Picosecond fluorescence kinetics in spinach chloroplasts at room temperature. Effects of phosphorylation

We have used single-photon timing with picosecond resolution to investigate the effect of phosphorylation on the fluorescence decay from broken spinach chloroplasts. Phosphorylation of spinach thylakoids causes a quenching of the slow decay phase (equivalent to a quenching of variable fluorescence) and an increase in the yield of the middle phase decay component. In addition, phosphorylation alters the intensity dependence of fluorescence in a manner which indicates a decreased antenna size of Photosystem II. The observed changes are indicative of a State 1-State 2 transition and show a clear reversal when the membranes are dephosphorylated.     

Potentiometric titration of Photosystem II fluorescence decay kinetics in spinach chloroplasts

The fluorescence yield of chloroplasts reflects the redox state of the electron acceptor of the Photosystem II reaction center, with increasing yield as the acceptor is reduced. Chemical reductive titrations of fluorescence yield in chloroplasts at room temperature indicate two distinct midpoint potentials, suggesting the possibility of Photosystem II electron acceptor heterogeneity. We have carried out a potentiometric titration of the fluorescence decay kinetics in spinach chloroplasts using a continuous mode-locked dye laser with low-intensity excitation pulses and a picosecond-resolution single-photon timing system. At all potentials the fluorescence decay is best described by three exponential components. As the potential is lowered, the slow phase changes 30-fold in yield with two distinct midpoint potentials, accompanied by a modest (3-fold) increase in the lifetime. The titration curve for the slow component of the fluorescence decay of spinach chloroplasts is best characterized by two single-electron redox reactions with midpoint potentials at pH 8.0 of +119 and ?350 mV, with corresponding relative contributions to the fluorescence yield of 49 and 51%, respectively. There is little change in the fast and middle components of the fluorescence decay. We found that the oxidized form of the redox mediator 2-hydroxy-1,4-naphthoquinone preferentially quenches the fluorescence, causing an anomalous decrease in the apparent midpoint of the high-potential transition. This effect accounts for a significant difference between the midpoint potentials that we observe and some of those previously reported. The selective effect of reduction potentials on particular fluorescence decay components provides useful information about the organization and distribution of the Photosystem II electron acceptor.     

Picosecond fluorescence kinetics in spinach chloroplasts at room temperature. Effects of Mg

Single-photon timing with picosecond resolution is used to investigate the effect of Mg²⁺ on the room-temperature fluorescence decay kinetics in broken spinach chloroplasts. In agreement with an earlier paper (Haehnel, W., Nairn, J.A., Reisberg, P. and Sauer, K. (1982) Biochim. Biophys. Acta 680, 161–173), we find three components in the fluorescence decay both in the presence and in the absence of Mg²⁺. The behavior of these components is examined as a function of Mg²⁺ concentration at both the F₀ and the F_max fluorescence levels, and as a function of the excitation intensity for thylakoids from spinach chloroplasts isolated in the absence of added Mg²⁺. Analysis of the results indicates that the subsequent addition of Mg²⁺ has effects which occur at different levels of added cation. At low levels of Mg²⁺ (less than 0.75 mM), there appears to be a decrease in communication between Photosystem (PS) II and PS I, which amounts to a decrease in the spillover rate between PS II and PS I. At higher levels of Mg²⁺ (about 2 mM), there appears to be an increase in communication between PS II units and an increase in the effective absorption cross-section of PS II, probably both of these involving the chlorophyll light-harvesting antenna.     

