on the other hand, are similar to those observed with the inactive chloroplasts from the variegated tobacco: the fluorescence rise is small, and the fluorescence yield is not changed very much by the addition of a reducing agent like hydrosulfite, or by addition of an oxidant like ferricyanide, or by an inhibition of the electron flow in Photosystem II with 3(3,4-dichlorophenyl)-1,1-dimethylurea.

2. Determinations of the amount of the primary electron acceptors associated with Photosystem II point to a 2- to 3-fold larger electron acceptor pool in chloroplasts of young Su/su plants than in chloroplasts of old Su/su plants and of various green leaves, including those from green tobacco. This finding agrees with recently published data on the size of the photosynthetic unit in tobacco mutants and normal green plants.

3. The different fluorescence characteristics of all four types of chloroplasts under study are discussed on the basis of their structure and their activity in photosynthetic O₂ evolution.     

Some effects of inorganic phosphate on O2 evolution by isolated chloroplasts

1. After an initial lag, isolated spinach chloroplasts evolved O₂ in illuminated reaction mixtures containing bicarbonate but no added phosphate. This evolution soon ceased but could be restarted by the addition of phosphate.

2. The phosphate requirement could be met by orthophosphate, inorganic pyrophosphate, ATP or ADP but not by AMP. Approx. 3 molecules of O₂ were evolved for each molecule of orthophosphate added and approx. 6 for each molecule of pyrophosphate.

3. With CO₂ as the sole added substrate the extent of the initial lag in O₂ evolution was not greatly affected by small quantities of added orthophosphate but as the concentration of orthophosphate was increased there was a progressive increase in the lag and a progressive decrease in the maximum rate. Pyrophosphate failed to produce these effects at a 100 times the concentration and in the presence of pyrophosphate the orthophosphate inhibition was less severe. There was little or no orthophosphate inhibition in the presence of substrate quantities of 3-phosphoglycerate or ribose 5-phosphate and CO₂.

4. There was also a requirement for phosphate by chloroplasts evolving O₂ in the presence of 3-phosphoglycerate or ribose 5-phosphate plus CO₂. In the presence of endogenous phosphate only, added ribose 5-phosphate suppressed the O₂ evolution which normally followed the addition of 3-phosphoglycerate.

5. The results provide direct support for the proposed phosphate requirement of the photosynthetic carbon cycle and are discussed in this context. They also imply that orthophosphate, ribose 5-phosphate and 3-phosphoglycerate can penetrate the intact chloroplast envelope with considerable rapidity.     

Primary reactions, plastoquinone and fluorescence yield in subchloroplast fragments prepared with deoxycholate

1. In subchloroplast fragments prepared with the detergent deoxycholate the primary reactions of Photosystem II could be studied at room temperature, because the secondary reactions were largely or completely inhibited.

2. The main quencher of chlorophyll fluorescence in these particles was the photosynthetically active pool of plastoquinone in its oxidized form. Its photoreduction in the presence of artificial electron donors was accompanied by a shift of a chlorophyll a absorption band. Its reoxidation in the dark was very slow, even in the presence of ferricyanide.

3. Of all the artificial electron donors tested MnCl₂ was by far the most efficient.

4. Measurements at room temperature of the C550 absorbance change confirmed its correlation with the primary electron acceptor. Its difference spectrum was broader and its extinction coefficient correspondingly lower than at liquid-N₂ temperature. In chloroplasts the C550 concentration was about 1:360 chlorophylls.

5. In the dark C550 was largely in the reduced state and its oxidation by plastoquinone took place in the presence of an artificial electron donor only, suggesting that the redox potential of C550 was increased by accumulated positive charges at the donor side of the reaction center.

6. The free radical 1,1′-diphenyl-2-picrylhydrazyl oxidized C550 directly in a 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-insensitive reaction. A DCMU-insensitive oxidation of C550 was observed at high ferricyanide concentrations as well, but probably in this case an endogenous electron donor was oxidized, which in turn oxidized C550 via the back reaction of the photochemical reaction.

