Energy transfer and the distribution of excitation energy in the photosynthetic apparatus of spinach chloroplasts

Equations are derived from our model of the photochemical apparatus of photosynthesis to show that the yield of energy transfer from Photosystem II to Photosystem I, ?_T(II→Iz), can be obtained from measurements on an individual sample of chloroplasts frozen to ?196 °C by comparing the sum of two specifically defined fluorescence excitation spectra with the absorption spectrum of the sample. Then, given that value of ?_T(II→I), the fraction of the quanta absorbed by the photochemical apparatus which is distributed initially to Photosystem I, α, can be determined as a function of the wavelength of excitation from the same fluorescence excitation spectra. The results obtained in this study of individual samples of chloroplasts frozen to ?196 °C in the absence of divalent cations, namely, that ?_T(II→I) varies from a minimum value of 0.10 when the Photosystem II reaction centers are all open to a maximum value of 0.25 when the centers are all closed and that α has a value of about 0.30 which is almost independent of wavelength for wavelengths shorter than 675 nm (α increases rapidly toward unity at wavelengths longer than 675 nm), agrees quite well with results obtained previously from comparative measurements of chloroplasts frozen to ?196 °C in the presence and absence of divalent cations.     

Laser-flash-activated electron paramagnetic resonance studies of primary photochemical reactions in chloroplasts

Electron paramagnetic resonance studies of the primary reactants of Photosystems I and II have been conducted at cryogenic temperatures after laser-flash activation with monochromatic light.P-700 photooxidation occurs irreversibly in chloroplasts and in Photosystem I fragments after activation with a 730 nm laser flash at a temperature of 35 °;K. Flash activation of chloroplasts or Photosystem II chloroplast fragments with 660 nm light results in the production of a free-radical signal (g = 2.002, linewidth ～ 8 gauss) which decays with a half-time of 5.0 ms at 35 °;K. The half-time of decay is independent of temperature in the range of 10–77 °;K. This reversible signal can be eliminated by preillumination of the sample at 35 °;K with 660 nm light (but not by 730 nm light), by preillumination with 660 nm light at room temperature in the presence of 3-(3′, 4′-dichlorophenyl)-1,1′-dimethylurea (DCMU) plus hydroxylamine, or by adjustment of the oxidation-reduction potential of the chloroplasts to — 150 mV prior to freezing. In the presence of ferricyanide (20–50 mM), two free-radical signals are photoinduced during a 660 nm flash at 35 °;K. One signal decays with a half-time of 5 ms, whereas the second signal is formed irreversibly. These results are discussed in terms of a current model for the Photosystem II primary reaction at low temperature which postulates a back-reaction between P-680⁺ and the primary electron acceptor.     

The effect of temperature of the rate of photosynthetic electron transfer in chloroplasts of chilling-sensitive and chilling-resistant plants

