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.     

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.     

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).     

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

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

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

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

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

Control of excitation transfer in photosynthesis. IV. Kinetics of chlorophyll a fluorescence in Porphyra yezoensis

The kinetics of chlorophyll a fluorescence were measured at 685 nm in intact cells of Porphyra yezoensis during alternate illumination of the organism with two colors of light, one absorbed by phycoerythrin and the other by chlorophyll a. Two components of fluorescence change overlapping each other in time were separated; the fast component may be controlled by the rate of Photoreaction II which competes with the fluorescence emission process, and the slow component by the light-induced change in excitation transfer between two pigment systems as suggested in our previous study⁶. The kinetics of the slow change in fluorescence yield were extensively investigated.

Terms, “State I” and “State II” are used to describe the state of excitation transfer. In the State I a lesser amount of excitation energy is delivered in Pigment System I and greater to Pigment System II than in the State II. The conversion of the states is achieved by the selective illumination of pigment systems.

The conversion from the State I toward the State II occurred under Light II (light absorbed by Pigment System II) with a half time of about 10 sec, and it saturated at a light intensity of less than 1000 ergs×cm⁻²×sec⁻¹. The reverse conversion occurred under Light I (light absorbed by Pigment System I) with a half time of about 5 sec, and it saturated at about 10 000 ergs×cm⁻²×sec⁻¹.

Light I and Light II competed with each other in the interconversion of the states.     

Transfer and trapping of excitation energy in Photosystem II as studied by chlorophyll a2 fluorescence quenching by dinitrobenzene and carotenoid triplet. The matrix model

1. The curves representing the reciprocal fluorescence yield of chlorophyll a of Photosystem II (PS II) in Chlorella vulgaris as a function of the concentration of m-dinitrobenzene in the states P Q and P Q^-, are found to be straight parallel lines; P is the primary donor and Q the primary acceptor of PS II. In the weakly trapping state P Q^- the half-quenching of dinitrobenzene is about 0.2 mM, in vitro it is of the order of 10 mM. The fluorescence yield as a function of the concentration of a quencher is described for three models for the structure of pigment systems: the model of separate units, the model of limited energy transfer between the units, and the matrix model. If it is assumed that the rate constant of quenching by dinitrobenzene is high and thus the number of dinitrobenzene molecules per reaction center low, it can be concluded that the pigment system of PS II in C. vulgaris is a matrix of chlorophyll molecules in which the reaction centers are embedded. Theoretical and experimental evidence is consistent with such an assumption.

For Cyanidium caldarium the zero fluorescence yield Ф₀ and its quenching by dinitrobenzene were found to be much smaller than the corresponding quantities for C. vulgaris. Nevertheless, our measurements on C. caldarium could be interpreted by the assumption that the essential properties (rate constants, dinitrobenzene quenching) of PS II are the same for these two species belonging to such widely different groups.

2. The measured dinitrobenzene concentrations required for half-quenching in vivo and other observations are explained by (non-rate-limiting) energy transfer between the chlorophyll a molecules of PS II and by the assumptions that dinitrobenzene is approximately distributed at random in the membrane and does not diffuse during excitation.

3. The fluorescence kinetics of C. vulgaris during a 350 ns laser flash of variable intensity could be simulated on a computer using the matrix model. From the observed fluorescence quenching by the carotenoid triplet (C^T) and the measurement of the number of C^T per reaction center via difference absorption spectroscopy, the rate constant for quenching of C^T is calculated to be k_T = 3.3 · 10¹¹ s⁻¹ which is almost equal to the rate constant of trapping by an open reaction center (Duysens, L.N.M. (1979) CIBA Foundation Symposium 61 (New Series), pp. 323–340).

4. The fluorescence quenching by C^T in non-treated spinach chloroplasts after a 500 ns laser flash (Breton, J., Geacintov, N.E. and Swenberg, C.E. (1979) Biochim. Biophys. Acta 548, 616–635) could be explained within the framework of the matrix model when the value for k_T is used as given in point 3.

