Singlet oxygen production in thylakoid membranes during photoinhibition as detected by EPR spectroscopy

Exposure of isolated spinach thylakoids to high intensity illumination (photoinhibition) results in the well-characterized impairment of Photosystem II electron transport, followed by degradation of the D1 reaction centre protein. In the present study we demonstrate that this process is accompanied by singlet oxygen production. Singlet oxygen was detected by EPR spectroscopy, following the formation of stable nitroxide radicals from the trapping of singlet oxygen with a sterically hindered amine TEMP (2,2,6,6-tetramethylpiperidine). There was no detectable singlet oxygen production during anaerob photoinhibition or in the presence of sodium-azide. Comparing the kinetics of the loss of PS II function and D1 protein with that of singlet oxygen trapping suggests that singlet oxygen itself or its radical product initiates the degradation of D1.Abbreviations HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonle acid - PS Photosystem - TEMP 2,2,6,6-tetramethylpiperidine - TEMPO 2,2,6,6-tetramethylpiperidine-1-oxyl     

Scope and limitations of the TEMPO/EPR method for singlet oxygen detection: the misleading role of electron transfer

For many biological and biomedical studies, it is essential to detect the production of ¹O₂ and quantify its production yield. Among the available methods, detection of the characteristic 1270-nm phosphorescence of singlet oxygen by time-resolved near-infrared (TRNIR) emission constitutes the most direct and unambiguous approach. An alternative indirect method is electron paramagnetic resonance (EPR) in combination with a singlet oxygen probe. This is based on the detection of the TEMPO free radical formed after oxidation of TEMP (2,2,6,6-tetramethylpiperidine) by singlet oxygen. Although the TEMPO/EPR method has been widely employed, it can produce misleading data. This is demonstrated by the present study, in which the quantum yields of singlet oxygen formation obtained by TRNIR emission and by the TEMPO/EPR method are compared for a set of well-known photosensitizers. The results reveal that the TEMPO/EPR method leads to significant overestimation of singlet oxygen yield when the singlet or triplet excited state of the photosensitizer is efficiently quenched by TEMP, acting as electron donor. In such case, generation of the TEMP⁺ radical cation, followed by deprotonation and reaction with molecular oxygen, gives rise to an EPR-detectable TEMPO signal that is not associated with singlet oxygen production. This knowledge is essential for an appropriate and error-free application of the TEMPO/EPR method in chemical, biological, and medical studies.     

Hydroxyl radical scavenging and singlet oxygen quenching properties of polyamines

Polyamines (cadaverine, putrescine, spermidine, spermine) have been shown to be present in all prokaryotic and eukaryotic cells, and proposed to be important anti-inflammatory agents. Some polyamines at high concentrations are known to scavenge superoxide radicals in vitro. We have investigated the possible antioxidant properties of polyamines and found that polyamines, e.g., cadaverine, putrescine, spermidine and spermine do not scavenge superoxide radicals at 0.5, 1.0 and 2 mM concentrations. However, polyamines were found to be potent scavengers of hydroxyl radicals. Hydroxyl radicals were produced in a Fenton type reaction and detected as DMPO-OH adducts by electron paramagnetic resonance spectroscopic technique. Spermine, spermidine, putrescine and cadaverine inhibited DMPO-OH adduct formation in a dose dependent manner, and at 1.5 mM concentration virtually eliminated the adduct formation. The *OH-dependent TBA reactive product of deoxyribose was also inhibited by polyamines in a dose-dependent manner. Polyamines were also found to inhibit the 1O2-dependent 2,2,6,6-tetramethylpiperidine N-oxy 1 (TEMPO) formation. 1O2 was produced in a photosensitizing system using Rose Bengal or Methylene Blue as photosensitizers, and was detected as TEMP-1O2 adduct by EPR spectroscopy. Spermine or spermidine inhibited the 1O2-dependent TEMPO formation maximally to 50%, whereas putrescine or cadaverine inhibited this reaction only up to 15%, when used at 0.5 and 1 mM concentrations. These results suggest that polyamines are powerful. OH scavengers, and spermine or spermidine also can quench singlet oxygen at higher concentrations.     

