FLASH PHOTOLYSIS-ELECTRON SPIN RESONANCE STUDIES OF THE INTERACTION OF PHENAZINE METHOSULFATE WITH REACTION-CENTER PREPARATIONS FROM THE R-26 MUTANT OF RHODOPSEUDOMONAS SPHEROIDES*

Abstract— Using the technique of flash photolysis-electron spin resonance, we have shown, by means of a kinetic analysis, that phenazine methosulfate (PMS) interacts with reaction-center preparations from the blue-green mutant R26 of Rhodopseudomonas spheroides. At intermediate concentrations of PMS, biphasic decay kinetics of the P870⁺ ESR signal are observed demonstrating that the PMS radical interacts with reaction centers by a specific binding mechanism. With PMS bound to reaction centers, the P870⁺ ESR signal decays in ˜ 1 ms; whereas, in unbound reaction centers the decay is ˜ 120 ms. A model is proposed involving the interaction of PMS on the donor side of P870.     

PULSE RADIOLYSIS STUDIES IN MODEL LIPID SYSTEMS: FORMATION AND BEHAVIOR OF PEROXY RADICALS IN FATTY ACIDS

Abstract— Radiolytic formation and peroxidation of fatty acid radicals have been investigated by pulse radiolysis techniques in oleate, linoleate, linolenate and arachidonate systems. A strong absorption band at 280 nm associated with conjugated radicals, R_conj, formed in polyunsaturated fatty acid moieties has been used as a probe for kinetic processes occurring at doubly allylic sites in the hydrocarbon chain. Formation of R_conj by O^- has been found to be more efficient than the less selective OH radical. Peroxidation of R_conj is shown to be somewhat slower, ( k _R+ O₂˜ 3 × 10⁸ M ^-1 s^-1), than O₂ reactions with radicals in oleate ( k _R+ O₂= 1 × 10⁹ M ^-1 s^-1). Peroxy radicals generated in these reactions disappear slowly by essentially second order processes (2 k RO₁˜ 10⁷ M ^-1 s^-1). The superoxide radical, O^-₂, shows little if any reactivity towards 0.01 M linolenate or arachidonate over periods of 20 s.     

KINETIC ANALYSIS OF THE FLUORESCENCE INDUCTION IN 3-(3,4-DICHLOROPHENYL)-1,1-DIMETHYLUREA POISONED CHLOROPLASTS

Abstract— The size of the area over the fluorescence rise curve of chloroplasts is a measure of the total number of quanta utilized in photosystem II during the fluorescence induction, while the growth of the area reflects the progress of photochemical events. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), the growth kinetics of the area are affected by the reoxidation of the primary acceptor Q ^- with stored oxidizing charges on the donor side of system II.
At low light intensities, a slow component of this back reaction may limit the steady state fluorescence emission. At higher intensities, however, the fluorescence rise is limited solely by photochemical events, although fast thermochemical reactions like the immediate recombination of photochemically separated charges may affect the efficiency of the photochemistry.
A kinetic analysis of the area growth at moderate light intensities revealed that it occurred in two first order phases which were described by the rate constants k _α and k _β. The biphasic nature suggested a sequential two-electron reduction of the primary acceptor Q , or the presence of two different types of photochemical centers in system II. The rate constants were light intensity dependent. They also were affected by changes in pH, by an addition of NH₂OH, or by a preillumination with short flashes prior to addition of DCMU. It is suggested that the pH of the medium, the presence of NH₂OH, and the flash induced state S_n of the water splitting enzyme, control the values of k _α and k _β by changing the rate constants of electron carrier interactions in the reaction center complex, with a resulting modification of the frequency of back reactions between the primary donor and the primary acceptor.     

DELAYED FLUORESCENCE AND THE REVERSAL OF PRIMARY PHOTOCHEMISTRY IN RHODOPSEUDOMONAS VIRIDIS

Abstract— Delayed fluorescence from chromatophores of the photosynthetic bacterium Rhodopseudomonas viridis was measured at temperatures below 0°C. A component with a decay half-time of about 7 ms was found. Its intensity was directly proportional to the number of reaction centers in the P985⁺·A^- state. During prolonged illumination it faded as electrons moved forward along the electron transport chain from the primary acceptor, A, (P985⁺·A^-→P985⁺·A), and its decay in the dark paralleled the disappearence of the P985⁺ electron paramagnetic resonance absorption. The data suggest that this component of delayed fluorescence results from a direct reversal of the primary light reaction. While the rate of the P985⁺middot;A^-→P985·A reaction was almost independent of temperature, delayed fluorescence intensity displayed an apparent activation energy of 0°2 eV. It is concluded that the P985⁺·A^-→P985·A reaction proceeds by parallel radiative and nonradiative routes. The direct proportionality between delayed fluorescence and the concentration of P985⁺·A^- pairs seems to preclude an involvement of triplet-triplet annihilation or dependence of delayed fluorescence upon the variable prompt fluorescence yield.     

