Triplet state dynamics in peridinin-chlorophyll-a-protein: a new pathway of photoprotection in LHCs?

This work investigates the interaction of carotenoid and chlorophyll triplet states in the peridinin-chlorophyll-a-protein (PCP) of Amphidinium carterae using step-scan Fourier transform infrared spectroscopy. We identify two carotenoid triplet state lifetimes of approximately 13 and approximately 42 mus in the spectral region between 1800 and 1100 cm(-1) after excitation of the 'blue' and 'red' peridinin (Per) conformers and the Q(y) of chlorophyll-a (Chl-a). The fast and slow decaying triplets exhibit different spectral signatures in the carbonyl region. The fast component generated at all excitation wavelengths is from a major conformer with a lactone stretching mode bleach at 1745 cm(-1). One (1720 cm(-1)) and two (1720 cm(-1) and 1741 cm(-1)) different Per conformers are observed for the slow component upon 670- and 530-480-nm excitation, respectively. The above result implies that (3)Per triplets are formed via two different pathways, corroborating and complementing visible triplet-singlet (T-S) spectra (Kleima et al., Biochemistry (2000), 39, 5184). Surprisingly, all difference spectra show that Per and Chl-a modes are simultaneously present during the (3)Per decay, implying significant involvement of (3)Chl-a in the (3)Per state. We suggest that this Per-Chl-a interaction via a delocalized triplet state lowers the (3)Per energy and thus provides a general, photoprotection mechanism for light-harvesting antenna complexes.     

Triplet formation on a monomeric chlorophyll in the photosystem II reaction center as studied by time-resolved infrared spectroscopy

The process of formation of the triplet state of chlorophyll in the photosystem II (PS II) reaction center complex was studied by means of time-resolved infrared (IR) spectroscopy. Using a dispersive-type IR spectrometer with a time resolution of approximately 55 ns, transient spectra in the C=O stretching region (1760--1600 cm(-1)) were measured at 77 K. The data were analyzed by singular-value decomposition and subsequent least-squares fitting. Two distinct spectral components having different kinetic behaviors were resolved. One had spectral features characterized by negative peaks at 1740 and 1680 cm(-1) and an overall positive background and was assigned to the P(680)(+)Phe(-)/P(680)Phe radical pair by static FTIR measurements of the P(680)(+)/P(680) and Phe(-)/Phe differences. The other had prominent negative and positive peaks at 1668 and 1628 cm(-1), respectively, which were previously assigned to the keto C==O change upon triplet formation of the monomeric chlorophyll denoted as Chl(T) [Noguchi, T., Tomo, T., and Inoue, Y. (1998) Biochemistry 37, 13614-13625]. The former component of P(680)(+)Phe(-)/P(680)Phe exhibited a multiphasic decay with time constants of 77 ns (75%), 640 ns (18%), 8.3 micros (4%), and 0.3 ms (3%), while the latter component of (3)Chl(T)/Chl(T) was formed with a single-exponential rise with a time constant of 57 ns and had a lifetime of 1.5 ms. From the observations that only the two spectral components were resolved without any other triplet intermediates and the time constant of (3)Chl(T) formation roughly agreed with or seemed even faster than that of the major phase of the P(680)(+)Phe(-) decay, two alternative mechanisms of triplet formation are proposed. (i) (3)Chl(T) is directly formed from P(680)(+)Phe(-) by charge recombination at Chl(T), and (ii) (3)P(680) is formed, and then the triplet is transferred to Chl(T) with a time constant of much less than 50 ns. The location of Chl(T) in the D1 subunit as the monomer chlorophyll corresponding to the accessory bacteriochlorophyll in the L subunit of purple bacteria is favored to explain the former mechanism as well as the triplet properties reported in the literature. The physiological role of the triplet formation on Chl(T) is also discussed.     

EPR characterisation of the triplet state in photosystem II reaction centers with singly reduced primary acceptor Q(A)

The triplet states of photosystem II core particles from spinach were studied using time-resolved cw EPR technique at different reduction states of the iron--quinone complex of the reaction center primary electron acceptor. With doubly reduced primary acceptor, the well-known photosystem II triplet state characterised by zero-field splitting parameters |D|=0.0286 cm(-1), |E|=0.0044 cm(-1) was detected. When the primary acceptor was singly reduced either chemically or photochemically, a triplet state of a different spectral shape was observed, bearing the same D and E values and characteristic spin polarization pattern arising from RC radical pair recombination. The latter triplet state was strongly temperature dependent disappearing at T=100 K, and had a much faster decay than the former one. Based on its properties, this triplet state was also ascribed to the photosystem II reaction center. A sequence of electron-transfer events in the reaction centers is proposed that explains the dependence of the triplet state properties on the reduction state of the iron--quinone primary acceptor complex.     

