ELECTRON AND ENERGY TRANSFER IN ILLUMINATED POROUS VYCOR GLASS

Abstract— Porous Vycor glass samples containing adsorbed molecules were illuminated at 77 K by a mercury lamp jacketed by a filter cutting off wavelengths below 250 nm. Oxygen or carbon dioxide on Vycor produces an asymmetric electron paramagnetic resonance (EPR) signal best described as holes trapped in the glass. Methyl bromide produces an identical EPR signal plus four other lines due to methyl radicals. Evidence is presented that the products result from excitonic energy transfer from the Vycor to the adsorbed materials. Triphenylamine (TPA) adsorbed on Vycor can also be photoionized by similar illumination, and the cation radical TPA⁺ can be stabilized at 77 K if an electron acceptor is also adsorbed. Attachment of the photoejected electron by carbon dioxide forms CO₂^-, and that by methyl bromide leads to methyl radicals. The CH₃ radical yield is dependent on the surface separation between the electron donor (TPA) and the acceptor (CH₃Br). By monitoring the relative quantum yield of the methyl radicals as a function of distance separating the TPA and CH₃Br, it is shown that the photoelectron is capable of migrating on the Vycor glass surface.     

THE ELECTROPHOTOLUMINESCENCE OF CHLOROPLASTS

Abstract— Delayed light emission emanating from preilluminated chloroplasts can be perturbed with pulsed DC electric fields (200–4000 V cm^-1), The perturbation produces a strong stimulation of chlorophyll luminescence. During the field perturbation the stimulated emission rises to a maximum, typically within 100μs. and then decays. Two kinetic components, R (rapid) and S (slow)†, are distinguished on the basis of their rise and decay times and their field-dependence. The R component increases exponentially at high fields, decays within 100–300μs during the field pulse and collapses with t _1/2= 15 μs at the end of the field pulse. The S component occurs at low fields, exhibits near saturation at 500 V cm^-1, decays with t _1/2 about 3 ms during the field pulse, and collapses with t _1/2= 38μs at the end of the field pulse. Studies using inhibitors, ionophores, electron donors and electron acceptors associate the R component with ion transport processes. The relation to electron transport associated with Photosystem II is discussed.     

MAGNESIUM ION EFFECTS ON CHLOROPLAST PHOTOSYSTEM II FLUORESCENCE AND PHOTOCHEMISTRY

Abstract. –Although there have been several reports that divalent cations, especially Mg²⁺, can significantly affect chloroplast photoprocesses, the molecular mechanism of cation interaction is not well understood. We have investigated the interaction of Mg²⁺with Photosystem II photoprocesses by studying cation effects on chloroplast fluorescence and the Hill reaction. Our results are summarized as follows.
1. Mg²⁺stimulation of background fluorescence (20–30%) saturates at about 0.5 mM Mg²⁺, while Mg²⁺stimulation of variable fluorescence (250%) saturates at about 2.5 mM Mg²⁺.
2. Addition of Mg²⁺to chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea or dithionite causes a doubling in the amount of total (background + variable) fluorescence.
3. Studies on chloroplasts in the presence of 2,6-dichlorophenolindophenol indicate that Mg²doubles the relative yield of variable fluorescence under light-limiting conditions.
4. Mg²causes large (70–120%) increases in the light-limited rate of the DCIP Hill reaction.
We interpret these results in terms of a model involving two components of chloroplast emission. Our analysis indicates that Mg²⁺increases the effective absorption cross section (size) of the pigment array associated with Photosystem II photochemistry.     

