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The triplet state of the carotenoid peridinin, populated by triplet-triplet energy transfer from photoexcited chlorophyll triplet state, in the reconstituted Peridinin-Chlorophyll a-protein, has been investigated by ODMR (Optically detected magnetic resonance), and pulse EPR spectroscopies. The properties of peridinins associated with the triplet state formation in complexes reconstituted with Chl a and Chl d have been compared to those of the main-form peridinin-chlorophyll protein (MFPCP) isolated from Amphidinium carterae. In the reconstituted samples no signals due to the presence of chlorophyll triplet states have been detected, during either steady state illumination or laser-pulse excitation. This demonstrates that reconstituted complexes conserve total quenching of chlorophyll triplet states, despite the biochemical treatment and reconstitution with the non-native Chl d pigment. Zero field splitting parameters of the peridinin triplet states are the same in the two reconstituted samples and slightly smaller than in native MFPCP. Analysis of the initial polarization of the photoinduced Electron-Spin-Echo detected spectra and their time evolution, shows that, in the reconstituted complexes, the triplet state is probably localized on the same peridinin as in native MFPCP although, when Chl d replaces Chl a, a local rearrangement of the pigments is likely to occur. Substitution of Chl d for Chl a identifies previously unassigned bands at approximately 620 and approximately 640 nm in the Triplet-minus-Singlet (T-S) spectrum of PCP detected at cryogenic temperature, as belonging to peridinin.

The photoexcited triplet state of the carotenoid peridinin in the high-salt peridinin–chlorophyll a -protein (HSPCP) of the dinoflagellate Amphidinium carterae was investigated by ODMR (optically detected magnetic resonance), pulse EPR and pulse ENDOR spectroscopies. The properties of peridinins associated to the triplet state formation in HSPCP were compared to those of peridinins involved in triplet state population in the main-form peridinin–chlorophyll protein (MFPCP), previously reported. In HSPCP no signals due to the presence of chlorophyll triplet state have been detected, during either steady-state illumination or laser-pulse excitation, meaning that peridinins play the photo-protective role with 100% efficiency as in MFPCP. The general spectroscopic features of the peridinin triplet state are very similar in the two complexes and allow drawing the conclusion that the triplet formation pathway and the triplet localization in one specific peridinin in each subcluster are the same in HSPCP and MFPCP. However some significant differences also emerged from the analysis of the spectra. Zero field splitting parameters of the peridinin triplet states are slightly smaller in HSPCP and small changes are also observed for the hyperfine splittings measured by pulse ENDOR and assigned to the β-protons belonging to one of the two methyl groups present in the conjugated chain, ( a iso = 10.3 MHz in HSPCP vs a iso = 10.6 MHz in MFPCP). The differences are explained in terms of local distortion of the tails of the conjugated chains of the peridinin molecules, in agreement with the conformational data resulting from the X-ray structures of the two complexes.

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 α- and β-protons of the peridinin conjugated chain. Assignments of β-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 π-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.

The mechanism of triplet–triplet energy transfer in the peridinin–chlorophyll–protein (PCP) from Amphidinium carterae was investigated by time-resolved EPR (TR-EPR). The approach exploits the concept of spin conservation during triplet–triplet energy transfer, which leads to spin polarization conservation in the observed TR-EPR spectra. The acceptor (peridinin) inherits the polarization of the donor (chlorophyll) in a way which depends on the relative geometrical arrangement of the donor–acceptor couple. Starting from the initially populated chlorophyll triplet state and taking the relative positions among Chls and peridinins from the X-ray structure of PCP, we calculated the expected triplet state polarization of any peridinin in the complex. Comparison with the experimental data allowed us to propose a path for triplet quenching in the protein. The peridinin–chlorophyll pair directly involved in the triplet–triplet energy transfer coincides with the one having the shortest center to center distance. A water molecule, which is coordinated to the central Mg atom of the Chl, is also placed in close contact with the peridinin. The results support the concept of localization of the triplet state mainly in one specific peridinin in each of the two pigment subclusters related by a pseudo C2 symmetry.

A comparative electron paramagnetic resonance (EPR) study has been performed on a series of structurally related molecular triads which undergo photoinduced electron transfer and differ one from the other in terms of the acceptor or donor moieties. The molecular triads, C-P-C60, TTF-P-C60 and C-P-PF, share the same free-base, tetraarylporphyrin (P) as the primary electron donor, which after light excitation initiates the electron transfer process, but differ either in terms of the electron acceptor (fullerene derivative, C60, versus fluorinated free-base porphyrin, PF), or in terms of the final electron donor (carotenoid polyene, C, versus tetrathiafulvalene, TTF). All these molecular triads can be considered artificial photosynthetic reaction centers in their ability to mimic several key properties of the reaction center primary photochemistry. Photoinduced charge separation and recombination have been followed by time-resolved EPR in a glass of 2-methyltetrahydrofuran and in the nematic phase of the uniaxial liquid crystal E-7. All the triads undergo photoinduced electron transfer, with the generation of charge-separated states in both the low-dielectric environment of the 2-methyl-tetrahydrofuran glass and in anisotropic E-7 medium. Different photochemical pathways have been recognized depending on the specific donor and acceptor moieties constituting the molecular triads. In the presence of the tetrathiafulvalene electron donor singlet- and triplet-initiated electron transfer routes are concurrently active. Recombination to the low-lying carotenoid triplet state occurs in the carotene-based triads, while singlet recombination is the only active route for the TTF-P-C60 triad, where a low-lying triplet state is lacking. Long-lived charge separation has been observed in the case of TTF-P-C60: about 8 μs for the singlet-born radical pair in the glassy isotropic matrix and about 7 μs for the triplet-born radical pair in the nematic phase of E-7. For all the molecular triads, a weak exchange interaction (J≊1 G) between the electrons in the final spin-correlated radical pair has been evaluated by simulation of the EPR spectra, providing evidence for superexchange electronic interactions mediated by the tetraarylporphyrin bridge.