Reaction center triplet states in photosystem I and photosystem II

A photosystem I (PS I) particle has been prepared by lithium dodecyl sulfate digestion which lacks the acceptor X, and iron-sulfur centers B and A. Illumination of these particles at liquid helium temperature results in the appearance of a light-induced spin-polarized triplet signal observed by EPR. This signal is attributed to the triplet state of P-700, the primary donor, formed by recombination of the light induced radical pair P-700+ A1- (where A1 is the intermediate acceptor). Formation of the triplet does not occur if P-700 is oxidized or if A1 is reduced, prior to the illumination. A comparison of the P-700 triplet with that of P-680, the primary donor of Photosystem II, shows several differences. (1) The P-680 triplet is 1.5 mT (15 G) wider than the P-700 triplet. This is reflected by the zero-field splitting parameters, which indicate that P-700 is a slightly larger species than P-680. The zero-field splitting parameters do not indicate that either P-700 or P-680 are dimeric. (2) The P-700 triplet is induced by red and far-red light, while the P-680 triplet is induced only by red light. (3) The temperature dependences of the P-700 triplet and the P-680 triplet are different.     

Protonation and hydrogen-bonding state of the distal histidine in the CO complex of horseradish peroxidase as studied by ultraviolet resonance Raman spectroscopy

Ultraviolet resonance Raman (UVRR) spectroscopy has been used to characterize the structure and hydrogen bonding state of the distal histidine (His42) in horseradish peroxidase (HRP) complexed with carbon monoxide (HRP-CO). The HRP-CO - HRP UVRR difference spectrum in D(2)O solution at pD 7.0 shows two positive peaks at 1408 and 1388 cm(-)(1), which are ascribable to medium-to-weak and strong hydrogen bonding states, respectively, of the protonated imidazolium side chain of His42 in HRP-CO. Both His42 peaks decrease in intensity with increase of pD with a midpoint of transition at pD 8.8, indicating that the pK(a) of His42 in HRP-CO is 8.8. The CO ligand exhibits two C-O stretching Raman peaks at 1932 and 1902 cm(-)(1), the latter of which diminishes at alkaline pD and is assignable to a strong hydrogen-bonded state. It is most probable that the imidazolium side chain of His42 forms a strong hydrogen bond with CO, giving a His42 peak at 1388 cm(-)(1) and a CO peak at 1902 cm(-)(1), in one conformer. The other hydrogen bonding state of His42, giving the 1408 cm(-)(1) peak, is ascribed to another conformer forming a medium-to-weak hydrogen bond with a water molecule in the distal cavity. The present finding that His42 can act as a strong proton donor to CO and decrease the CO bond order is consistent with the role of His42 as a general acid to cleave the O-O bond of hydrogen peroxide, a specific oxidizing agent, in the catalytic cycle of HRP.     

Chemiluminescence in L-tyrosine-H2O2-horseradish peroxidase system: possible formation of tyrosine cation radical

Tyrosine-H2O2-horseradish peroxidase system at pH 7.4 emitted light in visible region. Phenolic compounds other than tyrosine were also emissive, whereas methoxy phenylalanine and phenyl compounds were not, in H2O2-peroxidase systems. Chemiluminescence spectrum of tyrosine of tyrosine-H2O2-horseradish peroxidase system showed two prominent peaks at 478 nm and 500 nm (Luminescence 1) and additional two or three peaks near 550 and 610 nm (Luminescence 2). Luminescence 1 is quite similar to the phosphorescence originated from an excited tyrosine in triplet state, while Luminescence 2 is quite similar to the phosphorescence originated from an indole in triplet state. Possible formation of tyrosine cation radical (a precursor of the excited tyrosine) and indole cation radical in the enzyme protein (a precursor of the excited tryptophan residue) were discussed.     