A PHOTOCHEMICAL TECHNIQUE TO ENHANCE SENSITIVITY OF DETECTION OF FLUORESCENCE RESONANCE ENERGY TRANSFER

Abstract: A photochemical kinetic method of measuring small values of efficiency of fluorescence resonance energy transfer (FRET) between special probes is proposed. The FRET efficiency (ω) is determined from kinetics of the photochemical reaction of the energy acceptor sensitized by FRET from the energy donor. The choice of an appropriate donor-acceptor pair permits the minimization of background reactions. Application of the method is demonstrated by the detection of FRET from 2,5- bis (5- tert -butyl-2-benzoxasolyl)thiophen (BBOT) to acridine orange (AO) in phospholipid vesicles. Photobleaching of AO in the presence of CBr₄ was applied as a photochemical reaction of the acceptor. The reaction was monitored by steady-state fluorescence measurements. The FRET measurements were carried out by the proposed technique when the probe/lipid ratio and ω were as small as 1.1 × 10⁻⁵ M/M and 0.0017, respectively. Under these conditions, the rate constant of AO photobleaching was increased by 26% as compared with that of the reference sample without BBOT. The results suggest that applications of the technique may be useful in the study of the membrane topography.     

SYNTHESIS OF AMPHIPATHIC PORPHYRINS AND THEIR PHOTOINDUCED ELECTRON TRANSFER REACTIONS AT THE LIPID BILAYER-WATER INTERFACE

A one flask synthesis of cis -substituted amphipathic porphyrins is reported. These porphyrins were used to study electrostatic effects on photoinduced electron transfer across the lipid bilayer-water interface. A neutral porphyrin undergoes only dynamic interfacial electron transfer reactions irrespective of charge of the acceptor, although ionic strength effects indicate a negative charge on the porphyrin donor species. A dianionic porphyrin forms an interfacial static complex with a dicationic electron acceptor, methyl viologen, at low ionic strength. The electron transfer rate within the complex is slow, 10⁵∼ 10⁶ s^-1, which is attributed to a near orthogonal orientation between the donor and the acceptor ∼ orbitals.     

PHYSICAL AND PHOTOCHEMICAL PROPERTIES OF A FLUORESCENT CHLOROPHYLL COLLOID

Abstract— A fluorescent colloid of chlorophyll a , of which some qualitative properties were noted by Krasnovsky and Brin⁽¹¹⁾, has been quantitatively characterized. The colloid is formed in neutral PO₄ buffer containing 0 1 to 8.0% Tween 20, and is stable in darkness. The extinction coefficient is 7.8 × 10⁴ 1. mole cm^-1 at the red absorption peak (668 mμ), the yield of fluorescence is ˜ 0.25, and the yield of photoautooxidative bleaching is ˜ 2 times 10^-4. The colloid sensitizes the autooxidation of paratoluenediamine with a yield of ˜0.01 to ˜0.3 depending on light intensity and substrate concentration. The yield is independent of detergent and chlorophyll concentrations. In all respects—except the dependence of yield on illumination—the colloid appears to be physically and photochemically equivalent to dissolved chlorophyll, as known in dilute solutions in organic solvents. The light dependence—the yield is inversely proportional to the cube root of absorbed intensity—could be due to a bimolecular back reaction of a chlorophyll or substrate derivative.     