7. The oxidized form of the primary electron donor, P680⁺, accumulated in the light in the presence of deoxycholate and a low ferricyanide concentration. In chloroplasts the P680 concentration was about 1:360 chlorophylls.

8. The P 680 absorption difference spectrum and electron spin resonance could be explained by the oxidation of a chlorophyll a dimer. Repeated deoxycholate treatments progressively changed the spectra to those of a monomer. The monomer was still photochemically active.

9. A new interpretation of the difference spectrum of P700 is proposed: it may be the same as that of the difference spectrum of P680 if the bleaching at 700 nm is attributed to a band shift.     

Anthroyl stearate as a fluorescent probe of chloroplast membranes

1. A reversible light-induced enhancement of the fluorescence of a “hydrophobic fluorophore”, 12-(9-anthroyl)-stearic acid (anthroyl stearate), is observed with chloroplasts supporting phenazine methosulfate, cyclic or 1,1′-ethylene-2,2′-dipyridylium dibromide (Diquat) pseudo-cyclic electron flow; no fluorescence change is observed when methyl viologen or ferricyanide are used as electron acceptors. The stearic acid moiety of anthroyl stearate is important for its localization and fluorescence response in the thylakoid membrane, since structural analogs of anthroyl stearate lacking this group do not show the same response.

2. This effect is decreased under phosphorylating conditions (presence of ADP, P_i, Mg²⁺), and completely inhibited by the uncoupler of phosphorylation NH₄Cl (5–10 mM), as well as the ionophores nigericin and gramicidin-D (both at 5 · 10⁻⁸ M). The MgCl₂ concentration dependence of the anthroyl stearate enhancement effect is identical to that previously observed for cyclic photophosphorylation, as well as for the formation of a “high energy intermediate”. The anthroyl stearate fluorescence enhancement is inhibited by increasing concentrations of ionophores in parallel with the decrease in ATP synthesis, but is essentially unaffected by specific inhibitors (Dio-9 and phlorizin) of photophosphorylation; thus, it appears that anthroyl stearate monitors a component of the “high energy state” of the thylakoid membrane rather than a terminal phosphorylation step.

3. The light-induced anthroyl stearate fluorescence enhancement is suggested to monitor a proton gradient in the energized chloroplast because (a) similar enhancement can be produced by sudden injection of hydrogen ions in a solution of anthroyl stearate; (b) when the proton gradient is dissipated by gramicidin or nigericin light-induced anthroyl stearate fluorescence is eliminated; (c) when the proton gradient is dissipated by tetraphenylboron, light-induced anthroyl stearate fluorescence decreases, and (d) light-induced anthroyl stearate fluorescence change as a function of pH is qualitatively similar to that observed with other probes for a proton gradient (e.g. 9-aminoacridine). Furthermore, anthroyl stearate does not monitor H⁺ uptake per se because (a) the pH dependence of H⁺ transport is different from that of the anthroyl stearate fluorescence change, and (b) tetraphenylboron, which does not inhibit H⁺ uptake, reduces anthroyl stearate fluorescence.

Thus, anthroyl stearate appears to be a useful probe of a proton gradient supported by phenazine methosulfate or Diquat catalyzed electron flow and is the first “non-amine” fluorescence probe utilized for this purpose in chloroplasts.     

Light-dependent changes of the Mg concentration in the stroma in relation to the Mg dependency of CO2 fixation in intact chloroplasts

(1) Light-dependent changes of the Mg²⁺ content of thylakoid membranes were measured at pH 8.0 and compared with earlier measurements at pH 6.6. In a NaCl and KCl medium, the light-dependent decrease in the Mg²⁺ content of the thylakoid membranes at pH 8.0 is found to be 23 nmol Mg²⁺ per mg chlorophyll, whereas in a sorbitol medium it is 83 nmol Mg₂₊ per mg chlorophyll.