Picosecond fluorescence kinetics and energy transfer in chloroplasts and algae

Single-photon timing with picosecond resolution is used to investigate the kinetics of the fluorescence emission of chlorophyll a in chloroplasts from spinach and pea and in the algae Chlorella pyrenoidosa and Chlamydomonas reinhardii. The fluorescence decay is best described by three exponential components in all species. At low light intensity and with open reaction centers of Photosystem II (F₀), we find lifetimes of approx. 100, 400 and 1100 ps for the three components. Closing the reaction centers by addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea plus hydroxylamine and by increasing light intensity produces only minor changes in the almost constant fast- and medium-lifetime components; however, there is a dramatic increase in the yield of the slow component, by a factor of about 20, accompanied by only a modest increase in the lifetime to 2200 ps (F_max). In good agreement with previous fluorescence lifetime measurements, we find an increase in the averaged lifetime of the three components from 0.5 to 2.0 ns, which is proportional to the 4-fold increase in the total fluorescence yield. Our time-resolved results are inconsistent with models which are based on the proportionality between lifetime and yield and which involve a homogeneous origin of fluorescence that is sensitive to the state of the reaction centers. We conclude that the variable part of the fluorescence, which is dominated by the slow phase, reflects the kinetics of charge recombination in the reaction center, as proposed previously (Klimov, V.V., Allakhverdiev, S.I. and Paschenko, V.Z. (1978) Dokl. Akad. Nauk S.S.S.R. 242, 1204–1207). The modest increase in lifetime of the slow phase indicates the presence of some energy transfer between photosynthetic units.     

Photoconversion kinetics of chloroplast Photosystems I and II. Effect of Mg2+

The effect of divalent cations on the primary photoconversion kinetics of chloroplast Photosystems (PS) I and II was investigated by absorbance difference spectrophotometry in the ultraviolet (ΔA₃₂₀) and red (ΔA₇₀₀) regions and by fluorescence at room temperature. Three main chlorophyll (Chl) a fluorescence emission components were identified. Addition of 5 mM MgCl₂ to unstacked chloroplasts caused a 5–7-fold increase in F_vα, the variable fluorescence yield controlled by the α-centers. The fluorescence yield F_vβ controlled by the β-centers and the nonvariable fluorescence yield F₀ were only slightly changed by the treatment. The absolute number of α- and β-centers remained unchanged and independent of divalent cations. The rate constants K_α, K_β and K_P-700 determined from the photoconversion kinetics of Q_α, Q_β and P-700 were also unchanged by divalent cations, suggesting a constancy of the respective absorption cross-sections. Evidence is presented that the Mg²⁺ effect on Chl a fluorescence is not due simply to unstacking. Conclusion: (1) In the absence of divalent cations from the chloroplast suspending medium, the variable fluorescence yield is not complementary to the rate of PS II photochemistry. (2) A spillover of excitation from PS II to PS I in the absence of Mg²⁺ cannot account for the 7-fold lowering of the variable fluorescence yield F_vα at room temperature. The results are discussed in view of a model of excitation transfer and fluorescence emission in the pigment bed of PS II_α and PS II_β.     

Photoinduced quenching of chlorophyll fluorescence in intact chloroplasts and algae. Resolution into two components

The light-induced decline of chlorophyll a fluorescence from a peak (P) to a low stationary level (S) in intact, physiologically active isolated chloroplasts and in intact Chlorella cells is shown to be predominantly composed of two components: (1) fluorescence quenching by partial reoxidation of the quencher Q, the primary acceptor of Photosystem II and (2) energy-dependent fluorescence quenching related to the photoinduced acidification of the intrathylakoid space. These two mechanisms of fluorescence quenching can be distinguished by the different kinetics of the relaxation of quenching observed upon addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). The relaxation of quenching by addition of DCMU is biphasic. The fast phase with a half-time of about 1 s is attributed to the reversal of Q-dependent quenching. The slow phase with a half-time of about 15 s in chloroplasts and 5 s in Chlorella cells is ascribed to relaxation of energy-dependent quenching. As shown by fluorescence spectroscopy at 77 K, the energy-dependent fluorescence quenching essentially is not caused by increased transfer of excitation energy to Photosystem I. By analyzing the energy- and Q-dependent components of quenching, information on the energy state of the thylakoid membranes and on the redox state of Q under various physiological conditions is obtained.     

