QUANTITATION OF PHOTOSYSTEM II ACTIVITY IN SPINACH CHLOROPLASTS. EFFECT OF ARTIFICIAL QUINONE ACCEPTORS

Quantitation of photosystem II (PSII) activity in spinach chloroplasts is presented. Rates of PSII electron-transport were estimated from the concentration of PSII reaction-centers (Chl/PSII = 380:1 when measured spectrophotometrically in the ultraviolet [ΔA₃₂₀] and green [ΔA_540–550] regions of the spectrum) and from the rate of light utilization by PSII under limiting excitation conditions. Rates of PSII electron-transport were measured under the same light-limiting conditions using 2,5-dimethylbenzoquinone or 2,5-dichlorobenzoquinone as the PSII artificial electron acceptors. Evaluation is presented on the limitations imposed in the measurement of PSII electron flow to artificial quinones in chloroplasts. Limitations include the static quenching of excitation energy in the pigment bed by added quinones, the fraction of PSII centers (PSII_β) with low affinity to native and added quinones, and the loss of reducing equivalents to molecular oxygen. Such artifacts lowered the yield of steady-state electron transport in isolated chloroplasts and caused underestimation of PSII electron-transport capacity. The limitations described could explain the low PSII concentration estimates in higher plant chloroplasts (Chl/PSII = 600 ± 50) resulting from proton flash yield and/or oxygen flash-yield measurements. It is implied that quantitation of PSII by repetitive flash-yield methods requires assessment of the slow turnover of electrons by PSII_β and, in the presence of added quinones, assessment of the PSII quantum yield.     

AFFINITY FOR OXYGEN IN PHOTOREDUCTION OF MOLECULAR OXYGEN AND SCAVENGING OF HYDROGEN PEROXIDE IN SPINACH CHLOROPLASTS

Abstract— The apparent K _m for O₂ in the photoreduction of molecular oxygen by spinach class II chloroplasts and photosystem I subchloroplast fragments was determined. In both cases, a value of 2 ∼ 3 μ M O₂ was obtained. The reaction rate constant between O₂ and P-430, the primary electron acceptor of PS I, is estimated to be ∼ 1.5 × 10⁷ M ^-1 s^-1 and the factors affecting the production of superoxide by the photoreduction of O₂ in chloroplasts are discussed. Preliminary evidence is presented indicating the occurrence of an azide-insensitive scavenging system for H₂O₂ in chloroplast stroma.     

THE 518-mμ ABSORBANCE CHANGE AND ITS RELATION TO THE PHOTOSYNTHETIC PROCESS

Abstract— A study of the 518-mμ light-induced absorbance change in green cells and a comparison with photosynthetic O₂ evolution were made. The effect of various chemical agents was also investigated. On the one hand, no antagonistic two-light effect was observed on the absorbance change, and DCMU had only a partial inhibitory effect on it. On the other hand, it was possible to observe in some cases an indirect kinetic relationship between O₂ evolution and 518-mμ change. It is suggested that probably at least two substances absorb around 518-mμ, one ( X _I) belonging to system I and another ( X _II) to system II. The existence and the function of X _I are hypothetical and the identity of X _II with the first photoproduct of system II is yet not well established.     

EFFECT OF AGGREGATION ON THE HEMATOPORPHYRIN-SENSITIZED PRODUCTION OF SINGLET MOLECULAR OXYGEN

Abstract— The hematoporphyrin-sensitized production of singlet molecular oxygen, O₂(¹Δ_g), has been investigated in methanol and in aqueous solution. The quantum yield for formation of O₂(¹Δ_g) (Φ_Δ) has been measured by both steady-state (oxygen consumption) and time-resolved (near-infrared luminescence) methods. In methanol, both techniques indicate that Φ_Δ= 0.76 and the value remains independent of sensitizer concentration over a wide range. This finding is consistent with the dye persisting in a monomelic form in methanol solution. In contrast, Φ_Δ decreases markedly with increasing sensitizer concentration in water due to dimerization of the dye. Analysis of the steady-state data indicates Φ_Δ values of 0.74 and 0.12, respectively, for monomer and dimer. It is further shown that the efficiency whereby quenching of the triplet state by O₂ results in generation of O₂(¹Δ_g) is substantially lower for the dimer than for the corresponding monomer. Because monomer and dimer possess quite different absorption spectral profiles, the efficacy for photodynamic action with hematoporphyrin exhibits a pronounced wavelength dependence.     

