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.     

EFFECTS OF HIGH PRESSURE ON PHOTOCHEMICAL REACTION CENTERS FROM RHODOPSEUDOMONAS SPHEROIDES*

Abstract— Photochemical reaction centers from Rhodopseudomonas spheroides were subjected to pressures ranging from 1 to 6000 atm. Optical absorption, fluorescence and photochemical activities were studied under these conditions. Absorption spectra showed bathychromic shifts of the long-wave bands attributed to bacteriopheophytin and bacteriochlorophyll (the latter as P800 and P870). The quantum efficiency of photochemical oxidation of P870 was diminished at high pressure. The quantum yield of P870 fluorescence showed a parallel decline, as if high pressure introduced a quenching process that competed with both photochemistry and fluorescence. The original efficiencies were largely restored when the pressure was returned to 1 atm. The efficiency of oxidation of mammalian cytochrome c, coupled to the photochemical oxidation of P870 in reaction centers, was lowered by high pressure. This effect was more pronounced than the effect on P870 oxidation, and was irreversible. The kinetics of recovery of P870 following its photochemical oxidation showed effects of high pressure. The main effect was the appearance, at high pressure, of slow recovery suggesting the trapping of electrons. This effect was partly irreversible.     

FLASH PHOTOLYSIS-ELECTRON SPIN RESONANCE STUDIES OF THE DYNAMICS OF PHOTOSYSTEM I IN GREEN-PLANT PHOTOSYNTHESIS-I. EFFECTS OF ACCEPTORS AND DONORS IN SUBCHLOROPLAST PARTICLES*

Abstract —Transient decay kinetics of Signal I in spinach subchloroplast particles have been studied in the presence of artificial donors [5-methyl phenazonium methyl sulfate (PMS), 2, 6-dichlorophenolindophenol (DPIP), N, N, N', N'-tetramethyl-p-phenylenediamine (TMPD) and ascorbate], artificial acceptors [benzyl viologen (BV) and methyl viologen (MV)] and various natural donor and acceptor moieties, using the technique of flash photolysis-electron spin resonance spectroscopy. The results are discussed in the framework of three mechanisms for reduction of P700⁺: (1) direct electron return from the primary acceptor to P700⁺, (2) cyclic electron flow via an electron carrier, and (3) noncvclic electron flow.     

FLASH PHOTOLYSIS-ELECTRON SPIN RESONANCE STUDIES OF THE DYNAMICS OF PHOTOSYSTEM I IN GREEN-PLANT PHOTOSYNTHESIS—II. INTACT AND BROKEN SPINACH CHLOROPLASTS*

Abstract —The transient oxidation and subsequent reduction of P700⁺ in spinach chloroplasts has been monitored by flash photolysis-electron spin resonance spectroscopy in the presence of various donors and acceptors. In general, the results obtained correlate well with results on Photosystem I subchloroplast particles, with two major differences. For Type A and B intact chloroplasts in the presence of 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU), the electron acceptor methyl viologen has no effect on the decay kinetics. This phenomenon is interpreted in terms of a functioning cyclic electron flow path around Photosystem I. Also, the photoresponse of Signal I depends on the length of the photolyzing flash. This is interpreted in terms of the existence of a primary electron donor to P700⁺ with a transfer time of ? 10 μs.     

ULTRAVIOLET LIGHT-INDUCED FREE RADICAL FORMATION IN SKIN: AN ELECTRON PARAMAGNETIC RESONANCE STUDY

It has been suggested that ultraviolet light induces free radical formation in skin, leading to photoaging and cancer. We have demonstrated by electron paramagnetic resonance that the ascorbate free radical is naturally present in unexposed skin at a very low steady state level. When a section of SKH-1 hairless mouse skin in an EPR cavity is exposed to UV light (4,500 J m⁻²−1, Xe lamp, 305 nm cutoff and IR filters), the ascorbate free radical signal intensity increases. These results indicate that UV light increases free radical oxidative stress, consistent with ascorbate's role as the terminal, small-molecule antioxidant. The initial radicals produced by UV light would have very short lifetimes at room temperature; thus, we have applied EPR spin trapping techniques to detect these radicals. Using α-[4-pyridyl 1-oxide]-N- tert -butyl nitrone (POBN), we have for the first time spin trapped a UV light-produced carbon-centered free radical from intact skin. The EPR spectra exhibited hyperfine splittings that are characteristic of POBN/alkyl radicals, a^N= 15.56 G and a^H= 2.70 G, possibly generated from membrane lipids as a result of β-scission of lipid alkoxyl radicals. Iron can act as a catalyst for free radical oxidative reactions; chronic exposure of skin to UV radiation causes increased iron deposition. Using our spin trapping system, we have shown that topical application of the iron-chelator, Desferal, to a section of skin reduces the UV light-induced POBN adduct radical signal. These results provide direct evidence for free radical generation and a role for iron in UV light-induced dermatopathology. We suggest that iron chelators can serve as photoprotective agents by preventing these oxidations.     

