The mechanism of the oxidation of ascorbate and Mn by chloroplasts: The role of the radical superoxide

1. 1. The mechanism of the photooxidation of ascorbate and of Mn²⁺ by isolated chloroplasts was reinvestigated.

2. 2. Our results suggest that ascorbate or Mn²⁺ oxidation is the result of the Photosystem I-mediated production of the radical superoxide, and that neither ascorbate nor Mn²⁺ compete with water as electron donors to Photosystem II nor affect the rate of electron transport through the two photosystems: The radical superoxide is formed as a result of the autooxidation of the reduced forms of low potential electron acceptors, such as methylviologen, diquat, napthaquinone, or ferredoxin.

3. 3. In the absence of ascorbate or Mn²⁺ the superoxide formed dismutases either spontaneously or enzymatically producing O₂ and H₂O₂. In the presence of ascorbate or Mn²⁺, however, the superoxide is reduced to H₂O₂ with no formation of O₂. Consequently, in the absence of reducing compounds, in the reaction H₂O to low potential acceptor one O₂ (net) is taken up per four electrons transported where as in the presence of ascorbate, Mn²⁺ or other suitable reductants up to three molecules O₂ can be taken up per four electrons transported.

4. 4. This interpretation is supported by the following observations: (a) in a chloroplast-free model system containing NADPH and ferredoxin-NADP reductase, methylviologen can be reduced to a free radical which is autooxidizable in the presence of O₂; the addition of ascorbate or Mn²⁺ to this system results in a two fold stimulation of O₂ uptake, with no stimulation of NADPH oxidation. The stimulation of O₂ uptake is inhibited by the enzyme superoxide dismutase; (b) the stimulation of light-dependent O₂ uptake in the system H₂O → methylviologen in chloroplasts is likewise inhibited by the enzyme superoxide dismutase.

5. 5. In Class II chloroplasts in the system H₂O → NADP upon the addition of ascorbate or Mn²⁺ an apparent inhibition of O₂ evolution is observed. This is explained by the interaction of these reductants with the superoxide formed by the autooxidation of ferredoxin, a reaction which proceeds simultaneously with the photoreduction of NADP. Such an effect usually does not occur in Class I chloroplasts in which the enzyme superoxide dismutase is presumably more active than in Class II chloroplasts.

6. 6. It is proposed that since in the Photosystem I-mediated reaction from reduced 2,4-dichlorophenolindophenol to such low potential electron acceptor as methylviologen, superoxide is formed and results in the oxidation of the ascorbate present in the system, the ratio ATP/2e in this system (when the rate of electron flow is based on the rate of O₂ uptake) should be revised in the upward direction.

Pigment systems and electron transport in chloroplasts I. Quantum requirements for the two light reactions in spinach chloroplasts

From studies of electron-transport reactions of isolated spinach chloroplasts, we observe the following quantum requirements: (A) For the photoreduction of NADP⁺, measured both aerobically and anaerobically, in a 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) poisoned system with ascorbate and reduced 2,6-dichlorophenolindophenol (DCIPH₂) present as electron donors, the quantum requirements are 1.0 ± 0.05 at wavelengths longer than 700 nm of actinic light, and 1.5–2.5 for wavelengths between 620 and 680 nm. (B) For the photoreduction of 2,6-dichlorophenolindophenol (DCIP) with water as the electron donor, the quantum requirements are 1.0 ± 0.05 in the range 630–660 nm. (C) For the photoreduction of NADP⁺ with water as the electron donor, the quantum requirements are 2.0 ± 0.1 in the wavelength range 640–678 nm of actinic light, increasing to 6 or greater at wavelengths beyond 700 nm. These results are shown to be inconsistent with the “separate package” model for the two pigment systems in higher plant photosynthetic electron transport. The evidence is most easily interpreted using a “controlled spillover” model, in which the transfer of electronic excitation energy from one pigment system to the other is under the control of incompletely identified factors in the reaction mixture.

