Triplet state of 4-methoxybenzyl alcohol chemisorbed on silica nanoparticles

The knowledge of photochemical kinetics in colloidal systems is important in understanding environmental photochemistry on dispersed solid surfaces. As model materials for the chemically sorbed organic compounds present in natural environments, modified silica nanoparticles (NPs) were obtained here by condensation of the silanol groups of fumed silica nanoparticles with 4-methoxybenzyl alcohol. These particles were characterized by different techniques. To evaluate their toxicity, the inhibition of the natural luminescence emission of the marine bacterium Vibrio fischeri in suspensions of the particles was measured. Laser flash-photolysis experiments (λ(exc) = 266 nm) performed with NP suspensions in acetonitrile-aqueous phosphate buffer mixtures showed the formation of the lowest triplet excited state of the chemisorbed organic groups (λ(max) = 390 nm). DFT calculations of the absorption spectrum of this radical support the assignment. From the calculated triplet energy, a thermodynamically favorable energy transfer from these triplet states to oxygen to yield singlet molecular oxygen is predicted. A value of 0.09 was measured for the quantum yield of singlet molecular oxygen generation by air-saturated suspensions of the nanoparticles in the mixture of solvents acetonitrile-aqueous phosphate buffer. The quantum yield of singlet molecular oxygen generation by the free 4-methoxybenzyl alcohol in the same solvent is 0.31.     

Synthesis and EPR investigations of new aminated hypocrellin derivatives

Hypocrellins are novel photodynamic agents. A recent advance in the synthesis of hypocrellin congeners results in the production of two amino-substituted hypocrellin B derivatives in high yield via photochemical reaction. Both compounds exhibit similar photodynamic activity as hypocrellin B in terms of type I and type II mechanisms. In anaerobic media, semiquinone anion radicals can be detected by electron paramagnetic resonance (EPR) under irradiation; while superoxide anion radical, hydroxyl radical and singlet oxygen are photoproduced when oxygen was present. The quantum yields of singlet oxygen by these two new compounds are determined to be 0.72 and 0.64, respectively, similar to that of hypocrellin B. The comparison of the photosensitization chemistry of compounds 1 and 2 in liposomes with that in homogeneous solution has also been made. In liposomes, the type II photoprocess was favored and predominant over the type I photoprocess due to the decreased interactions between dye molecules. Both compounds exhibit much stronger red light absorption than the parent hypocrellin and therefore, merit investigation as photosensitizers.     

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.     

Photophysical properties of a photocytotoxic fluorinated chlorin conjugated to four beta-cyclodextrins

A meso-tetrakis(pentafluorophenyl)-chlorin with the reduced pyrrole ring linked to an isoxazolidine ring (FC) has been conjugated to four beta-cyclodextrins (CDFC). The CDFC exhibits excellent water solubility and is a potent photosensitizer towards proliferating NCTC 2544 human keratinocytes. The study by conventional steady state absorption and fluorescence spectroscopies and by time-resolved femto- and nanosecond laser flash spectroscopies suggests that in ethanol and pH 7 buffer the beta-cyclodextrins embed the highly hydrophobic tetrakis(pentafluorophenyl)-chlorin macrocycle and strongly interact with the chlorin rings in the singlet and triplet manifolds. In these solvents, femtosecond spectroscopy suggests that the conjugate undergoes a rapid relaxation in the upper excited singlet states induced by photochemical and/or conformation change(s) at a rate of about 5 ps(-1) to fluorescent states whose lifetime is approximately 8 ns. This interaction is destroyed upon addition of Triton X100 to buffer. Both FC and CDFC strongly fluoresce (Phi(F) approximately 0.5) in micelles. Similar behavior is observed at the triplet level. In ethanol and water, the initial transient triplet state absorbance decays within 1-3 mus yielding a longer lived triplet with spectral properties indistinguishable from that of original difference absorbance spectra. The determination of the molar absorbance in the 440-460 nm region ( approximately 35 000 M(-1) cm(-1)) leads to an estimate of approximately 0.2 for the triplet formation quantum yield of FC in toluene and of FC and CDFC in Triton X100 micelles. Quenching of the CDFC triplets by dioxygen in buffer produces (1)O(2) in a good yield consistent with the effective photocytotoxicity of the chlorin-cyclodextrins conjugate towards cultured NCTC 2544 human keratinocytes. By contrast, FC which aggregates in buffer produces little if any (1)O(2).     