Vasoactive intestinal peptide, a singlet oxygen quencher

The neuropeptide vasoactive intestinal peptide (VIP), a highly basic 28-amino acid peptide, has a widespread distribution in the body. The functional specificity of this peptide not only includes its potent vasodilatory activity, but also its role in protecting lungs against acute injury, in preventing T-lymphocyte proliferation and in modulating immune function. We have investigated the possible antioxidant properties of VIP and found that VIP does not have significant O2-, OH., or H2O2 scavenging ability. However, VIP was found to inhibit, in a dose-dependent manner, the 1O2-dependent 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) formation. 1O2 was produced in photosensitizing systems using rose bengal or methylene blue as sensitizers and was detected as TEMP-1O2 product (TEMPO) by electron paramagnetic resonance (EPR) spectroscopic techniques. The formation of TEMPO signal was strongly inhibited by known singlet quenchers, e.g. beta-carotene, histidine as well as azide, but not by catalase (20 micrograms/ml) which removes H2O2 and mannitol (6 mM) or ethanol (5.9 mM) which remove OH.. Superoxide dismutase (2.5 micrograms/ml) inhibited the photoreaction up to 20% by removing O2- and most probably by blocking the secondary charge transfer pathway of 1O2 formation. These results suggest that the formation of nitroxide radical by 1O2 attack on TEMP may be used as a simple and specific assay for 1O2, and VIP can serve as an effective 1O2 scavenger/quencher, thus it may modulate the oxidative tissue injury caused by this reactive species of oxygen.     

Scavenging of photogenerated oxidative species by antimuscarinic drugs: atropine and derivatives

The quenching ability of photogenerated oxidative species by some antimuscarinic drugs generically named atropines (e.g. atropine [I] eucatropine [II], homatropine [III] and scopolamine [IV]) have been investigated employing stationary photolysis, polarographic detection of dissolved oxygen, stationary and time-resolved fluorescence spectroscopy, and laser flash photolysis. Using Rose Bengal as a dye sensitiser for singlet molecular oxygen, O(2)((1)Delta(g)), generation, compounds I-IV behave as moderate chemical plus physical quenchers of the oxidative species. Correlation between kinetic and electrochemical data indicates that the process is possibly driven by a charge-transfer interaction. The situation is somewhat more complicated employing the natural pigment riboflavin (Rf) as a sensitiser. Compounds I and II complex Rf ground state, diminishing the quenching ability towards singlet and triplet excited state of the pigment. On the other hand, compounds III and IV effectively quench Rf excited states, protecting the pigment against photodegradation. Under anaerobic conditions, semireduced Rf (Rf(.-)) is formed through quenching of excited triplet Rf. Nevertheless, although Rf(.-) is a well-known generator of the reactive species superoxide radical anion by reductive quenching in the presence of oxygen, the process of O(2)((1)Delta(g)) production prevails over superoxide radical generation, due to the relatively low rate constants for the quenching of triplet Rf by the atropines (in the order of 10(7) M(-1)s(-1) for compounds III and IV) in comparison to the rate constant for the quenching by ground state oxygen, approximately two orders of magnitude higher, yielding O(2)((1)Delta(g)). Compound I is the most promising O(2)((1)Delta(g)) physical scavenger, provided that it exhibits the higher value for the overall quenching rate constant and only 11% of the quenching process leads to its own chemical damage.     

Insulin-like growth factor I receptor is expressed at normal levels in Nijmegen breakage syndrome cells

Curcumin (diferuloylmethane) is a major component of food flavoring turmeric (Curcuma longa), and has been reported to be anticarcinogenic and anti-inflammatory. Although curcumin was shown to have antioxidant properties, its exact antioxidant nature has not been fully investigated. In this report we have investigated the possible antioxidant properties of curcumin using EPR spectroscopic techniques. Curcumin was found to inhibit the (1)O(2)-dependent 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) formation in a dose-dependent manner. (1)O(2) was produced in a photosensitizing system using rose bengal as sensitizer, and was detected as TEMP-(1)O(2) adducts by electron paramagnetic resonance (EPR) spectroscopic techniques using TEMP as a spin-trap. Curcumin at 2.75 microM caused 50% inhibition of TEMP-(1)O(2) adduct formation. However, curcumin only marginally inhibited (24% maximum at 80 microM) reduction of ferricytochrome c in a xanthine-xanthine oxidase system demonstrating that it is not an effective superoxide radical scavenger. Additionally, there was minor inhibition of DMPO-OH adduct formation by curcumin (solubilized in ethanol) when an ethanol control was included in the EPR spin-trapping study, suggesting that curcumin may not be an effective hydroxyl radical scavenger. Together these data demonstrate that curcumin is able only to effectively quench singlet oxygen at very low concentration in aqueous systems.     

