Composition of the photosystems and chloroplast structure in extreme shade plants

Chloroplasts were isolated from leaves of three species of tropical rainforest plants, Alocasia macrorrhiza, Cordyline rubra and Lomandra longifolia; these species are representative of extreme “shade” plants. It was found that shade plant chloroplasts contained 4–5 times more chlorophyll than spinach chloroplasts. Their chlorophyll a/chlorophyll b ratio was 2.3 compared with 2.8 for spinach. Electron micrographs of leaf sections showed that the shade plant chloroplasts contained very large grana stacks. The total length of partitions relative to the total length of stroma lamellae was much higher in Alocasia than in spinach chloroplasts. Freeze-etching of isolated chloroplasts revealed both the small and large particles found in spinach chloroplasts.

Despite their increased chlorophyll content, low chlorophyll a/chlorophyll b ratio, and large grana, the shade plant chloroplasts were fragmented with digitonin to yield small fragments (D-144) highly enriched in Photosystem I, and large fragments (D-10) enriched in Photosystem II. The degree of fragmentation of the shade plant chloroplasts was remarkably similar to that of spinach chloroplasts, except that the subchloroplast fragments from the shade plants had lower chlorophyll a/chlorophyll b ratios than the corresponding fragments from spinach. The D-10 fragments from the shade plants had chlorophyll a/chlorophyll b ratios of 1.78-2.00 and the D-144 fragments ratios of 3.54–4.07. We conclude that Photosystems I and II of the shade plants have lower proportions of chlorophyll a to chlorophyll b than the corresponding photosystems of spinach. The lower chlorophyll a/chlorophyll b ratio of shade plant chloroplasts is not due to a significant increase in the ratio of Photosystem II to Photosystem I in these chloroplasts.

The extent of grana formation in higher plant chloroplasts appears to be related to the total chlorophyll content of the chloroplast. Grana formation may simply be an means of achieving a higher density of light-harvesting assemblies and hence a more efficient collection of light quanta.     

Intersystem exciton transfer in isolated chloroplasts

The following arguments in favor of exciton transfer between the two photosystems are presented:

1. (1) MgCl₂ (1–10 mM range) decreases the intersystem transfer but does not modify the partition of absorbed photons between the photosystems. MgCl₂ addition causes a simultaneous increase of excitation life time (τ) and of fluorescence intensity (F). The same linear relationship is obtained with or without added Mg²⁺.

2. (2) The deactivation of Photosystem II by the Photosystem II to Photosystem I transfer increases with the level of reduced Photosystem II traps. When all Photosystem II traps are closed, half of Photosystem II excitons are deactivated by transfer to Photosystem I.

3. (3) From the relative values of the 685-nm fluorescence yield and System II electron transport rate in limiting light, measured with and without MgCl₂, the values of rate constants of Photosystem II deactivation were calculated.

4. (4) The intersystem transfer determines a 715-nm variable fluorescence, which is lowered by MgCl₂ addition. When this transfer is decreased by MgCl₂ the efficiency of the transfer between Photosystem II-connected units is enhanced, and a more sigmoidal fluorescence rise is obtained.

A double-layer model of the thylakoid membrane where each photosystem is restricted to one leaflet is proposed to explain the decrease of the intersystem transfer after adding cations. It is suggested that MgCl₂ decreases the thickness of the Photosystem I polar region, increasing the distance between the pigments of the two photosystems.     

Isolation and properties of particles containing the reactioncenter complex of Photosystem II from spinach chloroplasts

By density gradient centrifugation of the 80000 × g supernatant of digitonintreated spinach chloroplasts two main green bands and one minor green band were obtained. The purification and properties of the particles present in the main bands, which were shown to be derived from Photosystem I and Photosystem II, have been described previously; those of the particles in the minor fraction will be described in the present paper.

After purification, these particles show Photosystem II activity but are devoid of Photosystem I activity. They have a high chlorophyll a/chlorophyll b ratio and are enriched in β-carotene and cytochrome b₅₅₉. At liquid nitrogen temperature, photoreduction of C550 and photooxidation of cytochrome-b₅₅₉ can be observed. At room temperature, cytochrome b₅₅₉ undergoes slight photooxidation.

These properties indicate that this particle may be the reaction-center complex of Photosystem II. It is suggested that, in vivo, the Photosystem II unit is made up of a reaction-center complex and an accessory complex, the latter being found in one of the main green bands of the density gradient.     