The carotenoid triplet populations associated with the fluorescence emission chlorophyll forms of Photosystem II have been investigated in isolated spinach thylakoid membranes by means of fluorescence detected magnetic resonance in zero field (FDMR). The spectra collected in the 680-690 nm emission range, have been fitted by a global analysis procedure. At least five different carotenoid triplet states coupled to the terminal emitting chlorophyll forms of PS II, peaking at 682 nm, 687 nm and 692 nm, have been characterised. The triplets associated with the outer antenna emission forms, at 682 nm, have zero field splitting parameters |D| = 0.0385 cm-1, |E| = 0.00367 cm-1; |D| = 0.0404 cm-1, |E| = 0.00379 cm-1 and |D| = 0.0386 cm-1, |E| = 0.00406 cm-1 which are very similar to those previously reported for the xanthophylls of the isolated LHC II complex. Therefore the FDMR spectra recorded in this work provide insights into the organisation of the LHC II complex in the unperturbed environment represented by thylakoid membranes. The additional carotenoid triplet populations, detected by monitoring the chlorophyll emission at 687 and 692 nm, are assigned to carotenoids bound to inner antenna complexes and hence attributed to beta-carotene molecules.

Photoinduced charge separation and recombination in a carotenoid-porphyrin-fullerene triad C-P-C 6 0 (Bahr et al., 2000) have been followed by time-resolved electron paramagnetic resonance. The electron-transfer process has been characterized in a glass of 2-methyltetrahydrofuran and in the nematic phase of two uniaxial liquid crystals (E-7 and ZLI-1167). In all the different media, the molecular triad undergoes two-step photoinduced electron transfer, with the generation of a long-lived charge-separated state (C . + -P-C 6 0 . - ), and charge recombination to the triplet state, localized in the carotene moiety, mimicking different aspects of the photosynthetic electron-transfer process. The magnetic interaction parameters have been evaluated by simulation of the spin-polarized radical pair spectrum. The weak exchange interaction parameter (J = +1.7 ′ 0.1 G) provides a direct measure of the dominant electronic coupling matrix element V between the C . + -P-C 6 0 . - radical pair state and the recombination triplet state 3 C-P-C 6 0 . Comparison of the estimated values of V for this triad and a structurally related triad differing only in the porphyrin bridge (octaalkylporphyrin vs tetraarylporphyrin) explains in terms of an electronic coupling effect the ∼6-fold variation of the recombination rate induced by the modification of the porphyrin bridge as derived by kinetic experiments (Bahr et al., 2000).

Photoinduced electron transfer has been observed in a molecular triad, consisting of a porphyrin (P) covalently linked to a tetrathiafulvalene (TTF) and a fullerene derivative (C(60)), in the different phases of the liquid crystal E-7 and in a glass of 2-methyltetrahydrofuran (2-MeTHF) by means of time-resolved electron paramagnetic resonance (EPR) spectroscopy. In both solvents, an EPR signal observed immediately after excitation has been assigned to the radical pair TTF(*+)-P-C(60)(*-), based on its magnetic interaction parameters and spin polarization pattern. In the 2-MeTHF glass and the crystalline phase of E-7, the TTF(*+)-P-C(60)(*-) state is formed from the TTF-(1)P-C(60) singlet state via an initial TTF-P(*+)-C(60)(*-) charge-separated state. Long-lived charge separation ( approximately 8 mus) for the singlet-born radical pair is observed in the 2-MeTHF glass at cryogenic temperatures. In the nematic phase of E-7, a high degree of ordering in the liquid crystal is achieved by the molecular triad. In this phase, both singlet- and triplet-initiated electron transfer routes are concurrently active. At room temperature in the presence of the external magnetic field, the triplet-born radical pair (T)(TTF(*+)-P-C(60)(*-)) has a lifetime of approximately 7 mus, while that of the singlet-born radical pair (S)(TTF(*+)-P-C(60)(*-)) is much shorter (1 mus). The difference in lifetimes is ascribed to spin dynamic effects in the magnetic field.

