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A series of Gd3+ complexes (Gd1-Gd3) with the general formula GdL3(EtOH)2, where L is a β-diketone ligand with polycyclic aromatic hydrocarbon substituents of increasing size (1-3), was studied by combining time-resolved electron paramagnetic resonance (TR-EPR) spectroscopy and DFT calculations to rationalize the anomalous spectroscopic behavior of the bulkiest complex (Gd3) through the series. Its faint phosphorescence band is observed only at 80 K and it is strongly red-shifted (∼200 nm) from the intense fluorescence band. Moreover, the TR-EPR spectral analysis found that triplet levels of 3/Gd3 are effectively populated and have smaller |D| values than those of the other compounds. The combined use of zero-field splitting and spin density delocalization calculations, together with spin population analysis, allows us to explain both the large red shift and the low intensity of the phosphorescence band observed for Gd3. The large red shift is determined by the higher delocalization degree of the wavefunction, which implies a larger energy gap between the excited S1 and T1 states. The low intensity of the phosphorescence is due to the presence of C-H groups which favor non-radiative decay. These groups are present in all complexes; nevertheless, they have a relevant spin density only in Gd3. The spin population analysis on NaL models, in which Na+ is coordinated to a deprotonated ligand, mimicking the coordinative environment of the complex, confirms the outcomes on the free ligands.

The energy transfer (ET) from carotenoids (Cars) to chlorophylls (Chls) in photosynthetic complexes occurs with almost unitary efficiency thanks to the synergistic action of multiple finely tuned channels whose photophysics and dynamics are not fully elucidated yet. We investigated the energy flow from the Car peridinin (Per) to Chl a in the peridinin chlorophyll a protein (PCP) from marine algae Amphidinium carterae by using two-dimensional electronic spectroscopy (2DES) with a 10 fs temporal resolution. Recently debated hypotheses regarding the S2-to-S1 relaxation of the Car via a conical intersection and the involvement of possible intermediate states in the ET were examined. The comparison with an N89L mutant carrying the Per donor in a lower-polarity environment helped us unveil relevant details on the mechanisms through which excitation was transferred: the ET yield was conserved even when a mutation perturbed the optimization of the system thanks to the coexistence of multiple channels exploited during the process.

The photo-excited triplet state of Zn-protoporphyrin IX located in the heme pocket of human neuroglobin has been investigated by time-resolved EPR coupled to magnetophotoselection. The triplet state in the protein matrix has been compared with the model complex in organic glass, considering both non-coordinating and coordinating solvent mixtures. The protein matrix plays an important role in stabilizing the coordination of the embedded chromophore, resulting in a more homogeneous environment relative to that of the chromophore in a glassy solvent, even in the presence of an axial nitrogenous ligand like pyridine. The EPR spectral parameters point out a slow Jahn–Teller interconversion between slightly different triplet states, both in organic solvent and in the protein matrix. The EPR-magnetophotoselection allows us to propose a reinterpretation of the assignment of the Q bands in the electronic absorption spectrum.

The photoexcited triplet state of the “primary donors” in the two photosystems of oxygenic photosynthesis has been investigated by means of electron-nuclear double resonance (ENDOR) at Q-band (34 GHz). The data obtained represent the first set of1H hyperfine coupling tensors of the3P700 triplet state in PSI and expand the existing data set for3P680. We achieved an extensive assignment of the observed electron-nuclear hyperfine coupling constants (hfcs) corresponding to the methine α-protons and the methyl group β-protons of the chlorophyll (Chl) macrocycle. The data clearly confirm that in both photosystems the primary donor triplet is located on one specific monomeric Chl at cryogenic temperature. In comparison to previous transient ENDOR and pulse ENDOR experiments at standard X-band (9–10 GHz), the pulse Q-band ENDOR spectra demonstrate both improved signal-to-noise ratio and increased resolution. The observed ENDOR spectra for3P700 and3P680 differ in terms of the intensity loss of lines from specific methyl group protons, which is explained by hindered methyl group rotation produced by binding site effects. Contact analysis of the methyl groups in the PSI crystal structure in combination with the ENDOR analysis of3P700 suggests that the triplet is located on the Chlaʹ (PA) in PSI. The results also provide additional evidence for the localization of3P680 on the accessory ChlD1in PSII.