A possible mechanism of the generation of singlet molecular oxygen in nadph-dependent microsomal lipid peroxidation.

A simplified system, consisting of NADPH, Fe3+-ADP, EDTA, liposomes, NADPH-cytochrome c reductase and Tris - HCl buffer (pH 6.8), has been employed in studies of the generation of singlet oxygen in NADPH-dependent microsomal lipid peroxidation. The light emitted by the system involves 1deltag type molecular oxygen identifiable by its characteristic emission spectrum and its behavior with beta-carotene. The generation of another excited species (a compound in the triplet state) could be demonstrated in this system by changes of light intensity and emission spectra which arise from photosensitizer (9,10-dibromoanthracene sulfonate, eosin, Rose-Bengal)-mediated energy transfers. Chemiluminescence in the visible region was markedly quenched by various radical trappers and by an inhibitor of NADPH-cytochrome c reductase, but not by superoxide dismutase. During the early stage of lipid peroxidation, the intensity of chemiluminescence was proportional to the square of the concentration of lipid peroxide. These characteristics suggest that singlet oxygen and a compound in the triplet state (probably a carbonyl compound) are generated by a self-reaction of lipid peroxy radicals.     

Antibody-targeted photolysis: in vitro immunological, photophysical, and cytotoxic properties of monoclonal antibody-dextran-Sn(IV) chlorin e6 immunoconjugates.

A set of anti-melanoma immunoconjugates were prepared which contained chlorin e6: antibody molar ratios of 18.9:1, 11.2:1, 6.8:1, and 1.7:1. All immunoconjugates retained antigen binding activity regardless of the chromophore:antibody substitution ratio that was attained. In contrast, the ground-state absorption spectra of the immunoconjugates showed features which appeared to be dependent on the chromophore:antibody molar ratio. In addition, the quantum yield of singlet oxygen generated by the conjugated chromophores was observed to be significantly less than that observed with the unbound dye. Time-resolved absorbance spectroscopy of the chromophore excited triplet state indicated that the loss of singlet oxygen quantum yield resulted from diminished chromophore triplet yield. Analysis of data obtained from in vitro photolysis of target melanoma cells, in combination with that obtained from the immunochemical and photochemical studies, indicates that the observed immunoconjugate phototoxicity can be reasonably quantitatively represented by (1) the ability of the immunoconjugate to bind SK-MEL-2 cell surface antigen, (2) the amount of chromophore localized at the target cells by immunoconjugate binding, (3) the delivered dose of light at 634 nm, and (4) the singlet oxygen quantum yield of the antibody-bound photosensitizer. Though these data argue strongly for photolysis by the cumulative dosage of singlet oxygen at the cell membrane, nonetheless, the concurrent photoinduced release of other cytotoxic agents should not be ruled out.     

Photophysical properties of non-homoconjugated 1,2-dihydro, 1,2,3,4-tetrahydro and 1,2,3,4,5,6-hexahydro-C60 derivatives.