(2) A light dependent increase in the Mg²⁺ content of the stroma was detected when chloroplasts were subjected to osmotic shock, amounting to 26 nmol/mg chlorophyll. Furthermore, a rapid and reversible light-dependent efflux of Mg²⁺ has been observed in intact chloroplasts when the divalent cation ionophore A 23 187 was added, indicating a light-dependent transfer of about 60 nmol of Mg²⁺ per mg chlorophyll from the thylakoid membranes to the stroma.

(3) CO₂ fixation, but not phosphoglycerate reduction, could be completely inhibited when A 23 187 was added to intact chloroplasts in the absence of external Mg²⁺. If Mg²⁺ was then added to the medium, CO₂ fixation was restored. Half of the maximal restoration was achieved with about 0.2 mM Mg²⁺, which is calculated to reflect a Mg²⁺ concentration in the stroma of 1.2 mM. The further addition of Ca²⁺ strongly inhibits CO₂ fixation.

(4) The results suggest that illumination of intact chloroplasts causes an increase in the Mg²⁺ concentration of 1–3 mM in the stroma. Compared to the total Mg²⁺ content of chloroplasts, this increase is very low, but it appears to be high enough to have a possible function in the light regulation of CO₂ fixation.     

Regulation of fructose-1,6-bisphosphatase activity in intact chloroplasts. Studies of the mechanism of inactivation

1. The aim of this work was to investigate the mechanism of dark inactivation of fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) in isolated intact chloroplasts of Triticum aestivum.

2. Dark inactivation of the enzyme, which was rapid under aerobic conditions, was prevented under anaerobic conditions when chloroplasts were incubated in the absence of an electron acceptor. Electron acceptors such as oxaloacetate readily brought about inactivation under anaerobic conditions whether chloroplasts were illuminated or in the dark. Inactivation of the enzyme also occurred if illuminated or darkened anaerobic chloroplasts were exposed to oxygen.

3. Pyocyanine, which catalyses a cyclic electron flow around Photosystem I, also caused inactivation of the enzyme in illuminated, anaerobic chloroplasts.

4. It is proposed that the activity of fructose-1,6-bisphosphatase is regulated by the availability of electrons, and thus by electron acceptors, and that dark inactivation may occur by a direct reversal of the activation process.     

Transport in C4 mesophyll chloroplasts. Evidence for an exchange of inorganic phosphate and phosphoenolpyruvate

1. Mesophyll chloroplasts of the C₄ plant Digitaria sanguinalis contain endogenous phosphoenolpyruvate which appears to distribute across the envelope according to the existing pH gradient. The phosphoenolpyruvate remaining in the stroma can be rapidly released by external inorganic phosphate or 3-phosphoglycerate while external pyruvate did not affect the distribution.

2. Phosphoenolpyruvate (PEP) was a competitive inhibitor (K_i(PEP) = 450 μM) of ³²P_i uptake (K_m(P_i) = 200 μM) by chloroplasts in the dark and also reduced the steady-state internal concentration of ³²P_i, which is consistent with phosphate and phosphoenolpyruvate sharing a common carrier.

3. Phosphoenolpyruvate formation by chloroplasts in the light in the presence of pyruvate but in the absence of inorganic phosphate was slow and the concentration ratio of phosphoenolpyruvate (internal/external) was high. Addition of 0.1 mM phosphate induced a high rate of phosphoenolpyruvate formation and the concentration ratio (internal/external) decreased 15-fold. It is proposed that external phosphate is required both for phosphoenolpyruvate formation and efflux from the chloroplast.     

Role of the superoxide free radical ion in photosynthetic ascorbate oxidation and ascorbate-mediated photophosphorylation

The mechanism of ascorbate photooxidation in isolated chloroplasts has been studied. The enzyme superoxide dismutase has been used as a tool to show that ascorbate is oxidized by the superoxide free radical ion, which is formed during the autooxidation of a low-potential electron acceptor.

In the absence of an artificial, low-potential electron acceptor, addition of ascorbate stimulates photophosphorylation in isolated chloroplasts. This effect of ascorbate is abolished by superoxide dismutase, indicating that both the superoxide free radical ion and ascorbate are responsible for the stimulation of photophosphorylation. In this case, the superoxide free radical ion seems to be formed during the autooxidation of an endogenous electron acceptor.