At moderate light intensities the steady state rate of the [ascorbate + DCIPH₂ → NADP⁺] reaction (A) in the presence of DCMU and added ferredoxin can be increased more than 3 times when saturating amounts of plastocyanin and ferredoxin-NADP reductase are added to the chloroplasts. Similarly, the steady-state rate of the [H₂O → DCIP] Hill reaction (B) is increased about 3-fold by added MgCl₂ and plastocyanin, but added ferredoxin or ferredoxin-NADP reductase have no effect on this reaction. Plastocyanin appears to be the electron transport component which couples to DCIP, either in the oxidized or in the reduced form, in the reaction media. The steady-state rate of the [H₂O → NADP⁺] reaction (C) with saturating amounts of ferredoxin can be further increased more than 3-fold when MgCl₂, plastocyanin and ferredoxin-NADP reductase are added.     

Effects of monovalent cations on light energy distribution between two pigment systems of photosynthesis in isolated spinach chloroplasts

The effects of monovalent cations on the light energy distribution between two pigment systems of photosynthesis were studied in isolated spinach chloroplasts by measuring chlorophyll a fluorescence and photochemical reactions.

The addition of NaCl to the chloroplast suspension produced a 40–80% increase in fluorescence yield measured at 684 nm at room temperature. The fluorescence increase was completed about 5 min after the addition. The effect saturated at 100 mM NaCl. Low-temperature fluorescence spectra showed that NaCl increased the yields of two fluorescence bands of pigment system II at 684 and 695 nm but decreased that of pigment system I at 735 nm. Similar effects on chlorophyll a fluorescence at room and at low temperatures were obtained with NaBr, NaNO₃, Na₂SO₄, LiCl, KCl, RbCl, CsCl, NH₄Cl and CH₃NH₃Cl.

NaCl suppressed the quantum efficiency of NADP⁺ reduction supported by the ascorbate-2,6-dichlorophenolindophenol (DCIP) couple as an electron donor system in the presence of 3-(3′,4′-chlorophenyl)-1,1-dimethylurea (DCMU). On the other hand, NaCl only slightly enhanced the quantum yield of photoreaction II measured by the Hill reaction with DCIP.

It is concluded that the monovalent cations tested suppressed the excitation transfer from pigment system II to pigment system I; the effects were the same as those of alkaline earth metals and Mn²⁺ (refs. 1, 2).     

Interaction of oxidized and reduced N-methylphenazonium methosulfate (PMS) with Photosystem II

In 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) poisoned chloroplasts, the restoration of the fluorescence induction is presumed to be due to a back reaction of the reduced primary acceptor (Q⁻) and the oxidized primary donor (Z⁺) of Photosystem II. Carbonylcyanide m-chlorophenylhydrazone (CCCP) is known to inhibit this back reaction. The influence of reduced N-methylphenazonium methosulfate (PMS) in the absence of CCCP and of oxidized PMS in the presence of CCCP on the back reaction was investigated and the following results were obtained:

1. (1) Reduced PMS at the concentration of 1 μM inhibits the back reaction as effectively as hydroxylamine, suggesting an electron donating function of reduced PMS for System II.

2. (2) The inhibition of the back reaction by CCCP is regenerated to a high degree by oxidized PMS which led to assume a cyclic System II electron flow catalysed by PMS.

3. (3) At concentrations of reduced PMS higher than 1 μM it is shown that both the fast initial emission and more significantly the variable emission are quenched.

Wavelength-dependent quantum yield of ATP synthesis and NADP reduction in normal and dichlorodimethylphenylurea-poisoned chloroplast

At short wavelengths (525–690 mμ) the direct measurement of the quantum yield of the photoreduction of NADP⁺ in normal O₂-evolving spinach chloroplasts is constant ( approx. 0.3 equiv/hv). At short wavelengths (<690 mμ) the quantum yield for NADP⁺ reduction in 3(3,4-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts supplied with the ascorbate-2,6-dichlorophenolindophenol couple (donor system) is approx. half as efficient as the normal system. At long wavelengths the quantum yield of NADP⁺ reduction in the donor system increases by a factor of 2 ( approx. 0.3 equiv/hv) when compared with the corresponding yield for the donor system at short wavelengths ( approx. 0.15 equiv/hv).