The triplet states populated under illumination in the monomeric light-harvesting complex II (LHCII) were analyzed by EPR and Optically Detected Magnetic Resonance (ODMR) in order to fully characterize the perturbations introduced by site-directed mutations leading to the removal of key chlorophylls. We considered the A2 and A5 mutants, lacking Chls a612(a611) and Chl a603 respectively, since these Chls have been proposed as the sites of formation of triplet states which are subsequently quenched by the luteins. Chls a612 and Chl a603 belong to the two clusters determining the low energy exciton states in the complex. Their removal is expected to significantly alter the excitation energy transfer pathways. On the basis of the TR- and pulse EPR triplet spectra, the two symmetrically related pairs constituted by Chl a612/Lut620 and Chl a603/Lut621 were both possible candidate for triplet-triplet energy transfer (TTET). However, the ODMR results clearly show that only Lut620 is involved in triplet quenching. In the A5 mutant, the Chl a612/Lut620 pair retains this pivotal photoprotective role, while the A2 mutant was found to activate an alternative pathway involving the Chl a603/Lut621pair. These results shows that LHCII is characterized by a robust photoprotective mechanism, able to adapt to the removal of individual chromophores while maintaining a remarkable degree of Chl triplet quenching. Small amounts of unquenched Chl triplet states were also detected. The analysis of the results allowed us to assign the sites of "unquenched" chlorophyll triplets to Chl a610 and Chl a602.

An anthraquinone modified with a nitroxide radical and able to intercalate into DNA has been synthesized to obtain a molecule the spin state of which can be manipulated by visible light and DNA binding. The doublet ground state of the molecule can be photo-switched to either a strongly coupled spin state (quartet + doublet), when isolated, or to an uncoupled spin state (triplet and doublet), when bound to DNA. The different spin state that is obtained upon photoexcitation depends on the intercalation of the quinonic core into double-stranded DNA which changes the conformation of the molecule, thereby altering the exchange interaction between the excited state localized on the quinonic core and the nitroxide radical. The spin state of the system has been investigated using both continuous-wave and time-resolved EPR spectroscopy.

The type-I, homodimeric photosynthetic reaction center (RC) of Heliobacteria (HbRC) is the only known RC in which bacteriochlorophyll g (BChl g) is found. It is also simpler than other RCs, having the smallest number of protein subunits and bound chromophores of any type-I RC. In the presence of oxygen, BChl g isomerizes to 81-hydroxychlorophyll aF (Chl aF). This naturally occurring process provides a way of altering the chlorophylls and studying the effect of these changes on energy and electron transfer. Transient absorbance difference spectroscopy reveals that triplet-state formation occurs in the antenna chlorophylls of HbRCs but does not provide site-specific information. Here, we report on an extended optically detected magnetic resonance (ODMR) study of the antenna triplet states in HbRCs with differing levels of conversion of BChl g to Chl aF. The data reveal pools of BChl g molecules with different triplet zero-field splitting parameters and different susceptibilities to chemical oxidation. By relating the detailed spectroscopic characteristics derived from the ODMR data to the recently solved crystallographic structure, we have tentatively identified BChl g molecules in which the probability of triplet formation is high and sites at which BChl g conversion is more likely, providing useful information about the fate of the excitation in the complex.

Frataxin is a highly conserved protein whose deficiency results in the neurodegenerative disease Friederich’s ataxia. Frataxin’s actual physiological function has been debated for a long time without reaching a general agreement; however, it is commonly accepted that the protein is involved in the biosynthetic iron-sulphur cluster (ISC) machinery, and several authors have pointed out that it also participates in iron homeostasis. In this work, we use site-directed spin labeling coupled to electron paramagnetic resonance (SDSL EPR) to add new information on the effects of ferric and ferrous iron binding on the properties of human frataxin in vitro. Using SDSL EPR and relating the results to fluorescence experiments commonly performed to study iron binding to FXN, we produced evidence that ferric iron causes reversible aggregation without preferred interfaces in a concentration-dependent fashion, starting at relatively low concentrations (micromolar range), whereas ferrous iron binds without inducing aggregation. Moreover, our experiments show that the ferrous binding does not lead to changes of protein conformation. The data reported in this study reveal that the currently reported binding stoichiometries should be taken with caution. The use of a spin label resistant to reduction, as well as the comparison of the binding effect of Fe2+ in wild type and in the pathological D122Y variant of frataxin, allowed us to characterize the Fe2+ binding properties of different protein sites and highlight the effect of the D122Y substitution on the surrounding residues. We suggest that both Fe2+ and Fe3+ might play a relevant role in the context of the proposed FXN physiological functions.