Pressure dependence of human fibrinogen correlated to the conformational alpha-helix to beta-sheet transition: an Fourier transform infrared study microspectroscopic study

We used Fourier transform infrared (FTIR) microspectroscopy to investigate pressure-induced conformational changes in secondary structure of fibrinogen (FBG). Solid state FBG was compressed on a KBr pellet (1KBr method) or between two KBr pellets (2KBr method). The peak positions of the original and second-derivative ir spectra of compressed FBG samples prepared by the 1KBr method were similar to FBG sample without pressure. When FBG was prepared by the 2KBr method and pressure was increased up to 400 kg/cm(2), peaks at 1625 (intermolecular beta-sheet) and 1611 (beta-sheet aggregates structure and/or the side-chain absorption of the tyrosine residues) cm(-1) were enhanced. The peaks near 1661 (beta-sheet) and 1652 (alpha-helix) cm(-1) also exhibited a marked change with pressure. A linear correlation was found between the peak intensity ratio of 1611/1652 cm(-1) (r = 0.9879) or 1625/1652 cm(-1) (r = 0.9752) and applied pressure. The curve-fitted compositional changes in secondary structure of FBG also indicate that the composition of the alpha-helix structure (1657-1659 cm(-1)) was gradually reduced with the increase in compression pressure, but the composition of the beta-sheet structure (1681, 1629, and 1609 cm(-1)) gradually increased. This indicates that pressure-induced conformational changes in FBG include not only transformations from alpha-helix to beta-sheet structure, but also unfolding and denaturation of FBG and the formation of aggregates.     

Ru(bpy)(3)](2+) as a reference in transient absorption spectroscopy: differential absorption coefficients for formation of the long-lived (3)MLCT excited state

Transient absorption spectroscopy and other time-resolved methods are commonly used to study chemical reactions and biological processes induced by absorption of light. In order to scale the signal amplitude or to compare results obtained under different conditions, it is advisable to use a reference system, a standard of convenient and well-defined properties. Finding Tris(bipyridine)ruthenium(ii), [Ru(bpy)(3)](2+), a suitable candidate for a transient-absorption spectroscopy reference due to its favourable photochemical properties, we have determined accurate relative values of differential molar absorption coefficients (Δε) for light-induced formation of the metal-to-ligand charge transfer (MLCT) excited triplet state at several relevant wavelengths (wavelengths of commercially available lasers) in the UV and visible regions. We have also attempted to determine the absolute value of Δε close to the wavelength of maximum bleaching (～450 nm) and we propose to narrow down the interval of conceivable values for Δε(450) from the broad range of published values (-0.88 × 10(4) M(-1)cm(-1) to -1.36 × 10(4) M(-1)cm(-1)) to -1.1 × 10(4) M(-1)cm(-1)± 15%. Having ourselves successfully applied [Ru(bpy)(3)](2+) as a standard in a recent time-resolved study of enzymatic DNA repair, we would like to encourage other scientists to use this convenient tool as a reference in their future spectroscopic studies on time scales from picoseconds to hundreds of nanoseconds.     

Protic solvent effects on the photophysical properties of O=Ti(IV)TSPP: photoinduced electron transfer.

The photophysical properties of oxotitanium(IV)meso-tetra(4-sulfonatophenyl) porphyrin (O=Ti(IV)TSPP) have been investigated in water and methanol by laser spectroscopic techniques. The fluorescence emission spectrum of O=Ti(IV)TSPP in methanol exhibits two strong emission bands at 610 and 670 nm at room temperature with the decay time of ca. 310 +/- 10 ps and the rise time shorter than 30 ps, in contrast to the extremely weak emission with the decay time of ca. 27 +/- 4 ps in water, indicating that the fluorescence emissive states are different in the two solvents as supported by the solvent dependences of the excitation spectrum. The transient Raman spectra of O=Ti(IV)TSPP in water has been observed to exhibit a remarkable enhancement of phenyl-related mode at 1599 cm(-1), while in methanol, the Raman frequencies of the porphyrin skeletal modes (upsilon2 and upsilon4) are down-shifted without any apparent enhancement of the phenyl-related mode, indicating different interactions of the two solvents with the excited O=Ti(IV)TSPP. These Raman studies reveal that methanol molecule interacts with the photoexcited O=Ti(IV)TSPP more strongly than water, forming the exciplex, O=Ti(IV)TSPP(MeOH)*, suggesting that the two different emissive states are the singlet Franck-Condon state and the exciplex state in methanol and water, respectively. A broad triplet transient absorption of O=Ti(IV)TSPP has been also observed at 480 nm in water as well as in methanol, which is decreased upon addition of methyl viologen (MV2+) with appearance of a new absorption band at 620 nm. This indicates that the photoinduced electron transfer (PET) takes place from the porphyrin to MV2+ in both solvents. The kinetic analysis of the transient absorption band exhibits the PET rate constants of 4.76 x 10(5) s(-1) and 3,03 x 10(4) s(-1) in methanol and water, respectively. All these results infer that the PET takes place from the (d,pi) CT state and the triplet state of the excited porphyrin in methanol and water, respectively.     