TEMPERATURE DEPENDENCE OF DELAYED LIGHT EMISSION IN THE 6 to 340 MICROSECOND RANGE AFTER A SINGLE FLASH IN CHLOROPLASTS

Abstract. The delayed light emission decay rate (up to 120 μs) and the rise in chlorophyll a fluorescence yield (from 3 to 35 μs) in isolated chloroplasts from several species, following a saturating 10 ns flash, are temperature independent in the 0–35°C range. However, delayed light in the 120–340 μs range is temperature dependent. Arrhenius plots of the exponential decay constants are: (a) linear for lettuce and pea chloroplasts but discontinuous for bush bean (12–17°C) and spinach (12–20°C) chloroplasts; (b) unaffected by 3-(3,4 dichlorophenyl)-1,1-dimethylurea (inhibitor of electron flow), gramicidin D (which eliminates light-induced membrane potential) and glutaraldehyde fixation (which stops gross structural changes).
The discontinuities, noted above for bush bean and spinach chloroplasts, are correlated with abrupt changes in (a) the thylakoid membrane lipid fluidity (monitored by EPR spectra of 12 nixtroxide stearate, 12NS) and (b) the fluidity of extracted lipids (monitored by differential calorimetry and EPR spectra of 12 NS). However, no such discontinuity was observed in (a) chlorophyll a fluorescence intensity of thylakoids and (b) fluorescence of tryptophan residues of delipidated chloroplasts.
Microsecond delayed light is linearly dependent on light intensity at flash intensities as low as one quantum per 2 times 10⁴ chlorophyll molecules. We suggest that this delayed light could originate from a one quantum process in agreement with the hypothesis that recombination of primary charges leads to this light emission. A working hypothesis for the energy levels of Photosystem II components is proposed involving a charge stabilization step on the primary acceptor side, which is in a lipid environment.
Finally, the redox potential of P680 (the reaction center for chlorophyll of system II) is calculated to be close to 1.0–1.3 V.     

LASER PHOTOLYSIS STUDIES OF SUPEROXIDE ADDUCTS OF COBALT(II) AND ZINC(II) PORPHYRINS IN DIMETHYLFORMAMIDE

Abstract— Photochemistry of superoxide adducts of cobalt(II) and zinc(II) porphyrins has been studied by laser photolysis. It was found that the former in dimethlformamide photodissociates the superoxide anion radical, O₂^-, with the quantum yield of 0.5 ± 0.05 at the excitation wavelenths 355 and 532 nm, and the latter gives flurescence and the triple state without giving rise to the photodissociation of O₂^-     

PROPERTIES OF THE CHLOROPLAST FILM ELECTRODE IMMOBILIZED ON AN SnO2-COATED GLASS PLATE

Abstract— Type C chloroplasts were deposited on the surface of an SnO₂ optically transparent electrode glass plate with polyvinyl alcohol plus bovine serum albumin as immobilizing supports. This electrode, on illumination of 250 J/m² in an electrolyte solution, generated anodic photo current more than 150 nA per 10 μg chlorophyll/cm² of the SnO₂ glass plate at a potentiostatic condition of + 0.5 V against a saturated calomel electrode, and gave rise to an open circuit potential up to 300 mV. The photocurrent output was enhanced as high as 60-fold under the short circuit condition by the addition of an artificial electron carrier, l-methoxy-5-methylphenazinium methyl sulfate, to the electrolyte solution. With the electrode poised at +0.5 V against a saturated calomel electrode, the enhancement effect was exhibited as high as 13-fold in the presence of 2,6-dichlorophenol indophenol. A photocurrent spectrum coincides well with an absorption spectrum of the chloroplast film electrode. Effects of heat-treatment, photosynthetic inhibitors, and electrolyte's pH on the magnitude of the photocurrent were studied in detail. Water molecule, a primary electron donor in the chloroplast photosystems, contributes to the large majority of photocurrent generation. A minor output was observed with the electrode coated with completely inactivated chloroplasts, probably due to the chlorophyll photosensitization.     

PHOTOSYNTHETIC NITRITE REDUCTION BY DITHIOERYTHRITOL AND THE EFFECT OF NITRITE ON ELECTRON TRANSPORT IN ISOLATED CHLOROPLASTS