In this study, triplet quenching, the major photoprotection mechanism in antenna proteins, has been studied in the light-harvesting complex of photosystem I (LHC-I). The ability of carotenoids bound to LHC-I subunit Lhca4, which is characterized by the presence of the red-most absorption components at wavelength >700 nm, to protect the system through quenching of the chlorophyll triplet states, has been probed, by analyzing the induction of carotenoid triplet formation. We have investigated this process at low temperature, when the funneling of the excitation toward the low-lying excited states of the Chls is stronger, by means of optically detected magnetic resonance (ODMR), which is well-suited for investigation of triplet states in photosynthetic systems. The high selectivity and sensitivity of the technique has made it possible to point out the presence of specific interactions between carotenoids forming the triplet states and specific chlorophylls characterized by red-shifted absorption, by detection of the microwave-induced Triplet minus Singlet (T-S) spectra. The effect of the red forms on the efficiency of triplet quenching was specifically probed by using the Asn47His mutant, in which the red forms have been selectively abolished (Morosinotto, T., Breton, J., Bassi, R., and Croce, R. (2003) J. Biol. Chem. 278, 49223-49229). Lack of the red forms yields into a reduced efficiency of the triplet quenching in LHC-I thus suggesting that the "red Chls" play a role in enhancing triplet quenching in LHC-I and, possibly, in the whole photosystem I.

Optically detected magnetic resonance of chlorosomes-containing membranes from the green sulfur bacterium Chlorobium limicola has been performed both by fluorescence and absorption detection. Triplet states localized in the chlorosomes and in the FMO complex have been characterized. After chemical reduction with dithionite followed by illumination at 200 K, a recombination triplet state localized in the primary donor P840 becomes populated under illumination at low temperature. A reaction center triplet state characterized by slightly different ZFS parameters, grows up irreversibly after prolonged illumination at low temperature in the presence of reductant. We were able to obtain the T−S spectra of the FMO complex and of the primary donor P840 in their native environment and to compare them to the spectra obtained in isolated complexes previously published, revealing differences in the spectra. Fluorescence detected magnetic resonance measurements demonstrate that the BChl c antenna pigments are connecte...

Optically detected magnetic resonance of chlorosome-containing membranes from the green filamentous bacterium Chloroflexus aurantiacus has been performed both by fluorescence and absorption detection. Triplet states localized in the chlorosomes and in the B808–866 complex have been characterized. After chemical reduction with ascorbate followed by illumination at 200 K, recombination triplet state localized in the primary donor becomes largely populated under illumination at low temperature while all the antenna triplet states, which are localized in carotenoids and BChl a molecules, are strongly quenched. We were able to obtain the T-S spectrum of the primary donor P870 surrounded by all the antenna complexes connected to the RC via energy transfer and then in its intact environment. We found clear spectroscopic evidence for exciton interaction between the RC and the B808–866 antenna complex. This evidence was provided by the comparison of the T−S spectrum of P870 in the membranes with that of isolated RC. The analogy of some features of the difference spectra with those previously found in the same kind of experiments for Rb. sphaeroides, allows to predict a similar coupling among the primary donor and the nearby antenna BChl a molecules, assembled as circular aggregate.

Chemical oxidation of the chlorosomes from Chloroflexus aurantiacus and Chlorobium tepidum green bacteria produces bacteriochlorophyll radicals, which are characterized by an anomalously narrow EPR signal compared to in vitro monomeric BChl c (.+) [Van Noort PI, Zhu Y, LoBrutto R and Blankenship RE (1997) Biophys J 72: 316-325]. We have performed oxidant concentration and temperature-dependent X-band EPR measurements in order to elucidate the line narrowing mechanism. The linewidth decreases as the oxidant concentration is increased only for Chloroflexus indicating that for this system Heisenberg spin exchange is at least partially responsible for the EPR spectra narrowing. For both species the linewidth is decreasing on increasing the temperature. This indicates that temperature-activated electron transfer is the main narrowing mechanism for BChl radicals in chlorosomes. The extent of the electron transfer process among different BChl molecules has been evaluated and a comparison between the two species representative of the two green bacteria families has been made. In parallel, high frequency EPR experiments have been performed on the oxidized chlorosomes of Chloroflexus and Chlorobium at 110 and 330 GHz in the full temperature range investigated at X-band. The g-tensor components obtained from the simulation of the 330 GHz EPR spectrum from Chlorobium show the same anisotropy as those of monomeric Chl a (.+) [Bratt PJ, Poluektov OG, Thurnauer MC, Krzystek J, Brunel LC, Schrier J, Hsiao YW, Zerner M and Angerhofer A (2000) J Phys Chem B 104: 6973-6977]. The spectrum of Chloroflexus has a nearly axial g-tensor with reduced anisotropy compared to Chlorobium and monomeric Chl a in vitro. g-tensor values and temperature dependence of the linewidth have been discussed in terms of the differences in the local structure of the chlorosomes of the two families.

We have found that the addition of N -methylfulleropyrrolidine to poly(methylmethacrylate) (PMMA), during the thermal polymerization route, produces a new fullerene adduct that has been characterized by time-resolved optical and EPR methods. In fact, a new component appears in the differential absorption spectrum with a lifetime of 350 μs and a maximum at 850 nm besides the typical component due to fullerene adducts centered around 700 nm. The new component has been attributed to a fullerene adduct crosslinked to the PMMA chains, supporting the active role of such a polymer matrix in modifying the optical properties of fullerene. Time-resolved electron paramagnetic resonance (TREPR) spectrum of this new species exhibits zfs parameters and spin polarization that are similar to those observed for a trans 3– trans 3– trans 3 adduct in a toluene matrix.