Photosynthetic membrane development studied using picosecond fluorescence kinetics

Using measurements of the kinetics of chlorophyll a fluorescence emission, we have investigated the development of the photosynthetic membrane during etioplast-to-chloroplast differentiation. The chlorophyll fluorescence decay kinetics of pea chloroplasts from plants grown under intermittent (2 min light-118 min dark) and continuous light regimes were monitored with a single-photon timing system with picosecond resolution. We have associated the changes in the fluorescence yields and decay kinetics with known structural and organizational developmental phenomena in the chloroplast. This correlation provides a more detailed assignment of the origins of the fluorescence decay components than has been previously obtained by studying only mature chloroplasts. In particular, our analysis of the variable kinetics and multiexponential character of the fluorescence emission during thylakoid development focuses on the organization of photosynthetic units and the degree of communication between reaction centers in the same photosystem. Our results further demonstrate that the age of etiolated tissue is critical to plastid development.     

Picosecond time-resolved study of MgCl2-induced chlorophyll fluorescence yield changes from chloroplasts

The MgCl₂-induced chlorophyll fluorescence yield changes in broken chloroplasts, suspended in a cation-free medium, treated with 3,-(3′,4′-dichlorophenyl)-1,1-dimethylurea and pre-illuminated, has been investigated on a picosecond time scale. Chloroplasts in the low fluorescing state showed a fluorescence decay law of the form exp $\text{?At}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, where A was found to be 0.052 $\text{ps}{}^{\mathrm{<mtext>?1</mtext><mtext>2</mtext>}}$, and may be attributed to the rate of spillover from Photosystem II to Photosystem I. Addition of 10 mM MgCl₂ produced a 50% increase in the steady-state fluorescence quantum yield and caused a marked decrease in the decay rate. The fluorescence decay law was found to be predominantly exponential with a 1/e lifetime of 1.6 ns. These results support the hypothesis that cation-induced changes in the fluorescence yield of chlorophyll are related to the variations in the rate of energy transfer from Photosystem II to Photosystem I, rather than to changes in the partitioning of absorbed quanta between the two systems.     

Picosecond fluorescence kinetic studies of electron acceptor Q redox heterogeneity

We have investigated the influence of chloroplast organization on the nature of chemical reductive titrations of Photosystem II fluorescence decay kinetics in spinach chloroplasts. Structural changes of the chloroplast membrane system were induced by varying the ionic environment of the thylakoids. A single-photon timing system with picosecond resolution monitored the kinetics of the chlorophyll a fluorescence emission. At all ionic concentrations studied, we have observed biphasic potentiometric titration curves of fluorescence yield; these have been interpreted to be suggestive of electron acceptor Q heterogeneity (Karukstis, K.K. and Sauer, K. (1983) Biochim. Biophys. Acta 722, 364–371; Cramer, W.A. and Butler, W.L. (1969) Biochim. Biophys. Acta 172, 503–510). A direct relation is observed between the E_m value of the low-potential component of Q and the Mg²⁺ concentration of the chloroplast suspending medium. We have attributed these midpoint potential variations to the thylakoid structural rearrangements involved in cation-regulated grana stacking. Ionic effects on the fluorescence decay kinetics at the redox transitions are discussed in terms of the heterogeneity of Photosystem II units (α- and β-centers) and the mechanism of deexcitation at a closed reaction center (fluorescence or nonradiative decay).     

External electric field effects on prompt and delayed fluorescence in chloroplasts

An electric field pulse was applied to a suspension of osmotically swollen spinach chloroplasts after illumination with a saturating flash in the presence of DCMU. In addition to the stimulation of delayed fluorescence by the electric field, discovered by Arnold and Azzi (Arnold, W.A. and Azzi, R. (1971) Photochem. Photobiol. 14, 233–240) a sudden drop in fluorescence yield was observed. The kinetics of this fluorescence change were identical to those of the integrated delayed fluorescence emission induced by the pulse. The S-state dependence of the stimulated emission was very similar to that of the normal luminescence. We assume that the membrane potential generated by the pulse changes the activation energy for the back reaction in Photosystem II. On this basis, and making use of data we obtained earlier from electrochromic absorbance changes induced by the pulse, the kinetics of the field-induced prompt and delayed fluorescence changes, and also the amplitude of the fluorescence decrease, which was about 12% for a nearly saturating pulse, are explained. Our results indicate that in those reaction centers where a decrease of the activation energy occurs the effect of a pulse can be quite spectacular: the back reaction, which normally takes seconds, is completed in a few hundred microseconds when a sufficiently strong pulse is applied. Measurements of the polarization of the stimulated luminescence supported the interpretation given above.Only 2.8% of the back reaction was found to proceed via transition of reexcited chlorophyll to the ground state, both during the field pulse and in the absence of the field.     