Changes in secondary structures and acidic side chains of melibiose permease upon cosubstrates binding

Infrared difference spectroscopy analysis of the purified melibiose permease of Escherichia coli reconstituted into liposomes was carried out as a function of the presence of the two symporter substrates (Na(+), melibiose) in either H(2)O or in D(2)O media. Essentially, the data first show that addition of Na(+) induces appearance of peaks assigned to changes in the environment and/or orientation of alpha-helical domains of purified melibiose permease. Likewise, melibiose addition in the presence of Na(+) produces peaks corresponding to additional changes of alpha-helix environment or tilt. In addition to these changes, a pair of peaks (1599 (+) cm(-1)/1576 (-) cm(-1)) appearing in the Na(+)-induced difference spectrum is assigned to the antisymmetric stretching of COO(-) groups, since they show practically no shift upon H/D exchange. It is proposed that these acidic groups participate in Na(+) co-ordination. A corresponding pair of peaks, again fairly insensitive to H/D substitution (1591 (-) cm(-1)/1567 (+) cm(-1)), appear in the melibiose-induced difference spectra, and may again be assigned to COO(-) groups. The latter carboxyl groups may correspond to part or all of the acidic residues interacting with Lys or Arg in the resting state that become free upon melibiose binding.     

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).     

Major signal increase in fluorescence microscopy through dark-state relaxation

We report a substantial signal gain in fluorescence microscopy by ensuring that transient molecular dark states with lifetimes >1 micros, such as the triplet state relax between two molecular absorption events. For GFP and Rhodamine dye Atto532, we observed a 5-25-fold increase in total fluorescence yield before molecular bleaching when strong continuous-wave or high-repetition-rate pulsed illumination was replaced with pulses featuring temporal pulse separation >1 micros. The signal gain was observed both for one- and two-photon excitation. Obeying dark or triplet state relaxation in the illumination process signifies a major step toward imaging with low photobleaching and strong fluorescence fluxes.     

Spectroscopic characterization of triplet forming states in photosystem II.

Fluorescence and electron paramagnetic resonance (EPR) measurements have been applied to characterize chlorophyll triplet formation in the reaction center of photosystem II (PSII). A highly triplet forming state was generated in PSII membranes by chemical double reduction of the primary electron acceptor QA. In triplet forming PSII centers, the steady-state yield of chlorophyll fluorescence decreased to about 70% of the maximal fluorescence yield observed in closed PSII centers in which QA is singly reduced. The results are well interpreted in the framework of a model where the charge state of QA electrostatically controls the yield of primary charge separation [Schatz, G. H., Brock, H., & Holzwarth, A. R. (1988) Biophys. J. 54, 397-405]. Thus, high triplet yield and decreased, although still quite high, fluorescence indicate a charge-neutralized state of PSII in which QA is singly or doubly reduced and protonated or absent. The EPR signal of the triplet primary chlorophyll donor, 3P680, is suppressed by illumination at 77 K concomitant with the formation of a cationic radical (g = 2.0025-2.0027, and 0.92 mT wide) that is stable in the dark. This is attributed to the oxidation of an accessory chlorophyll (Chl) in the vicinity of P680. Electrostatic repulsion between Chl+ and P680+ is likely to prevent primary charge separation, and in turn triplet formation, providing a further example of electrostatic control of primary charge separation. The triplet P680 EPR signal is also suppressed in the presence of oxygen. This effect, which is almost completely reversible by removing the oxygen, is attributed to the interaction of triplet P680 with triplet O2.     

A light-induced spin-polarized triplet detected by EPR in photosystem II reaction centers

A light-induced spin-polarized triplet state has been detected in a purified Photosystem II preparation by electron paramagnetic resonance spectroscopy at liquid helium temperature. The electron spin polarization pattern is interpreted to indicate that the triplet originates from radical pair recombination between the oxidized primary donor chlorophyll, P-680+, and the reduced intermediate pheophytin, I-, as has been previously demonstrated in bacterial reaction centers. The dependence of the triplet signal on the redox state of I and the primary acceptor, Q, are consistent with the origin of the triplet signal from the triplet state of P-680. Redox-poising experiments indicate the presence of an endogenous donor (or donors) which operates at 3-5 K and 200 K. The zero field-splitting parameters of the triplet are very similar to those of monomeric chlorophyll a however, this alone does not allow a distinction to be made between monomeric and dimeric structures for P-680.     