Fucoxanthin chlorophyll-binding proteins (FCPs) are the major light-harvesting complexes of diatoms. In this work, FCPs isolated from Cyclotella meneghiniana have been studied by means of optically detected magnetic resonance (ODMR) and time-resolved electron paramagnetic resonance (TR-EPR), with the aim to characterize the photoprotective mechanism based on triplet-triplet energy transfer (TTET). The spectroscopic properties of the chromophores carrying the triplet state have been interpreted on the basis of a delved analysis of the recently solved crystallographic structures of FCP. The results point toward a photoprotective role for two fucoxanthin molecules exposed to the exterior of the FCP monomers. This shows that FCP has adopted a structural strategy different from that of related light-harvesting complexes from plants and other microalgae, in which the photoprotective role is carried out by two highly conserved carotenoids in the interior of the complex.

Carotenoids represent the first line of defence of photosystems against singlet oxygen (1O2) toxicity, because of their capacity to quench the chlorophyll triplet state (3Chl) through a physical mechanism based on the transfer of triplet excitation (triplet–triplet energy transfer, TTET). In previous works, we showed that the antenna LHCII is characterised by a robust photoprotective mechanism, able to adapt to the removal of individual chlorophylls while maintaining a remarkable capacity for 3Chl quenching. In this work, we investigated the effects on this quenching induced in LHCII by the replacement of the lutein bound at the L1 site with violaxanthin and zeaxanthin. We studied LHCII isolated from the Arabidopsis thaliana mutants lut2—in which lutein is replaced by violaxanthin—and lut2 npq2, in which all xanthophylls are replaced constitutively by zeaxanthin. We characterised the photophysics of these systems via optically detected magnetic resonance (ODMR) and time-resolved electron paramagnetic resonance (TR-EPR). We concluded that, in LHCII, lutein-binding sites have conserved characteristics, and ensure efficient TTET regardless of the identity of the carotenoid accommodated.

Optically detected magnetic resonance of triplet states populated by photoexcitation in water-soluble chlorophyll proteins (WSCPs) from Lepidium virginicum has been performed using both absorption and fluorescence detection. Well resolved triplet-singlet (T-S) spectra have been obtained and interpreted in terms of electronic interactions among the four chlorophylls (Chls), forming two dimers in the WSCP tetramer. Localization of the triplet state on a single Chl leads to a redistribution of the oscillator strength in the remaining three Chls of the complex. By comparing the spectra with those obtained on a substoichiometric WSCP complex containing only 2 Chls per protein tetramer, we proved that, to interpret the optical spectra of the WSCP fully loaded with 4 Chls, the interactions between the two dimers have to be taken into account and cannot be considered negligible. The results show that the WSCP may well be considered as an ideal model system to study Chl-Chl interactions, also in view of the possibility to modify the number and molecular structure of the bound porphyrin chromophores.

Bacteria overexpress, under condition of starvation or oxidative stress, Dps (DNA-binding proteins from starved cells), hollow sphere formed by 12 identical subunits endowed with ferritin-like activity. The iron oxidation and incorporation in Dps take place using H2O2 produced under starvation as preferred iron oxidant, thereby protecting bacteria from oxidative damage. Even if the role of Dps is well known, the mechanism of iron oxidation and incorporation remain to be elucidated. Here, we have used the EPR technique to shed light on the Fe(II) binding and oxidation mechanism at the ferroxidase center using both the wild-type (wt) protein and mutants of the iron ligands (H31G, H43G and H31G-H43G-D58A). The EPR titration of wt Dps and the H31G mutant with Fe(II) upon H2O2 addition shows that Fe(II) is oxidized with the increase of the signal at g = 4.3, reaching a maximum for 12 Fe(II)/subunit. The EPR signal becomes negligible when the titration is carried out on the triple mutant. These experiments indicate that the iron firstly occupied the A site at the ferroxidase center and confirm that the residues H31, H43 and D58 have a key role in the iron oxidation and incorporation process. Moreover, the data indicate that the ferroxidase center, upon mutation of H31 or H43 to Gly, changes the mode of iron binding. Finally, we demonstrate here that, when the iron micelle forms, the EPR signal at g = 4.3 disappears indicating that iron leaves the ferroxidase center to reach the inner cavity.