Anisotropy of the emission and absorption bands of spinach chloroplasts measured by fluorescence polarization and polarized excitation spectra at low temperature

Spectra of fluorescence polarization were measured between 4 and 120 K of spinach chloroplasts, oriented in a magnetic field. At least seven emission bands were observed. The well known bands near 685 nm (‘F-685’) and 735–740 nm (‘F-735’) and the band near 680 nm (‘F-680’) were strongly polarized parallel to the plane of the thylakoid membrane, whereas emission bands near 695 nm (‘F-695’), 710, 730–735 and 760 nm showed perpendicular polarization. Assuming perfect orientation of the thylakoid membranes, we calculated orientation angles of 64, 47 and 66.5° for the emission dipoles of F-685, F-695 and F-735, respectively, with respect to the normal of the membrane. Excitation spectra of F-695 and F-735 in polarized light at 4 K provided information about the orientation of the absorption dipoles of chlorophylls a and b. The spectra thus obtained were in very good agreement with the linear dichroism spectrum. Moreover, they allowed us to distinguish between the pigments associated with Photosystems I and Ii, which is not possible from measurement of linear dichroism alone. The results indicate that a high degree of orientation is not confined to the long-wave absorbing bands, but also bands at shorter wavelength show a clear anisotropy. The calculated orientations were in quantitative agreement with the hypothesis that F-685 and F-735 are associated with chlorophylls absorbing at 676 and 710–715 nm, respectively.     

Measurement of degree of chlorophyll fluorescence polarization in relation to the regulation of excitation energy transfer between Photosystems I and II in pea chloroplasts

The degree of chlorophyll fluorescence polarization (p) at 740 nm was measured at room temperature for pea chloroplasts subjected to various conditions. (1) In agreement with previous published observations, p decreased when the Photosystem (PS) II traps were closed by illumination in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. (2) Under these conditions, the magnitude of p was also sensitive to the presence of salts. Under conditions when ‘spillover’ of the excitation energy from PS II to PS I was low, p was also low, being consistent with increased migration of energy between the PS II light-harvesting chlorophylls. In contrast, when spillover was at a maximum p increased. (3) The change in p in the presence of salts was dependent on the concentration and valency of the cations in such a way as to suggest the changes were mediated through electrostatic forces. The dependency of p on ionic composition of the experimental medium was closely related to the associated changes in fluorescence yield. (4) Membrane stacking, caused by lowering pH of the chloroplast suspension, did not induce a significant change in p, suggesting that this pH-induced process is different from the membrane stacking brought about by manipulating the salt levels. (5) Incubation of thylakoids with ATP induces light-dependent phosphorylation of the light-harvesting chlorophyll-protein complexes, and regulates excitation energy transfer between PS I and PS II (Bennett, J., Steinback, K.R. and Arntzen, C.J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5253–5257). Under conditions which bring about this phosphorylation it was found that p increased to a value indicative of spillover.     

Induction kinetics of delayed light emission in spinach chloroplasts

Induction curves of the delayed light emission in spinach chloroplasts were studied by measuring the decay kinetics after each flash of light. This study differs from previous measurements of the induction curves where only the intensities at one set time after each flash of light were recorded. From the decay kinetics after each flash of light, the induction curves of the delayed light emission measured 2 ms after a flash of light were separated into two components: one component due to the last flash only and one component due to all previous flashes before the last one. On comparing the delayed light induction curves of the two components with the fluorescence induction curves in chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and in chloroplasts treated with hydroxylamine and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the component due to the last flash only is found to be dependent on the concentration of open reaction centers and the component due to all previous flashes except the last is dependent on the concentration of closed reaction centers. This implies that the yield of the fast decaying component of the delayed light emission is dependent on the concentration of open reaction centers and the yield of the slow decaying component is dependent on the concentration of closed reaction centers.     