In the presence of ferredoxin and NADP⁺, photophosphorylation in isolated chloroplasts stops as soon as the available NADP⁺ is fully reduced. If ascorbate is present in this system, however, a linear rate of photophosphorylation is maintained in spite of the fact, that NADP⁺ is fully reduced. This ascorbate-mediated photophosphorylation again is abolished by superoxide dismutase.

During the catalysis of this oxygen-dependent photophosphorylation, ascorbate consumption is not observed. These findings support the idea, that in chloroplasts ascorbate together with the superoxide free radical ion may function in providing additional ATP by an oxygen-dependent photophosphorylation.     

Photooxidation of cytochrome b-559 in leaves and chloroplasts at room temperature

1. Light-induced absorbance changes of cytochrome b-559 and cytochrome f in the -band region were examined in leaves and in isolated chloroplasts.

2. Absorbance changes of cytochrome b-559 were not detected in untreated leaves or in most preparations of isolated chloroplasts. After treatment of leaves or chloroplasts with carbonyl cyanide m-chlorophenylhydrazone, high rates of photooxidation of cytochrome b-559 were obtained, both in far-red (>700 nm) and red actinic light. Cytochrome f was photooxidized in far-red light, but in red light it remained mainly in the reduced state. The initial rates of photooxidation of cytochrome b-559 in leaves or chloroplasts treated with carbonyl cyanide m-chlorophenylhydrazone were considerably decreased by 3-(3′,4′-dichlorophenyl)-1,1-dimethyl urea.

3. A slow photoreduction of cytochrome b-559 was observed in aged mutant pea chloroplasts in red light.

4. The results do not support the view that cytochrome b-559 is a component of the electron transport chain between the light reactions. It is suggested that cytochrome b-559 is located on a side path from Photosystem II, but with a possible additional link to Photosystem I.     

The site of phosphorylation associated with Photosystem I

1. 1. Small particles prepared from spinach chloroplasts after treatment with digitonin, exhibited Photosystem I reactions, including phosphorylation, at rates as high as those in chloroplasts, whereas electron flow from water to NADP⁺ or ferricyanide through Photosystem II was completely lost. Mediators of cyclic electron flow, such as pyocyanine, or N-methylphenazonium methosulfate in red light, had to be reduced to support photophosphorylation.Diaminodurene at high concentrations catalyzed cyclic phosphorylation under anaerobic conditions without addition of a reductant. In fact, addition of ascorbate gave rise to a marked inhibition which was released by addition of a suitable electron acceptor such as methylviologen.

2. 2. Under aerobic conditions a low O₂ uptake, observed in the presence of diaminodurene, was stimulated several-fold upon addition of methylviologen and was stimulated again several-fold on further addition of ascorbate. The rate of phosphorylation, however, remained the same. The low P/2e ratio obtained under these conditions was not decreased at lower light intensities.

3. 3. These findings suggest a phosphorylation site associated with cyclic electron flow through Photosystem I without participation of the electron carriers of Photosystem II. A non-cyclic electron flow to O₂ can be induced in this system by addition of methylviologen which effectively competes with the electron acceptors of cyclic flow. This non-cyclic electron flow still involves the same phosphorylation site. A scheme for electron transport and for the location of phosphorylation sites in chloroplasts is proposed.

Chlorophyll a fluorescence and photochemical activities of chloroplast fragments

1. Comparative studies were made on the fluorescence characteristics of chlorophyll a at 20° and −193°, and quantum efficiencies for P 700 oxidation and NADP⁺ reduction were measured in chloroplasts and chloroplast fragments obtained after incubation with 0.5% digitonin.

2. Differences in the flurescence yield of chlorophyll a in flowing and stationary suspensions of untreated chloroplasts and of the large fragments are indicative of light-induced photoreduction of the quencher Q of chlorophyll a, associated with pigment System 2 (chlorophyll a₂). The relatively low constant fluorescence yield of chlorophyll a in the small fragments indicates the absence of fluorescent chlorophyll a₂ from these fragments and suggests that the low fluorescence is due to chlorophyll a, associated with pigmen System 1 (chlorophyll a₁). The ratio of the fluorescence yields of chlorophyll a₁ and chlorophyll a₂ is 0.45:1. In the large particles the concentration ratio of pigment System 1 and System 2 is 1:3.