1. Photochemical activities as a function of temperature have been compared in chloroplasts isolated from chilling-sensitive (below approximately 12 °C) and chilling-resistant plants.2. An Arrhenius plot of the photoreduction of NADP⁺ from water by chloroplasts isolated from tomato (Lycopersicon esculentum var. Gross Lisse), a chilling-sensitive plant, shows a change in slope at about 12 °C. Between 25 and 14 °C the activation energy for this reaction is 8.3 kcal·mole^?1. Between 11 and 3 °C the activation energy increases to 22 kcal·mole^?1. Photoreduction of NADP⁺ by chloroplasts from another chilling-sensitive plant, bean (Phaseolus vulgaris var. brown beauty), shows an increase in activation energy from 5.9 to 17.5 kcal·mole^?1 below about 12 °C.3. The photoreduction of NADP⁺ by chloroplasts isolated from two chilling-resistant plants, lettuce (Lactuca sativa var. winter lake) and pea (Pisum sativum var. greenfeast), shows constant activation energies of 5.4 and 8.0 kcal·mole^?1, respectively, over the temperature range 3–25 °C.4. The effect of temperature on photosynthetic electron transfer in the chloroplasts of chilling-sensitive plants is localized in Photosystem I region of photosynthesis. Both the photoreduction of NADP⁺ from reduced 2,6-dichlorophenol-indophenol and the ferredoxin-NADP⁺ reductase (EC 1.6.99.4) activity of choroplasts of chilling-sensitive plants show increases in activation energies at approximately 12 °C whereas Photosystem II activity of chloroplasts of chilling-sensitive plants shows a constant activation energy over the temperature range 3–25 °C. The photoreduction of Diquat (1,1′-ethylene-2,2′-dipyridylium dibromide) from water by bean chloroplasts, however, does not show a change in activation energy over the same temperature range. The activation energies of each of these reactions in chilling-resistant plants is constant between 3 and 25 °C.5. The effect of temperature on the activation energy of these reactions in chloroplasts from chilling-sensitive plants is reversible.6. In chilling-sensitive plants, the increased activation energies below approximately 12 °C, with consequent decreased rates of reaction for the photoreduction of NADP⁺, would result in impaired photosynthetic activity at chilling temperatures. This could explain the changes in chloroplast structure and function when chilling-sensitive plants are exposed to chilling temperatures.     

Two temperature-sensitive photosystem II mutants of pea

Two nuclear gene mutants of pea, chlorotica-887 and chlorina-5756, are temperature-sensitive in the development of photosystem II activity. Low temperature flourescence emission spectra of leaves show that the peak at 697 nm from the reaction center of photosystem II is present when the mutants have been grown at 18°C, but absent when they have been grown at 30°C. For leaves of chlorina-5756 grown at 18°C the relative size of the peak at 697 nm is reduced compared to that of leaves of the wild type or chlorotica-887 grown at this temperature. Flourescence induction curves of leaves from wild type plants and chlorotica-887 grown at 18°C possess two steps, while those of leaves from chlorina-5756 grown at 18°C or 30°C and chlorotica-887 grown at 30°C show at fast rise to the maximal level of fluorescence. Measurements on chloroplasts isolated from the mutants indicated that the photosystem I activity per g leaf material is comparable for plants grown at 18°C and plants grown at 30°C. In contrast, no photosystem II activity was detected when the mutants had been grown at 30°C. It is suggested that these mutants are affected in a component required for the assembly of functional photosystem II complexes.     

Luminescence of bacteriorhodopsin from Halobacterium halobium and its connection with the photochemical conversions of the chromophore

Red luminescence of purple membranes from Halobacterium halobium cells in suspension, dry film or freeze-dried preparations was studied and its emission, excitation and polarization spectra are reported. The emission spectra have three bands at 665–670, 720–730 and at 780–790 nm. The position (maximum at 580 nm) and shape of the excitation spectra are close to those of the absorption spectra. The spectra depend on experimental conditions, in particular on pH of the medium. Acidification increases the long wavelength part of the emission spectra and shifts the main excitation maximum 50–60 nm to the longer wavelength side. Low-temperature light-induced changes of the absorption, emission and excitation spectra are presented. Several absorbing and emitting species of bacteriorhodopsin are responsible for the observed spectral changes. The bacteriorhodopsin photoconversion rate constant was estimated to be about 1 · 10¹¹ s^?1 at ? 196°C from the quantum yields of the luminescence (1 · 10^?3) and photoreaction (1 · 10^?1). The temperature dependence of the luminescence quantum yield points to the existence of two or three quenching processes with different activation energies. High degree of luminescence polarization (about 45–47%) throughout the absorption and fluorescence spectra and its temperature independence show that there is no energy transfer between bacteriorhodopsin molecules and no chromophore rotation during the excitation lifetime. In carotenoid-containing membranes, energy migration from the bulk of carotenoids to bacteriorhodopsin was not found either. Bacteriorhodopsin phosphorescence was not observed in the 500–1100 nm region and the emission is believed to be fluorescence by nature.     