Abstract. Photosynthetic reduction of nitrite to ammonia with type C chloroplasts from the heterocont alga Bumilleriopsis filiformis was investigated using 3,6-diaminodurene/ascorbate and 3,6-diaminodurene/dithioerythritol (DAD/DTE) as electron donor couple. Rates approach 6–10 μmol NO^-₂ reduced/mg chlorophyll/h and are steady for up to 30 min. The presence of oxygen or NADP⁺ only slightly diminished the rates of nitrite reduction obtained with DAD/DTE. Illuminated chloroplasts reduce oxygen in the presence of DAD/DTE at 135 μmol/mg chlorophyll/h without acceptor supplied. Photosynthetic oxygen uptake by this system in the presence of ferredoxin and NO^-₂, however, is inhibited to 42% by nitrite reductase with concurrent nitrite reduction. NO^-₃ and NO^-₂ have no effect on photosystem I-mediated NADP⁺ reduction, NO^-₂ (10 m M ) inhibits ferricyanide-mediated oxygen evolution to 72%. Also photosystem II reactions assayed e.g. with silicomolybdate are inhibited significantly by NO^-₂ (1 m M ), but only slightly by NO^-₃. Nitrite reductase is inhibited by p -chloromercuribenzoate ( p CMB), and this inhibition is prevented by DTE. Results suggest that photosynthetic nitrite reduction can cope with low concentrations of either compound, provided relevant thiol groups are protected.     

DYE-SENSITIZED PHOTOREDUCTION OF METHYL VIOLOGEN

Abstract— The kinetics of the proflavine-sensitized photoreduction of methyl viologen (MV²⁺) to the blue radical cation (MV^.+) are presented. The triplet excited state of proflavine accepts an electron from EDTA to form the singly-reduced species of proflavine (PH^.); this species donates an electron to either the oxidized (MV^.+) or the singly–reduced (MV^.+) species of the bipyridyl. MV²⁺ can be reoxidized by oxygen to MV²⁺ but is decomposed by H₂O₂. The doubly-reduced form can not be reoxidized either by oxygen or by peroxide. Potassium iodide inhibits the photoreduction of MV²⁺ by competing with it as reactant for the singly-reduced form of proflavine (PH^.). The mechanism presented may be analogous to that occurring in the reduction of MV²⁺ by illuminated spinach chloroplasts; its herbicidal action can not be ascribed to the formation of peroxides.     

A SINGLE PULSE PICOSECOND LASER STUDY OF EXCITON DYNAMICS IN CHLOROPLASTS

Abstract. Using single picosecond laser pulses at 610 nm, the fluorescence yield (φ) of spinach chloroplasts as a function of intensity ( I ) (10¹²-10¹⁶ photons/pulse/cm²) was studied in the range of 21–300 K. The quantum yield decreases with increasing intensity and the φ vs I curves are identical at the emission maxima of 685 and 735 nm. This result is interpreted in terms of singlet exciton-exciton annihilation on the level of the light-harvesting pigments which occurs before energy is transferred to the Photosystem I pigments which emit at 735 nm.
The yield φ is decreased by factors of 12 and 43 at 300 and 21 K, respectively. The shapes of the φ vs I curves are not well accounted for in terms of a model which is based on a Poisson distribution of photon hits in separate photosynthetic units, but can be satisfactorily described using a one-parameter fit and an exciton-exciton annihilation model. The bimolecular annihilation rate constant is found to be γ= (5–15) times 10^-9cm³s^-1 and to exhibit only a minor temperature dependence. Lower bound values of the singlet exciton diffusion coefficient (≥ 10^-3cm²s^-1), diffusion length (≥ 2 times 10^-6cm) and Förster energy transfer rates (≥ 3 ≥ 10¹⁰s^-1) are estimated from γ using the appropriate theoretical relationships.     

INTENSITY AND TIME-DEPENDENCE OF THE CAROTENOID TRIPLET QUENCHING UNDER LIGHT FLASHES OF RECTANGULAR SHAPE IN CHLORELLA

Abstract— As is known the chlorophyll fluorescence of photosynthetizing plants is strongly quenched by carotenoid triplet states if the exciting light intensity is high enough (> 10 kW/m²). This light-induced quenching process was studied by measuring the relative yield of chlorophyll fluoresccncc excited with a pulsed argon laser at 20 C in light adapted algae as function of time (within lo μ s ) and exciting light intensity (<400 kW/m²). The experimental yield against time and yield against intensity curves have been adequately explained by a statistical model of Photosystem 2 (PS 2) units based upon the existence of freely moving excitons according to which the carotenoid triplet quenchers are randomly distributed and are perfect traps for excitons. Accepting the hypothesis that carotenoid triplet quenching occurs only in PS 2 units. it could be concluded that the height of the true zero level of PS 2 fluorescence is somewhat lower than the half value of the fluorescence level of the dark adapted state.     