Singlet oxygen generation from phosphatidylcholine hydroperoxide in the presence of copper

This study pursued whether singlet oxygen ((1)O2) is generated from phosphatidylcholine hydroperoxide (PCOOH), the oxidized modification product of a major constituent of biomembranes and serum lipoproteins. The (1)O2 formation was detected, by utilizing the oxidation of 2,2,6,6-tetramethyl-4-piperidone (TMPD) by (1)O2 to yield 2,2,6,6-tetramethyl-4-piperidone-1-oxyl (TEMPONE), which generates electron spin resonance (ESR) signals. The TEMPONE signal was detected in human plasma with addition of PCOOH by ESR determination after introducing copper(II). The TEMPONE formation was proportional to the amounts of PCOOH added according to moles of active oxygen. The TEMPONE signal intensity was weakened significantly in the presence of beta-carotene and histidine in a concentration-dependent manner, but was not at all decreased by mannitol, Mn-superoxide dismutase and catalase. In addition, HPLC-chemiluminescence analysis demonstrated that incubation with the PCOOH/Cu(II) combination oxidized cholesterol, a relatively oxidation-resistant component, to the cholesterol hydroperoxide. These results reveal that (1)O2 is generated from PCOOH in contact with copper(II). In conclusion, this in-vitro study provides directly the (1)O2 formation in living organisms following the advancement of peroxidation of constitutive lipids.     

Irradiation of titanium dioxide generates both singlet oxygen and superoxide anion.

Although photoexcited TiO2 has been known to initiate various chemical reactions, such as the generation of reactive oxygen species, precise mechanism and chemical nature of the generated species remain to be elucidated. The present work demonstrates the generation of singlet oxygen by irradiated TiO2 in ethanol as measured by ESR spectroscopy using 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TMP) as a 1O2-sensitive trapping agent. Under identical conditions, the superoxide ion was also detected by spin trapping agent 5,5-dimethyl-pyrroline-N-oxide (DMPO). Kinetic analysis in the presence of both 4-oxo-TMP and DMPO revealed that singlet oxygen is produced directly at the irradiated TiO2 surface but not by a successive reaction involving superoxide anion. The basis for this view is the fact that DMPO added in the mixture increased the signals responsible for 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO), a reaction product of 4-oxo-TMP and 1O2. The detailed mechanism for the generation of 1O2 and superoxide ion by irradiated TiO2 and reactions between these species and DMPO are discussed.     

Thioredoxin, a singlet oxygen quencher and hydroxyl radical scavenger: redox independent functions

Thioredoxin is a ubiquitous small protein known to protect cells and tissues against oxidative stress. However, its exact antioxidant nature has not been elucidated. In this report, we present evidence that human thioredoxin is a powerful singlet oxygen quencher and hydroxyl radical scavenger. Human thioredoxin at 3 microM caused 50% inhibition of TEMP-(1)O(2) (TEMPO) adduct formation in a photolysis EPR study. In contrast, Escherichia coli thioredoxin caused 50% inhibition of TEMPO formation at 80 microM. Both E. coli thioredoxin and human thioredoxin inhibited (*)OH dependent DMPO-OH formation as demonstrated by EPR spectrometry. The quenching of (1)O(2) or scavenging of (*)OH was not dependent upon the redox state of thioredoxin. Using a human thioredoxin in which the structural cysteines were mutated to alanine, Trx-C3A, we show that structural cysteines that do not take part in the catalytic functions of the protein are also important for its reactive oxygen scavenging properties. In addition, using a quadruple mutant Trx-C4A, where one of the catalytic cysteines, C35 was mutated to alanine in addition to the mutated structural cysteines, we demonstrated that catalytic cysteines are also required for the scavenging action of thioredoxin. Identification of thioredoxin as a (1)O(2) quencher and (*)OH scavenger may be of significant importance in explaining various redox-related antioxidant functions of thioredoxin.     