COOPERATION OF CHARGES IN PHOTOSYNTHETIC O2 EVOLUTION–I. A LINEAR FOUR STEP MECHANISM

Abstract— Using isolated chloroplasts and techniques as described by Joliot and Joliot[6] we studied the evolution of O₂ in weak light and light flashes to analyze the interactions between light induced O₂ precursors and their decay in darkness. The following observations and conclusions are reported: 1. Light flashes always produce the same number of oxidizing equivalents either as precursor or as O₂. 2. The number of unstable precursor equivalents present during steady state photosynthesis is ∼ 1.2 per photochemical trapping center. 3. The cooperation of the four photochemically formed oxidizing equivalents occurs essentially in the individual reaction centers and the final O₂ evolution step is a one quantum process. 4. The data are compatible with a linear four step mechanism in which a trapping center, or an associated catalyst, ( S ) successively accumulates four + charges. The S ⁴⁺ state produces O₂ and returns to the ground state S ₀. 5. Besides S ₀ also the first oxidized state S ⁺ is stable in the dark, the two higher states, S²⁺ and S³⁺ are not. 6. The relaxation times of some of the photooxidation steps were estimated. The fastest reaction, presumably S *₁← S ₂, has a (first) half time ≤ 200 μsec. The S *₂ state and probably also the S *₀ state are processed somewhat more slowly (˜ 300–400 μsec).     

THE INFLUENCE OF MOLECULAR CONFORMATION ON THE STABILITY OF ULTRAVIOLET STABILIZERS TOWARD DIRECT AND DYE-SENSITIZED PHOTOIRRADIATION: THE CASE OF 2-(2'-HYDROXY-5'-METHYPHENYL)BENZOTRIAZOLE (TIN P)

Abstract— The mechanism for photodegradation of the ultraviolet photostabilizer 2-(2'-hydroxy-5'-methylphenyl)benzotriazole (TIN P) upon direct and dye-sensitized (singlet molecular oxygen [O₂(1Δ_g)]-mediated) irradiation was studied. From the experimental TIN P photodegradation rate data, and low temperature (77 K) fluorescence and phosphorescence quantum yields, one can conclude that the photodegradative process involves phosphorescent states of TIN P. The open conformer of TIN P quenches O₂(¹Δ_g) by physical scavenging with a rate constant (k_q) in dimethylsulfoxide of 2.8 times 10⁶ M ^-1 s^-1. The intramolecular hydrogen-bonded conformer does not appreciably interact with O₂(¹Δ_g). In the presence of a relatively high concentration of OH- (either 5 times 10^-2 M KOH in ethanol or water at pH 13), the ionic form of TIN P (with an ionized phenol group) physically and chemically quenches O₂(¹Δ_g). The reaction rate constant ( k _r) is 1 times 10 ⁸ M ^-1 s^-1, and the ratio k _q/ k _r is approximately three in alkaline aqueous media.     

UPCONVERSION OF THE SINGLET OXYGEN 1269 nm EMISSION TO THE VISIBLE

Abstract— Experiments are described that enable the kinetic behavior of singlet oxygen, O₂(^IΔ_g), to be monitored in the time-resolved mode using a photomultiplier to detect deep orange light (γ_max 660 nm). This orange light is a consequence of the upconversion of the natural emission of O₂(^IΔ_g) at 1269 nm.     