Light-induced changes of fluorescence and absorbance in spinach chloroplasts at −40 °C

1. 1. Spinach chloroplasts were stored in the dark for at least 1 h, rapidly cooled to −40 °C, and illuminated with continuous light or short saturating flashes. In agreement with the measurements of Joliot and Joliot, chloroplasts that had been preilluminated with one or two flashes just before cooling showed a less efficient increase in the yield of chlorophyll a fluorescence upon illumination at −40 °C than dark-adapted chloroplasts. The effect disappeared below −150 °C, but reappeared again upon warming to −40 °C. Little effect was seen at room temperature in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), added after the preillumination.

2. 2. Light-induced absorbance difference spectra at −40 °C in the region 500–560 nm indicated the participation of two components, the socalled 518-nm change (P518) and C-550. After preillumination with two flashes the absorbance change at 518 nm was smaller, and almost no C-550 was observed. After four flashes, the bands of C-550 were clearly visible again.

3. 3. The fluorescence increase and the absorbance change at 518 nm showed the same type of flash pattern with a minimum after the second and a maximum at the fourth flash. In the presence of 100 μM hydroxylamine, the fluorescence response was low after the fourth and high again after the sixth flash, which confirmed the hypothesis that the flash effect was related to the so-called S-state of the electron transport pathway from water to Photosystem 2.

4. 4. The kinetics of the light-induced absorbance changes were the same at each wavelength, and, apart from the size of the deflection, they were independent of preillumination. Flash experiments indicated that the absorbance changes were a one-quantum reaction. This was also true for the fluorescence increase in dark-adapted chloroplasts, but with preilluminated chloroplasts several flashes were needed to approximately saturate the fluorescence yield.

5. 5. The results are discussed in terms of a mechanism involving two electron donors and two electron acceptors for System 2 of photosynthesis.

Evidence for the role of the light-harvesting chlorophyll a/b protein complex in photosystem II heterogeneity

Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll a/b protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II and PS II_β centres. On excitation at 550 nm the major contribution was from PS II_β centres, that from PS II centres was only minimal. Mg²⁺ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS II_β centres being the major contributors; the PS II contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll a/b ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II and PS II_β centres. Mg²⁺ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II and PS II_β to the fluorescence induction kinetics. PS II characteristics are produced by LHCP association with PS II, whereas PS II_β characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.     

The effects of carbon dioxide concentration on oxygen evolution and fluorescence transients in synchronous cultures of Chlorella pyrenoidosa

1. 1. The steady-state fluorescence yield of Chlorella pyrenoidosa is strongly affected by CO₂ concentration: the yield is approximately 2-fold higher in the presence than in the absence of CO₂. During induction, in the presence of saturating CO₂, accelerating oxygen evolution is paralleled by rising fluorescence (M₂-P₃ transient); in the absence of CO₂, fluorescence yield remains at the low M₂ level.

2. 2. Both illumination and CO₂ content are important in determining the steady-state fluorescence yield: at lower illuminations, lower concentrations of CO₂ are required to obtain a maximum fluorescence yield.

3. 3. The slow fluorescence transients are not affected directly by pH but only indirectly through the CO₂ concentration.

4. 4. The CO₂-dependent fluorescence rise (M₂-P₃ transient) is most readily observed in cells harvested early in the light period of a synchronous culture, but it can also be elicited in cells harvested during the dark period.

5. 5. Addition of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) to CO₂-deprived cells raises the fluorescence yield approximately 4-fold, that is to the same high level as cells supplied with CO₂ and DCMU.

6. 6. The effects of CO₂ provide a new example of a marked parallelism between photosynthetic electron transport and fluorescence. To explain such parallelism, it seems necessary to postulate large changes in the de-excitation processes within Photosystem II units or in the distribution of excitation between Photosystems I and II.

Effect of lincomycin on the chlorophyll protein complex I content and Photosystem I activity of greening leaves

1. 1. Greening barley and pea leaves treated with lincomycin have a reduced chlorophyll content. Lincomycin does not alter the proportion of chlorophyll in chlorophyll-protein complex II (CPII) but greatly reduces that in chlorophyll-protein complex I (CPI).

2. 2. Difference spectra show that chloroplasts from lincomycin-treated leaves are deficient in at least two long wavelength forms of chlorophyll a. These have maxima at 77 K of 683 and 690 nm.

3. 3. The chemically determined P-700/chlorophyll ratio of chloroplasts is unaffected by lincomycin but the photochemical P-700/chlorophyll ratio is less than half of that of the control. It is less affected than the chlorophyll-protein complex I content.