The photophysical properties of a novel series of non-homoconjugated 1,2-di-, 1,2,3,4-tetra-, and 1,2,3,4,5,6-hexasubstituted fullerenes (compounds 1, 2, and 3, respectively) have been systematically investigated. In this report, we examine the effect of substitution pattern of non-homoconjugated derivatized fullerenes on the ground state UV-Vis absorption, triplet state properties (lifetime, quantum yield, extinction coefficient), and singlet oxygen quantum yield. The non-homoconjugated fullerene derivatives 1-3 exhibit higher singlet oxygen quantum yield than analogous homoconjugated Bingel adducts with the same number of saturated C[double bond, length as m-dash]C bonds and exhibit decreasing quantum yield of singlet oxygen generation upon increasing the degree of functionalization on a single six member ring on the fullerene cage. This trend is similar for triplet quantum yield and triplet lifetime. The triplet extinction coefficient increases with functionalization. A detailed discussion comparing 1, 2, and 3 with functionalized homoconjugated systems and with other non-homoconjugated derivatives is presented.     

Production of singlet oxygen-derived hydroxyl radical adducts during merocyanine-540-mediated photosensitization: analysis by ESR-spin trapping and HPLC with electrochemical detection.

Activated oxygen species produced during merocyanine 540 (MC540)-mediated photosensitization have been examined by electron spin resonance (ESR) spin trapping and by trapping reactive intermediates with salicylic acid using HPLC with electrochemical detection (HPLC-EC) for product analysis. Visible light irradiation of MC540 associated with dilauroylphosphatidylcholine liposomes in the presence of the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave an ESR spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO/.OH). Addition of ethanol or methanol produced additional hyperfine splittings due to the respective hydroxyalkyl radical adducts, indicating the presence of free.OH.DMPO/.OH formation was not significantly inhibited by Desferal, catalase, or superoxide dismutase (SOD). Production of DMPO/.OH was strongly inhibited by azide and enhanced in samples prepared with deuterated phosphate buffer (PB-D2O), suggesting that singlet molecular oxygen (1O2) was an important intermediate. When MC540-treated liposomes were irradiated in the presence of salicylic acid (SA), HPLC-EC analysis indicated almost exclusive formation of 2,5-dihydroxybenzoic acid (2,5-DHBA), with production of very little 2,3-DHBA, in contrast to .OH generated by uv photolysis of H2O2, which gave nearly equimolar amounts of the two products. 2,5-DHBA production was enhanced in PB-D2O and inhibited by azide, again consistent with 1O2 intermediacy. 2,5-DHBA formation was significantly reduced in samples saturated with N2 or argon, and such samples showed no D2O enhancement. Ethanol had no effect on 2,5-DHBA production, even when present in large excess. Catalase and SOD also had no effect, and only a small inhibition was observed with Desferal. DMPO inhibited 2,5-DHBA production in a concentration-dependent fashion and enhanced formation of 2,3-DHBA. We propose that 1O2 reacts with DMPO to give an intermediate which decays to form DMPO/.OH and free.OH, and that the reaction between 1O2 and SA preferentially forms the 2,5-DHBA isomer. This latter process may provide the basis for a sensitive analytical method to detect 1O2 intermediacy. Singlet oxygen appears to be the principle activated oxygen species produced during MC540-mediated photosensitization.     

Characterization of the transient species generated by the photoexcitation of C-phycocyanin from Spirulina platensis: a laser photolysis and pulse radiolysis study

Nanosecond laser flash photolysis and pulse radiolysis were used to generate and characterize the triplet state and cation radical of C-phycocyanin (C-PC) from Spirulina platensis. The transient absorption spectra of C-PC were measured from direct excitation and acetone sensitization in aqueous solution at room temperature by KrF (248 nm) laser flash photolysis. Laser-induced transient species have been characterized by the method of acetone sensitization and one-electron oxidation. In nitrous oxide-saturated phosphate buffer saline (pH = 7.0) of C-PC, the produced intermediates are assigned to the excited triplet state and the radical cation. Using acetone as photosensitizer, the C-PC excited triplet states produced via triplet-triplet energy transfer and the C-PC radical cation from electron transfer reaction were further confirmed. Furthermore, the corresponding kinetic parameters were determined. To our knowledge, the transient absorption spectra of C-PC have been reported for the first time.     