Hydrogen evolution by co-immobilized chloroplasts and Clostridium butyricum

Spinach chloroplasts and Clostridium butyricum cells were immobilized in 2% agar gel. Crude ferredoxin isolated from spinach and benzyl viologen were used as electron carriers. The optimum pH for both NADP reduction by immobilized chloroplasts and for hydrogen evolution by immobilized Cl. butyricum was 8.0. The optimum temperature was between 25 and 30°C for NADP reduction by immobilized chloroplasts, and 37°C for hydrogen evolution by immobilized cells. The total amount of hydrogen evolved in 6 h was 41 μmol/mg Chl for the immobilized chloroplast-benzyl viologen-immobilized Cl. butyricum system, and 11 μmol/mg Chl for the immobilized chloroplast-ferredoxin-Cl. butyricum system. The systems evolved only a trace amount of hydrogen when dichlorophenyldimethylurea was added. The immobilized chloroplast-benzyl viologen-immobilized Cl. butyricum system evolved hydrogen continuously for 6 h, and immobilized Cl. butyricum retained the initial hydrogenase activity. However, the photoreduction activity of chloroplasts decreased to 30% of the initial activity after 6 h of reaction.     

pH dependent changes in the reactivity of the primary electron acceptor of System II in spinach chloroplasts to external oxidant and reductant

Changes in the rates of dark oxidation and reduction of the primary electron acceptor of System II by added oxidant and reductant were investigated by measuring the induction of chlorophyll fluorescence under moderate actinic light in 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-inhibited chloroplasts at pH values between 3.6 and 9.5. It was found that:

1. (1) The rate of dark oxidation of photoreduced primary acceptor was very slow at all the pH values tested without added electron acceptor.

2. (2) The rate was accelerated by the addition of ferricyanide in the whole pH range. It was dependent approximately on the 0.8th power of the ferricyanide concentration.

3. (3) The rate constant for the oxidation of the primary acceptor by ferricyanide was pH-dependent and became high at low pH. The value at pH 3.6 was more than 100 times that at pH 7.8.

4. (4) The pH-dependent change in the rate constant was almost reversible when the chloroplasts were suspended at the original pH after a large pH change (acid treatment).

5. (5) An addition of carbonylcyanide m-chlorophenylhydrazone or heavy metal chelators had little effect on the rate of dark oxidation of the primary acceptor by ferricyanide.

6. (6) The dark reduction of the primary acceptor by sodium dithionite also became faster at low pH.

From these results it is concluded that at low pH the primary acceptor of System II becomes accessible to the added hydrophilic reagents even in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea.     

Oxygen exchange associated with electron transport and photophosphorylation in spinach thylakoids

O₂ uptake in spinach thylakoids was composed of ferredoxin-dependent and -independent components. The ferredoxin-independent component was largely 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) insensitive (60%). Light-dependent O₂ uptake was stimulated 7-fold by 70 μM ferredoxin and both uptake and evolution (with O₂ as the only electron acceptor) responded almost linearly to ferredoxin up to 40 μM. NADP⁺ reduction, however, was saturated by less than 20 μM ferredoxin. The affinity of O₂ uptake for for O₂ was highly dependent on ferredoxin concentration, with $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(O}{}_2\text{)}$ of less than 20 μM at 2 μM ferredoxin but greater than 60 μM O₂ with 25 μM ferredoxin. O₂ uptake could be suppressed up to 80% with saturating NADP⁺ and it approximated a competitive inhibitor of O₂ uptake with a K_i of 8–15 μM. Electron transport in these thylakoids supported high rates of photophosphorylation with NADP⁺ (600 μmol ATP/mg Chl per h) or O₂ (280 μmol/mg Chl per h) as electron acceptors, with $\text{ATP}\text{2}\text{e}$ ratios of 1.15–1.55. Variation in $\text{ATP}\text{2}\text{e}$ ratios with ferredoxin concentration and effects of antimycin A indicate that cyclic electron flow may also be occurring in this thylakoid system. Results are discussed with regard to photoreduction of O₂ as a potential source of ATP in vivo.