Triplet state characteristics and singlet oxygen generation properties of anthracyclines

The triplet states of adriamycin (Ad), daunomycin (D) and two daunomycin analogues, daunomycinone (Dc) and daunomycin N-trifluoroacetamide (DAc), have been studied using laser flash photolysis and pulse radiolysis techniques. Triplet lifetimes, molar absorption coefficients, energy levels and quantum yields have been obtained for Dc and DAc, and estimated for D and Ad. Time-resolved near-infrared singlet oxygen luminescence measurements have been carried out on D, Ad and 5-iminodaunomycin (5-ID) in 2H2O solution and Dc in benzene solution at room temperature. Singlet oxygen quenching by the water-soluble anthracyclines was observed and a second-order rate constant of approx. 10(8) M-1.s-1 obtained. Electron spin resonance experiments have demonstrated that D photoexcited at lambda less than or 365 nm gives rise to singlet oxygen as shown by its reaction with 2,2,6,6-tetramethyl-4-piperidone to give the corresponding nitroxyl radical. Although all the anthracyclines studied have the ability to photosensitize the formation of singlet oxygen, the quantum yields are very low (phi delta approximately 0.02-0.03), suggesting that these anthracyclines would be poor photodynamic sensitisers.     

Dark production of reactive oxygen species in photosystem II membrane particles at elevated temperature: EPR spin-trapping study

In our study, EPR spin-trapping technique was employed to study dark production of two reactive oxygen species, hydroxyl radicals (OH.) and singlet oxygen ((1)O2), in spinach photosystem II (PSII) membrane particles exposed to elevated temperature (47 degrees C). Production of OH., evaluated as EMPO-OH adduct EPR signal, was suppressed by the enzymatic removal of hydrogen peroxide and by the addition of iron chelator desferal, whereas externally added hydrogen peroxide enhanced OH. production. These observations reveal that OH. is presumably produced by metal-mediated reduction of hydrogen peroxide in a Fenton-type reaction. Increase in pH above physiological values significantly stimulated the formation of OH., whereas the presence of chloride and calcium ions had the opposite effect. Based on our results it is proposed that the formation of OH. is linked to the thermal disassembly of water-splitting manganese complex on PSII donor side. Singlet oxygen production, followed as the formation of nitroxyl radical TEMPO, was not affected by OH. scavengers. This finding indicates that the production of these two species was independent and that the production of (1)O2 is not closely linked to PSII donor side.     

Anti-oxidant and pro-oxidant behaviour of bucillamine.

Bucillamine (BUC) is used clinically for the treatment of rheumatoid arthritis. Some of the pharmacological action of BUC has been reported as being dependent on the production of reactive oxygen species (ROS). In this paper the reactivity of BUC with superoxide anion radical (O(2) (*-)) generated from potassium superoxide/18-crown-6 ether dissolved in DMSO, hydroxyl radical (HO(*)) produced in the Cu(2+)-H(2)O(2) reaction, peroxyl radical (ROO(*)) from 2,2'-azobis (2-amidino-propane) dichloride decomposition, and singlet oxygen ((1)O(2)) from a mixture of alkaline aqueous H(2)O(2) and acetonitrile, have been investigated. Chemiluminescence, fluorescence, electron paramagnetic resonance (EPR) spin-trapping techniques and the deoxyribose and oxygen radical absorbance capacity towards ROO(*) (ORAC(ROO)) assays were used to elucidate the anti- and pro-oxidative behaviours of BUC towards ROS. The results indicated that BUC efficiently inhibited chemiluminescence from the O(2) (*-)-generating system at relatively high concentrations (0.5-2 mmol/L); however, at lower concentrations (<0.5 mmol/L) the drug enhanced light emission. The behaviour of BUC was correlated with a capacity to decrease the chemiluminescence signal from the Cu(2+)-H(2)O(2) system; scavenging HO(*) was effective only at high concentrations (1-2 mmol/L) of the drug. Bucillamine also prevented deoxyribose degradation induced by HO(*) in a dose-dependent manner, reaching maximal inhibition (24.5%) at a relative high concentration (1.54 mmol/L). Moreover, BUC reacts with ROO(*); the relative ORAC(ROO) was found to be 0.34 micromol/L Trolox equivalents/micromol sample. The drug showed quenching of (1)O(2)-dependent 2,2,6,6-tetramethylpiperidine-N-oxide radical formation from 2,2,6,6-tetramethyl-piperidine (e.g. 90% inhibition was found at 1 mmol/L concentration). The results showed that BUC may directly scavenge ROS or inhibit reactions generating them. However, the drug may have pro-oxidant activity under some reaction conditions.     