The radical anions of three C60 derivatives (N-methyl-3,4-fulleropyrrolidine, N-methyl-2-(3,6,9-trioxadecyl)3,4-fulleropyrrolidine, and bis-N-methyl-3,4-fulleropyrrolidine are respectively MFP - , tegMFP - , and bisMFP - ) have been investigated by continuous wave (cw) and pulsed EPR in methyltetrahydrofuran and tetrahydrofuran solutions. The EPR spectra for MFP - and tegMFP - show a 1:1:1 triplet, due to the hyperfine coupling with the pyrrolidine nitrogen. The EPR spectrum of bisMFP - shows a superimposition of two signals, each due to a 1:2:3:2:1 quintet. Each quintet is due to the hyperfine coupling with the two pyrrolidine nitrogen atoms. The two signals are attributed to two different conformers of the radical. A direct transfer of spin density from the C60 sphere to the nitrogen atoms is discussed as arising from the bent conformation of the pyrrolidine rings. All the spectra show several weaker satellite lines, due to the radical anions containing a 13 C atom. From their analysis a C 2V symmetry for the spin distribution is obtained in the case of MFP - and tegMFP - . The EPR line widths of these latter radicals at room temperature are larger than those expected on the basis of spin relaxation due to rotational diffusion in solution, and the line widths decrease upon decreasing the temperature. Pulsed EPR measurements of T1 in liquid and frozen solutions show that the EPR line widths are determined by the anomalously short lifetimes of the spin states. The temperature dependence of T1 obeys to the Arrhenius law, giving an activation energy of 7 kJ/mol. The latter results are compared with those obtained in the past for the lone C60 . They are attributed to the closeness of the electronic levels, due to the weak perturbation of the pyrrolidine ring on the symmetrical electronic distribution of C 60. On the other hand, the spin relaxation properties of bisMFP - are shown those expected for a nonsymmetrical radical. The comparison of cw- and pulsed-EPR spectra of MFP - in frozen solutions of MeTHF and THF indicates the presence of aggregates of interacting radicals in THF.

A series of carotenoporphyrin dyads, in which the carotenoid is covalently linked to a tetraarylporphyrin at the ortho, meta or para position of one of the meso aromatic rings, has been studied using Time-Resolved Electron Paramagnetic Resonance (TREPR) spectroscopy. In parallel, an investigation has been carried, on two different photosynthetic antenna systems, the B800–B850 complex ofR. acidophila and the LHCII complex of higher organisms. The initial spin polarization of the carotenoid triplet-state, populated indirectly by laser excitation, has been detected. It has been demonstrated that the initial polarization is not a characteristic property of the carotenoid triplet-state, as previously stated, but depends on the donor-acceptor mutual orientation. The triplet energy transfer to the carotenoid from a chlorophyll or porphyrin triplet state is discussed on the basis of the observed spin polarization.

Carotenoid triplets play a photoprotective role in natural photosynthesis. The main process of carotenoid triplet formation is known to be triplet-triplet energy transfer from chlorophyll triplets. The structural requirements for high transfer yields are still a matter of discussion and the presence of competitive triplet formation pathways has not been excluded. Transient EPR measurements of triplet states formed by photoexcitation allow detection of the initial spin polarization. This pattern derives from the mechanism of triplet formation. In the case of triplet-triplet energy transfer, if the condition of spin angular momentum conservation is fulfilled, simulation of the EPR spectra gives information about the donor-acceptor mutual orientation. We describe transient EPR experiments on two artificial photosynthetic dyads, consisting of a carotenoid covalently-linked to a free-base or zinc substituted pyropheophorbide moiety and we discuss the results in terms of possible dyad conformations.

The EPR spectra of excited triplet states of two fullerene derivatives, C60O and C60C2H4N(CH3), in the glassy matrices of toluene and of polymethylmethacrylate (PMMA) and as films were examined at low temperature and the magnetic parameters were measured. In films, species with zfs parameters smaller than those in toluene were found and they were assigned to triplet excitations visiting more than one molecule in the crystallites that form the film. When C60C2H4N(CH3) was excited in PMMA grown by in sity thermal polymerization a new triplet species was originated characterized by a dipolar splitting constant D about three times larger than that measured in toluene. Evidence was gained that such species participates to cross-linking in the polymer. The effect of PMMA matrix on the molecular dynamics of 3C70 was also investigated. It was found that a dynamical model holds based on the pseudorotation of the molecule around the axis of larger dipolar splitting. Motion is activated with activation energy of 210 ± 50 cm−1 a value comparable with that obtained for pseudorotation of 3C70 in cyclohexane.