Pulse ENDOR and density functional theory on the peridinin triplet state involved in the photo-protective mechanism in the peridinin-chlorophyll a-protein from Amphidinium carterae

The photoexcited triplet state of the carotenoid peridinin in the Peridinin-chlorophyll a-protein of the dinoflagellate Amphidinium carterae has been investigated by pulse EPR and pulse ENDOR spectroscopies at variable temperatures. This is the first time that the ENDOR spectra of a carotenoid triplet in a naturally occurring light-harvesting complex, populated by energy transfer from the chlorophyll a triplet state, have been reported. From the electron spin echo experiments we have obtained the information on the electron spin polarization dynamics and from Mims ENDOR experiments we have derived the triplet state hyperfine couplings of the alpha- and beta-protons of the peridinin conjugated chain. Assignments of beta-protons belonging to two different methyl groups, with aiso=7.0 MHz and aiso=10.6 MHz respectively, have been made by comparison with the values predicted from density functional theory. Calculations provide a complete picture of the triplet spin density on the peridinin molecule, showing that the triplet spins are delocalized over the whole pi-conjugated system with an alternate pattern, which is lost in the central region of the polyene chain. The ENDOR investigation strongly supports the hypothesis of localization of the triplet state on one peridinin in each subcluster of the PCP complex, as proposed in [Di Valentin et al. Biochim. Biophys. Acta 1777 (2008) 186-195]. High spin density has been found specifically at the carbon atom at position 12 (see Fig. 1B), which for the peridinin involved in the photo-protective mechanism is in close contact with the water ligand to the chlorophyll a pigment. We suggest that this ligated water molecule, placed at the interface between the chlorophyll-peridinin pair, is functioning as a bridge in the triplet-triplet energy transfer between the two pigments.     

Photophysical properties of Pr(III) and Er(III) complexes of poly(pyrazolyl)borates.

The complexes [M(L(1))(2)(NO(3))] and [M(L(2))(NO(3))(2)](M = Pr, Er; L(1)= the tetradentate ligand dihydrobis-[3-(2-pyridyl)pyrazolyl]borate; L(2)= the hexadentate ligand hydrotris-[3-(2-pyridyl)pyrazolyl]borate) were prepared and their structural and photophysical properties studied. All complexes are 10-coordinate. Crystallographic analysis of [M(L(1))(2)(NO(3))](M = Pr, Er) showed that for the smaller Er(iii) ions steric congestion at the metal centre results in two of the Er-N(pyridyl) distances being particularly long, which does not occur with the larger Pr(iii) ion that is better able to accommodate 10-fold coordination. On UV irradiation, both Pr(iii) complexes show, in the visible region of their luminescence spectra, transitions originating from both the (3)P(0) level (at ca. 21,000 cm(-1)) and the (1)D(2) level (at ca. 17,000 cm(-1)), a consequence of the fact that the lowest triplet state of the coordinated pyrazolylborate ligands lies at ca. 24,000 cm(-1) in each case so is high enough in energy to populate both levels. This contrasts with Pr(iii) complexes based on diketonate ligands in which the lower triplet energies of the ligands result in emission from the (1)D(2) level only. At longer wavelengths, near-infrared luminescence arising from the (1)D(2) emissive level is observed with lifetimes (in both the solid state and solution) being in the range 50-110 ns. For both Er(iii) complexes, luminescence at 1530 nm occurs following UV excitation of ligand-centred transitions. In CH(2)Cl(2) both complexes gave dual-exponential luminescence, with the major component having a lifetime characteristic of an intact Er(iii) complex (approximately 1.5 micros) and the minor component being much shorter lived (0.2-0.5 micros), suggestive of a species in which a ligand is partially detached and the metal is solvated, with the two forms interconverting slowly. This behaviour is consistent with the steric congestion and long M-N(pyridyl) bonds that were observed in [Er(L(1))(2)(NO(3))]. In the solid state both Er(iii) complexes gave very weak luminescence, which could be fitted to a single exponential decay with a lifetime similar to the longer-lived of the solution components.     

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.     

