Excited complexes in oxidation-reduction photoreactions of pigments. I. Spectral detection of triplet state exciplex in the chlorophyll oxidation reaction]

Photooxidation of chlorophyll "a" with p-benzoquinone in toluene and dioxane is studied by the method of flash photolysis. It is shown that in nonpolar medium the triplet exiplex is the product of this reaction. The experiments on competitive quenching of chlorophyll triplet state with naphthacene prove the diffusion mechanism of exiplex formation. The spectrum of triplet-triplet absorption of the exiplex is presented and the rate constant of intercombination degradation equalling 1.103 sec(-1) is measured. Possible structure of the exiplex is discussed.     

Synthesis and photophysical properties of zinc myoglobin appending an ethidium ion as a DNA intercalator

In order to elucidate the intramolecular photoinduced electron-transfer or energy-transfer mechanisms of the zinc myoglobin (ZnMb) dyad and to construct a photoreaction system within a Mb–DNA complex, we newly prepared ZnMb appending an ethidium ion (Et⁺). The steady-state fluorescence of ZnMb–Et⁺ at 600 nm and its lifetime (2.2 ns) indicate that the excited singlet state of ¹(ZnMb)* is not quenched by the Et⁺ moiety, whereas the lifetime of the excited triplet state of ³(ZnMb)*–Et⁺ was shorter (τ = 4.3 ms) than those of ZnMb and the intermolecular (ZnMb + ethidium) system. Upon photoirradiation of Et⁺, fluorescence studies indicated the intramolecular quenching reactions from the excited singlet state, ¹(Et⁺)*, to ZnMb, the process of which is likely the photoinduced energy-transfer reaction via a through-space mechanism. We also demonstrate the photophysical and spectroscopic properties of ZnMb–Et⁺ in the presence of calf thymus (CT) DNA. The changes in the absorption and fluorescence spectra of ZnMb–Et⁺ on the addition of CT-DNA up to 15 equiv were very small, indicating that there are no major changes in the heme pocket. However, we observed a longer lifetime of ³(ZnMb)*–Et⁺ in the presence of CT-DNA (τ = 5.3 ms) by single flash photolysis. This was induced by noncovalent interactions between Et⁺ and CT-DNA, followed by a conformational change of Et⁺ at the surface of ZnMb, where the donor–acceptor distance was probably elongated by CT-DNA. The synthetic manipulation at the Mb surface, by using a DNA intercalator coupled with photoinduced reaction, may provide a sensitive transient signal for DNA and valuable information to construct new Mb–DNA complex. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of the Light-induced Transient States of the Chlorophyll Proteins 668 and 743 from Atriplex rosea

The light-induced transient states of chlorophyll-protein 668 (Cp668) and its photoconverted from Cp743 were investigated using flash photolysis. Short lived transient species induced by a short flash were detected in both Cp668 and Cp743. The Cp668 transient had a half decay time of 2.0 milliseconds and showed a broad absorption band at 460 nanometers. The Cp743 transient had a half decay time of only 0.6 millisecond and had a major absorption peak at 410 nanometers in addition, to a broad absorption band around 530 nanometers. Both transient signals were quenched by oxygen. Cp668 had a temperature-dependent delayed fluorescence at room temperature with a half-life of 2.0 milliseconds, the same as the life-time of the absorption transient. This suggests that the transient species observed was a triplet state of chlorophyll.     

Aggregation of LHCII Leads to a Redistribution of the Triplets over the Central Xanthophylls in LHCII