The interplay between active molecules and the protein environment in light-harvesting complexes tunes the photophysics and the dynamical properties of pigment–protein complexes in a subtle way, which is not fully understood. Here we characterized the photophysics and the ultrafast dynamics of four variants of the water-soluble chlorophyll protein (WSCP) as an ideal model system to study the behavior of strongly interacting chlorophylls. We found that when coordinated by the WSCP protein, the presence of the formyl group in chlorophyll b replacing the methyl group in chlorophyll a strongly affects the exciton energy and the dynamics of the system, opening up the possibility of tuning the photophysics and the transport properties of multichromophores by engineering specific interactions with the surroundings.

An investigation of the photoexcited triplet state of chlorophyll (Chl) a in the water-soluble chlorophyll protein (WSCP) of Brassica oleracea has been carried out by means of electron-nuclear double resonance (ENDOR), achieving a complete assignment of the observed hyperfine couplings corresponding to methine protons and methyl groups of Chl a triplet state. The triplet-state properties, and in particular the hyperfine couplings, were found to be similar to those previously reported for Chl a in the WSCP of Lepidium virginicum. Therefore, the porphyrin ring deformation observed in Brassica oleracea WSCP seems to only slightly affect the spin density of 3Chl a. This may be relevant when considering the robustness of triplet–triplet energy transfer mechanisms, relying on wavefunction overlap, in systems, such as the photosynthetic light-harvesting complexes, in which Chl triplet states with distorted geometries are involved.

The Water-Soluble Chlorophyll Protein (WSCP) of Brassicaceae is a remarkably stable tetrapyrrole-binding protein that, by virtue of its simple design, is an exceptional model to investigate the interactions taking place between pigments and their protein scaffold and how they affect the photophysical properties and the functionality of the complexes. We investigated variants of WSCP from Lepidium virginicum (Lv) and Brassica oleracea (Bo), reconstituted with Chlorophyll (Chl) b, to determine the mechanisms by which the different Chl binding sites control their Chl a/b specificities. A combined Raman and crystallographic investigation has been employed, aimed to characterize in detail the hydrogen-bond network involving the formyl group of Chl b. The study revealed a variable degree of conformational freedom of the hydrogen bond networks among the WSCP variants, and an unexpected mixed presence of hydrogen-bonded and not hydrogen-bonded Chls b in the case of the L91P mutant of Lv WSCP. These findings helped to refine the description of the mechanisms underlying the different Chl a/b specificities of WSCP versions, highlighting the importance of the structural rigidity of the Chl binding site in the vicinity of the Chl b formyl group in granting a strong selectivity to binding sites.

An investigation of the photoexcited triplet state of chlorophyll (Chl)

Water-soluble chlorophyll proteins (WSCPs) of class IIa from Brassicaceae form tetrameric complexes containing one chlorophyll (Chl) per apoprotein but no carotenoids. The complexes are remarkably stable toward dissociation and protein denaturation even at 100 °C and extreme pH values, and the Chls are partially protected against photooxidation. There are several hypotheses that explain the biological role of WSCPs, one of them proposing that they function as a scavenger of Chls set free upon plant senescence or pathogen attack. The biochemical properties of WSCP described in this paper are consistent with the protein acting as an efficient and flexible Chl scavenger. At limiting Chl concentrations, the recombinant WSCP apoprotein binds substoichiometric amounts of Chl (two Chls per tetramer) to form complexes that are as stable toward thermal dissociation, denaturation, and photodamage as the fully pigmented ones. If more Chl is added, these two-Chl complexes can bind another two Chls to reach the fully pigmented state. The protection of WSCP Chls against photodamage has been attributed to the apoprotein serving as a diffusion barrier for oxygen, preventing its access to triplet excited Chls and, thus, the formation of singlet oxygen. By contrast, the sequential binding of Chls by WSCP suggests a partially open or at least flexible structure, raising the question of how WSCP photoprotects its Chls without the help of carotenoids.

