Influence de l'oxygene sur les transferts d'electrons de la photosynthese. II. Influence de tres faibles concentrations en oxygene sur la reduction du NADP par les chloroplastes isoles

Influence of oxygen on the electron transfers of photosynthesis. II. Influence of very low oxygen concentration on the NADP⁺ reduction by isolated chloroplasts

The influence of very low O₂ concentration on the NADP⁺ reduction by isolated spinach chloroplasts has been studied.

The results show that in the presence of very low O₂ concentration (< 0.3%) NADP⁺ reduction is partially inhibited. This inhibition may be partially reversed under some conditions, especially when, in spite of the presence of an O₂ trap (glucose plus glucose oxidase (EC 1.1.3.4)) an O₂ evolution is observed.     

Etude de la photophosphorylation endogene des chloroplastes isoles d'epinardStudy on endogenous photophosphorylation of isolated spinach chloroplasts

Whole spinach chloroplasts were able to perform photophosphorylation under nitrogen without the addition of any redox cofactor. This “endogenous” phosphorylation was totally insensitive to 3-(p-chlorophenyl)-1,1-dimethylurea. After osmotic shock endogenous ATP formation decreased but the addition of 3-(p-chlorophenyl)-1,1-dimethylurea stimulated it.

Under a stream of nitrogen, whole chloroplasts reduced NADP⁺ after an osmotic shock, in the absence of added ferredoxin. The resulting ATP/NADPH ratios were high (approx. 2 or 3). They decreased to 1 in the presence of either exogenous ferredoxin, 3-(p-chlorophenyl)-1,1-dimethylurea or limiting light: i.e. high ATP/NADPH ratios were observed only when the terminal step of NADP⁺ reduction was limiting.

The endogenous anaerobic phosphorylation was inhibited by antimycin A to the same extent as the O₂-dependent endogenous non-cyclic phosphorylation.

A direct inhibition of electron transport by antimycin A has never been observed.     

Wavelength-dependent quantum yields of chloroplast phosphorylation catalyzed by phenazine methosulphate

Studies of phenazine methosulphate (PMS)-catalyzed O₂ exchange and phosphorylation in spinach chloroplasts reveal that at short wavelengths (<680 mμ) PMS acts at a reduced quantum efficiency as an oxidant for O₂ evolution with concomitant phosphorylation. The quantum yield profile of phosphorylation obtained with PMS differs markedly from the yield profile of phosphorylation for normal chloroplasts with NADP⁺ or ferricyanide as oxidant. Between 525 and 680 mμ the quantum yield of phosphorylation (ATP) catalyzed by PMS is less than half the constant maximum ATP of the normal system. The maximum ATP value for the normal system is approx. 0.16 ATP/hv. With the PMS system a peak in the yield at 690 mμ is obtained approaching the ATP value of the normal system. This yield falls again at longer wavelengths (>700 mμ).

The addition of ascorbate to the PMS phosphorylating system decreases the short-wavelength (<680 mμ) phosophorylation activity but increases the long-wavelength (>690 mμ) phosphorylation activity. The quantum yield profile of this system, showing a long-wavelength rise in phosphorylation efficiency is obtained with or without the addition of 3(3,4-dichlorophenyl)-1, 1-dimethylurea.

These experiments have been interpreted as indicating two separate electrontransfer processes catalyzed by PMS, one in which PMS acts at a reduced efficiency as a Hill oxidant, and the other in which PMS acts as an electron donor and acceptor in a cyclic fashion in sensitizing and essentially long-wavelength phosphorylation process.     