A possible mechanism of the generation of singlet molecular oxygen in NADPH-dependent microsomal lipid peroxidation

A simplified system, consisting of NADPH, Fe³⁺-ADP, EDTA, liposomes, NADPH-cytochrome c reductase and Tris · HCl buffer (pH 6.8), has been employed in studies of the generation of singlet oxygen in NADPH-dependent microsomal lipid peroxidation.The light emitted by the system involves ¹Δg type molecular oxygen identifiable by its characteristic emission spectrum and its behavior with β-carotene. The generation of another excited species (a compound in the triplet state) could be demonstrated in this system by changes of light intensity and emission spectra which arise from photosensitizer (9, 10-dibromoanthracene sulfonate, eosin, Rose-Bengal)-mediated energy transfers.Chemiluminescence in the visible region was markedly quenched by various radical trappers and by an inhibitor of NADPH-cytochrome c reductase, but not by superoxide dismutase. During the early stage of lipid peroxidation, the intensity of chemiluminescence was proportional to the square of the concentration of lipid peroxide.These characteristics suggest that singlet oxygen and a compound in the triplet state (probably a carbonyl compound) are generated by a self-reaction of lipid peroxy radicals.     

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.     

Primary steps of the photochemical reactions of 2-cyano-10-(3-[dimethylamino, N-oxide]-2-methylpropyl)-5-oxide-phenothiazine, the photoproduct of cyamemazine, a phototoxic neuroleptic: comparison with the sulfoxide.

The UVA-absorbing photoproduct resulting from the oxidation of the sulfur atom and of the side chain nitrogen of the phototoxic drug cyamemazine (CMZ) (2-cyano-10-(3-[dimethylamino]-2 methylpropyl)-phenothiazine) is a potent photodynamic photosensitizer. The photophysical and photochemical properties of this photoproduct (P) (2-cyano-10-(3-[dimethylamino, N-oxide]-2-methylpropyl)-5-oxide-phenothiazine)) have been investigated in neutral buffered aqueous solutions and in ethanol and compared to those of the sulfoxide (S) (2-cyano-10-(3-[dimethylamino]-2 methylpropyl)-5-oxide-phenothiazine), a CMZ oxidation product of cells. The fluorescence quantum yield (PhiF) of P is 0.25 and 0.21 in pH 7 phosphate buffer and ethanol, respectively. By contrast, S (PhiF = 0.14 in buffer) is practically unfluorescent in alcohol. In buffer, the fluorescence lifetimes of P and S are 10.5 and 11.8 ns, respectively. The transient absorbance of the first excited triplet state (3P1) with a characteristic absorption band peaking at 660 nm (epsilon = 5,300 M(-1) cm(-1)) has been observed by 355 nm laser flash spectroscopy of deaerated phosphate buffer or ethanol solutions. In buffer, the 3P1 lifetime is 0.5 micros. The energy transfer which occurs from the 3P1 to naproxen suggests that the 3P1 energy is greater than 62 kcal mol(-1). Triplet quenching by dioxygen occurs at rate 2.3 x 10(9) M(-1) s(-1). With the triplet benzophenone as actinometer, the 3P1 formation quantum yield is found to be 0. 40 in buffer. The 3P1 state is quenched by ethanol and 2-propanol with bimolecular reaction rate constants of 1.6 and 2.4 x 10(6) M(-1) s(-1), respectively. In buffer, P and S triplet states react with tryptophan, indole and cysteine at rate constants of the order of 10(9) M(-1) s(-1) for Trp and indole and 10(8) M(-1) s(-1) for Cys.     