5. The observations mentioned under point 1 indicate that the fluorescence yield Ф₀ for centers in trapping state P Q is probably for a fraction exceeding 0.8 emitted by PS II.     

A study of chlorophyll b in vivo

1. The absorption spectrum of chlorophyll b in vivo at 77°K is presented as the difference spectrum between preparations of spinach and chlorophyll b-free Vischeria stellata chloroplasts.

2. A shoulder on this spectrum around 662 nm is due to a component different from chlorophyll b. This component may well be identical with the chlorophyll a form, chlorophyll a (665).

3. The 77°K chlorophyll b absorption spectra in the nonfractionated photosyn-thetic pigment apparatus and in fractions mainly representing Photosystems 1 or 2 are not significantly different.

4. The aerobic irreversible photobleaching of chlorophyll b was studied in the intact pigment complex as well as in fractions mainly consisting of Photosystem 1 or 2. A two-step photobleaching was observed in all cases. The time-course of this bleaching was not significantly different for chlorophyll b in both fractions.

5. These results do not indicate that more than a single chlorophyll b complex occurs in vivo.     

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.     

Quantum yield and rate of formation of the carotenoid triplet state in photosynthetic structures

The formation of the triplet state of carotenoids (detected by an absorption peak at 515 nm) and the photo-oxidation of the primary donor of Photosystem II, P-680 (detected by an absorption increase at 820 nm) have been measured by flash absorption spectroscopy in chloroplasts in which the oxygen evolution was inhibited by treatment with Tris. The amount of each transient form has been followed versus excitation flash intensity (at 590 or 694 nm). At low excitation energy the quantum yield of triplet formation (with the Photosystem II reaction center in the state Q⁻) is about 30% that of P-680 photo-oxidation. The yield of carotenoid triplet formation is higher in the state Q⁻ than in the state Q, in nearly the same proportion as chlorophyll a fluorescence. It is concluded that, for excited chlorophyll a, the relative rates of intersystem crossing to the triplet state and of fluorescence emission are the same in vivo as in organic solvent. At high flash intensity the signal of P-680⁺ completely saturates, whereas that of carotenoid triplet continues to increase.

The rate of triplet-triplet energy transfer from chlorophyll a to carotenoids has been derived from the rise time of the absorption change at 515 nm, in chloroplasts and in several light-harvesting pigment-protein complexes. In all cases the rate is very high, around 8 · 10⁷ s⁻¹ at 294 K. It is about 2–3 times slower at 5 K. The transitory formation of chlorophyll triplet has been verified in two pigment-protein complexes, at 5 K.     

Light-induced movement of magnesium ions in intact chloroplasts. Spectroscopic determination with Eriochrome Blue SE

The metallochromic indicator Eriochrome Blue SE was used to measure light-induced internal movement of Mg²⁺ in intact chloroplasts. By dual-wavelength spectroscopy (measuring wavelength 554 nm, reference 592 nm) a light-induced, dark-reversible absorbance increase of Eriochrome Blue in samples of isolated intact chloroplasts was observed. The light/dark difference spectrum of Eriochrome Blue between 550 and 590 nm (reference wavelength 562 nm) indicated that this absorbance increase was caused by an increased concentration of free Mg²⁺ in a neutral or slightly alkaline chloroplast compartment.

The signal was seen only with intact, but not with broken, envelope-free chloroplasts, which had lost most of their divalent cations. This is interpreted to show that the indicator responds to an increase of Mg²⁺ concentration in the chloroplast stroma, which represents an efflux of Mg²⁺ from the intra-thylakoid space caused by light-dependent proton pumping.

As calculated from corrected values of the absorbance increase of Eriochrome Blue, the light-induced internal release of Mg²⁺ was close to 100 nequiv per mg chlorophyll at pH 7.6 and 250 nequiv at pH 7.1. This corresponds to a light-dependent increase in the concentration of free Mg²⁺ in the stroma of about 2 and 5 mM, respectively.     

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.     