We present laser flash-induced triplet-minus-singlet (TmS(flash)) and absorbance-detected-magnetic-resonance (TmS(ADMR)) measurements on the light-harvesting chlorophyll a/b pigment-protein complex (LHCII) from pea. We investigated the influence of LHCII aggregation on xanthophyll triplet formation. The effect of aggregation was previously studied using TmS(ADMR) [van der Vos et al. (1994) Biochim. Biophys. Acta 1208, 243-250] for LHCII from spinach, and it was concluded that aggregation leads to a large increase of the amount of intertrimer triplet transfer. However, a similar study on LHCII from pea with the use of TmS(flash) measurements [Barzda et al. (1998) Biochemistry 37, 546-561] showed much smaller effects. To resolve this apparent discrepancy and to compare the results of TmS(ADMR) and TmS(flash) measurements, we used both techniques to study LHCII from pea, applying an identical aggregation procedure in both cases. It appears that aggregation does not lead to an increase of intertrimer triplet transfer as thought before but to a redistribution of the triplets over the two central xanthophylls (mainly lutein) that are present in each monomeric subunit of LHCII. Moreover, it is argued that the TmS band at 525 nm is due to lutein instead of violaxanthin as was reported in earlier studies. It is concluded that aggregation leads to a change in chlorophyll-xanthophyll interactions, which might explain the large change in excited-state lifetime of chlorophyll a in LHCII upon aggregation. This change in lifetime is possibly related to the phenomenon of nonphotochemical quenching in green plants, which is an important protective regulatory mechanism, that lowers the probability of photoinhibition.     

Predictors of Seasonal Oxygen Levels in Small Florida Lakes: The Importance of Color

This study examines the relationship of profundal oxygen concentrations in 55 shallow Florida lakes to humic color, trophic state, and lake size during different seasons. The data set represented a broad range of color and trophic state. The percent saturation of dissolved oxygen remained relatively constant during the fall (mean 78.4%), winter (mean 81.3%), and spring (mean 82.5%), but declined markedly during summer (mean 65.2%). Chlorophyll a concentrations were highest during the winter (mean 2.52 mg m^–3) and lowest during the fall (mean 1.17 mg m^–3), while color peaked during the fall (mean 30.1 mg Pt l^–1) and was lowest during the summer (mean 12.7 mg Pt l^–1). The relative importance of lake size, chlorophyll a, and color in explaining variation in percent oxygen saturation was examined using multiple regression. Percent oxygen saturation was negatively correlated with color during the winter, spring, and summer, and positively correlated with lake size in the winter and spring. However, percent oxygen saturation showed no relationship with chlorophyll a during any season. These results suggest that colored Florida lakes are naturally oxygen depleted and that profundal oxygen values have little relationship to lake trophic state. This revised version was published online in July 2006 with corrections to the Cover Date.     

Triplet excited states of polycyclic aromatic compounds as probes of their microenvironment in serum albumin complexes.

Using flash photolysis techniques, the triplet excited states of benzo(a)pyrene, pyrene, benz(a)anthracene and other aromatic hydrocarbons have been detected in complexes of bovine (and human) serum albumin dissolved in aqueous solutions at room temperature. The triplet lifetimes can be adjusted to any value within the microsecond-millisecond time domains by varying the partial pressure of oxygen from zero to one atmosphere, thus providing a useful probe on these time scales. Local oxygen concentrations as low as ～ 2 × 10^?7M can be detected. In air saturated solutions, the triplet lifetimes are sensitive to pH dependent conformational changes of the host bovine serum albumin molecules.     

Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy.

The investigation in this report aimed at providing photophysical evidence that the long-lived triplet excited state plays an important role in the non-single-exponential photobleaching kinetics of fluorescein in microscopy. Experiments demonstrated that a thiol-containing reducing agent, mercaptoethylamine (MEA or cysteamine), was the most effective, among other commonly known radical quenchers or singlet oxygen scavengers, in suppressing photobleaching of fluorescein while not reducing the fluorescence quantum yield. The protective effect against photobleaching of fluorescein in the bound state was also found in microscopy. The antibleaching effect of MEA let to a series of experiments using time-delayed fluorescence spectroscopy and nanosecond laser flash photolysis. The combined results showed that MEA directly quenched the triplet excited state and the semioxidized radical form of fluorescein without affecting the singlet excited state. The triplet lifetime of fluorescein was reduced upon adding MEA. It demonstrated that photobleaching of fluorescein in microscopy is related to the accumulation of the long-lived triplet excited state of fluorescein and that by quenching the triplet excited state and the semioxidized form of fluorescein to restore the dye molecules to the singlet ground state, photobleaching can be reduced.     