Between 525 and 690 mμ, the phosphorylation yield for the normal system is constant ( = 0.15 ATP/hv), maintaining a constant P/2e ratio of unity. The P/2e ratios indicate a tight coupling between phosphorylation and electron transport encompassing a single phosphorylation site for the transfer of two electrons.

Between 525 and 680 mμ, the phosphorylation yield for the donor system is constant ( approx. 0.04 ATP/hv), maintaining a P/2e ratio of approx. 0.5. At longer wavelengths (>690 mμ) the phosphorylation yield of the donor system rises ( approx. 0.07–0.08 ATP/hv) concomitant with the rise in the yield of electron flow.

These experiments suggest the possibility that two types of phosphorylation processes operate in chloroplasts, (1) a short-wavelength process coupled to the normal O₂-evolving activity, and (2) a long-wavelength process coupled to the electron-donor activity of reagents such as DCIP.     

Formation of singlet molecular oxygen in illuminated chloroplasts. Effects on photoinactivation and lipid peroxidation

The formation of singlet molecular oxygen (¹O₂) in illuminatedchloroplasts and the effects of ¹O₂ on oxidation or destructionof components and functional integrity of chloroplasts werestudied. The rate of photoreduction of 2,6-dichloroindophenol(DCIP) and the extent of the 515-nm absorbance change were decreasedby light irradiation and by xanthine oxidase treatment. Malondialdehyde(MDA) formation, an indicator of lipid peroxidation, was observedin the light-irradiated chloroplast fragments, but not in thexanthine-xanthine oxidase-treated chloroplast fragments. MDAformation was absent under anaerobic conditions. MDA formation was stimulated when electron transfer on the oxidizingside of photosystem II (or I) was inhibited or inactivated bycarbonylcyanide m-chlorophenylhydrazone (CCCP), Tris-treatment,prolonged illumination, etc. MDA formation was also stimulatedby 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) when electrontransfer between water and the reaction center of photosystemII was intact. CCCPor DCMU-stimulated MDA formation was inhibitedby 1,4-diazabicyclo[2.2.2]octane, a quencher of singlet molecularoxygen (¹O₂). DCMU and electron donors for photosystem II, suchas ascorbate, hydroquinone and semicarbazide, inhibited MDAformation by illumination of the Tris-washed or CCCP-poisonedchloroplast fragments. Reduced DCIP, an electron donor for photosystemI, also inhibited MDA formation in the presence of DCMU. These results lead to the conclusion that MDA formation wasinitiated by ¹O₂ formed in illuminated chloroplasts. Of thethree mechanisms discussed for ¹O₂ generation in illuminatedchloroplasts, the formation by the electron transfer reactionbetween superoxide anion radical and the oxidant formed on theoxidizing side of photosystem II (or I) is mostimportant. (Received March 31, 1975; )     

Inhibition of O2 evolution by NADP in isolated potato tuber chloroplast and its identity with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea inhibition site.

Chloroplast from greening potato tuber showed good photosynthetic capacity. The evolution of O₂ was dependent upon the intensity of light. A light intensity of 30 lux gave maximum O₂ evolution. At higher intensities inhibition was observed. The presence of bicarbonate in the reaction mixture was essential for O₂ evolution. NADP was found to be a potent inhibitor of O₂ evolution in this system. NADP and 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) inhibited the O₂ evolution completely at a 3 μm concentration level, which was reversed by oxidized 2,6-dichlorophenol-indophenol (DCIP). Cyanide (CN)-treated chloroplasts showed full O₂ evolution capacity, when a lipophilic electron acceptor like N-tetramethyl-p-phenylenediamine (TMPD) or DCIP was used along with ferricyanide. Ferricyanide alone showed only 20% reduction. NADP or DCMU could inhibit O₂ evolution only when TMPD was the acceptor but not with DCIP. Photosystem II (PS II) isolated from these chloroplasts also showed inhibition by NADP or DCMU and its reversal by DCIP. Here also the evolution of O₂ with only TMPD as acceptor was sensitive to NADP or DCMU. In the presence of added silicotungstate in PS II NADP or DCMU did not affect ferricyanide reduction or oxygen evolution. The chloroplasts were able to bind exogenously added NADP to the extent of 120 nmol/mg chlorophyll. It is concluded that the site of inhibition of NADP is the same as in DCMU, and it is between the DCIP and TMPD acceptor site in the electron transport from the quencher (Q) to plastoquinone (PQ).     