Protein-Protein interactions of light-harvesting pigment protein from spinach chloroplasts. I. Ca binding and its relation to protein association

The role of divalent cations in the regulation of the distribution of excitation energy between the two photosystems involved in green plant photosynthesis has led us to search for a better understanding of how such phenomena might occur at the molecular level. Since small changes in orientation of and distance between pigment molecules could greatly affect the distribution of excitation energy, we have decided to study the effects of ions on the light-harvesting pigment protein from spinach chloroplasts. The light-harvesting pigment protein is shown to have two types of binding sites for Ca²⁺. Binding studies and analytical ultracentrifugation indicate that site I (K_d = 2.5 μM, n = 1.5−4.0 μmol Ca²⁺ bound/mg chlorophyll) is lost as the protein associates. Site II (K_d = 32 μM, n = 9.5 μmol Ca²⁺/mg chlorophyll) is not affected by the association of the protein. This site is responsible, however, for a further divalent cation-dependent association of the protein. The possible role of this protein in grana stacking and control of spillover is 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.     

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.     

Studies on the exchange of G-actin-bound calcium with bivalent cations

1. The possibility of the replacement of G-actin-bound calcium by various bivalent cations has been investigated. After the reaction with all cations studied, with the exception of Cu²⁺, action remains active, i.e., contains bound ATP and polymerizes in 0.1 M KCl.

2. The amount of G-actin-bound calcium, as well as the sum of bivalent cation after replacement, not removable by short-time Dowex-50 treatment, accounts to about 1 mole per 50000 g of G-actin.

3. The rate of exchange is of the same order for bivalent cations studied, including calcium.

4. G-actin-bound Ca²⁺ is fully replaced, besides free Ca²⁺, by free Mn²⁺ and Cd²⁺. The replacement with Mg²⁺, Co²⁺, Ni²⁺ and Zn²⁺ is not complete, and there is practically no reaction with Ba²⁺ and Sr²⁺.

5. Assuming the affinity constant of Ca²⁺ as 1, the following affinity constants for other bivalent cations were obtained: Mn²⁺, 0.90; Cd²⁺, 1.07; Mg²⁺, 0.27; Zn²⁺, 0.22; Co²⁺, 0.18; Ni²⁺, 0.08.

6. The results obtained show that there exists a close correlation between the ionic radius of a particular bivalent cation, and its ability to replace bound Ca²⁺.     

Polarized light spectroscopy of photosynthetic membranes in magneto-oriented whole cells and chloroplasts. Fluorescence and dichroism

1. The wavelength dependence of the fluorescence polarization (FP) ratio and dichroism has been studied with magneto-oriented (10–13 kG) whole cells of Chlorella pyrenoidosa, Scenedesmus obliquus, Euglena gracilis and spinach chloroplasts suspended in their aqueous growth media (or Tris-buffered sucrose solution in the case of the chloroplasts) under physiological conditions. The FP ratio is defined as the fluorescence intensity polarized parallel divided by the intensity polarized perpendicular to the membrane planes.

2. The FP ratio is typically in the range of 1.2–1.9 in Chlorella, 1.20–1.25 in Scenedesmus and 1.4–1.5 in spinach chloroplasts at fluorescence wavelengths above 690 nm. Below 690 nm the FP ratio decreases steadily with decreasing wavelength and may be as low as approx. 1.05 at 660 nm. These results are interpreted in terms of the orientation of the Q_y transition moment vectors of the different spectroscopic forms of chlorophyll. For the chlorophyll a 680 form these vectors are inclined at angles of 30° or less (in Chlorella) with respect to the membrane planes, while the shorter wavelength chlorophyll a 670 forms appear to be not nearly as well oriented.

3. The Euglena fluorescence peak is red shifted to 714 nm (in the other algae and chloroplasts it is situated at 685 nm) and the FP ratio is approx. 1.20 in the 720–730 nm region and decreases with decreasing wavelength below 720 nm and is only 1.05 at 690 nm. This wavelength dependence is in good qualitative agreement with the fluorescence microscope studies of single chloroplasts of Euglena by Olson, R. A., Butler, W. H. and Jennings, W. H. ((1961) Biochim. Biophys. Acta, 54, 615–617).