Time-resolved absorption, circular dichroism, and emission of tRNA. Evidence that the photo-cross-linking of 4-thiouridine in tRNA occurs from the triplet state

The time-resolved optical density (TROD) and time-resolved circular dichroism (TRCD) spectra of the lowest triplet state of 4-thiouridine (4t-Urd) in aqueous solutions of tRNA are reported. The TROD spectrum is consistent with the triplet state being primarily in the thione tautomer. The intersystem crossing yield to the triplet is 0.35 and 0.27 (+/- 10%), respectively, with and without 10(-2) Mg2+ added to the solution. Upon addition of increasing amounts of I- to solutions of tRNA, the initial triplet yield decreases, the rate of the observed triplet decay increases, and the quantum yield of internal photo-cross-linking decreases for the 4t-Urd chromophore. The results show quantitatively that the near-UV-induced photo-cross-linking reaction in tRNA occurs from the triplet state of 4t-Urd. From the TRCD spectrum the dissymmetry factor (delta epsilon/epsilon) of some of the triplet-triplet absorption bands is shown to be significantly larger than for any of the ground-state absorption bands. Two CD transitions are seen in the triplet-triplet spectrum which are obscured in the TROD spectrum by the strong ground-state bleaching signal near 335 nm. This shows that TRCD may be useful, in some cases, in locating electronic transitions that are not observed in TROD spectra.     

Photochemistry of 6-chloro and 6-bromopicolinic acids in water. Heterolytic vs. homolytic photodehalogenation.

The photochemistry of 6-chloro and 6-bromopicolinate ions ( and , respectively) was investigated by product studies and ns laser flash photolysis (LFP). In deoxygenated pH 5.4 water, yields 6-hydroxypicolinic acid (70%) and a substituted pyrrole. In 2-propanol-water (1 : 1) mixture, the reaction yields, very unselectively, 6-hydroxypicolinic acid, 2-carboxypyridine, pyridine and bipyridines. Photolysis of aqueous leads to 6-hydroxypicolinic acid (78%) and hydroxybipyridines. Oxygen suppresses the photolysis of but does not affect that of . By LFP, we detected a short-lived transient at the pulse end from (lambda(max)= 305 nm, k=(2.8 +/- 0.2)x 10(5) s(-1), epsilonphi= 2200 +/- 200 dm3 mol(-1) cm(-1)). This is quenched either by oxygen or methyl acrylate and thus assigned to the triplet excited state. The triplet excited state of is detected at pH 1 only (lambda(max)= 320 nm, k > 3 x 10(7) s(-1)). The radical ion Cl2- could be successfully detected by photolysing in 2-propanol-water (1 : 1) in the presence of Cl-. Similarly, Br2- could be detected by irradiating aqueous in the presence of Br-. These results show that the photodehalogenation of is heterolytic in water and mainly homolytic in 2-propanol-water mixtures while that of is both heterolytic and homolytic in water. A mechanism in which the triplet excited state undergoes homolysis of the C-X bond and subsequent electron transfer from the carboxypyridyl radical to the halogen atom to form an ion pair may account for these observations.     

Chlorophyll triplet states associated with photosystem II of thylakoids

The analysis of FDMR thylakoid spectra, determined at multiple emission wavelengths, by a global decomposition technique, has revealed the presence of three previously undescribed triplet populations at emission wavelengths characteristic of Photosystem II chlorophyll/protein complexes. Their zero-field splitting parameters have been determined in order to compare them with the well-studied PSII recombination triplet state. None of these triplets have the zero-field splitting parameters characteristic of the recombination triplet and are therefore probably not generated directly in the reaction center. On the basis of their microwave-induced emission spectra, it is suggested that two are probably generated in the core complex(es) while the third may be generated in the external antenna. These triplets are formed under nonreducing redox conditions, when the recombination triplet is undetectable. It is suggested that they may be involved in the photoinhibitory damage of Photosystem II. The triplet-minus-singlet spectrum associated with the recombination triplet state has been determined for thylakoids after reduction of the secondary acceptors. Its main peak is at 685 nm, slightly red shifted with respect to earlier reports, with a weak signal, of opposite sign at approximately 675 nm. The 685 nm peak indicates that at cryogenic temperatures, the triplet is located on the long-wavelength chlorophyll state present in the reaction center complex of Photosystem II (D1.D2.Cytb(559) complex). From the absence of a clear structure in the 680 nm absorption region, this long-wavelength absorbing state does not appear to be strongly coupled to P(680), though it must be associated with one of the "inner core" pigments recently identified in the photosystem II crystallographic structure [Zouni et al. (2001) Nature 408, 739-743].