Fluorescence properties of the envelope membranes from spinach chloroplasts. Detection of protochlorophyllide

At 77 K, under excitation at 440 nm, two major fluorescence emission peaks were observed in envelope membranes from spinach chloroplasts at 636 and 680 nm. A narrow range of wavelengths around 440 nm and a wider range of wavelengths between 390 and 440 nm, respectively, were responsible for excitation of the 636 and 680 nm fluorescence emissions which, in marked contrast with thylakoid fluorescence emission, were devoid of any exciting components between 460 and 500 nm. In acetonic extract of envelope membranes, two fluorescence emission peaks were observed at 635 and 675 nm. After extraction of the acetonic solution by nonpolar solvents (petroleum ether or hexane), the 675 nm fluorescence emission was partitioned between the polar and nonpolar phases whereas the 635 nm fluorescence emission was solely recovered in the polar phase. All together, the results obtained suggest that envelope membranes contain low amounts of pigments having the absorption and fluorescence spectroscopic properties, together with the behavior in polar/nonpolar solvents, of protochlorophyllide and chlorophyllide. In addition, modulation of the level of fluorescence at 636 and 680 nm could be obtained by addition of NADPH to envelope membranes under illumination. The presence of protochlorophyllide in chloroplast envelope membranes together with its possible photoconversion into chlorophyllide could have major implication for the understanding of chlorophyll biosynthesis in mature chloroplasts.     

Influence of temperature,ethylene and cyanide on the occurrence of alternative respiration in mitochondria from iris bulbs

Mitochondria, isolated from iris (Iris hollandica cv. Ideal) bulbs that have been treated for early flowering with high temperatures (14 days at 35°C followed by 3 days at 40°C) or with ethylene (10–500 ppm), show an induction of alternative respiratory capacity and a rise in state III respiration. Mitochondria from untreated bulbs (stored at 30°C) do not have an alternative pathway capacity and state III respiration is low. Induction of the alternative respiration by ethylene is maximal after 24 h, while induction by high temperature (> 36°C) is much slower. In the temperature range from 36–40°C, the extent of the induced alternative respiratory capacity increases with higher temperatures. A temperature of 42°C is lethal within 5 days. Bulbs stored at 30°C and 35°C before 40°C treatment reach the same values for alternative respiratory capacity. A treatment of the bulbs with 2.2 mM HCN (30°C) leads to an induction of alternative respiration concomitant with a decrease in state III respiration, after a lag time of 2–3 days. A treatment of 5 days with 2.2 mM HCN or longer is lethal.     

A demonstration of energy transfer from photosystem II to photosystem I in chloroplasts

Photosystem I activity of Tris-washed chloroplasts was measured at room temperature as the rate of photoreduction of NADP and as the rate of oxygen uptake mediated by methyl viologen in both cases using dichlorophenolindophenol plus ascorbate as the source of electrons for Photosystem I. With both assay systems the rate of electron transport by Photosystem I was stimulated approx. 20 % by the addition of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea which caused the Photosystem II reaction centers to close. Photosystem I activity of chloroplasts was measured at low temperature as the rate of photooxidation of P-700. Chloroplasts suspended in the presence of hydroxylamine and 3-(3,4-dichlorophenyl)-1, 1-dimethylurea were frozen to ?196 °C after adaptation to darkness or after a preillumination at room temperature. The Photosystem II reaction centers of the frozen dark-adapted sample were all open; those of the preilluminated sample were all closed. The rate of photooxidation of P-700 at ?196 °C with the preilluminated sample was approx. 25 % faster than with the dark-adapted sample. We conclude from both the room temperature and the low temperature experiments that there is greater energy transfer from Photosystem II to Photosystem I when the Photosystem II reaction centers are closed and that these results are a direct demonstration of spillover.     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37°C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20°C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20°C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freezethaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Temperature-sensitivity of D1 protein metabolism in isolated Zea mays chloroplasts