Properties of photoreductions by photosystem II in isolated chloroplasts. III. The effect of uncouplers on phenylenediamine shuttles across the membrane in the presence of dibromothymoquinone

In the presence of the plastoquinone antagonist dibromothymoquinone the photoreduction of ferricyanide by isolated chloroplast membranes is attributed to Photosystem II. The reaction is stimulated by the addition of phenylenediamine or C-substituted phenylenediamines (which may form a diimine on oxidation) but not of N-substituted phenylenediamines (which form a stable radical on oxidation). Phenylenediamines also restore NADP reduction (and O₂ evolution) in 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)-treated chloroplasts. In this bypassing of the inhibition site, N-substituted phenylenediamines are very effective, whereas p-phenylenediamine and C-substituted phenylenediamines are inefficient. Uncouplers exhibit a surprising effect on these systems. Even under coupling conditions uncouplers inhibit electron flow to ferricyanide mediated by phenylenediamine in the pH range 7.3–8.0, whereas the restoration of the NADP system is stimulated.

For the interpretation of the results the side of the membrane involved is considered. It is proposed that in ferricyanide reduction by Photosystem II, a phenylenediimine/diamine shuttle operates which moves reducing equivalents from the inside to the outside across the membrane. This shuttle requires a pH gradient across the membrane because of different optimal ratios of diimine/diamine inside and outside. This pH difference is abolished by the uncoupler, accounting for the observed inhibition.

The restoration of electron flow from water to NADP in DBMIB-treated chloroplasts is assumed to be a bypass of the inhibition site inside the membrane via a phenylenediamine. Because the imine/amine ratio brought about by the pH gradient is not favorable for the inside oxidation an uncoupler stimulates NADP reduction even under coupling conditions.

Also in photoreductions by Photosystem I, for example NADP reduction at the expense of P-phenylenediamine/ascorbate, a shuttle of reducing equivalents across the membrane occurs but this time from outside to inside.     

Isolation of Photosystem II enriched membrane vesicles from spinach chloroplasts by phase partition

Partition in an aqueous Dextran-polyethylene glycol two-phase system has been used for the separation of chloroplast membrane vesicles obtained by press treatment of a grana-enriched fraction after unstacking in a low salt buffer.

The fractions obtained were analysed with respect to chlorophyll, photochemical activities and ultrastructural characteristics. The results reveal that the material partitioning to the Dextran-rich bottom phase consisted of large membrane vesicles possessing mainly Photosystem II properties with very low contribution from Photosystem I. Measurements of the H₂O to phenyl-p-benzoquinone and ascorbate-Cl₂Ind to NADP⁺ electron transport rates indicate a ratio of around six between Photosystem II and I.

The total fractionation procedure could be completed within 2–3 h with high recovery of both the Photosystem II water-splitting activity and the Photosystem I reduction of NADP⁺.

These data demonstrate that press treatment of low-salt destabilized grana membranes yields a population of highly Photosystem-II enriched membrane vesicles which can be discriminated by the phase system. We suggest that such membrane vesicles originate from large regions in the native grana membrane which contain virtually only Photosystem II.     

Fluorescence induction and activity of ferredoxin-NADP reductase in Bryopsis chloroplasts

Effects of medium osmolarity on the rate of CO₂ fixation, the rate of the NADP⁺-Hill reaction, and the DPS₁ transient of chlorophyll fluorescence were measured in intact Bryopsis chloroplasts. Upon decreasing the sorbitol concentration from 1.0 M (the isoosmotic conditions) to 0.25 M, the envelopes of the chloroplasts became leaky to small molecules, resulting in a considerable depression of the CO₂-fixation rate and a higher rate of the NADP⁺-Hill reaction whereas the DPS₁ transient was unaffected. This DPS₁ transient of chlorophyll fluorescence is thought to be caused by the photoactivation of electron flow on the reducing side of Photosystem I at a site occurring after ferredoxin and probably before the reduction of NADP⁺ (Satoh, K. and Katoh, S. (1980) Plant and Cell Physiol. 21, 907–916). Little effect of NADP⁺ on the DPS₁ transient and a marked lag in NADP⁺ photo-reduction in dark-adapted (inactivated) chloroplasts support the hypothesis that the site of dark inactivation is prior to the reduction site of NADP⁺, and therefore, that ferredoxin-NADP⁺ reductase is inactivated in the dark and activated in the light. Moreover, at 0.25 M sorbitol, the activity of ferredoxin-NADP⁺ reductase itself (2,6-dichlorophenolindophenol reduction by NADPH) was shown to increase according to dark-light transition of the chloroplasts. At low osmolarities (below 0.1 M sorbitol), the difference in the diaphorase activity between dark-and light-adapted chloroplasts and the lag time observed in the NADP⁺ photoreduction were lowered. This may correspond to a less pronounced DPS₁ transient at low concentrations of sorbitol. The mechanism of the photo-activation is discussed.     