INCREASED QUANTUM YIELD OF PHOTOREDUCTION OF DYES (A CATALYTIC EFFECT)

Abstract— While studying the photoreduction of some dyes (D) by reducing agents (R), it was observed that the quantum yield of the photoreduction increases considerably upon addition of a third substance (C), whereas it is very small when the dye is photoreduced by C alone (catalytic effect), (see Table 1).
The system thionine (D), allylthiourea (R), and azulene (C) was investigated in detail using both flash photolysis and continuous illumination. On photolysis, thionine is converted into its photo-reduced form, leucothionine. Azulene reacts with the basic form of the thionine triplet ³ TH ⁺ to produce the semithionine radical. In the system thionine and azulene, most of these radicals revert back to thionine. When ATU (˜ 10^2- M ) is added to thionine and azulene (3 × 10^-4 M ), the semithionine radicals are reduced to leucothionine; the quantum yield of this reduction is considerably higher than in the system thionine and allylthiourea. Flash experiments demonstrate that allylthiourea does not react with the semithionine radicals.
At very high ATU concentrations (≥ 10^-1 M ), however, the primary reaction is between thionine triplet and allylthiourea; under these conditions the quantum yield is not influenced by azulene (3 × 10^-4 M ).     

LIGHT-DRIVEN REDOX PROCESSES IN FUNCTIONAL MICELLAR UNITS—III. Zn-TETRAPHENYLPORPHYRIN SENSITIZED REACTIONS IN METHYL VIOLOGENE SURFACTANT ASSEMBLIES

Abstract— The photoreduction of methyl viologene by ZnTPP triplet excited states was studied in mixed micelles containing the functional surfactant JV-dodecyl, N -methyl viologene (C₁₂MV²⁺) as one and Cetyltrimethylammoniumchloride (CTAC) as the other component. Using the laser photolysis technique the kinetics of the redox reaction were studied at various CTAC/C₁₂MV²⁺ ratios. The average time for the electron transfer decreases linearly with the C₁₂MV²⁺ occupancy. The back transfer from C₁₂MV²⁺ to ZnTPP²⁺ can be intercepted if a donor such as NADH is cosolubilized in the micelle. In such a system irreversible methyl viologene reduction is achieved which in the presence of a suitable redox catalyst can be used to produce hydrogen from water.     

AN 125I-LABELED N6-SUBSTITUTED AZIDO ANALOG OF NAD+ FOR THE PHOTOAFFINITY LABELING OF NAD+-LINKED ENZYMES

Abstract ¹²⁵I- N ⁶-(N-[6-N-{5-iodo-4-azidosalicyl}-aminohexyl]-aminocarbamoylmethyl)-nicotinamide adenine dinucleotide (¹²⁵I- N ⁶-I-ASA-AH-NAD⁺) was synthesized by coupling N ⁶ -([6-aminohexyl]-carbamoylmethyl)-NAD⁺ with 4-azidosalicylic acid N-hydroxysuccinimide ester followed by radioiodination. The utility of ¹²⁵I-N ⁶ -I-ASA-AH-NAD⁺ as an effective site-directed photoprobe was demonstrated by the photolabeling of both glutamate dehydro-genase and 15-hydroxyprostaglandin dehydrogenase. Both enzymes can be saturated with labeled probe with apparent dissociation constants comparable to those reported for NAD⁺. Photoincorporation of the probe into both enzymes was found to be protected specifically by NAD⁺. These results indicate that ¹²⁵I- N ⁶-I-ASA-AH-NAD⁺ can be a specific photoprobe for NAD⁺-linked enzymes.     

KINETIC PROPERTIES OF THE TRIPLET STATES OF METHYLENE BLUE AND OTHER PHOTOSENSITIZING DYES

Abstract— The long-lived (> 1 μsec) transients formed in the flash excitation of the representative photosensitizers methylene blue, eosin Y and pyrene have been investigated and various criteria have been used to distinguish between triplet state intermediates and chemical intermediates. Previous assignments of the triplet transients of methylene blue appeared less secure in view of the photochemical reactivity of this dye and its lack of phosphorescence. Earlier assignments of monomeric and dimeric triplet transients of methylene blue are substantiated, however, by the observations that the rate constant for quenching by oxygen is approximately 1/9^th diffusion controlled and the formation rates are commensurate with singlet decay rates and by the observation of triplet-triplet annihilation. Additional evidence in support of monomer triplet assignments for methylene blue and eosin Y is provided by the effect of heavy atom quenchers Cs⁺, Hg²⁺ and T1⁺ on decay rates. Due to chemical reactivity, quenching by I⁻appears less suitable as a diagnostic test for triplet state intermediates. The effect of N₃⁻, which is known to quench singlet oxygen molecules and to alter the course of photosensitized oxidations, on the triplet decay of methylene blue, eosin Y and pyrene is also investigated.     