The effects of low temperature on the synthesis and stability of the 32 kDa D1 protein of photosystem II were investigated in chloroplasts isolated from maize (Zea mays cv. LG11) leaves. The synthesis of D1 by intact chloroplasts in vitro was strongly dependent on temperature; the Q₁₀ for the initial rate of incorporation of [³⁵S]-methionine into D1 was ca. 2.6 over the range 13–25°C. The synthesis of other thylakoid polypeptides exhibited a similar temperature dependence, whilst synthesis of stromal proteins was considerably less temperature-dependent, with the exception of two polypeptides of ca. 56 and 59.5 kDa. The stability of newly-synthesized D1 in the thylakoid membranes was dependent both on the temperature at which the plants were grown and on the temperature during the pulse-labelling period when the protein was synthesized. In chloroplasts isolated from maize leaves grown at 25°C, D1 that was synthesized and assembled at 25 °C in vitro was rapidly degraded during the chase period. At lower chase temperatures the protein was more stable. When chloroplasts from 25°C-grown leaves were pulse-labelled at 13°C, the stability of D1 was markedly enhanced at all temperatures during the chase period. This effect was even more pronounced in chloroplasts isolated from plants grown at 14°C. The implications of these results are discussed with regard to the ability of maize to recover from photoinhibitory damage at low temperatures.     

Temperature-sensitive formation of chloroplast protrusions and stromules in mesophyll cells of Arabidopsis thaliana

Summary. In leaf mesophyll cells of transgenic Arabidopsis thaliana plants expressing GFP in the chloroplast, stromules (stroma-filled tubules) with a length of up to 20 μm and a diameter of about 400–600 nm are observed in cells with spaces between the chloroplasts. They appear extremely dynamic, occasionally branched or polymorphic. In order to investigate the effect of temperature on chloroplasts, we have constructed a special temperature-controlled chamber for usage with a light microscope (LM-TCC). This LM-TCC enables presetting of the temperature for investigation directly at the microscope stage with an accuracy of ±0.1 °C in a temperature range of 0 °C to +60 °C. With the LM-TCC a temperature-dependent appearance of chloroplast protrusions has been found. These structures have a considerably smaller length-to-diameter ratio than typical stromules and reach a length of 3–5 μm. At 5–15 °C (low temperatures), almost no chloroplast protrusions are observed, but they appear with increasing temperatures. At 35–45 °C (high temperatures), numerous chloroplast protrusions with a beaklike appearance extend from a single chloroplast. Interaction of stromules with other organelles has also been investigated by transmission electron microscopy. At 20 °C, transverse sections of stromules are frequently observed with a diameter of about 450 nm. A close membrane-to-membrane contact of stromules with the nucleus and mitochondria has been visualised. Golgi stacks and microbodies are found in the spatial vicinity of stromules. At 5 °C, virtually no chloroplast protrusions or stromules are observed. At 35 °C, chloroplast protrusions are present as broader thylakoid-free stroma-filled areas, resulting in an irregular chloroplast appearance. Correspondence and reprints: Department of Physiology and Cell Physiology of Alpine Plants, Institute of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck, Austria.     

Electric surface charge dynamics of chloroplast thylakoid membranes. Temperature dependence of electrokinetic potential and aminoacridine interaction

Electric surface charge dynamics of unstacked broken chloroplasts at low-ionic strength were studied by free-flow electrophoresis and aminoacridine fluorescence and binding changes over the temperature range 4–36°C. Both illumination and ATP hydrolysis in the dark cause a significant increase of net negative surface charge. The light and dark electrokinetic (ζ) potentials have a broad temperature optimum between 20 and 36°C. The decline at lower temperature shows a transition at about 18°C. The ATP-induced increase of the ζ potential requires preactivation of the ATPase and is dicyclohexylcarbodiimide sensitive. Aminoacridine binding shows a quite different temperature dependence. At lower temperatures there is an increased number of binding sites with a decreased affinity and the binding becomes positively cooperative. It is demonstrated that aminoacridines aggregate to dimers upon binding to the membranes. This phenomenon is stimulated by light and favoured at lower temperatures. The light-dependent extra binding increases sigmoidally with increasing temperature, similar to the increase of ζ potential, but with a less abrupt transition. The different effects of temperature on the electrokinetic and binding data are explained in terms of surface charge screening in the electric double-layer of the thylakoid membrane.     