CAROTENOID TRIPLET STATE AND CHLOROPHYLL FLUORESCENCE QUENCHING IN CHLOROPLASTS AND SUBCHLOROPLAST PARTICLES

Abstract— Absorption changes attributed to the triplet state of carotenoids and to primary electron donors (P-700. P-680)^: and fluorescence quenching at several wavelengths have been measured with a single apparatus. following flash excitation with a dye or a ruby laser. Spinach chloroplasts as well as subchloroplast particles enriched in Photosystem-1 (F₁), Photosystem-2 (F₁) or the light-harvesting Chl a/h (F_III) have been examined at temperatures varying between 5 and 294 K.
The triplet state of carotenoids has been identified on the basis of its difference spectrum (having a peak at 515 nm) and decay kinetics (⋍ 7 µs at low temperature; accelerated by O₂ at 294 K). It is formed in all of the materials studied. The quantum yield of carotenoid triplet formation in chloroplasts increases at low temperature, but less than the fluorescence yield.
In most cases the fluorescence quenching recovers approximately with the same kinetics as the decay of the carotenoid triplets. The fluorescence recovery is, however, significantly faster for chloroplasts at 730 nm. Fluorescence quenching occurs in all types of materials. The ratio of fluorescence quenching to the concentration of carotenoid triplets varies with the material, being maximum in chloroplasts and minimum in F_m particles.
We conclude that the formation of the carotenoid triplet state is not limited to a few sites in the chloroplast and that a carotenoid triplet is a quencher of chlorophyll fluorescence. A detailed comparison of carotenoid triplets and fluorescence quenching gives some information concerning the organization of the pigments in the photosynthetic apparatus.     

POLYMER BOUND PYRROLE COMPOUNDS–VI. PHOTOPHYSICAL PROPERTIES OF MONOMERIC MODELS FOR POLYSTYRENE-BOUND PORPHYRINS

Abstract— Several porphyrin esters used as models for polystyrene-bound porphyrins have been prepared and their excited states have been studied by laser flash photolysis, IR phosphorescence of singlet molecular oxygen, O₂(¹Δ_g), and steady-state fluorescence. The photophysical properties of the porphyrin esters in solution are affected by the presence of nitro group(s) in the chain. In this case, an important decrease in φ_f, φ_T and φ_δ (to ca 0.7–0.4 of the value for the parent dimethyl ester) is observed. This is mainly due to intramolecular electron-transfer quenching [by the nitro group(s)] of the first excited singlet state of the porphyrin. The thermodynamic feasibility of this deactivation pathway has been confirmed polarographically. Quenching of the porphyrin triplet state and of O₂(¹Δ_g) by the nitro groups is negligible. The present conclusions explain also the results obtained previously for the photooxidation of bilirubin sensitized by the parent insoluble polystyrene-bound porphyrins. In that case the photooxidation rates were correlated directly with the quantum yield of O₂(¹Δ_g) production by the sensitizer. The consequences of these results for the use of polystyrene-bound porphyrins in sensitized photooxidation processes are discussed.     

RATE PARAMETERS FOR THE QUENCHING OF SINGLET OXYGEN BY WATER-SOLUBLE AND LIPID-SOLUBLE SUBSTRATES IN AQUEOUS AND MICELLAR SYSTEMS

Abstract— The laser flash photolysis method has been used to determine the bimolecular rate constants for the reaction between O₂(¹Δ₉) and several lipid-soluble and water-soluble substrates. Values for lipid-soluble substrates have been obtained using aqueous dispersions of surfactants above the critical micelle concentration with 1,3 diphenylisobenzofuran as monitor of singlet oxygen. Under these conditions the hydrophobic substances are solubilized by the micellar phase. For substrates which are water-soluble, 9,10-anthracene dipropionic acid disodium salt was used as singlet oxygen monitor. For several substances, the values obtained are comparable to the values found in homogeneous nonaqueous solutions. In cases where significant differences have been found these have been rationalized according to the individual case. The only major unexpected result concerned β-carotene which, in micellar dispersion, failed to react at all with O₂(¹Δ₉) This may be due to multi-molecular aggregations occurring in the polar medium. The work described herein shows clearly that, under appropriate conditions, singlet oxygen kinetics can be effectively followed in aqueous solutions by time resolved methods. The indiscriminate use of β-carotene as a quencher of O₂(¹Δ₉)in mainly aqueous media is questioned.     

ELECTRIC LINEAR DICHROISM OF P700 CHLOROPHYLL U-PROTEIN COMPLEXES

Abstract— The P₇₀₀ chlorophyll a -protein complex (CPI) isolated from green plants was oriented in aqueous solutions using pulsed electric fields of up to 6700 V cm^-1. The electric linear dichroism spectrum is reported in the range of 400–720nm. Positive peaks in the linear dichroism Δ A = A _I - A ₁ (where A_I and A₁ are the absorbance components in which the polarizer orientation is parallel and perpendicular with respect to the electric field. respectively) are observed at 443 and 686 nm. The ΔA signal at 686 nm is discussed in terms of either a specialized chlorophyll form absorbing at 686 nm. or due to an exciton component absorbing at the same wavelength.     