EXCISION REPAIR OF PYRIMIDINE DIMERS IN MARSUPIAL CELLS

Monodelphis domestica was further characterized as a model for photobiological studies by measuring the excision repair capabilities of this mammal's cells both in vivo and in vitro. Excision repair capability of the established marsupial cell line, Pt K2 ( Potorous tridactylus ), was also determined. In animals held in the dark, we observed that ˜50% of the dimers were removed by 12 and 15 h after irradiation with 400 J m⁻² and 600 J m⁻², respectively, from an FS-40 sunlamp (280–400 nm). Cells from primary cultures of M. domestica excised ˜50% of the dimers by 24 h after irradiating with 50 J m⁻² and 36 h after exposure to 100 J m⁻² with no loss of dimers observed 24 h following a fluence of 300 J m⁻². Pt K2 cells were observed to have removed -50% of the dimers at -12 h after 50 J m⁻² with only -10% of the dimers removed at 24 h following 300 J m⁻². The observed loss of pyrimidine dimers from epidermal DNA of UV-irradiated animals and from fibroblasts in culture, held in the dark, suggests that these marsupial cells are capable of DNA excision repair.     

PHOTOCHEMICAL HYDROXYLATION OF 7,8-DIMETHYLALLOXAZINE (LUMICHROME)-A FLASH PHOTOLYSIS STUDY

Abstract— Flash photolysis experiments on the hydroxylation of lumichrome (L) in aqueous 0.5 M H₂SO₄ solution in the presence of O₂ or Ni²⁺ as triplet quenchers and quantum yield measurements confirm the assignment of the photoreactive species to the protonated form of the excited singlet state. A mechamism concerning the photochemical step is proposed, accounting for the formation of protonated 9-hydroxy-5,10-dihydrolumichrome (LOH₃⁺). This primary stable photoproduct was characterized by spectral and kinetic data. The dark reactions originating from LOH₃⁺ were investigated, and data regarding the successive steps are presented. The reaction LOH₃⁺ L→ LO + LH₃⁺ is demonstrated to be a two-electron reduction. The rate constant for the reaction of LH₂⁺ with O₂ is much larger than that for the oxidation of LH₃⁺ by oxygen.     

THE TRIPLET STATE OF 8-METHOXYPSORALEN

Abstract— Transient absorption spectra produced by laser flash photolysis of an aqueous solution of 8-methoxypsoralen (8-MOP) have been studied. The biphotonic production of hydrated electrons and of the radical ions, 8-MOP ⁺ and 8-MOP^- is reported. The hydrated electron was found to react with ground state 8-MOP with k ˜ 3 × 10¹⁰ M ^-1 s^-1. In order to obtain a true triplet-triplet absorption spectrum. contributions from the radical ions were subtracted from the overall transient absorption. In addition, contributions from e^-_aq to the transient spectrum were removed by using N₂O, low laser intensity to minimize photoionization or by measuring the transient O.D. after the electron has decayed. These three methods each produced the same triplet-triplet spectrum which differs in the red region from previously reported spectra.     

SOLID PHASE PHOTOREDUCTION OF METHYLVIOLOGEN ON CELLULOSE SENSITIZED BY TRIS(2,2'-BIPYRIDYL)RUTHENIUM COMPLEX

–Methylviologen (MV²⁺) adsorbed on cellulose could be reduced photochemically in the solid phase sensitized by tris(2,2'-bipyridyl)ruthenium(II) complex, [Ru(II)(bpy)₃], using disodium ethylene-diaminetetraacetic acid (Na₂EDTA) as a reducing agent. Formation of the cation radical (MV ⁺.) was confirmed by visible and EPR spectra. The MV⁺. formed on cellulose was remarkably stable against air oxidation and rapidly accumulated even by the irradiation under air. Water adsorbed on the cellulose greatly retarded the photoreaction. Action spectrum showed that the excitation of Ru(II)(byp)₃ is responsible for the photochemical reaction. The quenching of the emission from Ru(II)(bpy)*₃ by MV²⁺. indicated that a primary photochemical reaction occurs between Ru(II)(bpy)*₃ and MV²⁺. The main reaction path is the reduction of MV²⁺ by Ru(II)(bpy)₃, giving MV⁺. and Ru(III)(bpy)₃, followed by the reduction of Ru(III)(bpy)₃ to Ru(II)(bpy)₃ with Na₂EDTA, which in turn is oxidized to give carbon dioxide.     