Flash-induced absorption changes of the primary donor of photosystem II at 820 nm in chloroplasts inhibited by low pH or tris-treatment

A comparative study is made, at 15 °C, of flash-induced absorption changes around 820 nm (attributed to the primary donors of Photosystems I and II) and 705 nm (Photosystem I only), in normal chloroplasts and in chloroplasts where O₂ evolution was inhibited by low pH or by Tris-treatment.At pH 7.5, with untreated chloroplasts, the absorption changes around 820 nm are shown to be due to P-700 alone. Any contribution of the primary donor of Photosystem II should be in times shorter than 60 μs.When chloroplasts are inhibited at the donor side of Photosystem II by low pH, an additional absorption change at 820 nm appears with an amplitude which, at pH 4.0, is slightly higher than the signal due to oxidized P-700. This additional signal is attributed to the primary donor of Photosystem II. It decays ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 180 μs) mainly by back reaction with the primary acceptor and partly by reduction by another electron donor. Acid-washed chloroplasts resuspended at pH 7.5 still present the signal due to Photosystem II ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 120 μs). This shows that the acid inhibition of the first secondary donor of Photosystem II is irreversible.In Tris-treated chloroplasts, absorption changes at 820 nm due to the primary donor of Photosystem II are also observed, but to a lesser extent and only after some charge accumulation at the donor side. They decay with a half-time of 120 μs.     

Dominance of a 675?nm chlorophyll(ide) form upon selective 632.8 or 654?nm laser illumination after partial protochlorophyllide phototransformation

The phototransformation pathways of protochlorophyllide forms were studied in 8?C14-day-old leaves of dark-germinated wheat (Triticum aestivum L.) using white, 632.8?nm He?CNe laser and 654?nm laser diode light. The photon flux density (PFD) values (0.75?C360???mol photons?m^?2?s^?1), the illumination periods (20?ms?C10?s) and the temperature of the leaves (between ?60?°C and room temperature) were varied. The 77?K fluorescence spectra of partially phototransformed leaves showed gradual accumulation or even the dominance of the 675?nm emitting chlorophyllide or chlorophyll form at room temperature with 632.8?nm of PFD less than 200???mol photons?m^?2?s^?1 or with 654?nm of low PFD (7.5???mol photons?m^?2?s^?1) up to 1?s. Longer wavelength (685 or 690?nm) emitting chlorophyllide forms appeared at illuminations under ?25?°C with both laser lights or at room temperature when the PFD values were higher or the illumination period was longer than above. We concluded that the formation of the 675?nm emitting chlorophyllide form does not indicate the direct photoactivity of the 633?nm emitting protochlorophyllide form; it can derive from 644 and 657?nm forms via instantaneous disaggregation of the newly-produced chlorophyllide complexes. The disaggregation is strongly influenced by the molecular environment and the localization of the complex.     

Spectral characterization of a fluorescent nicotinamide adenine dinucleotide analog: 3-Aminopyridine adenine dinucleotide