Quenching of fluorescence by triplet excited states in chloroplasts

The fluorescence quantum yield in spinach chloroplasts at room temperature has been studied utilizing a 0.5–4.0 μs duration dye laser flash of varying intensities as an excitation source. The yield (Ф) and carotenoid triplet concentration were monitored both during and following the laser flash. The triplet concentration was monitored by transient absorption spectroscopy at 515 nm, while the yield Ф following the laser was probed with a low intensity xenon flash. The fluorescence is quenched by factors of up to 10–12, depending on the intensity of the flash and the time interval following the onset of the flash. This quenching is attributed to a quencher Q whose concentration is denoted by Q. The relative instantaneous concentration of Q was calculated from Ф utilizing the Stern-Volmer equation, and its buildup and decay kinetics were compared to those of carotenoid triplets. At high flash intensities (10¹⁶ photon · cm⁻²) the decay kinetics of Q are slower than those of the carotenoid triplets, while at lower flash intensities they are similar. Q is sensitive to oxygen and it is proposed that Q, at the higher intensities, is a trapped chlorophyll triplet. This hypothesis accounts well for the continuing rise of the carotenoid triplet concentration for 1–2 μs after the cessation of the laser pulse by a slow detrapping mechanism, and the subsequent capture of the triplet energy by carotenoid molecules.

At the maximum laser intensities, the carotenoid triplet concentration is about one per 100 chlorophyll molecules. The maximum chlorophyll ion concentration generated by the laser pulses was estimated to be below 0.8 ions/100 chlorophyll molecules. None of the observations described here were altered when a picosecond pulse laser train was substituted for the microsecond pulse.

A simple kinetic model describing the generation of singlets and triplets (by intersystem crossing), and their subsequent interaction leading to fluorescence quenching, accounts well for the observations. The two coupled differential equations describing the time dependent evolution of singlet and triplet excited states are solved numerically. Using a singlet-triplet bimolecular rate constant of γ_st = 10⁻⁸ cm³ · s⁻¹, the following observations can be accounted for: (1) the rapid initial drop in Ф and its subsequent levelling off with increasing time during the laser pulse, (2) the buildup of the triplets during the pulse, and (3) the integrated yield of triplets per pulse as a function of the energy of the flash.     

Rate of carotenoid triplet formation in solubilized light-harvesting complex II (LHCII) from spinach.

In the present study the rate of triplet transfer from chlorophyll to carotenoids in solubilized LHCII was investigated by flash spectroscopy using laser pulses of approximately 2 ns for both pump and probe. Special attention has been paid to calibration of the experimental setup and to avoid saturation effects. Carotenoid triplets were identified by the pronounced positive peak at approximately 507 nm in the triplet-singlet difference spectra. DeltaOD (507 nm) exhibits a monoexponential relaxation kinetics with characteristic lifetimes of 2-9 micros (depending on the oxygen content) that was found to be independent of the pump pulse intensity. The rise of DeltaOD (507 nm) was resolved via a pump probe technique where an optical delay of up to 20 ns was used. A thorough analysis of these experimental data leads to the conclusion that the kinetics of carotenoid triplet formation in solubilized LHCII is almost entirely limited by the lifetime of the excited singlet state of chlorophyll but neither by the pulse width nor by the rate constant of triplet-triplet transfer. Within the experimental error the rate constant of triplet-triplet transfer from chlorophyll to carotenoids was estimated to be kTT > (0.5 ns)-1. This value exceeds all data reported so far by at least one order of magnitude. The implications of this finding are briefly discussed.     

Quantum yield and rate of formation of the carotenoid triplet state in photosynthetic structures

The formation of the triplet state of carotenoids (detected by an absorption peak at 515 nm) and the photo-oxidation of the primary donor of Photosystem II, P-680 (detected by an absorption increase at 820 nm) have been measured by flash absorption spectroscopy in chloroplasts in which the oxygen evolution was inhibited by treatment with Tris. The amount of each transient form has been followed versus excitation flash intensity (at 590 or 694 nm). At low excitation energy the quantum yield of triplet formation (with the Photosystem II reaction center in the state Q⁻) is about 30% that of P-680 photo-oxidation. The yield of carotenoid triplet formation is higher in the state Q⁻ than in the state Q, in nearly the same proportion as chlorophyll a fluorescence. It is concluded that, for excited chlorophyll a, the relative rates of intersystem crossing to the triplet state and of fluorescence emission are the same in vivo as in organic solvent. At high flash intensity the signal of P-680⁺ completely saturates, whereas that of carotenoid triplet continues to increase.