ELECTRON SPIN RESONANCE OF TRIPLET STATES AND FREE RADICALS APPEARING IN PORPHYRINS, AROMATIC AMINO-ACIDS AND PROTEINS UNDER THE EFFECT OF LIGHT

Abstract— The electron paramagnetic resonance (EPR) of the triplet excited state of solutions of a series of porphyrins and aromatic amino-acids has been studied at 77°K. It has been shown that for some of the compounds it is possible to observe EPR for transitions with δ M =±1, ±2.
It has been shown that photoreactions in solid solutions of porphyrins and aromatic amino-acids proceed through the triplet excited state.
Reactions of photosensitized deamination of aliphatic amino-acids in solid aqueous solutions at 77°K by aromatic compounds have been studied.     

TRANSFER OF ENERGY FROM REACTION CENTER TO LIGHT HARVESTING CHLOROPHYLL IN A PHOTOSYNTHETIC BACTERIUM*

Abstract— Previous evidence indicates that energy transfer in photosynthetic bacteria can occur from reaction center to light harvesting chlorophyll (the reverse of the usually considered flow) and that the amount of this flow depends on the strain of bacteria. The present report demonstrates that the action spectrum for fluorescence of Rhodopseudomonas spheroides, strain R26, is changed by adding the strong reductant dithionite. This change indicates that the amount of reverse flow can be altered chemically. The amount of reverse flow inferred from these measurements is consistent with the amount predicted from the absorption and fluorescence spectra of chromatophores and isolated reaction centers, and from the relative fluorescence yields of these two. The measurements permit an estimate of the transfer rates describing the energy flow from light harvesting to reaction center chlorophyll as well as the reverse flow. The spectrum for delayed fluorescence of Rps. spheroides, strain Ga, was found to be similar to that of the variable part of the fluorescence. This is a necessary, but not sufficient, condition that the energy for delayed fluorescence originates in the reaction centers.     

ELECTRON SPIN RESONANCE STUDY OF PHOSPHORANYL RADICALS I. INFLUENCE OF STERIC AND ELECTRONIC EFFECTS ON RADICAL FORMATION IN SOLUTION

Abstract

An electron spin resonance study of phosphoranyl radicals, generated by u.v. irradiation of solutions containing a trialkylphosphite and a dialkylperoxide, enabled us to examine the influence of steric and electronic factors on radical formation. It was demonstrated that the influence of steric factors, such as α- or β-branching of the alkyl groups, on the magnitude of radical formation was present in both trialkyl- and methyldialkylphosphites, but not in dimethylalkylphosphites. Furthermore, the study of additional hetero atoms in the alkyl ligand of the dimethylalkylphosphites clearly revealed the influence of the gauche-effect. It was demonstrated that the presence of the gauche-effect, which causes a conformational preference in which a larger steric hindrance is produced, resulted in a decrease of radical formation.     

A STUDY OF LIGHT-INDUCED ELECTRON PARAMAGNETIC RESONANCE SIGNALS IN WALLS OF MOSS SPORES AND POLLEN GRAINS

Abstract —Light-induced EPR signals can be observed in moss spores and pollen grains. Their characteristics are studied in this paper. The modification of these signals during spore maturation or various physical and chemical treatments shows that they probably originate in the spore or pollen grain wall. This hypothesis is confirmed by the similarity between the spore (or isolated spore wall) absorption spectrum and the light-induced signal action spectrum.     

PHOTOCHEMICAL SENSITIZATION BY AZATHIOPRINE AND ITS METABOLITES. PART 3. A DIRECT EPR AND SPIN-TRAPPING STUDY OF LIGHT-INDUCED FREE RADICALS FROM 6-MERCAPTOPURINE AND ITS OXIDATION PRODUCTS