Photochemical properties of a Photosystem II subchloroplast fragment

1. The Photosystem II fraction (D-10) obtained by incubation of spinach chloroplasts with digitonin was further purified by incubation with Triton X-100. The resulting Photosystem II subchloroplast fragment (DT-10) contained 1 mole of cytochrome b-559 per 170 moles of chlorophyll. It lacked cytochrome f and cytochrome b₆ and its content of P700 was low.

2. The DT-10 fragment showed only traces of photochemical activity with water as electron donor, but it was active in a Photosystem II reaction with 2,6-dichlorophenolindophenol as electron acceptor and diphenyl carbazide as donor. Photoreduction of NADP⁺ with diphenyl carbazide as donor was negligible. There was some photoreduction of NADP⁺ with ascorbate plus 2,6 dichlorophenolindophenol as donor but this activity could be accounted for by contamination with Photosystem I. These results are consistent with the Z-scheme of photosynthesis with Photosystems I and II operating in series for the reduction of NADP⁺ from water. DT-10 subchloroplast fragments showed a light-induced rise in fluorescence yield at 20 °C in the presence of diphenyl carbazide. A light-induced fluorescence increase also was observed at 77 °K.

3. During the preparation of the DT-10 fragment, the high potential form of cytochrome b-559 was largely converted to a form of lower potential and C-550 was converted to the reduced state. A photoreduction of C-550 was observed at liquidnitrogen temperature, provided the C-550 was oxidised with ferricyanide prior to cooling. Some photooxidation of cytochrome b-559 was obtained at 77 °K if the preparation was reduced prior to cooling, but the degree of photooxidation was variable with different preparations. C-550 does not appear to be identical with the primary fluorescence quencher, Q.

4. Photosystem I subchloroplast fragments (D-144) released by the action of digitonin were compared with Photosystem I fragments (DT-144) released from D-10 fragments by Triton X-100. There were no significant differences between D-144 and DT-144 fragments either in chlorophyll a/b ratio or in P700 content.     

Transmembrane movements of artificial redox mediators in relation to electron transport and ionic currents in chloroplasts

Electrochemical data obtained with TMPD⁺-sensitive electrodes indicate that ammonium-uncoupled chloroplasts retain TMPD (N,N,N',N'-tetramethyl- p -phenylenediamine) mainly in the reduced form during illumination, whereas uncoupled DCMU-treated chloroplasts accumulate TMPD in the oxidized form (TMPD⁺). This observation indicates that the reduced plastoquinol is the preferred electron donor for photosystem I (PSI) and TMPD can only compete efficiently when plastoquinone reduction is blocked. After adding DCMU the formation of a transmembrane gradient for TMPD⁺ is reflected by a slow-down of the electrogenic electron transport and by the emerging of the overshoot of the membrane current in the light-off response. A light-dependent increase in photoelectric current generated by chloroplasts in the presence of NH₄Cl and TMPD is observed and considered to be caused by a reversible release of current limitation in the interfacial conductance barriers in the lumen.     

Comparison studies on the effects of ultraviolet irradiation on photosynthesis

1. 1. A comparison of chloroplasts from which plastoquinone had been extracted with ultraviolet irradiation supports the conclusion that plastoquinone destruction is not the major cause of ultraviolet inhibition of photosynthesis. No photodestruction of chloroplast lipids, carotenoids or soluble proteins by ultraviolet irradiation was detected.