The rate of triplet-triplet energy transfer from chlorophyll a to carotenoids has been derived from the rise time of the absorption change at 515 nm, in chloroplasts and in several light-harvesting pigment-protein complexes. In all cases the rate is very high, around 8 · 10⁷ s⁻¹ at 294 K. It is about 2–3 times slower at 5 K. The transitory formation of chlorophyll triplet has been verified in two pigment-protein complexes, at 5 K.     

Quenching of fluorescence by triplet excited states in chloroplasts

The fluorescence quantum yield in spinach chloroplasts at room temperature has been studied utilizing a 0.5-4.0 mus duration dye laser flash of varying intensities as an excitation source. The yield (phi) and carotenoid triplet concentration were monitored both during and following the laser flash. The triplet concentration was monitored by transient absorption spectoscopy at 515 nm, while the yield phi following the laser was probed with a low intensity xenon flash. The fluorescence is quenched by factors of up to 10-12, depending on the intensity of the flash and the time interval following the onset of the flash. This quenching is attributed to a quencher Q whose concentration is denoted by Q. The relative instantaneous concentration of Q was calculated from phi utilizing the Stern-Volmer equation, and its buildup and decay kinetics were compared to those of carotenoid triplets. At high flash intensities (greater than 10(16) photon . cm-2) the decay kinetics of Q are slower than those of the carotenoid triplets, while at lower flash intensities they are similar. Q is sensitive to oxygen and it is proposed that Q, at the higher intensities, is a trapped chlorophyll triplet. This hypothesis accounts well for the continuing rise of the carotenoid triplet concentration for 1-2 mus after the cessation of the laser pulse by a slow detrapping mechanism, and the subsequent capture of the triplet energy by carotenoid molecules. At the maximum laser intensities, the carotenoid triplet concentration is about one per 100 chlorophyll molecules. The maximum chlorophyll ion concentration generated by the laser pulses was estimated to be below 0.8 ions/100 chlorophyll molecules. None of the observations described here were altered when a picosecond pulse laser train was substituted for the microsecond pulse. A simple kinetic model describing the generation of singlets and triplets (by intersystem crossing), and their subsequent interaction leading to fluorescence quenching, accounts well for the observations. The two coupled differential equations describing the time dependent evolution of singlet and triplet excited states are solved numerically. Using a single-triplet bimolecular rate constant of gammast = 10(-8) cm3 . s-1, the following observations can be accounted for: (1) the rapid initial drop in phi and its subsequent levelling off with increasing time during the laser pulse, (2) the buildup of the triplets during the pulse, and (3) the integrated yield of triplets per pulse as a function of the energy of the flash.     

Conformational and electronic (AIM/NBO) study of unsubstituted A-type dimeric proanthocyanidin

The conformational space of the unsubstituted A-type dimeric proanthocyanidin was scanned using molecular dynamics at a semiempirical level, and complemented with functional density calculations. The lowest energy conformers were obtained. Electronic distributions were analysed at a higher calculation level, thus improving the basis set. A topological study based on Bader’s theory (AIM: atoms in molecules) and natural bond orbital (NBO) framework was performed. Furthermore, molecular electrostatic potential maps (MEPs) were obtained and analysed. NMR chemical shifts were calculated at ab initio level and further compared with previous experimental values; coupling constants were also calculated. The stereochemistry of the molecule is thoroughly discussed, revealing the key role that hyperconjugative interactions play in defining experimental trends. These results show the versatility of geminal spin–spin coupling ²J(C-1′,O) as a probe for stereochemical studies of proanthocyanidins.     

Laser flash photolysis study on antioxidant properties of hydroxycinnamic acid derivatives

The antioxidant properties of hydroxycinnamic acid derivatives (HCA) were studied by laser flash photolysis. The transient species with maximum absorption at 360 nm were assigned to the phenoxyl radical of HCA. The SO₄^•− induced oxidation of HCA was also investigated. It was shown that the interaction of SO₄^•− with HCA resulted in the formation of HCA phenoxyl radicals with rate constants of 2.0–3.9×10⁹ M⁻¹ s⁻¹. The reactions of HCA with triplet state of benzophenone were analyzed and quenching rate constants of 4.3–7.8×10⁹ M⁻¹ s⁻¹ were determined.     