Both absorption and fluorescence properties of 3-amino pyridine adenine dinucleotide (AAD) were examined. The shape of the AAD fluorescence emission spectrum, maximal at 425 nm, remains unchanged over the pH range 2 to 11, indicating that there is only one detectable emitting species. AAD fluorescence increases as the pH decreases, with an apparent pK_a of about 3.5. The absorption-pH profile indicates a pK_a of about 3.3 for the ground state of AAD. Effects of organic solvents on AAD fluorescence are somewhat diverse. The low fluorescence quantum yield of 0.022 corresponds well with the short lifetime of 1.15 ns at 23 °C in neutral aqueous solution. The steady-state polarization of AAD in water and that at infinite viscosity were determined at 23 °C to be 0.037 and 0.083, respectively. Since a smaller value of polarization for either donor or acceptor leads to a better estimate of the orientation factor for dipole-dipole interaction, AAD appears to be particularly suitable for energy transfer studies. Similar to NADH, AAD also assumes a folded conformation in aqueous solution. This is evident from effects of temperature and hydrolysis by phosphodiesterase on absorption and/or fluorescence properties of AAD. Energy transfer from the adenine group to the 3-aminopyridine ring has been detected to occur in aqueous solution at 23 °C with an efficiency of about 0.12, corresponding to a distance of 7.5 Å between these two ring moieties.     

Trapping at low temperature of oriented chloroplasts: Application to the study of antenna pigments and of the trap of photosystem-1

A technique is described for the preparation of oriented samples from spinach chloroplasts whose linear dichroism is then studied by (flash) absorption spectroscopy. The chloroplasts are suspended in a glycerol-containing medium, oriented in a magnetic field, and slowly cooled in the magnet until the medium is rigid enough to avoid disorientation effects. The absorption spectra in polarized light have been measured at ?50° and ?170°C. They allow the orientation of chlorophyll b to be resolved, and the red transition moment is found to be tilted out of the membrane plane. A study of the flash-induced absorption changes linked to Photosystem-1 activity reveals a progressive evolution of the difference spectra and of the linear dichroism with decreasing temperatures. At ?170°C, the difference spectrum of P700 in the red is well resolved. All transition moments are found to be largely parallel to the membrane plane. The potential use of the technique for other experiments by differential absorption spectroscopy and by EPR techniques is discussed.     

Light-induced absorbance changes due to Photosystems 1 and 2 in spinach chloroplasts at −50 °C

Absorbance changes in the region 500–565 nm and at 702 nm, brought about by excitation of Photosystems 1 and 2, respectively, were measured in spinach chloroplasts at ?50 °C. Either dark-adapted chloroplasts were used or chloroplasts preilluminated with a number of short saturating flashes just before cooling.Both photosystems were found to cause a light-induced increase of absorbance at 518 nm (due to “P518”). The System 1-induced change was not affected by preillumination. It decayed within 1 s in the dark and showed similar kinetics as P700. Experiments in the presence of external electron acceptors (methylviologen or Fe(CN)₆^3?) suggested that P518 was not affected by the redox state of the primary electron acceptor of System 1. The absorbance increase at 518 nm due to System 2 decayed in the dark with a half-time of several min. The kinetics were similar to those of C-550, the presumed indicator of the primary electron acceptor of System 2. After two flashes preillumination the changes due to P518 and C-550 were reduced by about 40%, and a relatively slow, System 2-induced oxidation of cytochrome b₅₅₉ occurred which proceeded at a similar rate as the increase in yield of chlorophyll a fluorescence. The results indicate that at ?50°C two different photoreactions of System 2 occur. One consists of a photoreduction of the primary electron acceptor associated with C-550, accompanied by the oxidation of an unknown electron donor; the other is less efficient and results in the photooxidation of cytochrome b₅₅₉.     

Analyses of absorption and fluorescence spectra of water-soluble chlorophyll proteins,pigment System II particles and chlorophyll a in diethylether solution by the curve-fitting method