2. 2. Phenazine methosulfate-mediated cyclic photophosphorylation and variable yield fluorescence were inhibited at the same rate as the Hill reaction. Examination of fluorescence emission spectra of chloroplasts and whole algal cells revealed decreases in both the 685-nm and long-wavelength emission peaks.

3. 3. Digestion of chloroplasts with lipase decreased fluorescence in a manner similar to ultraviolet irradiation. Hill reaction activity was also inhibited by lipase digestion.

4. 4. It is concluded that the inhibition of photosynthesis by ultraviolet irradiation is most likely due to a disruption of the structural integrity of the lamellar membranes which results in the loss of System II activity and associated reactions.

Transmembrane movements of artificial redox mediators in relation to electron transport and ionic currents in chloroplasts

Electrochemical data obtained with TMPD⁺-sensitive electrodes indicate that ammonium-uncoupled chloroplasts retain TMPD (N,N,N',N'-tetramethyl- p -phenylenediamine) mainly in the reduced form during illumination, whereas uncoupled DCMU-treated chloroplasts accumulate TMPD in the oxidized form (TMPD⁺). This observation indicates that the reduced plastoquinol is the preferred electron donor for photosystem I (PSI) and TMPD can only compete efficiently when plastoquinone reduction is blocked. After adding DCMU the formation of a transmembrane gradient for TMPD⁺ is reflected by a slow-down of the electrogenic electron transport and by the emerging of the overshoot of the membrane current in the light-off response. A light-dependent increase in photoelectric current generated by chloroplasts in the presence of NH₄Cl and TMPD is observed and considered to be caused by a reversible release of current limitation in the interfacial conductance barriers in the lumen.     

Activation of CO2 fixation in isolated spinach chloroplasts

1. 1. The effect of the Mg²⁺ concentration on the CO₂ fixation activity in situ in isolated and intact spinach chloroplasts upon suspension in hypotonic medium was examined. CO₂ fixation in the dark was activated 25–100 fold by 20 mM Mg²⁺ in the presence of added ATP plus either ribulose 5-phosphate or ribose 5-phosphate. 20 mM Mg²⁺-stimulated fixation only 2–3 fold in the presence of the substrate of fixation, ribulose 1,5-diphosphate. The highest Mg²⁺-stimulated rate of fixation in the dark observed with chloroplasts was 480 μmoles CO₂ fixed per mg chlorophyll per h.

2. 2. The concentration of bicarbonate at half of the maximal velocity (apparent K_m) during the Mg²⁺-stimulated fixation of CO₂ was 0.4 mM in the presence of ATP plus ribose 5-phosphate and 0.6 mM with ribulose 1,5-diphosphate.

3. 3. Dithioerythritol or light enhanced Mg²⁺-stimulated CO₂ fixation 1–3 fold in the presence of ATP plus ribose 5-phosphate but not ribulose 1,5-diphosphate.

4. 4. These results indicate that Mg²⁺ fluxes in the stroma of the chloroplast could control the activity of the phosphoribulokinase with a lesser effect on the ribulosediphosphate carboxylase. An increase in Mg²⁺ of 6–10 mM in the stroma region of the chloroplast would be enough to activate CO₂ fixation during photosynthesis.

Specific effect of manganese on the allantoinase of Vigna Radiata

Vigna radiata seedlings germinated in the presence of Mn²⁺ show an unusual increase in allantoinase activity which is proportional to Mn²⁺ concentration up to 5 mM. Though Mn²⁺ is not an activator for V. radiata allantoinase, it specifically protects allantoinase against thermal as well as papain-catalysed inactivation. Evidence is presented to show that the primary effect of Mn²⁺ is a protective one, both in vitro and in vivo, and that this is reflected in the observed enhancement of allantoinase activity in Mn²⁺ grown seedlings. That this unusual effect of Mn²⁺ is a specific one is indicated by the lack of a similar effect with Mg²⁺. Cu²⁺ is shown to destabilize V. radiata allantoinase in vitro as well as in vivo.     