4. 4. Photosystem I activity expressed on a chlorophyll basis is unaffected by lincomycin but the light intensity for half saturation is increased 8-fold.

5. 5. Chlorophyll-protein complex I apoprotein content is reduced by lincomycin. No evidence was found for an accumulation of its precursor(s). The relative abundance of major peptides of 18 000, 15 000 and 12 000 daltons in lincomycin-treated chloroplasts is attributed to a general inhibition of greening and associated membrane formation.

Relative stability of chlorophyll complexes in vivo

The time course of aerobic photobleaching of various chlorophyll-protein complexes in vivo at high light intensities was studied with isolated Aspidistra elatior chloroplasts.

1. 1. C_a680 bleaching starts with the onset of irradiation and, initially, proceeds linearly with time. Washing the chloroplasts causes a nearly constant increase of the bleaching rate throughout the experiment.

2. 2. C_a670 does not appreciably, if at all, bleach initially; subsequently, bleaching proceeds linearly with time and at a slightly higher rate than that for C_a680. Washing makes C_a670 bleach concomitantly with the onset of illumination, and at a nearly constant rate.

3. 3. Bleaching at 665 nm is likely to start only after a relatively long period of illumination. Washing shows no effects during this period. Once bleaching has started, washing causes its rate to increase.

4. 4. No indication of the occurrence of “short-wave” chlorophyll a forms other than C_a670 and C_a665 was obtained.

5. 5. C_b bleaching starts concomitantly with illumination at a low rate. The rate increases more or less exponentially with time. Washing enhances bleaching in two steps.

6. 6. The importance of the results is discussed.

The high-energy state of the thylakoid system as indicated by chlorophyll fluorescence and chloroplast shrinkage

Certain long-term fluorescence phenomena observed in intact leaves of higher plants and in isolated chloroplasts show a reverse relationship to light-induced absorbance changes at 535 nm (“chloroplast shrinkage”).

1. 1. In isolated chloroplasts with intact envelopes strong fluorescence quenching upon prolonged illumination with red light is accompanied by an absorbance increase. Both effects are reversed by uncoupling with cyclohexylammonium chloride.

2. 2. The fluorescence quenching is reversed in the dark with kinetics very similar to those of the dark decay of chloroplast shrinkage.

3. 3. In intact leaves under strong illumination with red light in CO₂-free air a low level of variable fluorescence and a strong shrinkage response are observed. Carbon dioxide was found to increase fluorescence and to inhibit shrinkage.

4. 4. Under nitrogen, CO₂ caused fluorescence quenching and shrinkage increase at low concentrations. At higher CO₂ levels fluorescence was increased and shrinkage decreased.

5. 5. In the presence of CO₂, the steady-state yield of fluorescence was lower under nitrogen than under air, whereas chloroplast shrinkage was stimulated in nitrogen and suppressed in air.

6. 6. These results demonstrate that the fluorescence yield does not only depend on the redox state of the quencher Q, but to a large degree also on the high-energy state of the thylakoid system associated with photophosphorylation.

Reactions of b-cytochromes in the red alga Porphyridium aerugineum

1. 1. The kinetics of light-induced absorbance changes due to oxidation and reduction of cytochromes were measured in a suspension of intact cells of the unicellular red alga Porphyridium aerugineum. Absorbance changes in the region 540–570 nm upon alternating far-red light and darkness indicated the oxidation of cytochrome ƒ and reduction of cytochrome b₅₆₃ upon illumination. The relative efficiencies of far-red and orange light indicated that both reactions were driven by Photosystem I.

2. 2. Experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), with anaerobic cells and in alternating far-red and orange light indicated that cytochrome b₅₆₃ reacts in a cyclic chain around Photosystem I, and that the reduced cytochrome does not react with oxygen or with another oxidized product of Photosystem II. The quantum requirement for the photoreduction was about 6 quanta/equiv at 700 nm. A low concentration of N-methylphenazonium methosulphate (PMS) enhanced the rate of reoxidation of cytochrome b₅₆₃ in the dark. In the presence of higher concentrations of PMS a photooxidation, driven by Photosystem I, instead of reduction was observed. These observations suggest that PMS enhances the rate of reactions between reduced cytochrome b₅₆₃ and oxidized products of Photosystem I.

3. 3. In the presence of carbonylcyanide m-chlorophenylhydrazone (CCCP) a light-induced decrease of absorption at 560 nm occurred. Spectral evidence suggested the photooxidation of cytochrome b₅₅₉ under these conditions. Inhibition by DCMU and a relatively efficient action of orange light suggested that this photooxidation is driven by Photosystem II.