Photoaffinity labeling with 8-azidoadenosine and its derivatives: chemistry of closed and opened adenosine diazaquinodimethanes

The reactive intermediate produced upon photolysis of 8-azidoadenosine was studied by chemical trapping studies, laser flash photolysis with UV-vis and IR detection, and modern computational chemistry. It is concluded that photolysis of 8-azidoadenosine in aqueous solution releases the corresponding singlet nitrene which rapidly tautomerizes to form a closed adenosine diazaquinodimethane in less than 400 fs. A perbenzoylated derivative of 8-azidoadenosine cannot undergo this tautomerization, and instead, it fragments upon photolysis to form an opened adenosine diazaquinodimethane. The singlet nitrene is too short-lived to be observed and, thus, to relax to the lowest triplet state or to become covalently attached to targeted biological macromolecules. The pivotal closed adenosine diazaquinodimethane, the product of nitrene tautomerization, has a lifetime of ca. 1 min or longer in water and in HEPES buffer at ambient temperature. However, this intermediate reacts rapidly with good nucleophiles such as amines, thiols, and phenolates, and significantly more slowly with weak nucleophiles such as alcohols and water. On the basis of these studies, it is clear that the closed adenosine diazaquinodimethane, and not the singlet or triplet nitrene, is the pivotal reactive intermediate involved in photolabeling and cross-linking studies using the 8-azidoadenosine family of photoaffinity labeling reagents.     

Vitamin B2-sensitised photooxidation of the ophthalmic drugs Timolol and Pindolol: kinetics and mechanism

The kinetics and mechanistic aspects of the riboflavin-photosensitised oxidation of the topically administrable ophthalmic drugs Timolol (Tim) and Pindolol (Pin) were investigated in water-MeOH (9:1, v/v) solution employing light of wavelength > 400 nm. riboflavin, belonging to the vitamin B(2) complex, is a known human endogenous photosensitiser. The irradiation of riboflavin in the presence of ophthalmic drugs triggers a complex picture of competitive reactions which produces the photodegradation of both the drugs and the pigment itself. The mechanism was elucidated employing stationary photolysis, polarographic detection of dissolved oxygen, stationary and time-resolved fluorescence spectroscopy, and laser flash photolysis. Ophthalmic drugs quench riboflavin-excited singlet and triplet states. From the quenching of excited triplet riboflavin, the semireduced form of the pigment is generated, through an electron transfer process from the drug, with the subsequent production of superoxide anion radical (O(2)(*-)) by reaction with dissolved molecular oxygen. Through the interaction of dissolved oxygen with excited triplet riboflavin, the species singlet oxygen (O(2)((1)Delta(g))) is also generated to a lesser extent. Both O(2)(*-) and O(2)((1)Delta(g)) induce photodegradation of ophthalmic drugs, Tim being approximately 3-fold more easily photooxidisable than Pin, as estimated by oxygen consumption experiments.     

Photoinactivation of Photosystem II by flashing light

Inhibition of Photosystem II (PS II) activity by single turnover visible light flashes was studied in thylakoid membranes isolated form spinach. Flash illumination results in decreased oxygen evolving activity of PS II, which effect is most pronounced when the water-oxidizing complex is in the S₂ and S₃ states, and increases with increasing time delay between the subsequent flashes. By applying the fluorescent spin-trap DanePy, we detected the production of singlet oxygen, whose amount was increasing with increasing flash spacing. These findings were explained in the framework of a model, which assumes that recombination of the S₂Q_B⁻ and S₃Q_B⁻ states generate the triplet state of the reaction center chlorophyll and lead to the production of singlet oxygen.     

Reaction mechanisms of riboflavin triplet state with nucleic acid bases.

ESR and laser flash photolysis studies have determined a reasonable order of reactivity of nucleotides with triplet riboflavin (3Rb*) for the first time. ESR detection of triplet state reactivity of Rb with nucleoside, polynucleotide and DNA has been obtained simultaneously. In addition, ESR spin elimination measurement of the reactivity of 3Rb* with nucleotides in good accord with laser flash photolysis determination of the corresponding rate constants offers a simple and reliable method to detect the reactivities of nucleic acids and its components with photoexcited flavins. Kinetic, ESR and thermodynamic studies have demonstrated that Rb should be a strong endogenous photosensitizer capable of oxidizing all nucleic acid bases, and preferentially two purine nucleotides with high rate constants.     