Difference in 2-thenoyltrifluoroacetone sensitivity of electron transport with and against the redox potential gradient

The effect of 2-thenoyltrifluoroacetone on electron transport with and against the redox potential gradient, with succinate or ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) as electron donor, was studied in rat liver mitochondria. It was found that 2-thenoyltrifluoroacetone inhibited succinate-linked intramitochondrial pyridine nucleotide reduction at low concentrations, which neither affected succinate oxidation in the controlled state nor interfered with intramitochondrial pyridine nucleotide reduction in the ascorbate plus TMPD case. The effect of 2-thenoyltrifluoroacetone on succinate-linked intramitochondrial pyridine nucleotide reduction is not attributable either to blocking of the overall rate of electron flow in the succinate dehydrogenase branch of the respiratory chain or to interference with energy transformation. Transition from the controlled to the active state enhanced the inhibitory effect of 2-thenoyltrifluoroacetone on succinate-linked respiration, and it became as sensitive to 2-thenoyltrifluoroacetone as the succinate-linked intramitochondrial pyridine nucleotide reduction. In the light of the above findings, the possibility is discussed that electrons from succinate enter the main branch of the respiratory chain by different routes, according to whether the flow is with or against the potential gradient.     

Effects of carbonyl cyanide m-chlorophenylhydrazone and hydroxylamine on the Photosystem II electron exchange mechanism in 3-(3,4-dichlorophenyl)-1,1-dimethylurea treated algae and chloroplasts

The effects of NH₂OH and carbonyl cyanide m-chlorophenylhydrazone (CCCP) on 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated algae and chloroplasts were studied. In the presence of DCMU, the photochemically separated charges can only disappear through a recombination back reaction; both substances induce an irreversible reduction of the donor side and after sufficient illumination their action in the presence of DCMU leads to the formation of a permanent fluorescent state.

In the DCMU + CCCP system, a fast fluorescence induction curve is observed. The fluorescence yield is brought to its maximum by two flashes. The luminescence emission is strongly inhibited and most centers reach their permanent fluorescent state after one flash.

In the DCMU + NH₂OH system, a slow fluorescence rise is observed and several saturating flashes are needed for the fluorescence yield to reach its maximum. The exhaustion of the NH₂OH oxidizing capacity and the complete transformation to a permanent fluorescent state also require a large number of flashes.

The reduction pathway catalyzed by CCCP appears to be a good competitor to the back reaction, while NH₂OH seems to be a relatively inefficient donor.

In addition the action of NH₂OH and CCCP on fluorescence suggests that the donor side influences the quenching properties of Photosystem II centers. A possible mechanism is proposed.     

Multiple sites of DCIP reduction by sonicated Oat chloroplasts: Role of plastocyanin

1. Oat chloroplasts, in the presence of 0.02 M methylamine, reduce 2,6 dichlorophenolindophenol (DCIP) at a rate of 350–500 μmoles/mg chl per h, in saturating light. Brief sonication for approx. 1 min lowers the rate to approx. 50 μmoles/mg chl per h; longer sonication does not reduce activity further. During brief sonication, plastocyanin is lost from the chloroplasts. When plastocyanin is added back to sonicated fragments, DCIP reduction is approximately doubled to 100 μmoles/mg chl per h.

2. When oxidized plastocyanin is added, a transient is observed when light is first turned on: this is due to a reduction of the plastocyanin before DCIP reduction begins. When reduced plastocyanin is added, a different transient occurs: this is due to a fast photoreduction of DCIP by the plastocyanin and is followed by the slower steady state reduction of DCIP by water. When light is turned off before complete reduction of DCIP, a transient reduction of oxidized plastocyanin by reduced DCIP is seen. Insensitivity of these transients to 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and the greater effectiveness of 710 nm light, along with the known capacity of plastocyanin to mediate electron transfer to System I, prove that an intrinsically fast reduction of DCIP occurs at a site close to the primary photoreduced product of System I.