Photogeneration of singlet oxygen (1O2) and free radicals (Sen·-, O·-2) by tetra-brominated hypocrellin B derivative

To improve photodynamic activity of the parent hypocrellin B (HB), a tetra-brominated HB derivative (compound 1) was synthesized in high yield. Compared with HB, compound 1 has enhanced red absorption and high molar extinction coefficients. The photodynamic action of compound 1, especially the generation mechanism and efficiencies of active species (Sen^·-, O^·-₂ and ¹O₂) were studied using electron paramagnetic resonance (EPR) and spectrophotometric methods. In the deoxygenated DMSO solution of compound 1, the semiquinone anion radical of compound 1 is photogenerated via the self-electron transfer between the excited and ground state species. The presence of electron donor significantly promotes the reduction of compound 1. When oxygen is present, superoxide anion radical (O^·-₂) is formed via the electron transfer from Sens^·- to the ground state molecular oxygen. The efficiencies of Sens^·- and O^·-₂ generation by compound 1 are about three and two times as much as that of HB, respectively. Singlet oxygen (¹O₂) can be produced via the energy transfer from triplet compound 1 to ground state oxygen molecules. The quantum yield of singlet oxygen (¹O₂) is 0.54 in CHCl₃ similar to that of HB. Furthermore, it was found that the accumulation of Sens^·- would replace that of O^·-₂ or ¹O₂ with the depletion of oxygen in the sealed system.     

Sulfate anion free radical formation by the peroxidation of (Bi)sulfite and its reaction with hydroxyl radical scavengers

The one-electron oxidation of (bi)sulfite is catalyzed by peroxidases to yield the sulfur trioxide radical anion (SO3-), a predominantly sulfur-centered radical as shown by studies with 33S-labeled (bi)sulfite. This radical reacts with molecular oxygen to form a peroxyl radical. The subsequent reaction of this peroxyl radical with (bi)sulfite has been proposed to form the sulfate anion radical, which is nearly as strong an oxidant as the hydroxyl radical. We used the spin trapping electron spin resonance technique to provide for the first time direct evidence for sulfate anion radical formation during (bi)sulfite peroxidation. The sulfate anion radical is known to react with many compounds more commonly thought of as hydroxyl radical scavengers such as formate and ethanol. Free radicals derived from these scavengers are trapped in systems where (bi)sulfite peroxidation has been inhibited by these scavengers.     

Singlet oxygen generation from the decomposition of alpha-linolenic acid hydroperoxide by cytochrome c and lactoperoxidase

Generation of singlet oxygen is first investigated in the decomposition of polyunsaturated lipid peroxide, alpha-linolenic acid hydroperoxide (LAOOH), by heme-proteins such as cytochrome c and lactoperoxidase. Chemiluminescence and electron spin resonance methods are used to confirm the singlet oxygen generation and quantify its yield. Decomposition products of LAOOH are characterized by HPLC-ESI-MS, which suggests that singlet oxygen is produced via the decomposition of a linear tetraoxide intermediate (Russell's mechanism). Free radicals formed in the decomposition are also identified by the electron spin resonance technique, and the results show that peroxyl, alkyl, and epoxyalkyl radicals are involved. The changes of cytochrome c and lactoperoxidase in the reaction are monitored by UV-visible spectroscopy, revealing the action of a monoelectronic and two-electronic oxidation for cytochrome c and lactoperoxidase, respectively. These results suggest that cytochrome c causes a homolytic reaction of LAOOH, generating alkoxyl radical and then peroxyl radical, which in turn releases singlet oxygen following the Russell mechanism, whereas lactoperoxidase leads to a heterolytic reaction of LAOOH, and the resulting ferryl porphyryl radical of lactoperoxidase abstracts the hydrogen atom from LAOOH to give peroxyl radical and then singlet oxygen. This observation would be important for a better understanding of the damage mechanism of cell membrane or lipoprotein by singlet oxygen and various radicals generated in the peroxidation and decomposition of lipids induced by heme-proteins.     