Abstract Sunlight has been implicated in the high incidence of skin cancer found in patients receiving 6-mercaptopurine (PSH) in the form of its pro-drug azathioprine. In this study we have used EPR spectroscopy in conjunction with the spin-trapping technique to determine whether PSH and its metabolic or photochemical oxidation products generate highly reactive free radicals upon UV irradiation. When an aqueous anaerobic solution (pH 5 or 9) of PSH (pK₂= 7.7) and either 2-methyl-2-nitrosopropane (MNP) or nitromethane (NM) were irradiated (λ > 300 nm) with a Xe arc lamp, the corresponding purin-6-thiyl (PS^.) radical adduct and the reduced form of the spin trap (MNPIH’or CH₃N0₂) were observed. However, no radical adducts were detected when PSH and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were irradiated (λ= 320 nrn) in oxygen-free buffer. These findings suggest that PSH does not photoionize but that instead MNP and NM are reduced by direct electron transfer from excited state PSH, ^1,3(PSH)*. In aerobic solution, oxygen can act as an electron acceptor and the O₂*- and PS radicals are formed and trapped by DMPO. 6-Mercaptopurine did photoionize when irradiated with a Nd:YAG laser at 355 nm as evidenced by the appearance of the DMPO/H (e_eq+ H⁺) adduct, which decreased in intensity in the presence of N₂O. ^1,3(6-Mercap-topurine)* oxidized ascorbate, formate and reduced glutathione to the corresponding ascorbyl, CO₂.- or glutathiyl radicals. The photochemical behavior of 6-thioxanthine and 6-thiouric acid was similar to PSH. However, the excited states of these metabolic oxidation products exhibited stronger reducing properties than ^1,3(PSH)*. Photolysis of PSH photoproducts purine-6-sulfonate or purine-6-sulfinate resulted in homolysis of the C-S bond and the appearance of the SO₃′- and SO₂^?_-- radicals, respectively, which were detected by direct EPR. These studies demonstrate that UV irradiation of PSH, its photoproducts and metabolites generates a variety of free radicals that may be involved in the etiology of skin cancer induced by azathioprine.     

CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY: FLASH PHOTOLYSIS STUDIES OF THE CHLOROPHYLL-QUINONE REACTION IN ALCOHOL SOLVENTS*

Abstract— Flash photolysis of chlorophyll a alone in CBE (cyclohexanol-t-butanol-ethanol) yields a difference spectrum similar to those obtained upon steady illumination of chlorophyll a-quinone mixtures in this solvent. Decay kinetics in CBE and dimethylsulfoxide are faster at the Soret band than at 460–580 nm and red band regions. This difference is not obtained in other solvents (CHCI₃, CCI₄, t-butanol, ethanol), implying that two or more species are obtained in CBE and DMSO. β-Carotene in CBE increases the rate of decay of the flash-induced chlorophyll transients at 430 and 660 nm but only decreases the magnitude of the signal at 470 nm. This implies that the 470 nm absorbance is due to a product formed from the triplet state. This effect is not observed in ethanol. Adding quinone to chlorophyll solutions results in slowly decaying species being generated by flash excitation in CBE. Three components can be distinguished: the first (t_1/2? 0.2 msec) corresponds to the triplet state; the second (t_1/2= 5–10 msec) is quinone concentration and species independent; the third (t_1/2= several seconds) is dependent upon quinone concentration and species (rate is faster for higher concentrations and lower potential quinones). The ESR signal decay rate is approximately equal to the third component flash decay rate when the chlorophyll and quinone concentrations are equal. With excess quinone, the flash decay rate becomes faster, and the ESR decay rate decreases slightly. These slowly-decaying species are not produced when quinone is added to chlorophyll a in ethanol or t-butanol, or to pheophytin in CBE. One observes merely a decrease in signal height with no accompanying increase in decay rate. Mechanisms to account for all of these phenomena are presented which involve an initial chlorophyll triplet-solvent reaction with the subsequent formation of several species of chloro-phyll-quinone radical complexes.     

MECHANISM OF THE PHOTOHYDRATION OF PYRIMIDINES: A FLASH PHOTOLYSIS STUDY*

Abstract— Based on comparisons between the microsecond flash photolysis of uracil and substituted uracils and the radiation chemistry of the corresponding 5,6-dihydro-2,4-dioxopyrimidines, it is proposed that the mechanism of photohydration of pyrimidine bases involves the formation of a pyrimidine carbocation. The effects of substitution in the pyrimidine ring and of pH are consistent with a proton transfer from water to the excited singlet state at theC–5 position of the pyrimidine ring. The resultant carbocation is thought to undergo solvolysis to form the photohydrate or eliminate a proton at N-l producing an intermediate isomeric form of the pyrimidine (isopyrimidine) which re-arranges to the parent pyrimidine by a first-order process.     