Bipyridylium quaternary salts and related compounds. V. Pulse radiolysis studies of the reaction of paraquat radical with oxygen. Implications for the mode of action of bipyridyl herbicides

1. Rate constants for reduction of paraquat ion (1,1′-dimethyl-4,4′-bipyridy-lium, PQ²⁺) to paraquat radical (PQ⁺·) by e⁻_aq and CO₂⁻· have been measured by pulse radiolysis. Reduction by e⁻_aq is diffusion controlled (k = 8.4·10¹⁰ M⁻¹·s⁻¹) and reduction by CO₂⁻· is also very fast k = 1.5·10¹⁰ M⁻¹·s⁻¹).

2. The reaction of paraquat radical with oxygen has been analysed to give rate constants of 7.7·10⁸ M⁻¹·s⁻¹ and 6.5·10⁸ M⁻¹·s⁻¹ for the reactions of paraquat radical with O₂ and O₂⁻·, respectively. The similarity in these rate constants is in marked contrast to the difference in redox potentials of O₂ and O₂⁻· (− 0.59 V and + 1.12 V, respectively).

3. These rate constants, together with that for the self-reaction of O₂⁻·, have been used to calculate the steady-state concentration of O₂⁻· under conditions thought to apply at the site of reduction of paraquat in the plant cell. On the basis of these calculations the decay of O₂⁻· appears to be governed almost entirely by its self-reaction, and the concentration 5 μm away from the thylakoid is still 90% of that at the thylakoid itself. Thus, O₂⁻· persists long enough to diffuse as far as the chloroplast envelope and tonoplast, which are the first structures to be damaged by paraquat treatment. O₂⁻· is therefore sufficiently long-lived to be a candidate for the phytotoxic product formed by paraquat in plants.     

Characterization of oxygen-tolerant Chinese hamster ovary cells: II. Energy metabolism and antioxidant status

Further characteristics of an oxygen-tolerant variant of Chinese hamster ovary cells (CHO-99) capable of stable proliferation at 99% O₂/1% CO₂, an O₂ level that is lethal to the parental line (CHO-20), are described. Previous work has revealed that CHO-99 cells have 2- to 4-fold increased activities of superoxide dismutases, catalase and glutathione peroxidase, and substantially increased relative volumes of mitochondria and peroxisomes. To document possible additional mechanisms of O₂ tolerance we compared CHO-20 cells growing at 20% O₂ (normoxia) and CHO-99 cells at 99% O₂ (normobaric hyperoxia). We show the following: (1) the estimated total (oxidative and glycolytic) ATP production in CHO-99 cells was 36% decreased. ATP production through oxidative phosphorylation was 52% lower in CHO-99 cells, while the relative contribution from glycolysis was increased from 6% to 30%. The ATP content was 29% lower in CHO-99 cells, the adenylate energy charge being also significantly decreased, indicating that energy production through oxidative phosphorylation is compromised in CHO-99 cells. Cyanide-resistant respiration was 4-fold higher in CHO-99 cells, probably reflecting, at least partly, the increased peroxisomal activity in these cells. (2) The level of reduced glutathione was several fold increased in CHO-99 cells, oxidized glutathione being unaltered; (NADPH + NADP⁺) levels were elevated 2.7-fold, while the ratio of NADPH to NADP⁺ was increased almost two-fold. These changes were associated with a 50% increased metabolism of glucose through the hexose monophosphate pathway. (3) No evidence was obtained for an increased steady-state level of endogenous lipid peroxidation in CHO-99 cells, in spite of a 50% increased content of polyunsaturated fatty acids in the phospholipid fraction.     