Electron transport between plastoquinone and chlorophyll aI in chloroplasts

After preillumination with System I light spinach chloroplasts were excited by one flash or a group of saturating flashes. During the following dark period the time courses of the oxidation of plastohydroquinone and of the simultaneous reduction of oxidized cytochrome f and chlorophyll a_I (P700) have been measured.

1. 1. From a correlation of these kinetics it can be concluded that at least 85% of the electrons from plastohydroquinone are transferred to chlorophyll a_I.

2. 2. After one flash 93% of the oxidized chlorophyll a_I is reduced. This suggests a high equilibrium constant between chlorophyll a_I and its donor as well as an equilibration between different chlorophyll a_I molecules.

3. 3. Cytochrome f is also reduced by plastohydroquinone. A ratio of active cytochrome f to chlorophyll a_I of 0.4:1 is observed. The half-life time of the reduction of cytochrome f is 17 ms. The time course indicates that in the dark cytochrome f does not transfer electrons to chlorophyll a_I and that no more than 15% of the electron transport passes cytochrome f. Therefore cytochrome f should be situated in a side path of the linear electron transport.

4. 4. The electrons which are released from plastohydroquinone and are not accepted by oxidized cytochrome f and chlorophyll a_I have been calculated. From this difference properties of an electron carrier, as yet not identified, between plastoquinone and chlorophyll a_I are predicted.

Respiratory cytochromes of Euglena gracilis

1. 1. Difference spectra of whole cells and of a particulate fraction of a streptomycin-bleached strain of Euglena gracilis showed the presence of a b-type cytochrome, cytochrome b (561 Euglena), and an a-type cytochrome, cytochrome a-type (609 Euglena). The cytochromes were characterized by pyridine hemochromogen formation and were found associated with a particulate fraction enriched with mitochondria.

2. 2. Both b-type and a-type cytochromes were reduced by succinate, oxidized by oxygen and reacted with a soluble c-type cytochrome, cytochrome c-type (556 Euglena), in reversible oxidation-reduction reactions. The steady-state level of reduction for each cytochrome was 92, 22 and 5% of the anaerobic level for the b-type, c-type and a-type cytochrome, respectively.

3. 3. Oxidation of c-type and a-type cytochromes was completely inhibited by cyanide, although respiration of a particulate fraction was only 60% inhibited by the same concentration of cyanide. Antimycin A inhibited respiration by up to 70% but completely inhibited reduction of the c-type cytochrome.

4. 4. The data suggest that electron transfer in the respiratory pathway of Euglena involves the b-, c- and a-type cytochrome in a direct sequence. The cyanide and antimycin A-insensitive oxidation pathway is considered to involve a more direct oxidation of the b-type cytochrome.

Excitation spectra for Photosystem I and Photosystem II in chloroplasts and the spectral characteristics of the distribution of quanta between the two photosystems

The parameters listed in the title were determined within the context of a model for the photochemical apparatus of photosynthesis.

The fluorescence of variable yield at 750 nm at −196 °C is due to energy transfer from Photosystem II to Photosystem I. Fluorescence excitation spectra were measured at −196 °C at the minimum, F_O, level and the maximum, F_M, level of the emission at 750 nm. The difference spectrum, F_M–F_O, which represents the excitation spectrum for F_V is presented as a pure Photosystem II excitation spectrum. This spectrum shows a maximum at 677 nm, attributable to the antenna chlorophyll a of Photosystem II units, with a shoulder at 670 nm and a smaller maximum at 650 nm, presumably due to chlorophyll a and chlorophyll b of the light-harvesting chlorophyll complex.

Fluorescence at the F_O level at 750 nm can be considered in two parts; one part due to the fraction of absorbed quanta, , which excites Photosystem I more-or-less directly and another part due to energy transfer from Photosystem II to Photosystem I. The latter contribution can be estimated from the ratio of F_O/F_V measured at 692 nm and the extent of F_V at 750 nm. According to this procedure the excitation spectrum of Photosystem I at −196 °C was determined by subtracting 1/3 of the excitation spectrum of F_V at 750 nm from the excitation spectrum of F_O at 750 nm. The spectrum shows a relatively sharp maximum at 681 nm due to the antenna chlorophyll a of Photosystem I units with probably some energy transfer from the light-harvesting chlorophyll complex.