Triplet-triplet energy transfer from chlorophylls to carotenoids is the mechanism underlying the photoprotective role played by carotenoids in many light harvesting complexes, during photosynthesis. The peridinin–chlorophyll-a protein (PCP) is a water-soluble light harvesting protein of the dinoflagellate Amphidinium carterae, employing peridinin as the main carotenoid to fulfil this function. The dipolar coupling of the triplet state of peridinin, populated under light excitation in isolated PCP, to the MTSSL nitroxide, introduced in the protein by site-directed mutagenesis followed by spin labeling, has been measured by Pulse ELectron-electron DOuble Resonance (PELDOR) spectroscopy. The triplet-nitroxide distance derived by this kind of experiments, performed for the first time in a protein system, allowed the assignment of the triplet state to a specific peridinin molecule belonging to the pigment cluster. The analysis strongly suggests that this peridinin is the one in close contact with the water ligand to the chlorophyll a, thus supporting previous evidences based on ENDOR and time resolved-EPR.

Frataxin is a highly conserved protein whose deficiency results in the neurodegenerative disease Friederich&rsquo, s ataxia. Frataxin&rsquo, s actual physiological function has been debated for a long time without reaching a general agreement, however, it is commonly accepted that the protein is involved in the biosynthetic iron-sulphur cluster (ISC) machinery, and several authors have pointed out that it also participates in iron homeostasis. In this work, we use site-directed spin labeling coupled to electron paramagnetic resonance (SDSL EPR) to add new information on the effects of ferric and ferrous iron binding on the properties of human frataxin in vitro. Using SDSL EPR and relating the results to fluorescence experiments commonly performed to study iron binding to FXN, we produced evidence that ferric iron causes reversible aggregation without preferred interfaces in a concentration-dependent fashion, starting at relatively low concentrations (micromolar range), whereas ferrous iron binds without inducing aggregation. Moreover, our experiments show that the ferrous binding does not lead to changes of protein conformation. The data reported in this study reveal that the currently reported binding stoichiometries should be taken with caution. The use of a spin label resistant to reduction, as well as the comparison of the binding effect of Fe2+ in wild type and in the pathological D122Y variant of frataxin, allowed us to characterize the Fe2+ binding properties of different protein sites and highlight the effect of the D122Y substitution on the surrounding residues. We suggest that both Fe2+ and Fe3+ might play a relevant role in the context of the proposed FXN physiological functions.

An investigation of the photoexcited triplet state of chlorophyll (Chl) b has been carried out by means of electron nuclear double resonance, both in a frozen organic solvent and in a protein environment provided by the water-soluble chlorophyll protein of Lepidium virginicum. Density functional theory calculations have allowed the complete assignment of the observed hyperfine couplings corresponding to the methine protons and the methyl groups, leading to a complete picture of the spin density distribution of the triplet state in the tetrapyrrole macrocycle. The triplet-state properties of Chl b are found to be similar, in many respects, to those previously reported for Chl a, although some specificities have been highlighted. Concerning the spin density distribution, the differences are mainly localized on the carbon atoms close to the formyl group which, in Chl b, replaces the methyl group of Chl a.

Electron paramagnetic resonance (EPR), coupled with site-directed spin labeling, has been proven to be a particularly suitable technique to extract information on the fraction of myosin heads strongly bound to actin upon muscle contraction. The approach can be used to investigate possible structural changes occurring in myosin of fiber s altered by diseases and aging. In this work, we labeled myosin at position Cys707, located in the SH1-SH2 helix in the myosin head cleft, with iodoacetamide spin label, a spin label that is sensitive to the reorientational motion of this protein during the ATPase cycle and characterized the biochemical states of the labeled myosin head by means of continuous wave EPR. After checking the sensitivity and the power of the technique on different muscles and species, we investigated whether changes in the fraction of strongly bound myosin heads might explain the contractile alterations observed in atrophic and hypertrophic murine muscles. In both conditions, the difference in contractile force could not be justified simply by the difference in muscle mass. Our results showed that in atrophic muscles the decrease in force generation was attributable to a lower fraction of strongly bound cross bridges during maximal activation. In contrast in hypertrophic muscles, the increase in force generation was likely due to several factors, as pointed out by the comparison of the EPR experiments with the tension measurements on single skinned fibers.