RED LIGHT-INDUCED REDUCTION OF A PARTICLE-ASSOCIATED b-TYPE CYTOCHROME FROM CORN IN THE PRESENCE OF METHYLENE BLUE*

Abstract— In the presence of methylene blue, red light causes the reduction of a h-type cytochrome in particulate fractions from corn coleoptiles. Two types of difference spectra for the cytochromes in these fractions are presented: (a) red light-minus-dark in the presence of methylene blue, and (b) dithionite-reduced-minus-oxidized. Comparison of these spectra shows that photoexcited methylene blue selectively reduces a b-type cytochrome which constitutes at most only 30% of the total dithionite-reducible cytochrome present in the most active fractions. The photoreducible cytochrome has an alpha band at room temperature near 557 nm. Bleaching of methylene blue precedes cytochrome reduction under appropriate conditions, suggesting that the photoreduced dye is donating an electron to the cytochrome. This electron transfer does not involve a flavin, at least as judged by the absence of light-induced spectral changes attributable to flavins. Preliminary kinetic studies suggest that EDTA provides the pool of electrons for the reaction. The cytochrome cannot be assigned exclusively either to mitochondria or to endoplasmic reticulum, as judged by its sedimentation properties. These results and the current literature are discussed in the context of the hypothesis that this b-type cytochrome may be involved in the photoreception mechanism for blue and uv light in vivo.     

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS–VIII. A SPIN-TRAPPING STUDY OF LIGHT INDUCED FREE RADICALS FROM CHLORPROMAZINE and PROMAZINE

Abstract— The clinically important phenothiazine drugs, particularly chlorpromazine, often elicit phototoxic and photoallergic reactions. We have used the spin traps 2-methyl-2-nitrosopropane (MNP) and 5,5-dimethyl-pyrroline-N-oxide (DMPO) to define the radical photolysis pathways of chlorpromazine and promazine. In the absence of oxygen the dechlorination product of chlorpromazine is trapped by MNP. The reactivity of the dechlorination product is similar to that of the phenyl radical as shown by its ability to extract hydrogen atoms from donors. Our results suggest that the dechlorination product is sufficiently reactive to account for the observation that chlorpromazine is more phototoxic than its parent promazine. In the presence of oxygen both chlorpromazine and promazine form a superoxide-dismutase-insensitive oxygen-centered intermediate which, when trapped by DMPO, rapidly decays to DMPO-OOH and subsequently to DMPO-OH. In addition, chlorpromazine readily undergoes photoelectron ejection only when it is excited into the second excited singlet state (Δ < 280 nra). This previously unknown wavelength dependence of photoionization should be considered in establishing the mechanism of chlorpromazine photosensitization.     

醇-二苯甲酮光化夺氢反应活泼自由基的ESR研究

本文用自由基捕捉剂2,3,4,6-四甲基亚硝基苯(ND)及苯亚甲基叔丁基氮氧化合物(PBN)与ESR相结合的方法研究了CnH_2n+1OH(n=1,2,…8)、(CH₃)₂CH(CH₂)_nOH(n=0,1,2)、CH₂=CHCH₂OH及C₆H₅CH₂OH等十三种醇与二苯甲酮的光化夺氢反应中的活泼自由基,结果表明: 1.用ND时,二苯酮分别夺取CnH_2n+1OH、(CH₃)₂CH(CH₂)_nOH及RCH₂OH(R=CH₂=CH、C₆H₅)中α-C、叔-C及α-C上的氢,而捕捉到C_n-1H_2n-1CHOH、(CH₃)₂CH(CH₂)_nOH及RCHOH自由基。 2.用PBN时,捕捉的自由基与ND捕获的相同。     

TRAPPING OF NITRIC OXIDE FORMED DURING PHOTOLYSIS OF SODIUM NITROPRUSSIDE IN AQUEOUS AND LIPID PHASES: AN ELECTRON SPIN RESONANCE STUDY

Abstract— Photolytic decomposition of sodium nitroprusside (SNP), a widely used nitrovasodilator, produced nitric oxide (NO), which was continuously monitored by electron spin resonance (ESR) spectroscopy. The NO present in the aqueous or the lipid phase was trapped by either a hydrophilic or a hydrophobic nitronyl nitroxide, respectively, to form the corresponding imino nitroxide. The conversion of nitronyl nitroxide to imino nitroxide was monitored by ESR spectrometry. The quantum yield for the generation of NO from SNP, measured from the rate of decay of nitronyl nitroxide, was 0.201 ± 0.007 and 0.324 ± 0.01 (¯± SD, n = 3) at 420 nm and 320 nm, respectively. The action spectrum for NO generation was found to overlap the optical absorption spectrum of SNP closely. A mechanism for the reaction between SNP and nitronyl nitroxide in the presence of light is proposed and computer-aided simulation of this mechanism using published rate constants agreed well with experimental data. The methodology described here may be used to assay NO production continuously during photoactivation of NO donors in aqueous and lipid environments. Biological implications of this methodology are discussed.     