4. By means of a model calculation it is shown that the high FP ratios observed with Chlorella are entirely consistent with the low values of the degree of polarization (0.01–0.06) determined by previous workers with unoriented cell suspensions.

5. The influence of reabsorption and the resulting distortion in the wavelength dependence of the FP ratio are described. The possibility that the fluorescence is polarized by scattering artifacts, rather than being a result of the intrinsic orientation of chlorophyll, is considered.

6. Linear dichroism studies with Chlorella and spinach chloroplasts confirm the orientation of the Q_y transition moment vectors deduced from the FP ratio. Furthermore, it appears that the porphyrin rings are tilted out of the membrane plane and that the carotenoid molecules tend to lie with their long axes in the lamellar plane.

7. In Euglena, dichroism studies indicate that chlorophyll a 680 is unoriented, while chlorophyll a 695 appears to be oriented similar to chlorophyll a 680 in Chlorella or spinach chloroplasts, a result which is also in accord with the measured FP ratio of Euglena.

8. The possibility that the magnetic field gives rise to the reorientation of individual chlorophyll molecules is shown to be highly unlikely.     

Inactivation of chloroplast photosynthetic electron-transport activity by Ni

Ni²⁺ inhibits electron-transport activity of isolated barley chloroplasts and this inhibition of electron transport by Ni²⁺ is distinctly different from other heavy metal ion (e.g., Pb²⁺, Cd²⁺, Zn²⁺)-induced inhibition of chloroplast function. Ni²⁺ inactivates Photosystem II (PS II) activity at a lower concentration than that required for the same extent of inhibition of Photosystem I (PS I)-mediated electron flow. Ni²⁺ induces changes in chlorophyll a (Chl a) emission characteristics and brings about a lowering of the Chl a fluorescence yield, and this lowering of Chl a fluorescence intensity is not relieved by the exogenously supplied electron donor NH₂OH which donates electrons very close to the PS II reaction centres. Immobilization of the chloroplast membrane structure with glutaraldehyde fails to arrest the Ni²⁺-induced loss of PS II activity. Also, Ni²⁺-treated chloroplasts do not regain the ability to photoreduce 2,6-dichlorophenolindophenol even after washing of chloroplasts with buffer. These results indicate that unlike Zn²⁺ or Pb²⁺, Ni²⁺ induces alterations in the chloroplast photosynthetic apparatus resulting in an irreversible loss of electron-transport activity.     

Fluorescence decay kinetics of mutants of corn deficient in photosystem I and photosystem II

The fast fluorescence decay kinetics of two photosynthetic mutants of corn (Zea mays) have been compared with those of normal corn. The fluorescence of normal corn can be resolved into three exponential decay components of lifetime 900–1500 ps (slow), 300–500 ps (middle) and 50–120 ps (fast), the yields of which are affected by light intensity and Mg²⁺ levels. The Photosystem II-(PS II)-defective mutant hcf-3 has similar decay lifetimes (approx. 1200, 450 and 100 ps) but is not affected by light intensity, reflecting the absence of PS II charge recombination. However, yields do respond to Mg²⁺ in a fashion typical of normal corn, which may be correlated with the presence of normal levels of light-harvesting chlorophyll a + b complex (LHCP). The PS I mutant hcf-50 also shows three-component decay kinetics. In conjunction with the results on the LHCP-deficient mutant of barley presented in a recent paper (Karukstis, K.K. and Sauer, K. (1984) Biochim. Biophys. Acta 766, 148–155), these data suggest that the slow component of normal chloroplasts is kinetically controlled by the decay processes of the LHCP and that the energy comes from one of two sources: (a) charge recombination in the reaction centre or (b) energy transferred within or between LHCP units only. The fast component appears to originate from both PS I and PS II. The complex response of the middle component to cations and light intensity, and its presence in all of the mutants, suggests that it also may have multiple origins.     

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.

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.