SPINACH FERREDOXIN REDUCTION BY MEMBRANE-BOUND CHLOROPHYLL TRIPLET STATE VIA A DIQUAT MEDIATOR

Abstract Laser flash photolysis experiments have shown that the diquat analog containing a propylene bridge (PDQ²⁺), when electrostatically bound to negatively-charged vesicles containing chlorophyll, is able to mediate the rapid reduction ( k = 1.1 × 10⁵ s^-1) of spinach ferredoxin via electron transfer quenching of triplet state chlorophyll. The kinetics of formation and decay of reduced ferredoxin are consistent with a mechanism involving complex formation between oxidized ferredoxin and vesicle-bound PDQ²⁺. Under optimal conditions, approximately 15% of the quenched triplets yield reduced ferredoxin. This process is a model for soluble ferredoxin reduction which occurs in green plant photosystem I, and results in an appreciable storage of electromagnetic energy in the reaction products.     

Bimanes 28: Extraction of sulfur from fluorescent models for biological thiols

Sulfur extraction from the tripeptide thiol, glutathione (α-glu-cys-gly) ( 1 ) via reaction with syn-(1-bromo-ethyl, methyl)bimane ( 2 ) yields glutathione slfide and the thiabridged bimane, μ(S)-syn-(methylmethylene, methyl)bimane ( 3 ) [1]. The reaction with 2 has been extended to dithiols as models for important biological thiols such as reduced trypanothione. The fluorescent dithiols were derived through reductive cleavage with triphenylphosphine (tetrahydrofuan, Hcl−Kbr solution, pH 1.5) of the dithiatriclic bimane esters, μ(O₂C(CH₂)SS(CH₂)_nCO₂)-syn-(CH₂,CH₃)B, n = 1, 2, 3, prepared from syn-(bromomethyl, methyl)bimane ( 4 ) and the corresponding dithiadicarboxylic acids. Sulfur extraction led to 3 and the cyclic sulfide derived from the dithiol in moderate yields. The dithiols, dithiothreitol and dithioerythritol, also yielded moderate amounts of 3 . Sterically hindered thiols (e.g., those in hemoglobin) gave 3 in lower yields. Treatment of human red blood cells and red cells membranes (hemoglobin-free ghosts) with 2 gave rise to some 3 . A side product in some reactions was the oxabridged bimane, μ(O)-syn-(methylmethylene, methyl)bimane.     

ALTERNATIVE MEASURES OF PHOTOSYSTEM II ELECTRON TRANSFER INHIBITION IN ANTHRAQUINONE-TREATED CHLOROPLASTS

We have previously used chlorophyll fluorescence measurements at Fmax conditions (i.e. with Photosystem II electron acceptor QA reduced) to monitor the action of 9,10-anthraquinones on photosynthetic electron transport in plant chloroplasts. The present investigation employs two additional techniques to characterize the extent of electron transport inhibition induced by the addition of substituted anthraquinones to the suspending medium of spinach chloroplasts. Results are presented for spectrophotometric assays of the rate of electron transfer to an exogenous electron acceptor, 2,6-dichloroindophenol (DCIP) and for electrochemical determinations of the rate of oxygen evolution in anthraquinone-treated chloroplasts. In general, amino-substituted anthraquinones are ineffective inhibitors, maintaining electron transfer rates to DCIP at levels ranging from 50 to 90% of normal rates and yielding rates of O2 evolution averaging at 70% of the rate in untreated chloroplasts. In contrast, hydroxy-substituted anthraquinones efficiently block Photosystem II electron transport, resulting in low rates of DCIP photoreduction ranging from 0 to 20% of normal values and reducing O2 evolution rates to an average of 30% of the rate observed for untreated chloroplasts. Relative rates of DCIP photoreduction for anthraquinone-treated chloroplasts show a strong linear correlation with the reported relative Fmax chlorophyll fluorescence intensities. Relative O2 evolution rates are observed to correlate with the Stern-Volmer fluorescence quenching parameter Ksv. We suggest that slight differences in the extent of inhibitory activity of an anthraquinone as measured by the three techniques are consistent with certain known Photosystem II heterogeneities. The similarities in relative rankings of inhibitory effects for the 9, 10-anthraquinones, however, demonstrate that the three techniques employed (measurements of Fmax chlorophyll fluorescence, DCIP photoreduction rates, and O2 evolution rates) are alternative assays of anthraquinone-induced Photosystem II electron transport inhibition.     