CHEMILUMINESCENCE AND THE FORMATION OF SINGLET OXYGEN IN THE OXIDATION OF CERTAIN POLYPHENOLS AND QUINONES

Abstract— The possibility of ¹O₂ (¹Δ_g) participation in the oxidation of polyphenols and quinones has been investigated in two systems: (1) the system involving autooxidation leading to oxidative polymerization and destruction, and (2) the modified Trautz-Schorigin reaction, i.e. oxidation of polyphenols and HCHO with H₂O₂ in concentrated alkaline solutions. The red band with maximum at 635 nm observed in chemiluminescence of pyrocatechol, adrenaline, pyrogallol, gallic acid, adrenochrome and p -benzoquinone corresponds to the transition 2O₂(¹Δ_g) → 2O₂(³Σ^-_g). Emission bands in the range 475–540 nm arise from the superposition of the 2O₂(¹Δ_g) → 2O₂(³Σ^-_g) transition and radiative deactivation of excited oxidation products. In system (2) chemiluminescence has a broad band from 580 nm beyond 800 nm and much higher intensity than in system (1). Formaldehyde was found to enhance light emission in system (1) by a factor of about 30. The influence of solvents, including D₂O in which ¹O₂ has varying lifetimes, on kinetics of chemiluminescence as well as quenching effect of β-carotene, hydroquinone, cysteine, bilirubin and biliverdin strongly support the involvement of ¹O₂ in the chemiluminescence of both systems.     

Effect of Magnesium and Calcium Complexation on the Photochemical Properties of Norfloxacin

Abstract— The fluoroquinolone antibiotics can induce skin photosensitivity in some patients and this has been ascribed to the generation of reactive oxygen species, such as singlet oxygen (O₂[¹Δ_g]). We have studied the photochemical properties of the different ionized forms of the fluoroquinolone norfloxacin upon complexation with Mg²+ and Ca²+ ions, as it is proposed that the antibiotic exists mainly as a complex in the blood plasma. We found that the norfloxacin cation (pH < 6) shows no photodegradation after UVA irradiation and has a low quantum yield of O₂(¹Δ_g) generation. The norfloxacin cation does not complex. Ca²+ or Mg²+ ions; when these ions are added to the solution, we observed no changes in the fluorescence quantum yields (φ_flu) and singlet oxygen yields (φ_Δ). In contrast, the neutral (6 ± pH > 8.5) and anionic (pH > 9) forms of norfloxacin are able to complex calcium and magnesium, and their generation of O₂ (¹Δ_g) is decreased by complexation. The neutral zwitterionic form and the anionic form also quench singlet oxygen by both chemical and physical pathways regardless of complex formation, while physical quenching is observed for the cation. At pH > 7.4, norfloxacin photobleaches and complexation to Ca²+ and Mg²+ increases the rate at which photobleaching occurs. Thus, both the pH of the medium and complexation with metal cations may affect the phototoxic potential of this antibiotic.     

PHOTOKINETIC AND PHOTOPHYSICAL MEASUREMENTS OF THE SENSITIZED PHOTOOXIDATION OF THE TRYPTOPHYL RESIDUE IN N-ACETYL TRYPTOPHANAMIDE AND IN HUMAN SERUM ALBUMIN

Abstract— The photosensitized oxidation of 10–100 μ M N -acetyl-L-tryptophanamide (NATA) in neutral aqueous solution and in the presence of various dyes proceeds by a pure O₂(¹Δ_g)-involving mechanism. Incorporation of the tryptophyl (Trp) residue into the polypeptide chain of human serum albumin (HSA) has no influence on the mechanism and efficiency of Trp photooxidation when sensitized either by methylene blue, a non-binding dye, or by rose bengal, a dye that gives non-covalent 1: 1 complexes with HSA. This is due to the location of the Trp residue in close proximity of the protein surface and, in the case of rose bengal, to the coincidence of the photophysical properties (including the quantum yield of O₂(¹Δ_g) generation) for the free and HSA-bound dye. Hematoporphyrin also binds to HSA with 1: 1 stoichiometry, although at a different site from rose bengal. Bound Hp again displays photophysical properties very similar with those of free Hp; however, the efficiency of Trp photo-oxidation in HSA is about 5-fold higher than in NATA owing to a limited rearrangement of the protein structure, induced by Hp binding, which enhances the probability of chemical quenching of O₂(¹Δ_g) by the indole ring.     