Samples of two fullerene derivatives, namely, C{sub 60}C{sub 2}H{sub 4}N(CH{sub 3}) and C{sub 60}C{sub 2}H{sub 4}N(Ph{sub 2}C{sub 6}H{sub 4}-4-OCH{sub 3}) in poly(methyl methacrylate) (PMMA), were prepared either by solvent evaporation from CHCl{sub 3} solutions or by thermal polymerization of methyl methacrylate monomers containing the fulleropyrrolidines. In the first case PMMA behaves as a neutral polymeric matrix, while in the second the material swells by absorption of the solvent and its glass transition temperature is shifted up by 8{degree}C with respect to undoped material. Both pieces of evidence are typical of cross-linking between the polymer chains that involves fulleropyrrolidines. EPR and ODMR spectroscopies are applied to the study of the cross-linked species. It is found its excited triplet state is characterized by unusually large electron dipolar splitting ({vert_bar}D{vert_bar} = 248x10{sup -4} cm{sup -1}). Calculations of D have been carried out using a model that considers {sup 3}C{sub 60} and the fullerene derivatives as a collection of fully localized double bonds. The experimental negative sign of D has been reproduced for {sup 3}C{sub 60}, and a positive sign of D is expected for cross-linked fulleropyrrolidine affected by cross-links in the equatorial region. Such a sign reversal can explain the different spin polarizationmore » patterns exhibited by their EPR spectra. 38 refs., 5 figs., 1 tab.« less

Fluorescence detected magnetic resonance (FDMR) is used to investigate peridinin-chlorophyll-a protein (PCP), the peripheral antenna of the photosystem of Amphidinium carterae . Peridinin triplet resonances are obtained monitoring chlorophyll-a fluorescence in agreement with efficient energy transfer between the two pigments. Two sites are revealed for peridinin having slightly different triplet energy (about 190 cm −1 ), and D values averaging 0.045 cm −1 . Spin exchange occurs between the two sites at a rate of about 5 × 10 7 exp (−67/ T ) s −1 .

Fluorescence detected magnetic resonance (FDMR) of various chlorophyll a triplets in PS II large particles was studied with the aim of identifying the nature of the different components underlying the complex line shape of the signals. The latter are deconvoluted into sums of Gaussians in different experimental conditions, and the charge recombination triplet is unequivocally identified as the source of two lines with negative signs at 722.5 and 992 MHz |D| = 0.0286 cm−1, |E| = 0.0043 cm−1). The origin of the other triplet signals from the integrated particles is discussed in terms of functional antenna components and products from alteration of the native environment of pigments. FDMR signals also arise from isolated reaction centre complexes and are all to be attributed to different triplet populations of the primary electron donor P680. The results are compared with those obtained by other authors on ADMR. The heterogeneity of P680 pigments in D1D2-cyt-b559 complexes is shown to arise during preparation procedures.

Biphenyl-TCNQ and biphenyl-tetrafluoro-TCNQ crystals, when excited with visible light, give rise to triplet excitons which were investigated by optical, EPR and zero-field (ZF) ODMR spectroscopies in a wide temperature range. The analysis of spin polarization carried by EPR and ZF ODMR spectra at room temperature enabled us to reveal the singlet depletion path S→ (T, T) which is thermally activated. The activation energies are Δ E = 1940 cm −1 for B-TCNQ and Δ E = 1720 cm −1 for B-TCNQF 4 . From the relation 2 E T = E S +Δ E the energies of the triplet state in the CT complexes were derived: E T =9770 ± 100 cm −1 for B-TCNQ and E T =8695 ± 100 cm −1 for B-TCNQF 4 . It was also established that the triplet species detected by ODMR at 1.8 K are populated by ISC.

The kinetics of electron transfer from phyllosemiquinone (PhQ(*-)) to the iron sulfur cluster F(X) in Photosystem I (PS I) are described by lifetimes of approximately 20 and approximately 250 ns. These two rates are attributed to reactions involving the quinones bound primarily by the PsaB (PhQ(B)) and PsaA (PhQ(A)) subunits, respectively. The factors leading to a approximately 10-fold difference between the observed lifetimes are not yet clear. The peptide nitrogen of conserved residues PsaA-Leu722 and PsaB-Leu706 is involved in asymmetric hydrogen-bonding to PhQ(A) and PhQ(B), respectively. Upon mutation of these residues in PS I of the green alga, Chlamydomonas reinhardtii , we observe an acceleration of the oxidation kinetics of the PhQ(*-) interacting with the targeted residue: from approximately 255 to approximately 180 ns in PsaA-L722Y/T and from approximately 24 to approximately 10 ns in PsaB-L706Y. The acceleration of the kinetics in the mutants is consistent with a perturbation of the H-bond, destabilizing the PhQ(*-) state, and increasing the driving force of its oxidation. Surprisingly, the relative amplitudes of the phases reflecting PhQ(A)(*-) and PhQ(B)(*-) oxidation were also affected by these mutations: the apparent PhQ(A)(*-)/PhQ(B)(*-) ratio is shifted from 0.65:0.35 in wild-type reaction centers to 0.5:0.5 in PsaA-L722Y/T and to 0.8:0.2 in PsaB-L706Y. The most consistent account for all these observations involves considering reversibility of oxidation of PhQ(A)(*-) and PhQ(B)(*-) by F(X), and asymmetry in the driving forces for these electron transfer reactions, which in turn leads to F(x)-mediated interquinone electron transfer.