AN FMN-PHOTOSYSTEM I PHOTOVOLTAIC CELL

Abstract— We have made a photovoltaic cell using Photosystem I subchloroplast particles isolated according to the method of Shiozawa et al. (1974). The particles were placed on a filter between two compartments one of which contained the electron donor, K₄Fe(CN)₆ and the other the electron acceptor, FMN. Upon illumination with white light ( I = 80 W/m²) a potential of 300 mV was observed across a 3000 Ω load resistance. Both Photosystem I photochemistry and direct photoreactions of FMN contribute to the process. A power output of 20 μW was observed for a 2 cm² filter containing 60 μg chlorophyll. This corresponds to 0.1 W/m². The power efficiency was 0.13%. The short circuit current was 108 μA.     

THE EFFECT OF LEVULINIC ACID ON THE LIGHT INDUCED DEVELOPMENT OF PHOTOSYSTEM I AND II ACTIVITIES IN GREENING MAIZE LEAVES*

Photosystem I and Photosystem II activities were measured in chloroplasts isolated after 0–20 h illumination from etiolated maize leaves in which chlorophyll synthesis was specifically inhibited by levulinic acid. In control leaves not treated with levulinic acid, Photosystem I activity/chlorophyll developed rapidly during the first 2h in light, then fell off, and reached a constant level after 6h of illumination. In levulinic acid treated leaves, in which chlorophyll accumulation was inhibited up to 60%, a similar initial rise in Photosystem I activity was observed. However, the decrease in activity was much slower and continued for at least 20 h. The development of Photosystem I activity calculated on a leaf fresh weight basis was similar for control leaves or leaves treated with levulinic acid. This indicates that development of Photosystem I activity may not be related to chlorophyll accumulation during greening. Photosystem II activity/chlorophyll in leaves treated with or without levulinic acid increased similarly during the first 6h and then remained constant. Activity of Photosystem II per leaf fresh weight increased linearly, after the first h, for 20 h in the control leaves; in levulinic acid treated leaves this development was reduced by about 60%. Thus, development of Photosystem II activity can be related to chlorophyll accumulation. SDS gel electrophoresis of plastid membranes from control leaves illuminated for 12 h showed the presence of chlorophyll-protein complex I as well as Chl-protein 11; in the case of levulinic acid treated leaves only Chl-protein complex I was detectable, while Chl-protein complex II was markedly reduced.     

STUDIES ON SOME INTERMEDIATES AND PRODUCTS OF THE PHOTOREDUCTION OF 9,10-ANTHRAQUINONE *,†

Abstract— The mechanism of the photoreduction of 9,10-anthraquinone (AQ) in alcohol and hexane has been studied by flash photolysis. The fluorescence spectrum of the photoproduct, 9,10-dihydroxy anthracene shows a large shift between hexane and ethanol. The quantum yields of photoreduction for AQ are solvent-dependent, the reaction between the solvent radical and AQ determining the quantum yield.
The absorption spectrum of the 9,10-anthrasemiquinone (AQH.) has a long-wavelength absorption band with peaks at 631 and 678 nm. The second-order decay constants for AQH. were estimated to be 1.3 × 10⁹, 6.7 × 10⁸ and 2.0 × 10⁸ M ^-1 sec^-1 in ethanol, 2-propanol and ethylene glycol, respectively.
A long-wavelength absorption band was observed for 9,10-anthrasemiquinone radical anion, having peaks at 776 and 860 nm; epsi;_max= 1900 at 776 nm. This spectrum is compared with the spectra of 9,10-dihydroxy anthracene mono- and di-anions. The 9,10-anthrasemiquinone radical anion was found to photoreduce quantitatively to 9,10-dihydroxy anthracene mono-anion with a quantum yield of 0.1.     