Absorption and fluorescence spectra in the red region of water-soluble chlorophyll proteins, Lepidium CP661, CP663 and Brassica CP673, pigment System II particles of spinach chloroplasts and chlorophyll a in diethylether solution at 25°C were analyzed by the curve-fitting method (French, C.S., Brown, J.S. and Lawrence, M.C. (1972) Plant Physiol. 49, 421–429). It was found that each of the chlorophyll forms of the chlorophyll proteins and the pigment System II particles had a corresponding fluorescence band with the Stokes shift ranging from 0.6 to 4.0 nm.The absorption spectrum of chlorophyll a in diethylether solution was analyzed to one major band with a peak at 660.5 nm and some minor bands, while the fluorescence spectrum was analyzed to one major band with a peak at 664.9 nm and some minor bands. A mirror image was clearly demonstrated between the resolved spectra of absorption and fluorescence. The absorption spectrum of Lepidium CP661 was composed of a chlorophyll b form with a peak at 652.8 nm and two chlorophyll a forms with peaks at 662.6 and 671.9 nm. The fluorescence spectrum was analyzed to five component bands. Three of them with peaks at 654.8, 664.6 and 674.6 nm were attributed to emissions of the three chlorophyll forms with the Stokes shift of 2.0–2.7 nm. The absorption spectrum of Brassica CP673 had a chlorophyll b form with a peak at 653.7 nm and four chlorophyll a forms with peaks at 662.7, 671.3, 676.9 and 684.2 nm. The fluorescence spectrum was resolved into seven component bands. Four of them with peaks at 666.7, 673.1, 677.5 and 686.2 nm corresponded to the four chlorophyll a forms with the Stokes shift of 0.6–4.0 nm. The absorption spectrum of the pigment System II particles had a chlorophyll b form with a peak at 652.4 nm and three chlorophyll a forms with peaks at 662.9, 672.1 and 681.6 nm. The fluorescence spectrum was analyzed to four major component bands with peaks at 674.1, 682.8, 692.0 and 706.7 nm and some minor bands. The former two bands corresponded to the chlorophyll a forms with peaks at 672.1 and 681.6 nm with the Stokes shift of 2.0 and 1.2 nm, respectively.Absorption spectra at 25°C and at ?196°C of the water-soluble chlorophyll proteins were compared by the curve-fitting method. The component bands at ?196°C were blue-shifted by 0.8–4.1 nm and narrower in half widths as compared to those at 25°C.     

Observation of two quenchers of chlorophyll fluorescence in chloroplasts at −196°C

Fluorescence induction at ?196°C has been monitored in chloroplasts rapidly frozen after poising at different redox potentials at room temperature. It was found that, as at room temperature, the initial level of fluorescence observed upon shutter opening (F_o), relative to the final level observed after 10 seconds of illumination (F_m) increased as the redox potential of the chloroplasts was lowered. Redox titration revealed the presence of two quenching components with E_m,7.8 at ?70 mV and ?275 mV accounting for approx. 75% and 25% of the variable fluorescence (F_v). Parallel observation of fluorescence yield at room temperature similarly gave two components, with E_m,7.8 at ?95 mV and ?290 mV, also accounting for approx. 75% and 25%. Simultaneous measurement of fluorescence emission at ?196°C at 695 nm and 735 nm indicated that both emissions are quenched by the same redox components.     

Two forms of intermediates of frog rhodopsin in rod outer segments

Using frog rod outer segments, we measured changes of the absorption spectrum during the conversion of rhodopsin to a photosteady-state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin by irradiation with blue light (440 nm) at ? 190°C and during the reversion of bathorhodopsin to a mixture of rhodopsin and isorhodopsin by irradiation with red light (718 nm) at ? 190°C. The reaction kinetics was expressed by one exponential in the former case and by two exponentials in the latter. These results suggest that rhodopsin is composed of a single molecular species, while bathorhodopsin is composed of two kinds of molecular species designated as batho₁-rhodopsin and batho₂-rhodopsin. On warming the two forms of bathorhodopsin, each bathorhodopsin converted to its own lumirhodopsin, metarhodopsin I and finally a free all-trans-retinal plus opsin. The absorption spectra of the two forms of bathorhodopsin, lumirhodopsin and metarhodopsin I were measured at ? 190°C. We infer that a rhodopsin molecule in the excited state relaxes to either batho₁-rhodopsin or batho₂-rhodopsin, and then converts to its own intermediates through one of the two parallel pathways.