We present an optically detected magnetic resonance (ODMR) and electron paramagnetic resonance (EPR) spectroscopic study on the quenching of photo-induced chlorophyll triplet states by carotenoids, in the intrinsic light-harvesting complex (LHC) from the dinoflagellate Amphidinium carterae.Two carotenoid triplet states, differing in terms of optical and magnetic spectroscopic properties, have been identified and assigned to peridinins located in different protein environment. The results reveal a parallelism with the triplet–triplet energy transfer (TTET) process involving chlorophyll a and luteins observed in the LHC-II complex of higher plants. Starting from the hypothesis of a conserved alignment of the amino acid sequences at the cores of the LHC and LHC-II proteins, the spin-polarized time-resolved EPR spectra of the carotenoid triplet states of LHC have been calculated by a method which exploits the conservation of the spin momentum during the TTET process. The analysis of the spectra shows that the data are compatible with a structural model of the core of LHC which assigns the photo-protective function to two central carotenoids surrounded by the majority of Chl a molecules present in the protein, as found in LHC-II. However, the lack of structural data, and the uncertainty in the pigment composition of LHC, leaves open the possibility that this complex posses a different arrangement of the pigments with specific centers of Chl triplet quenching.

Optically detected magnetic resonance (ODMR) of the light-harvesting complexes CP43 and CP47 of the spinach photosynthetic machinery revealed triplet states both from carotenoids and chlorophylls. The triplet state of the only carotenoid present in the complexes (β-carotene) is observed by ODMR using fluorescence detection (FDMR) in the main chlorophyll emission band in the 680 nm region, and absorption detection in the visible region. Chlorophyll triplet signals are also obtained by microwave sweeping in the chlorophyll emission and absorption regions. MIA spectra are obtained and discussed for both complexes.

Transient optical emission spectra of biphenyl-1,2,4,5-tetracyanobenzene crystal doped with TCNQ have been investigated in the (77÷300) K temperature range. Host prompt fluorescence lifetimes have been measured by cross-correlation phase fluorometry. The decay signal is fitted by a biexponential function whose coefficients are strongly dependent on temperature. A thermal activation energy of 300 cm−1 for the diffusion of singlet exciton has been determined.

Chlorophyll d (Chl d) is the major pigment in the antenna proteins of both photosystems (PSI and II) of the oxyphotobacterium Acaryochloris marina. This fact suggests that photosynthesis based upon Chl d rather than Chl a may be an interesting alternative in oxygenic photosynthesis. While a great deal of spectroscopic information relative to Chl a is available, both in vivo and in vitro, the literature on Chl d is scarce. In particular, the triplet state of Chl d has not been studied in vitro to date. Although triplet states do not represent the main excitation path in the photosynthetic process, they are involved in light stress events both in the antenna complexes and in the reaction centers and may also be used as endogenous paramagnetic probes of the molecular environment. In this paper we make use of both time-resolved EPR and ODMR to characterize, for the first time, the Chl d triplet state in the polar solvent methyl-tetrahydrofuran. Comparison with the spectra of Chl a obtained under the same expe...

The analysis of FDMR spectra, recorded at multiple emission wavelengths, by a global decomposition technique, has allowed us to characterise the triplet populations associated with Photosystem I and Photosystem II of thylakoids in the green alga Chlamydomonas reinhardtii. Three triplet populations are observed at fluorescence emissions characteristic of Photosystem II, and their zero field splitting parameters have been determined. These are similar to the zero field parameters for the three Photosystem II triplets previously reported for spinach thylakoids, suggesting that they have a widespread occurrence in nature. None of these triplets have the zero field splitting parameters characteristic of the Photosystem II recombination triplet observed only under reducing conditions. Because these triplets are generated under non-reducing redox conditions, when the recombination triplet is undetectable, it is suggested that they may be involved in the photoinhibition of Photosystem II. At emission wavelengths characteristic of Photosystem I, three triplet populations are observed, two of which are attributed to the P700 recombination triplet frozen in two different conformations, based on the microwave-induced fluorescence emission spectra and the triplet minus singlet difference spectra. The third triplet population detected at Photosystem I emission wavelengths, which was previously unresolved, is proposed to originate from the antenna chlorophyll of the core or the unusually blue-shifted outer antenna complexes of this organism.

Photoinduced charge separation and recombination were investigated in a triad consisting of a carotenoid (C), a tetraarylporphyrin (P) and a tris(heptafluoropropyl)porphyrin (PF), C–P–PF, by means of time-resolved electron paramagnetic resonance. The electron transfer process was studied in a glass of 2-methyltetrahydrofuran at 10 K, in the crystalline phase at 150 K and in the liquid nematic phase of the uniaxial LC E-7 at 295 K, and in the nematic phase of the LC ZLI-1167 at 300 K. In all the different media and in the different phases, the molecular triad undergoes two-step photoinduced electron transfer, with the generation of a long-lived charge-separated state (C•+–P– ), and charge recombination to the triplet state, localized in the carotene moiety 3C–P–PF. Low-temperature charge separation and triplet recombination are common features of both fullerene-based and diporphyrin molecular triads, proving that the large delocalized π-electron system of the porphyrin electron acceptor leads to low total ...