Calcium and photosynthetic oxygen evolution in cyanobacteria

Calcium activation of oxygen evolution from French-press preparations of Phormidium luridum is largely reversible upon removal of added Ca²⁺. Activation occurs via a first-order binding with a dissociation constant of 2.8 mM. An 8-fold increase in oxygen evolution rate observed upon Ca²⁺ addition is accounted for by a 4-fold increase in the number of active photosynthetic units, and a doubling of turnover rate. While both Ca²⁺ and Mg²⁺ stimulate turnover, unit activation is Ca²⁺ specific. Under optimal conditions, 30% of the units functioning in the intact cell can be recovered in the Ca²⁺-activated preparation.

The Ca²⁺ requirement of P. luridum preparations is not relieved by proton-carrying uncouplers, or by rate-saturating concentrations of the Hill acceptor, ferricyanide. Taken together with the reported stimulation by Ca²⁺ of oxygen evolution in the presence of DCMU (Piccioni, R.G. and Mauzerall, D.C. (1976) Biochim. Biophys. Acta 423, 605–609) these observations strongly suggest a site of Ca²⁺ action within Photosystem II.

The pronounced specificity of the Ca²⁺ requirement appears in preparations of other cyanobacteria (Anabaena flos-aquae and Anacystis nidulans) but not in the eucaryote Chlorella vulgaris. While milder cell-disruption methods bring about some Ca²⁺ dependence in P. luridum, French-press treatment is required for maximal expression of Ca²⁺-specific effects. French-press breakage causes a release of endogenous Ca²⁺ from cells, supporting the view that added Ca²⁺ restores oxygen evolution by satisfying a physiological requirement for the cation.     

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.

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).     

NADPH/NADP ratios in photosynthesizing reconstituted chloroplasts

Levels of reduced and oxidized triphosphopyridine nucleotides have been determined in reconstituted spinach chloroplasts and compared with levels in whole isolated chloroplasts during photosynthesis and darkness. The ratio of NADPH/NADP⁺ reaches values slightly above 1.0 at the beginning of photosynthesis, less than half the ratio attained with whole chloroplasts. Nonetheless these lower ratios are sufficient to maintain high rates of photosynthetic carbon dioxide fixation and reduction, which are comparable in the reconstituted chloroplasts to the rates found with whole chloroplasts. As with whole chloroplasts there is a decline in the ratio of NADPH/NADP⁺ as a function of time of photosynthesis. The effect of addition of bicarbonate (6 mM) in causing a transient drop in the ratio of NADPH/NADP⁺ is described and discussed in terms of the reversibility of the reduction of 3-phosphoglycerate to triose phosphate. The ratio NADPH/NADP⁺ can be improved by the addition of more lamellae either before or during the course of photosynthesis, and this improvement in ratio is accompanied by an improved rate of CO₂ fixation or a more sustained rate of CO₂ fixation with time of photosynthesis. The importance of NADPH/NADP⁺ ratio not only to the reduction of 3-phosphoglycerate to triose phosphate but also to the activation of the ribulose-1,5-diphosphate carboxylasemediated step is discussed.     