The wavelength dependence of was determined from fluorescence measurements at 692 and 750 nm at −196 °C. is constant to within a few percent from 400 to 680 nm, the maximum deviation being at 515 nm where shows a broad maximum increasing from 0.30 to 0.34. At wavelengths between 680 and 700 nm, increases to unity as Photosystem I becomes the dominant absorber in the photochemical apparatus.     

Energy transfer in the photochemical apparatus of flashed bean leaves

Fluorescence and energy transfer properties of bean leaves greened by brief, repetitive xenon flashes were studied at −196 °C. The bleaching of P-700 has no influence on the yield of fluorescence at any wavelength of emission. The light-induced fluorescence yield changes which are observed in both the 690 and 730 nm emission bands in the low temperature fluorescence spectra are due to changes in the state of the Photosystem II reaction centers. The fluorescence yield changes in the 730 nm band are attributed to energy transfer from Photosystem II to Photosystem I. Such energy transfer was also confirmed by measurements of the rate of photooxidation of P-700 at −196 °C in leaves in which the Photosystem II reaction centers were either all open or all closed. It is concluded that energy transfer from Photosystem II to Photosystem I occurs in the flashed bean leaves which lack the light-harvesting chlorophyll a/b protein.     

The P-700-chlorophyl a-protein complex and two major light-harvesting complexes of Acrocarpia paniculata and other brown seaweeds

Acrocarpia paniculata thylakoids were fragmented with Triton X-100 and the pigment-protein complexes so released were isolated by sucrose density gradient centrifugation. Three main chlorophyll-carotenoid-protein complexes with distinct pigment compositions were isolated.

1. (1) A P-700-chlorophyll a-protein complex, with a ratio of 1 P-700: 38 chlorophyll a: 4 ta-carotene molecules, had similar absorption and fluorescence characteristics to the chlorophyll-protein complex 1 isolated with Triton X-100 from higher plants, green algae and Ecklonia radiata.

2. (2) An orange-brown complex had a chlorophyll a : c₂ : fucoxanthin molar ratio of 2 : 1 : 2. This complex had no chlorophyll c₁ and contained most of the fucoxanthin present in the chloroplasts. This pigment complex is postulated to be the main light-harvesting complex of brown seaweeds.

3. (3) A green complex had a chlorophyll a : c₁ : c₂ : violaxanthin molar ratio of 8 : 1 : 1 : 1. This also is a light-harvesting complex.

The absorption and fluorescence spectral characteristics and other physical properties were consistent with the pigments of these three major complexes being bound to protein. Differential extraction of brown algal thylakoids with Triton X-100 showed that a chlorophyll c₂-fucoxanthin-protein complex was a minor pigment complex of these thylakoids.     

Effect of low temperature (−30 to −60°C) on the reoxidation of the Photosystem II primary electron acceptor in the presence and absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea

The fluorescence yield has been measured on spinach chloroplasts at low temperature (−30 to −60°C) for various dark times following a short saturating flash. A decrease in the fluorescence yield linked to the reoxidation of the Photosystem II electron acceptor Q is still observed at −60°C. Two reactions participate in this reoxidation: a back reaction or charge recombination and the transfer of an electron from Q⁻ to Pool A. The relative competition between these two reactions at low temperature depends upon the oxidation state of the donor side of the Photosystem II center:

1. (1) In dark-adapted chloroplasts (i.e. in States S₀+S₁ according to Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475), Q, reduced by a flash at low temperature, is reoxidized by a secondary acceptor and the positive charge is stabilized on the Photosystem II donor Z. Although this reaction is strongly temperature dependent, it still occurs very slowly at −60°C.

2. (2) When chloroplasts are placed in the S₂+S₃ states by a two-flash preillumination at room temperature, the reoxidation of Q⁻ after a flash at low temperature is mainly due to a temperature-independent back reaction which occurs with non-exponential kinetics.

3. (3) Long continuous illumination of a frozen sample at −30°C causes 6–7 reducing equivalents to be transferred to the pool. Thus, a sufficient number of oxidizing equivalents should have been generated to produce at least one O₂ molecule.

4. (4) A study of the back reaction in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) shows the superposition of two distinct non-exponential reactions one temperature dependent, the other temperature independent.