Superoxide, hydrogen peroxide and singlet oxygen in hematoporphyrin derivative-cysteine, -NADH and -light systems

Hematoporphyrin derivative and light in the presence of cysteine or glutathione were found to convert oxygen to superoxide and hydrogen peroxide at pH less than approx. 6.5, while at pH greater than 6.5 no superoxide or hydrogen peroxide production was observed. However, at pH values greater than 6.5 the rate of oxygen consumption increased. This rate paralleled the acid dissociation curve of the cysteine thiol group and is consistent with the chemical quenching of 1O2 by cysteine. The superoxide and hydrogen peroxide formation observed below pH 6.5 appeared not to be related to the singlet oxygen production of hematoporphyrin derivative. In addition, superoxide and hydrogen peroxide production was observed with hematoporphyrin derivative and light in the presence of NADH, both above and below pH 6.5. Direct detection of singlet oxygen luminescence at 1268 nm in the hematoporphyrin derivative-light system (2H2O as solvent) revealed an apparent linear increase in the singlet oxygen emission intensity as the p2H was raised from 7.0 to 10.0. Azide efficiently quenched this observed emission. In addition, at p2H 7.4, 1 mM cysteine resulted in a 40% reduction of the singlet oxygen luminescence, while at p2H 9.4 the signal was quenched by over 95% (under the experimental conditions employed). In total, we interpret these results as consistent with the chemical quenching of 1O2 by the ionized thiol group of cysteine.     

Generation of reactive oxygen species by photolysis of the ruthenium(II) complex Ru(NH3)5(pyrazine)2+ in oxygenated solution.

Metal-to-ligand charge transfer photolysis of the ruthenium(II) pyrazine complex Ru(NH3)5pz2+ (I) in pH 7.4 oxygenated phosphate buffer solution generates the Ru(III) analog Ru(NH3)5pz3+ plus the reactive oxygen species singlet oxygen and superoxide. Based on the very short MLCT lifetime (re-measured as approximately 250 ps in D2O) of I* and the quantum yield for singlet oxygen formation (0.01 for aerated D2O) the rate constant for oxygen quenching of I* was calculated to be approximately (3+/-1)x10(10) M-1 s-1.     

UVB photoprotective role of mycosporines in yeast: photostability and antioxidant activity of mycosporine-glutaminol-glucoside

Several yeast species are able to synthesize and accumulate UV-radiation-absorbing mycosporine metabolites that are of unclear physiological function. In this work we analyzed the relationship between mycosporine-glutaminol glucoside (MGG) production, cell survival after UVB irradiation, and formation of cyclobutane pyrimidine dimers (CPDs). We also assessed the photostability and singlet oxygen quenching activity of MGG. A set of nine isolates of the basidiomycetous yeast Cryptococcus steppossus cultured in both dark and light conditions was used for the studies. Survival of the UVB-irradiated isolates and MGG concentration had a linear relationship when the concentration was over 2.5 mg g(-1). CPD accumulation and MGG accumulation were inversely related. MGG in aqueous solution was photostable with a photodecomposition quantum yield of 1.16 × 10(-5). MGG quenching of singlet oxygen was also observed, and the rate constant for the process in D(2)O was 5.9 × 10(7) M(-1) s(-1). Our results support the idea that MGG plays an important role as a UVB photoprotective metabolite in yeasts by protecting against direct damage on DNA and probably against indirect damage by singlet oxygen quenching.     

In vitro photodynamic effects of phthalocyaninatosilicon covalently linked to 2,2,6,6-tetramethyl-1-piperidinyloxy radicals on cancer cells

In this paper, we have investigated the ability to sensitize the phototoxicity toward HeLa cells in vitro, of tetra-tert-butylphthalocyaninatosilicon (SiPc) covalently linked to one or two 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) radicals (R1c or R2c), which are shown as photosensitizers efficiently producing singlet oxygen (1Delta(g)). Addition of R1c or R2c encapsulated in liposomes to cultures, followed by irradiation with a 680-nm dye laser, resulted in a highly significant phototoxicity toward HeLa cells, in contrast to negligible phototoxicity observed with (dihydroxy)SiPc (R0). EPR measurements indicate that R1c and R2c exist in some degree as nitroxide radicals even in HeLa cells. Electronic absorption spectra indicate that the degree of aggregation increases in the order R2c相似文献   