THERMODYNAMIC FUNCTIONS FOR SINGLET MOLECULAR OXYGEN, O2(1△g) and O2 (1σ8+)

Abstract— A correction is offered to the approximate values previously given by Mendenhall (1978) for the enthalpy of formation and entropy of O₂(a₁Δ_g) and O₂(b₁₊) between 298 and 1500 K. Accurate values have been calculated for the functions together with the equilibrium constants for the formation of these species from O₂(X₃σ_g-).     

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.     

THE PRODUCTION OF SINGLET MOLECULAR OXYGEN BY ZINC(II) PHTHALOCYANINE IN ETHANOL AND IN UNILAMELLAR VESICLES. CHEMICAL QUENCHING AND PHOSPHORESCENCE STUDIES

Abstract— Zn(II)phthalocyanine (ZnPc) generates O₂(¹Δ_g) with a quantum yield of ca. 0.4 upon photocxcitation at 354 or 600 nm in ethanolic solution as determined by time-resolved phosphorescence studies at 1270 nm and photooxidation experiments using 1,3-diphenylisobenzofuran (DPBF) as substrate. The quantum yield of photooxidation slightly increases upon incorporation of ZnPc into unilamellar liposomes of dipalmitoylphosphatidylcholine. Under our irradiation conditions (600 nm, 18^°C, and short light exposure times), DPBF(5–50 μM) undergoes photooxidation by a pure Type II mechanism; the rate constant for the O₂(¹Δ_g) + DPBF reaction is (1.1 ±0.1) x 10⁹ M^-1 s^_1 in ethanol solution and determined to be about two orders of magnitude smaller when both ZnPc and DPBF are embedded into liposomes.     

SPECTRAL CHARACTERIZATION OF LIGHT-REDUCIBLE CYTOCHROME IN A PLASMA MEMBRANE-ENRICHED FRACTION AND IN OTHER MEMBRANES FROM CAULIFLOWER INFLORESCENCES

Abstract— Low temperature spectroscopy has been used to characterize microsomal fractions obtained from cauliflower inflorescences ( Brasska oleracea L.) by differential centrifugation and partition in an aqueous polymer two-phase system. The plasma membrane-enriched fraction (U₃) was found to contain one dominant b -cytochrome, which could be reduced both by blue light and by dithionite. An action spectrum of the blue light-induced absorbance change [LIAC, Δ(A₄₃₀—A₄₁₀)] associated with the reversible reduction of this b -type cytochrome indicated that the primary light-receptor was a flavin-like compound. Another microsomal fraction (L₃) containing membranes from mitochondria, endoplasmic reticulum and other organelles also contained light-reducible cytochrome. One of these could be identified as cytochrome c oxidase, and another may be identical to cytochrome b ₅ of the endoplasmic reticulum.     

ON THE NATURE OF THE INTERMEDIATE ELECTRON ACCEPTOR (A1) IN THE PHOTOSYSTEM I REACTION CENTER

Abstract— A sodium dodecyl sulfate-Photosystem I (PSI) complex has been prepared and characterized with respect to its electron acceptors. Component X and iron-sulfur centers A and B are absent from this preparation but the intermediate electron acceptor (A₁) is present. Flash-induced absorbance changes at 25°C show charge separation, followed by a back-reaction with a half-time of 5 µs. The spectrum of the flash-induced change from 350 to 550 nm indicates a contribution from the intermediate electron acceptor, A₁, as well as from P700⁺. EPR studies show that A₁ is associated with a free-radical signal having a g-value of 2.0025 and a linewidth of 12 gauss. A, would appear to be associated with a monomeric form of either Chi α or pheophytin a.     

SPECTRAL FEATURES OF THE BOUND ELECTRON ACCEPTOR A2 OF PHOTOSYSTEM I

Abstract— Difference spectrum for the reduction of A₂, a bound secondary electron acceptor of photo-system I, in the thylakoid membranes of a thermophilic blue-green alga, Synechococcus sp., was determined by subtracting the difference spectrum of P700 photooxidation from the difference spectrum for flash-induced absorption changes due to oxidation of P700 and reduction of A₂, or by measuring light-induced absorption changes under reducing conditions where reduced A₂ accumulates. The spectrum showing a broad bleaching with two maxima at 420 and 440 nm indicates that A₂ is an iron-sulfur center different from P430.