THE LIGHT REQUIREMENT OF ACID-BASE TRANSITION INDUCED LUMINESCENCE OF CHLOROPLASTS

Abstract— The luminescence that occurs when chloroplasts are taken from an acid environment to a basic one is shown to be dependent on prior illumination of the chloroplasts. The relation between the light absorbed and luminescence is given by the following equation L = L _max(1-e ^al where L and L _max are the light emitted and maximum light emission at high flash energy, respectively, J quanta absorbed per chlorlphyll molecule, and α a constant with a value of approximately 200 chlorophyll molecules per quanta absorbed. The action spectrum of the luminescence is consistent with that of photosystem II. The metastable state formed during illumination decays in the dark via a temperature dependent second order process.     

RIBOFLAVIN SENSITIZED PHOTOREDUCTION OF NITRO BLUE TETRAZOLIUM ION (NBT2+) IN DEGASSED AQUEOUS SOLUTION

Abstract— When sensitized by riboflavin, the reduction of NBT²⁺ (nitro blue tetrazolium ion) takes place in both aerated and degassed aqueous solution. Laser photolysis experiments revealed that the triplet riboflavin reduces NBT²⁺ to nitro blue tetrazolinyl radical, NBT²⁺. and it disappears according to pseudo-first order kinetics, with a bimolecular rate constant of (4.2 ± 0.2) X 10⁸ M ^-1s^-1. It is shown that NBT²⁺ is not always a good detecting reagent for the superoxide anion, when the formation of the anion is mediated by riboflavin.     

INTERFACIAL ELECTRON TRANSFER INVOLVING RADICAL IONS OF CAROTENE AND DIPHENYLHEXATRIENE IN MICELLES AND VESICLES

Abstract— The radical cations and anions of diphenylhexatriene have been produced and characterized in homogenous and micellar solutions by pulse radiolysis and laser flash photolysis techniques. Both types of radical ions were formed in cyclohexane on pulse radiolysis. The radical cation was formed in dichloroethane on pulse radiolysis, and by two photon photoionization in ethanol, dichloroethane, and various micelles. Both radical ions have intense ( 10⁵ M ^-1 cm^-1) absorption peaks at600–650nm. The cation peak occurs at slightly shorter wavelengths than that of the anion.
In micelles and vesicles the radical anion of carotene was formed by electron transfer from e_a– on pulse radiolysis. The radical cation was formed on pulse radiolysis of micellar solutions containing Br^-₂ as counterion, presumably by electron transfer to Br₂^-. The spectra agree with those of the radical cation and anion of carotene that have previously been obtained in homogenous solutions (Dawe and Land, 1975).
Electron transfer in micelles and vesicles from the radical anion of biphenyl to carotene and diphenylhexatriene, and from the radical anions of these to inorganic acceptors has been studied.     

LUMIFLAVIN-SENSITIZED PHOTOOXYGENATION OF INDOLE

Abstract— The lumiflavin-sensitized photooxygenation of indole in aqueous solutions has been investigated by means of steady light photolysis and flash photolysis. The semiquinone of lumiflavin and the half-oxidized radical of indole were formed by the reaction between triplet lumiflavin and indole (3.7 times 10⁹ M ^-1 s^-1). The semiquinone anion radical of lumiflavin reacted with oxygen to form superoxide radical. The triplet state of lumiflavin also reacted with oxygen forming singlet oxygen, ¹O₂. But the reaction between ¹O₂ and indole (7 times 10⁷ M^_l s^_1; estimated from steady light photolysis using Rose Bengal as a sensitizer) was far less efficient than the reaction between indole and triplet lumiflavin. The quantum yield of the lumiflavin-sensitized photooxygenation of dilute indole via radical processes was much higher than that via ¹O₂ processes, though appreciable ¹O₂ was formed.