Chemical oxidation of the chlorosomes from Chloroflexus aurantiacus and Chlorobium tepidum green bacteria produces bacteriochlorophyll radicals, which are characterized by an anomalously narrow EPR signal compared to in vitro monomeric BChl c (.+) [Van Noort PI, Zhu Y, LoBrutto R and Blankenship RE (1997) Biophys J 72: 316-325]. We have performed oxidant concentration and temperature-dependent X-band EPR measurements in order to elucidate the line narrowing mechanism. The linewidth decreases as the oxidant concentration is increased only for Chloroflexus indicating that for this system Heisenberg spin exchange is at least partially responsible for the EPR spectra narrowing. For both species the linewidth is decreasing on increasing the temperature. This indicates that temperature-activated electron transfer is the main narrowing mechanism for BChl radicals in chlorosomes. The extent of the electron transfer process among different BChl molecules has been evaluated and a comparison between the two species representative of the two green bacteria families has been made. In parallel, high frequency EPR experiments have been performed on the oxidized chlorosomes of Chloroflexus and Chlorobium at 110 and 330 GHz in the full temperature range investigated at X-band. The g-tensor components obtained from the simulation of the 330 GHz EPR spectrum from Chlorobium show the same anisotropy as those of monomeric Chl a (.+) [Bratt PJ, Poluektov OG, Thurnauer MC, Krzystek J, Brunel LC, Schrier J, Hsiao YW, Zerner M and Angerhofer A (2000) J Phys Chem B 104: 6973-6977]. The spectrum of Chloroflexus has a nearly axial g-tensor with reduced anisotropy compared to Chlorobium and monomeric Chl a in vitro. g-tensor values and temperature dependence of the linewidth have been discussed in terms of the differences in the local structure of the chlorosomes of the two families.

Optically detected magnetic resonance of chlorosome-containing membranes from the green filamentous bacterium Chloroflexus aurantiacus has been performed both by fluorescence and absorption detection. Triplet states localized in the chlorosomes and in the B808-866 complex have been characterized. After chemical reduction with ascorbate followed by illumination at 200 K, recombination triplet state localized in the primary donor becomes largely populated under illumination at low temperature while all the antenna triplet states, which are localized in carotenoids and BChl a molecules, are strongly quenched. We were able to obtain the T-S spectrum of the primary donor P870 surrounded by all the antenna complexes connected to the RC via energy transfer and then in its intact environment. We found clear spectroscopic evidence for exciton interaction between the RC and the B808-866 antenna complex. This evidence was provided by the comparison of the T-S spectrum of P870 in the membranes with that of isolated RC. The analogy of some features of the difference spectra with those previously found in the same kind of experiments for Rb. sphaeroides, allows to predict a similar coupling among the primary donor and the nearby antenna BChl a molecules, assembled as circular aggregate.

Photoinduced charge separation and recombination in a carotenoid-porphyrin-fullerene triad C-P-C(60)(1) have been followed by multifrequency time-resolved electron paramagnetic resonance (TREPR) at intermediate magnetic field and microwave frequency (X-band) and high field and frequency (W-band). The electron-transfer process has been characterized in the different phases of two uniaxial liquid crystals (E-7 and ZLI-1167). The triad undergoes photoinduced electron transfer, with the generation of a long-lived charge-separated state, and charge recombination to the triplet state, localized in the carotene moiety, mimicking different aspects of the photosynthetic electron-transfer process. Both the photoinduced spin-correlated radical pair and the spin-polarized recombination triplet are observed starting from the crystalline up to the isotropic phase of the liquid crystals. The W-band TREPR radical pair spectrum has allowed unambiguous assignment of the spin-correlated radical pair spectrum to the charge-separated state C(.+)-P-C(60)(.-). The magnetic interaction parameters have been evaluated by simulation of the spin-polarized radical pair spectrum and the spin-selective recombination rates have been derived from the time dependence of the spectrum. The weak exchange interaction parameter (J = +0.5 +/- 0.2 G) provides a direct measure of the dominant electronic coupling matrix element V between the C(.+)-P-C(60)(.-) radical pair state and the recombination triplet state (3)C-P-C(60). The kinetic parameters have been analyzed in terms of the effect of the liquid crystal medium on the electron-transfer process. Effects of orientation of the molecular triad in the liquid crystal are evidenced by simulations of the carotenoid triplet state EPR spectra at different orientations of the external magnetic field with respect to the director of the mesophase. The order parameter (S = 0.5 +/- 0.05) has been evaluated.

Photoinduced charge separation and recombination to the triplet state in a carotene (C) porphyrin (P) fullerene (C 60 ) triad have been followed by time-resolved electron paramagnetic resonance (EPR). The electron transfer process has been studied at different temperatures in both a glassy isotropic matrix (2-methyltetrahydrofuran) and uniaxial liquid crystal (E-7). In both media, the triad undergoes two-step photoinduced electron transfer, with the generation of a long-lived charge separated state (C ” + -P-C 60 ” m ), and charge recombination to the triplet state, localized in the carotene moiety. The carotenoid triplet state is initially polarized according to the mechanism of recombination of a radical pair with singlet precursor. Both the photoinduced spin-correlated radical pair and the carotene triplet are observed in E-7 starting from the glass to the liquid crystal phase. The exchange interaction between the electrons in the radical pair (J=1.2 Gauss) has been evaluated by simulation of the EPR s...

A comparative study on the isolated chlorosomes from Chloroflexus aurantiacus, a green filamentous photosynthetic bacterium and Chlorobium tepidum, a green sulfur photosynthetic bacterium, was done...

The photochemistry of a molecular triad consisting of a porphyrin (P) covalently linked to a carotenoid polyene (C) and a fullerene derivative (C60) has been studied at 20 K by time-resolved EPR sp...