3. After brief sonication and washing, no residual plastocyanin was detected in chloroplast fragments, and the rate of the slow DCIP reduction (about 50μmoles/mg chl per h) sustained by such fragments was essentially identical to that maintained by fragments of mutants lacking System I activity. Following et al.⁹, the simplest explanation for this slow DCIP reduction is that is occurs at a site close to System II and the system I is not involved.

4. A very slow transient reduction of DCIP occurs after extinguishing light; this presumably involves another reduction site close to System II, as suggested by ⁹.     

Effects of divalent metal ions on chlorophyll a fluorescence in isolated spinach chloroplasts

The effects of divalent metal ions on the yields of chlorophyll a fluorescence were investigated in isolated spinach chloroplasts at room and liquid nitrogen temperatures. Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺ and Mn²⁺ increased the yields of fluorescence emission at 684 and 695 nm from pigment system II and decreased that at 735 nm from pigment system I. Al³⁺ showed similar but less significant effects on the fluorescence yields. Zn²⁺ and Cd²⁺ showed no significant effect on the fluorescence yields at concentrations lower than 5 mM.

In accordance with the results of our previous study concerning the effects of Mg²⁺ on the excitation transfer in the chloroplasts, it was concluded that ions of alkaline earths and manganese suppress the excitation transfer from bulk chlorophylla of pigment system II to that of pigment system I.     

The Photochemical Activity of Chloroplasts and Subchloroplast Particles Isolated from Etiolated Bean Leaves Exposed to Continuous Light

The photochemical activity of chloroplasts and subchloroplastparticles isolated from primary bean leaves between the 4thand 24th hour of illumination of etiolated seedlings is thesubject of this paper. The photosystem I activity (oxygen uptakein the presence of MV, DCIP, ascorbate and DCMU), expressedon a unit chlorophyll basis, decreased approximately 10-foldbetween 4 and 8 h of greening. At the same time the photosystemII activity (DCIP photoreduction in the presence of DPC) wasreduced to a half. The photosystem I activity also decreasedin all hitherto investigated fractions which were isolated fromthe digitonin-treated chloroplasts. However, at the initialphase of greening this decrease was the most significant inthe fraction containing heavy particles. After 24 h of greening DCMU, at concentrations higher than 10^–10M, limited the rate of ferricyanide photoreduction by isolatedchloroplasts, whereas after 6 h of greening this effect wasobservable even in the presence of 10^–12 M DCMU. The resultsobtained demonstrated that under those conditions both photosystemswere active after 4 h of greening and PS I activity developedmore rapidly than that of PS II. It also follows from the presenteddata that the water splitting reactions were delayed in developmentas compared to the other reactions investigated, and that PSII units may limit the electron flow in chloroplasts at earlierstages of leaf greening.     

The Effect of Cadmium on Photosystem II in Spinach Chloroplasts

Cadmium ions, as an environmental pollution factor, significantly inhibited the photosynthesis especially, photosystem Ⅱ activity in isolated spinach chloroplasts. The presence of 5 mmol/l Cd2+ inhibited the O2-evolution to 53%. Cd2+ reduced the activity of photoreduction of DCIP and the variable fluorescence of chloroplasts and PSⅡ preparation. The inhibited DCIP photoreduction activity could only be restored slightly by the addition of an artificial electron donor of PSII, DPC, and the inhibited variable fluorescence could not be obviously recovered by the addition of NH2OH, another artificial electron donor of PSⅡ. It is considered that, besides the oxidizing side of PSI1, Cd2+ could also inhibit directly the PSⅡ reaction center. The inhibitory effect of Cd2+ on the whole chain electron transport (H2O→MV) was more serious than on O2-evolution (H2O→DCMU). It is suggested that the oxidizing side of PSⅡ is not the only site for Cd2+ action. There may be another site inhibited by Cd2+ in the electron transport chain between PSⅠ and PSⅡ.     

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.