Functional characterization of the PS II–LHC II supercomplex isolated by a direct method from spinach thylakoid membranes

Recently, a novel procedure to isolate a highly pure and active Photosystem II preparation directly from thylakoid membranes, referred to as PS II–LHC II supercomplex, was reported [Eshaghi et al. (1999) FEBS Lett 446: 23–26]. In addition to the reaction center core proteins, the supercomplex contains all the extrinsic proteins of the oxygen evolving complex and a set of chlorophyll a/b binding proteins. In this paper, the functional properties of this isolated supercomplex are further characterized by using EPR spectroscopy, thermoluminescence, fluorescence relaxation kinetics and flash induced oxygen yield measurements. The PS II–LHC II supercomplex contains, in addition to Q_A and Q_B, a small pool of plastoquinone (PQ). Although the isolated complex is no longer membrane bound, it has preserved functional characteristics of a well defined PS II preparation with the exception of some modification of Q_B sites. This revised version was published online in June 2006 with corrections to the Cover Date.     

On the nature of the two pathways in chlorophyll formation from protochlorophyllide

The kinetics of formation of esterified chlorophyll in etiolated barley (Hordeum vulgare L.) leaves after illumination with a single flash was studied. It was found that after partial (14–24%) and after full photoreduction of protochlorophyllide, the same quantity of esterified products appear during the first 5 s after the flash. The rest of formed chlorophyllide was esterified in a slow process during at least 30 min at 15 °C. The product of fast esterification can be correlated with ‘short-wavelength’ chlorophyll, characterized by a fluorescence emission peak at 673–675 nm. This is the only chlorophyll form detectable within 20 s after partial (14%) photoconversion, and it appears at the same time as the shoulder of the chlorophyll(ide) fluorescence after full photoconversion. The main product after full photoconversion shows a fluorescence at 689 nm shifting in darkness within 15 s to 693 nm and then within 30 min to 682 nm (Shibata shift). The slow esterification proceeds with similar kinetics as the Shibata shift. We propose that the fast esterification of only part of total chlorophyllide after full photoconversion of protochlorophyllide in etiolated leaves reflects the restricted capacity of the esterifying system. The slow esterification of the residual chlorophyllide may be time-limited by its release from protochlorophyllide oxidoreductase, by disaggregation of prolamellar bodies and by diffusion of tetraprenyl diphosphates towards chlorophyll synthase. This revised version was published online in June 2006 with corrections to the Cover Date.     

Lipid and lipoprotein oxidation: basic mechanisms and unresolved questions in vivo

Abstract

The kinetics and mechanistic aspects of the riboflavin-photosensitised oxidation of the topically administrable ophthalmic drugs Timolol (Tim) and Pindolol (Pin) were investigated in water–MeOH (9:1, v/v) solution employing light of wavelength > 400 nm. riboflavin, belonging to the vitamin B₂ complex, is a known human endogenous photosensitiser. The irradiation of riboflavin in the presence of ophthalmic drugs triggers a complex picture of competitive reactions which produces the photodegradation of both the drugs and the pigment itself. The mechanism was elucidated employing stationary photolysis, polarographic detection of dissolved oxygen, stationary and time-resolved fluorescence spectroscopy, and laser flash photolysis. Ophthalmic drugs quench riboflavin-excited singlet and triplet states. From the quenching of excited triplet riboflavin, the semireduced form of the pigment is generated, through an electron transfer process from the drug, with the subsequent production of superoxide anion radical (O₂^?–) by reaction with dissolved molecular oxygen. Through the interaction of dissolved oxygen with excited triplet riboflavin, the species singlet oxygen (O₂(¹Δ_g)) is also generated to a lesser extent. Both O₂^?– and O₂(¹Δ_g) induce photodegradation of ophthalmic drugs, Tim being ~3-fold more easily photooxidisable than Pin, as estimated by oxygen consumption experiments.