3. The efficiencies of quanta absorbed at 673, 683 and 705 nm for NADP⁺ reduction and P 700 oxidation in untreated chloroplasts and chloroplast fragments indicate that digitonin treatment results in a separation of System 2 from System 1 in the small fragments. Sonication does not cause such a separation. Under the conditions used P 700 oxidation and NADP⁺ reduction in the small fragments separated after digitonin treatment, occurred with maximal efficiency of 0.7 to 1.0 and 0.7, respectively.

4. The constancy of the fluorescence yield of chlorophyll a₁ in the small fragments, under conditions at which P 700 is oxidized and NADP⁺ is reduced, is interpreted as evidence either for the hypothesis that the fluorescence of chlorophyll a₁ is controlled by the redox state of the primary photoreductant XH, or alternatively for the hypothesis that energy transfer from fluorescent chlorophyll a₁ to P 700 goes via an intrinsically weak fluorescent, still unknown, chlorophyll-like pigment.

5. The low-temperature emission band around 730 nm is argued not to be due to excitation by System 1 only; the relatively large half width of the band, as compared to the emission bands at 683 and 696 nm, suggests that it is possibly due to overlapping emission bands of different pigments.     

Study of electrogenic electron transfer steps in chromatophore membrane of Chromatium vinosum by the response of merocyanin dye

1. Electrogenic steps in photosynthetic cyclic electron transport in chromatophore membrane of Chromatium vinosum were studied by measuring absorption changes of added merocyanin dye and of intrinsic carotenoid.

2. The change in dye absorbance was linear with the membrane potential change induced either by light excitation or by application of diffusion potential by adding valinomycin in the presence of K⁺ concentration gradient.

3. It was estimated that chromatophore membrane became 40–60 mV and 110–170 mV inside positive upon single and multiple excitations with single-turnover flashes, respectively, from the responses of the dye and the carotenoid.

4. Electron transfers between cytochrome c-555 or c-552 and reaction center bacteriochlorophyll dimer (BChl₂) and between BChl₂ and the primary electron acceptor were concluded to be electrogenic from the redox titration of the dye response.

5. No dye response which corresponded to the change of redox level of cytochrome b was observed in the titration curve. Addition of antimycin A slightly decreased the dye response.

6. The dye response was decreased under phosphorylating conditions.

7. From the results obtained localization of the electron transfer components in chromatophore membrane is discussed.     

A convenient electrochemical preparation of reduced methyl viologen and a kinetic study of the reaction with oxygen using an anaerobic stopped-flow apparatus

1. Single reduced methyl viologen (MV^.+) acts as an electron donor in a number of enzyme systems. The large changes in extinction coefficient upon oxidation (λ_max 600 nm; MV^.+, = 1.3 · 10⁴ M⁻¹ · cm⁻¹; oxidised form of methyl viologen (MV²⁺), = 0.0) make it ideally suited to kinetic studies of electron transfer reactions using stopped-flow and standard spectrophotometric techniques.

2. A convenient electrochemical preparation of large amounts of MV^.+ has been developed.

3. A commercial stopped-flow apparatus was modified in order to obtain a high degree of anaerobicity.

4. The reaction of MV^.+ with O₂ produced H₂O₂ (k > 5 · 10⁶ M⁻¹ · s⁻¹, pH 7.5, 25 °C). H₂O₂ subsequently reacted with excess MV^.+ (k = 2.3 · 10³ M⁻¹ · s⁻¹, pH 7.5, 25 °C) to produce water. The kinetics of this reaction were complex and have only been interpreted over a limited range of concentrations.

5. The results support the theory that the herbicidal action of methyl viologen (Paraquat, Gramoxone) is due to H₂O₂ (or radicals derived from H₂O₂) induced damage of plant cell membrane.