MCLA为化学发光探针测定氧化低密度脂蛋白中维生素C诱导的单线态氧产生

以一种海萤荧光素类似物MCLA〔2 methyl 6 (p methoxyphenyl) 3,7 dihydroimidazo [1,2 a]pyrazin 3 one〕作为高灵敏且有选择性的化学发光探针 ,用化学发光的方法直接观测到了少量Cu2 氧化的低密度脂蛋白 (Ox LDL)中维生素C诱导的单线态氧 (1O2 )的产生。实验中通过叠氮化钠 (NaN3 )对MCLA介导的化学发光的猝灭作用进一步证实了上述体系中1O2的形成。根据实验观察的结果 ,分析了这一体系中1O2 形成的可能途径 ,认为首先是维生素C将Cu2 转变为还原态 ,而自身失去一个电子转变为维生素C自由基 ,从而刺激了过氧自由基和烷氧自由基的形成 ,过氧自由基的双分子反应很可能就是体系内1O2 产生的反应机制     

Effect of ultrasound and ionizing radiation on a sterically hindered cyclic secondary amine: an ESR study.

The possible use of 2,2,6,6-tetramethyl-4-piperidone (TMPone) for the detection of singlet oxygen was investigated by gamma radiolysis and sonolysis of oxygen-saturated aqueous solutions. Formation of 2,2,6,6-tetra-methyl-4-piperidone-N-oxyl (TAN) was observed with both gamma radiolysis and sonolysis with a similar dependence on the concentration of TMPone up to 20 mM and a strong dependence on pH. In oxygen-saturated solutions the sonolysis of TMPone leads to the formation of the cyclic hydroxylamine (approx. 30% of the yield of TAN) while radiolysis does not. In the low pH range (5-6.5) and at high concentrations of OH radical scavengers (azide or formate), TAN is produced by sonolysis but not by radiolysis. Sonolysis of argon-saturated solutions of TMPone produces methyl radicals due to the high-temperature regions of the collapsing cavitation bubbles. The methyl radicals were detected by ESR (electron spin resonance) and spin trapping with 3,5-dibromo-2,6-dideuterio-4-nitroso-benzene sulfonate. Since the reaction of singlet oxygen with TMPone is also strongly dependent on pH, it does not seem likely that TMPone could be used for the detection of singlet oxygen in sonochemistry.     

Studies on the singlet oxygen scavenging mechanism of human macular pigment

It is thought that direct quenching of singlet oxygen and scavenging free radicals by macular pigment carotenoids is a major mechanism for their beneficial effects against light-induced oxidative stress. Corresponding data from human tissue remains unavailable, however. In the studies reported here, electron paramagnetic resonance (EPR) spectroscopy was used to measure light-induced singlet oxygen generation in post-mortem human macula and retinal pigment epithelium/choroid (RPE/choroid). Under white-light illumination, production of singlet oxygen was detected in RPE/choroid but not in macular tissue, and we show that exogenously added macular carotenoids can quench RPE/choroid singlet oxygen. When the singlet oxygen quenching ability of the macular carotenoids was investigated in solution, it was shown that a mixture of meso-zeaxanthin, zeaxanthin, and lutein in a ratio of 1:1:1 can quench more singlet oxygen than the individual carotenoids at the same total concentration.     

Generation of reactive oxygen species upon strong visible light irradiation of isolated phycobilisomes from Synechocystis PCC 6803

The mechanism of photodegradation of antenna system in cyanobacteria was investigated using spin trapping ESR spectroscopy, SDS-PAGE and HPLC-MS. Exposure of isolated intact phycobilisomes to illumination with strong white light (3500 micromol m(-2) s(-1) photosynthetically active radiation) gave rise to the formation of free radicals, which subsequently led to specific protein degradation as a consequence of reactive oxygen species-induced cleavage of the polypeptide backbone. The use of specific scavengers demonstrated an initial formation of both singlet oxygen (1O2) and superoxide (O2(-)), most likely after direct reaction of molecular oxygen with the triplet state of phycobiliproteins, generated from intersystem crossing of the excited singlet state. In a second phase carbon-based radicals, detected through the appearance of DMPO-R adducts, were produced either via O2(-) or by direct 1O2 attack on amino acid moieties. Thus photo-induced degradation of intact phycobilisomes in cyanobacteria occurs through a complex process with two independent routes leading to protein damage: one involving superoxide and the other singlet oxygen. This is in contrast to the mechanism found in plants, where damage to the light-harvesting complex proteins has been shown to be mediated entirely by 1O2 generation.