The effects of reduced oxygen tension on swine granulosa cell

Follicular growth is characterized by an augmented vascularization, possibly driven by a fall in the oxygen supply. The present study was undertaken to investigate the effects of hypoxia on swine granulosa cells. At first, we quantified oxygen partial pressure (pO₂) in follicular fluid from different size follicles; the granulosa cells collected from large follicles (>5 mm) were subjected for 18 h to normoxia (19% O₂), partial (5% O₂) or total hypoxia (1% O₂). The effects of these conditions were tested on the main parameters of granulosa cell function, steroidogenesis and cell proliferation, and on vascular endothelial growth factor (VEGF), nitric oxide (NO) and superoxide anion (O₂⁻) production. Oxygen tension in follicular fluid was negatively related to follicular size, pointing out a gradual reduction during follicular growth. Severe hypoxic conditions determined a reduction of both 17β estradiol and progesterone production, while partial hypoxia did not seem to affect them. Hypoxia increased VEGF as well as O₂⁻ production in swine granulosa cells without impairing cell growth; in addition, it decreased NO output.

We may conclude that physiological hypoxia could play a pivotal role in the follicular angiogenic process stimulating VEGF synthesis by granulosa cells. ROS are possibly involved in hypoxic signalling.     

Studies on the energy coupling sites of photophosphorylation IV. The relation of proton fluxes to the electron transport and ATP formation associated with Photosystem II

1. By using dibromothymoquinone as the electron acceptor, it is possible to isolate functionally that segment of the chloroplast electron transport chain which includes only Photosystem II and only one of the two energy conservation sites coupled to the complete chain (Coupling Site II, observed P/e₂ = 0.3–0.4). A light-dependent, reversible proton translocation reaction is associated with the electron transport pathway: H₂O → Photosystem II → dibromothymoquinone. We have studied the characteristics of this proton uptake reaction and its relationship to the electron transport and ATP formation associated with Coupling Site II.

2. The initial phase of H⁺ uptake, analyzed by a flash-yield technique, exhibits linear kinetics (0–3 s) with no sign of transient phenomena such as the very rapid initial uptake (“pH gush”) encountered in the overall Hill reaction with methylviologen. Thus the initial rate of H⁺ uptake obtained by the flash-yield method is in good agreement with the initial rate estimated from a pH change tracing obtained under continuous illumination.

3. Dibromothymoquinone reduction, observed as O₂ evolution by a similar flash-yield technique, is also linear for at least the first 5 s, the rate of O₂ evolution agreeing well with the steady-state rate observed under continuous illumination.

4. Such measurements of the initial rates of O₂ evolution and H⁺ uptake yield an H⁺/e⁻ ratio close to 0.5 for the Photosystem II partial reaction regardless of pH from 6 to 8. (Parallel experiments for the methylviologen Hill reaction yield an H⁺/e⁻ ratio of 1.7 at pH 7.6.)

5. When dibromothymoquinone is being reduced, concurrent phosphorylation (or arsenylation) markedly lowers the extent of H⁺ uptake (by 40–60%). These data, unlike earlier data obtained using the overall Hill reaction, lend themselves to an unequivocal interpretation since phosphorylation does not alter the rate of electron transport in the Photosystem II partial reaction. ADP, P_i and hexokinase, when added individually, have no effect on proton uptake in this system.

6. The involvement of a proton uptake reaction with an H⁺/e⁻ ratio of 0.5 in the Photosystem II partial reaction H₂O → Photosystem II → dibromothymoquinone strongly suggests that at least 50% of the protons produced by the oxidation of water are released to the inside of the thylakoid, thereby leading to an internal acidification. It is pointed out that the observed efficiencies for ATP formation (P/e₂) and proton uptake (H⁺/e⁻) associated with Coupling Site II can be most easily explained by the chemiosmotic hypothesis of energy coupling.     

Energy transduction in photosynthetic bacteria VI. Respiratory sites of energy conservation in membranes from dark-grown cells of Rhodopseudomonas capsulata

Membranes prepared from Rhodopseudomonas capsulata grown heterotrophically in the dark perform phosphorylation linked to oxidation of NADH and succinate, with P/2e⁻ ratios of about 0.5 and 0.15, respectively. The localization of the sites of energy conservation was investigated by observing the respiration-induced quenching of the fluorescence of atebrine.