Photoprocesses of chlorin e6 glucose derivatives

The ground and excited state processes of chlorin e6, the monomethyl ester C1, the glucose derivative C2 and the 3-heptylchlorin-glucose C3, were studied in solvents of lower and higher polarity. The excited singlet and lowest triplet states of C1-C3 were characterized by spectroscopic methods for several conditions. The quantum yields of formation of singlet molecular oxygen and the other triplet properties of the three chlorins and chlorin e6 are similar, whereas the fluorescence quantum yield decreases on going from C1 to C3. Time-resolved optoacoustic experiments revealed a ca. 30 kJ mol(-1) higher triplet level for C3 with respect to C1/2.     

Combined effects of singlet oxygen and hydroxyl radical in photodynamic therapy with photostable bacteriochlorins: evidence from intracellular fluorescence and increased photodynamic efficacy in vitro

Sulfonamides of halogenated bacteriochlorins bearing Cl or F substituents in the ortho positions of the phenyl rings have adequate properties for photodynamic therapy, including strong absorption in the near-infrared (λ(max) ≈ 750 nm, ε ≈ 10(5) M(-1) cm(-1)), controlled photodecomposition, large cellular uptake, intracellular localization in the endoplasmic reticulum, low cytotoxicity, and high phototoxicity against A549 and S91 cells. The roles of type I and type II photochemical processes are assessed by singlet oxygen luminescence and intracellular hydroxyl radical detection. Phototoxicity of halogenated sulfonamide bacteriochlorins does not correlate with singlet oxygen quantum yields and must be mediated both by electron transfer (superoxide ion, hydroxyl radicals) and by energy transfer (singlet oxygen). The photodynamic efficacy is enhanced when cellular death is induced by both singlet oxygen and hydroxyl radicals.     

Melatonin prevents the increase in hydroxyl radical-spin trap adduct formation caused by the addition of cisplatin in vitro

We studied the generation of reactive oxygen species (ROS) caused by cisplatin administration and the preventive effect of melatonin, the main secretory product of the pineal gland, on the reaction in vitro using electron spin resonance spectroscopy. Cisplatin induced generation of the hydroxyl radical (OH*) in phosphate buffer in pH 7.4 as a dose-dependent manner. However, OH* was not generated in phosphate buffer containing chloride ions at concentration exceeding 120 mM. The induction of OH. production by cisplatin was completely inhibited by the addition of melatonin, but not by the addition of 6-hydroxymelatonin, which is a hepatic metabolite of melatonin. Furthermore, melatonin was the most effective agent for preventing of OH* formation among various well-known antioxidants including mannitol and reduced glutathione. These results indicate that melatonin may scavenge OH. directly and thereby prevent renal tissue damage caused by OH* produced in response to cisplatin treatment.     

Dual role of triplet localization on the accessory chlorophyll in the photosystem II reaction center: photoprotection and photodamage of the D1 protein

Infrared absorption and electron spin resonance studies have shown that the excited triplet state of chlorophyll formed by radical pair recombination in the PSII reaction center is mainly localized on the accessory chlorophyll, which is most probably located in the D1 protein (Chl(1)). This triplet localization plays two contrasting roles, depending on the redox state of Q(A), in the process of acceptor-side photoinhibition of PSII. In the early stage of photoinhibition, in which singly reduced Q(A) is reversibly stabilized, the triplet state of Chl(1) ((3)Chl(1)*) is rapidly quenched (t(1/2) = 2-20 micro s) by the interaction with Q(A)(-), preventing formation of harmful singlet oxygen. In the next inhibitory stage, in which Q(A) is doubly reduced and then irreversibly released from the Q(A) pocket, the lifetime of (3)Chl(1)* becomes longer by more than two orders of magnitude (t(1/2) = 1-3 ms). As a result, singlet oxygen is produced around Chl(1) in the D1 protein, causing damage preferably to the D1 protein, which induces subsequent proteolytic degradation. Thus, (3)Chl(1)* functions as a switch to change from the protective to the degradative phase of the PSII reaction center by sensing either reversible or irreversible inhibited state at the Q(A) site.