Thylakoid polypeptide composition and light-independent phosphorylation of the chlorophyll a,b-protein in Prochloron, a prokaryote exhibiting oxygenic photosynthesis

Thylakoids of the prokaryote Prochloron, present as a symbiont in ascidians isolated from the Red Sea at Eilat (Israel), showed polypeptide electrophoretic patterns comparable to those of thylakoids from eukaryotic oxygen-evolving organisms. Low temperature, fluorescence spectroscopy of Prochloron, having a chlorophyll a/b ratio of 3.8–5, and frozen in situ, demonstrated the presence of Photosystem II chlorophyll-protein complex emitting at 686 and 696 nm, as well as the emission band of Photosystem I at 720 nm which was so far not observed in Prochloron species. The latter emission was absent, if the cells or thylakoids were isolated prior to freezing. Energy transfer from chlorophyll b to chlorophyll a could be demonstrated to occur in vivo. The chlorophyll a,b-protein complex of Photosystem II, isolated by non-denaturing polyacrylamide gel electrophoresis, contained one major polypeptide of 34 kDa. The polypeptide was phosphorylated in vitro by a membrane-bound protein kinase which was not stimulated by light. A light-independent protein kinase activity was also found in isolated thylakoids of another prokaryote, the cyanophyte Fremyella diplosiphon. State I–State II transition could not be demonstrated in Prochloron by measurements of modulated fluorescence intensity in situ. We suggest that the presence of a light-independent thylakoid protein kinase of Prochloron, collected in the Red Sea at not less than 30 m depth, might be the result of an evolutionary process whereby this organism has adapted to an environment in which light, absorbed preferentially by Photosystem II, prevails.     

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.     

The reduction kinetics of chlorophyll a1 as an indicator for proton uptake between the light reactions in chloroplasts

The flash-induced oxidation kinetics of the primary acceptor of light Reaction II (X-320) and the reduction kinetics of chlorophyll a₁ (P-700) after far-red preilluination have been studied with high time resolution in spinach chloroplasts.

1. 1. The kinetics of chlorophyll a₁ exhibits a pronounced lag phase of 2–3 ms at the onset of reduction as would be expected for the final product of consecutive reactions. Because the oxidation of the plastoquinone pool is the rate-limiting step for the electron transport between the two light reactions, the lag indicates the maximal electron transfer time over all preceding reactions after light Reaction II.

2. 2. The observation that the lag phase decreases with decreasing pH is evidence of an electron transfer step coupled to a proton uptake reaction.

3. 3. Protonation of X-320 after reduction in the flash is excluded because a slight increase of the decay time is found at decreasing pH values.

4. 4. The time course of plastohydroquinone formation is deduced from the first derivative of the reduction kinetics of chlorophyll a₁. This approach covers those plastohydroquinone molecules being available to the electron carriers of System I via the rate-limiting step. Direct measurements of absorbance changes would not allow to discriminate between these and functionally different plastohydroquinone molecules.

5. 5. The derived time course of plastohydroquinone at different pH gives evidence for an additional electron transfer step with a half time of about 1 ms following the proton uptake and preceding the rate-limiting step. It is tentatively attributed to the diffusion of neutral plastohydroquinone across the hydrophobic core of the thylakoid membrane.

6. 6. The lower limit of the rate constant for proton uptake by an electron carrier, consistent with the lag of chlorophyll a₁ reduction, is estimated as > 10¹¹ M⁻¹ · s⁻¹. The value is higher than that of the fastest diffusion controlled protonations of organic molecules in solution.

Possible mechanisms of linear electron transport between light Reaction II and the rate-limiting oxidation of neutral plastohydroquinone are thoroughly discussed.     

Excitation energy transfer in the light-harvesting chlorophyll

The “light-harvesting chlorophyll a/b · protein” described by Thornber has been prepared electrophoretically from spinach chloroplasts. The optical properties relevant to energy transfer have been measured in the red region (i.e. 600–700 nm). Measurements of the absorption spectrum, fluorescence excitation spectrum and excitation dependence of the fluorescence emission spectrum of this protein confirm that energy transfer from chlorophyll b to chlorophyll a is highly efficient, as is the case in concentrated chlorophyll solutions and in vivo. The excitation dependence of the fluorescence polarization shows a minimum polarization of 1.9 % at 650 nm which is the absorption maximum of chlorophyll b in the protein and rises steadily to a maximum value of 13.8 % at 695 nm, the red edge of the chlorophyll a absorption band. Analysis of these measurements shows that at least two unresolved components must be responsible for the chlorophyll a absorption maximum. Comparison of polarization measurements with those observed in vivo shows that most of the depolarization observed in vivo can take place within a single protein. Circular dichroism measurements show a doublet structure in the chlorophyll b absorption band which suggests an exciton splitting not resolved in absorption. Analysis of these data yields information about the relative orientation of the S₀→S₁ transition moments of the chlorophyll molecules within the protein.