Energization of the membrane can be demonstrated when NADH is oxidized by O₂, ferricyanide or Q₁, when succinate is oxidized by O₂ or by oxidized diaminodurene, and during the oxidation of reduced diaminodurene.

Antimycin A completely inhibits energization between succinate and O₂ or succinate and diaminodurene; however, it only inhibits partially NADH or succinate oxidases and energization between NADH and O₂. KCN inhibits NADH oxidase in a biphasic way: the first level of inhibition is observed at concentrations which block the oxidation of exogenous cytochrome c or of diaminodurene and energization between succinate or ascorbate-diaminodurene and O₂. The second level corresponds to the inhibition of the antimycin-insensitive oxidase.

The results are interpreted as evidence of the presence in these bacteria of a respiratory chain branching after the dehydrogenase system, one arm of the chain being sensitive to antimycin A and low concentrations of KCN and capable of energy conservation, the other being represented by a completely uncoupled system.     

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.     

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.     

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.     

Effect of chloride ions on the S-state distribution and deactivation kinetics in preparations of the cyanobacterium Anacystis nidulans

The roles of Ca²⁺ and Cl⁻ on the photosynthetic O₂ yield under flash illumination have been examined in EDTA-washed preparations of the cyanobacterium Anacystis nidulans. Especially the effect of Cl⁻ deficiency on the O₂ yield and on the S-state distribution was analyzed. As the results show, omission of both Ca²⁺ and Cl⁻ (Mn²⁺ present) almost totally inhibited O₂ evolution. When Ca²⁺ was replaced by Na⁺, a substantial reduction of the O₂ yield was observed, but only a minor change in the S-state distribution occurred. However, when Cl⁻ was displaced by NO⁻₃, which is equivalent to Cl⁻ deficiency of the water-splitting complex, a substantial reduction of the O₂ yield and in addition a significant change in the S-state distribution was observed. The comparison of deactivation kinetics in NO⁻₃ containing samples with those in control samples indicated that Cl⁻ deficiency allowed accumulation of oxidizing equivalents up to the S₃ state but modified the final step of O₂ evolution. Moreover, those centers which advanced to the S₃ state in the absence of Cl⁻ deactivated in a special way which involved a faster deactivation of S₂ and an increased formation of S₋₁.     

The reduction of fungal laccase at high pH

1. 1. The nature and mechanism of the reduction of fungal laccase (p-diphenol: O₂ oxidoreductase, EC 1.10.3.2) obtained on an increase in pH have been studied by optical and electron paramagnetic resonance (EPR) spectroscopy and by measurements of O₂ concentration.

2. 2. The decreases in the optical absorption and the EPR signal of the “blue” Type 1 Cu²⁺ at high pH indicate that this ion is reduced. This is confirmed by oxidation with hexachloroiridate(IV) which restores the blue color. The “nonblue” Type 2 Cu²⁺ remains divalent over the pH range studied, as seen from the EPR spectra.

3. 3. Approximately one equivalent of hexachloroiridate(IV) is sufficient to restore the color of a pH-bleached protein which suggests that the reduction involves a single electron. A comparison between the optical spectra at pH 5 and 8 shows that the two-electron accepting unit, which at pH 5.5 is reduced concomitantly with the Type 1 Cu²⁺, remains oxidized in the protein brought to high pH. This unit can be reduced at pH 8.3 by octacyanotungstate(IV), as shown by the fact that this reductant in anaerobic titrations is found to add about two electrons (and no more) to a protein already having the Type 1 copper reduced. Thus, an increase in pH introduces a difference in the reduction behavior of the electron acceptors in fungal laccase.

4. 4. Oxygraph experiments show that there is no production of O₂ with an increase in pH, as would occur if water was oxidized by laccase. On the contrary, there is a continuous consumption of O₂ at both pH 5 and 8, indicating that the protein preparation contains a reducing substance which is responsible for the pH-dependent reduction.