四氯乙烯光解活泼自由基及其与烃或醇的夺氢反应的ESR研究

本文用自由基捕捉剂2,3,5,6-四甲基亚硝基苯(ND)与ESR相结合的方法研究了四氯乙烯光解活泼自由基及其与烃或醇的夺氢反应, 结果表明:1. Cl_2C=CCl_2光解首先产生Cl_2C=CCl及Cl, 而又可进一步加成为Cl_2C-CCl_3.2. 对于CH_3(CH_2)_nOH(n=3,7)及(CH_3)_2CH(CH_2)_nOH(n=1,2)而言, Cl_2C=CCl(或Cl)分别夺取其α-碳及叔碳上的氢, 以形成CH_3(CH_2)_(n-1)CHOH及(CH_3)_2C(CH_2)_nOH, 并为ND所捕获。3. Cl_2C=CCl(或Cl)分别夺取CH_3(CH_2)_nCH3(n=3,4,5,6), C_6H_5-CH_2CH_3及(CH_3)_2CH(CH_2)_nCH_3(n=1,4), (C_2H_5)_2CHCH_3, C_6H_5CH(CH_3)_2中亚甲基及叔碳上的氢, 以形成CH_3(CH_2)_mCH(CH_2)_(n-m-1)CH_3, C_6H_5CHCH_3及(CH_3)_2C(CH_2)_nCH_3, (C_2H_5)_2CCH_3, C_6H_5C(CH_3)_2.     

CONFIGURATIONS OF NEUROSPORENE ISOMERS ISOLATED FROM THE REACTION CENTER AND THE LIGHT-HARVESTING COMPLEX OF Rhodobacter spheroides G1C. A RESONANCE RAMAN, ELECTRONIC ABSORPTION, AND 1H-NMR STUDY

Abstract— HPLC analysis of neurosporene extracted from the chromatophore membranes of Rhodobacter spheroides G1C showed two isomeric components 1 and 2. Extract from the light-harvesting complex (LH) gave only component 2, while extract from the reaction center (RC) mainly gave component 1. Both components were isolated, and their configurations were determined by means of (1) resonance Raman, (2) electronic absorption, and (3) ¹H-HMR spectroscopy. The configuration of component 2 originating from LH was determined to be all -trans and that of component 1 from RC was determined to be 15- cis . Thus, our previous configurational prediction of this particular carotenoid bound to RC, by means of resonance Raman spectroscopy, to be 15-cis [Y. Koyama, T. Takii, K. Saiki and K. Tsukida, Photobiochem. Photobiophys. 5 (1983)139–150] is confirmed.     

PHOTOSENSITIZATION OF PYRIMIDINES BY 2-METHYLNAPHTHOQUINONE IN WATER: A LASER FLASH PHOTOLYSIS STUDY

2-Methylnaphthoquinone (MQ) has been excited in water with a 20 ns laser flash at 353 nm and the resultant transient species have been observed optically. Triplet-state MQ (³MQ) decays on a sub-microsecond time scale. It has been characterized in terms of its absorption spectrum and quantum yield. Rate constants have been measured for the decay of ³MQ in infinitely dilute solution, for self-quenching by ground-state MQ, and for reactions of ³MQ with oxygen, thymine, uracil, 6-methyluracil, and orotic acid. The interaction of ³MQ with pyrimidines involves charge transfer to give the pyrimidine cation radical and the MQ : anion radical. These reactions are discussed in relation to the mechanism of pyrimidine photooxidation sensitized by MQ.     

A PHYTOCHROME PHOTOTRANSFORMATION STUDY USING TWO-LASER/TWO-COLOR FLASH PHOTOLYSIS: ANALYSIS OF THE DECAY MECHANISM OF I700*

The mechanism of I₇₀₀ decay, representing an early event in the phytochrome P_r→ P_fr phototransformation, was reanalyzed in the microsecond range by conventional laser flash photolysis as well as by two-laser/two-color flash photolysis. Three kinetic models that might describe the I₇₀₀ decay mechanism following P_r excitation were considered: a parallel, a sequential, and an equilibrium model. These models were used to mathematically simulate both the one- and two-laser flash experiments in an effort to select the model best describing the I₇₀₀ decay. The sequential model could be excluded already on the basis of the one-laser flash photolysis results alone. Discussion of the two-laser/two-color flash rcsults in the context of the equilibrium and the parallel models is presented.