Preparation and Evaluation of Arylazide-Substituted Pyridine Adenine Dinucleotides for Photoaffinity Labeling Experiments

Two pyridine-modified NAD'analogs, 3–(3-azido benzo-yl) pyridine adenine dinucleotide 1 and N -(3-azido-5-car-boxyl) phenyl nicotinamide adenine dinucleotide 2 have been prepared and evaluated for photoaffinity labeling experiments. The syntheses were accomplished via a mammalian NADase-catalyzed base exchange reaction. The new NAD⁺ analogs retained the carbonyl or carhox-amido functional group at the 3 position of the pyridine ring. The analog 1 is the first pyridine-modified azido derivative of NAD⁺ that has shown coenzyme activity in a stereospecific hydride transfer reaction catalyzed by a dehydrogenase. Both NAD⁺ analogs have shown potential for the study of active sites of NAD⁺-utilizing enzymes.     

INDUCTION OF LEAF UNROLLING BY PHYTOCHROME and ACETYLCHOLINE IN ETIOLATED WHEAT SEEDLINGS

Abstract-Phytochrome regulates the unrolling of primary leaf sections from 8-day-old dark-grown wheat ( Triticum aeslivum L. cv. Arminda) seedlings. Red light (R)-stimulated unrolling of leaf sections pretreated in 1 m M ethylene-bis-(β-aminoethylether)- N,N,N',N' -tetraacetic acid (EGTA) if 1 m M CaCl₂ was added during a 30 min treatment period including and following irradiation. Nifedipine at 1 μ M (a Ca²+-channel antagonist) applied 10 min before R prevented the R stimulation of leaf unrolling. The Ca²+-channel agonist Bay K-8644 (1 μ M ) and acetylcholine (ACh, 1 mY M ) stimulated unrolling of leaf sections prewashed in EGTA in darkness, if 1 m M CaCl₂ was present in the medium during a 30 min treatment period. Acetylcholine also induced leaf unrolling in the absence of Ca²+ when 100 μ M NaCl was present in the medium. Apart from ACh, only carbamylcholine out of the choline derivatives tested was active in induction of leaf unrolling in the presence of 1 m M Ca²+. The ACh receptor antagonists, atropine (10 μ M ) AND D-tubocurarine (10 μ M ), nullified the ACh-induced Ca²+- and Na+-dependent leaf unrolling, respectively. Muscarine and nicotine, agonists of ACh, at 1 μ M stimulated leaf unrolling in the presence of Ca²+ and Na+, respectively. The ACh-induced Ca²+-dependent leaf unrolling was reduced by 1 μ M Nifedipine, 10 μ M Li+ and 10 μ M "calmodulin" inhibitor, trifluoperazine (TFP), whereas only TFP was active in the reduction of the Na+-dependent ACh-induced leaf unrolling response. It is proposed that leaf unrolling of dark-grown primary wheat leaves can be regulated by phytochrome and by activation of two different types of ACh receptors.     

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).     

CHEMILUMINESCENT REACTIONS OF SO

Abstract— The rapid bimolecular reaction SO + O₃= SO₂+O₂+ 106 kcal/mole
yields electronically excited SO₂ in the ³B ₁ and ¹B₂ states with some vibrational excitation, as well as SO₂ in its electronic ground state. It is shown that k₁ = 1.5 x 10¹² exp (-2100/ RT ) cm² mole^-1 s-¹ and that the formation of electronically excited SO₂ involves higher activation energies.     

RATES OF REACTION OF SINGLET OXYGEN WITH SULFIDES

Abstract—Reaction rate constants for the reaction of singlet oxygen with a series of 24 sulfides in chloroform have been measured by inhibition of the self-sensitized photooxidation of rubrene. The reaction rate constant is sensitive to steric effects, decreasing as the carbons α- to sulfur become more highly substituted. Addition of a methyl group to each of the carbons α- to sulfur decreases the rate constant by about a factor of 10. From a series of p - and m -substituted thioanisoles, a ρ of -1.67 ± 0.09 was found. A much better correlation was found with σ than with σ⁺ indicating there is no resonance interaction with the reaction center. Typical rate constants are: di- n -butyl sulfide, 2.3 × 10⁷ M ^-1 s^-1; CBZ-L-methionine methyl ester, 1.4 × 10⁷; di-s-butyl sulfide, 1.8 × 10⁶; di- t -butyl sulfide, 1.3 × 10⁵; and thioanisole, 2.3 × 10⁶.