An ADMR T-S spectrum of the primary donor (P680) of photosystem II (PSII) was obtained from anaerobically photoreduced particles. The spectrum is the best resolved obtained so far having a main bleaching band at 684 nm with a linewidth of only 100 cm−1. The view that this spectrum is produced by native homogeneous P680 unlike those obtained before is defended. A small bleaching observed at 678 nm is discussed in terms of the reaction center structure. One possible interpretation of the observations is that P680 is a very loose dimer with an exciton splitting of only 144 cm−1 corresponding to a dimer center-to-center distance of roughly 11.5 Å.

Time decay of the ODMR signals obtained from light irradiated Biphenyl-TCNB crystals has been analyzed varying temperature and microwave power. New information are obtained on the triplet X-trap produced by illumination. It is also shown that time decay measurements are able to evidence in a simple way the cases in which the triplet is born in a non-stationary state.

Localized triplet excitations obtained by trapping of triplet excitons are investigated in the biphenyl (B)-tetracyanobenzene (TCNB) crystal by zero-field ODMR methods at low temperature (1.25 K). At high microwave power (1 W) the ODMR spectral lines are structured with new satellite lines appearing at the sides of the central peak due to forbidden transitions. Their analysis allowed us to obtain information on the quadrupole tensor components of the TCNB molecule in its lowest triplet state.

The photoexcited triplets in single crystals of the trans-stilbene-1,2,4,5-tetracyanobenzene (1:2) complex have been investigated by means of the ESR and DF ODMR techniques. The ESR lines attributed to localized species exhibit strong spin polarization whose dependence on the direction of the magnetic field has been examined. The zero-field splitting parameters have been measured: X = −308±15 MHz, Y = 2010±15 MHz, Z = −1702±15 MHz with the z axis directed along the normal to the t -stilbene phenyl rings. The negative sign of the DF ODMR signal has been attributed to annihilation of unlike triplets (exciton and X-trap), a process whose occurrence is supported also by decay measurements of the fluorescence.

The nuclear spin-lattice relaxation times of the nematogen PAA in the isotropic phase and of some nonmesomorphic azobenzene and azoxybenzene derivatives have been measured. A similar behaviour of the phenyl carbon T 1 , temperature dependence has been obtained for the mesogenic and non-mesogenic compounds. It is also shown that for PAA the reorientation rate of the methoxy group is comparable with the molecular diffusion rate.

The crystal and molecular structure of uranyl nitrate trimethylphosphate has been determined by three dimensional X-ray analysis, U---U intermolecular distances are correlated to solid state photochemical behavior in this class of compounds The mode of bonding of the nitrate ion to uranyl is compared with other known uranyl nitrate compounds, and it is confirmed that upon coordination the two NOcoordinate bonds are lengthened by 0.05 A, with a simultaneous reduction of the ONO coordinate angle from 120° to 112–115°. Extended Huckel calculations and VSEPR theory interpret this feature as being due to a donation of electron density from the NOcoordinate bonds to uranyl bonding orbitals.

Triplet excitons in electron donor—acceptor charge-transfer (CT) molecular crystals are generated through the intersystem crossing process by excitation in the CT visible band and give rise to delayed fluorescence. Delayed-fluorescence optically detected magnetic resonance (DF ODMR) in magnetic field is analyzed in terms of microwave-induced transitions between energy levels of either the isolated triplet excitons or the annihilating triplet exciton pair. The spin polarization of the triplet excitons plays an important role in the described phenomena. A comparison between DF ODMR and EPR spectra of the anthracene—tetracyanobenzene and biphenyl—tetracyanobenzene systems is presented. In the former case the microwave transitions occurring between free exciton sublevels are predominantly responsible of the DF ODMR signal, whereas the transitions between energy levels of the exciton pair are the most important for biphenyl—TCNB.

The crystal structure of Biphenylene and 1,2,4,5-Tetracyanobenzene (1: 1) charge-transfer (CT) complex has been determined at room temperature. The crystals belong to the monoclinic system with spa...

Uranyl complexes with triethylphosphate and trimethylphosphate dissolved in the corresponding alkyl phosphates are photoreduced by irradiation in their UV-VIS absorption band. The photoreaction proceeds through the formation of an intermediate species which was identified as U(V). The latter is quite stable in the dark, but undergoes a photochemical disproportionation giving U(IV) as the final product.

The EPR spectra of the biphenyl-TCNQ (B-TCNQ) photoexcited triplet trap in the single crystal of B-TCNB show optical spin polarization (OEP) that is ac

From 250 MHz 1H-NMR spectra it has been determined the conformation of DL-carnitine hydrochloride, (CH3)3 N + CH2CHOHCH2COOH Cl−, in D2O solutions. The 13C spin lattice relaxation times are also discussed.

The e.s.r. spectra of copper diselenocarbamate dissolved in various nematic solvents have been analyzed in terms of the orientational order parameters. Two Euler angles are found to be needed to specify the molecular orientation. The largest component of the g-tensor is confirmed to be parallel to the in-plane y-axis, as determined by the single crystal measurements, and the unusual lineshape of the spectra in both isotropic and nematic phase is interpreted on the basis of relaxation theories.