This paper investigates the reliability of blue-emitting phosphors for Near-UV (NUV) laser excitation. By means of a series of thermal stress experiments, and of stress under high levels of optical excitation, we have been able to identify the physical process responsible for the degradation of Eu2+-activated alkaline-earth halophosphate phosphors under typical and extreme operating conditions. In particular, for temperatures equal to or greater than 450 °C the material exhibited a time-dependent drop in the Photo-Luminescence (PL), which was attributed to the thermally induced ionization of the Eu2+ optically active centers. Several analytical techniques, including spatially and spectrally resolved PL, Electron Paramagnetic Resonance (EPR) and X-ray Photo-emission Spectroscopy (XPS) were used to support this hypothesis and to gain insight on the degradation process. By means of further tests, evidence of this degradation process was also found on samples stressed under a relatively low power density of 3 W/mm2 at 405 nm. This indicated that the optically (and thermally) induced ionization of the optically active species is the most critical degradation process for this family of phosphorescent material. The operating limits of a second-generation Eu-doped halophosphate phosphor were also investigated by means of short-term stress under optical excitation. The experimental data showed that a threshold excitation intensity for continuous pumping exists. Above this threshold, decay of the steady-state PL performance and non-recoverable degradation of the material were found to take place. This behavior is a consequence of the extremely harsh excitation regime, mainly due to the thermal management capabilities of the substrate material employed for our experimental purposes rather than from intrinsic properties of the phosphors.

The catalytic site of [FeFe]-hydrogenase, the “H-cluster”, composed by a [4Fe–4S] unit connected by a cysteinyl residue to a [2Fe] center coordinated by three CO, two CN− and a bridging dithiolate, is assembled in a complex maturation pathway, at present not fully characterized, involving three conserved proteins, HydG, HydE and HydF. In this contribution we review our studies on HydF, a protein which acts as scaffold and carrier for the [2Fe] unit of the H-cluster. HydF is a complex enzyme which contains one [4Fe–4S] cluster binding site, with three conserved cysteine residues and a non-Cys ligand. We have exploited EPR, HYSCORE and PELDOR spectroscopies to get insight into the structure and chemical role of HydF. On the basis of the results we discuss the possibility that the non-Cys ligated Fe atom of the [4Fe–4S] cluster, is the site where the [2Fe] subcluster precursor is anchored and finally processed to be delivered to the hydrogenase (HydA). Our PELDOR experiments on the isolated GTPase domain of HydF, have also suggested that interactions with HydG and HydE proteins may be regulated by the binding of the nucleotide.

The catalytic site of [FeFe]-hydrogenase, the "H-cluster", composed of a [4Fe-4S] unit connected by a cysteinyl residue to a [2Fe] center coordinated by three CO, two CN(-), and a bridging dithiolate, is assembled in a complex maturation pathway, at present not fully characterized, involving three conserved proteins, HydG, HydE, and HydF. HydF is a complex enzyme, which is thought to act as a scaffold and carrier for the [2Fe] subunit of the H-cluster. This maturase protein contains itself a [4Fe-4S] cluster binding site, with three conserved cysteine residues and a noncysteinyl fourth ligand. In this work, we have exploited 3p-ESEEM and HYSCORE spectroscopies to get insight into the structure and the chemical environment of the [4Fe-4S] cluster of HydF from the hyperthermophilic organism Thermotoga neapolitana. The nature of the fourth ligand and the solvent accessibility of the active site comprising the [4Fe-4S] cluster are discussed on the basis of the spectroscopic results obtained upon H/D exchange. We propose that the noncysteinyl ligated Fe atom of the [4Fe-4S] cluster is the site where the [2Fe] subcluster precursor is anchored and finally processed to be delivered to the hydrogenase (HydA).

This paper investigates the reliability of blue-emitting phosphors for Near-UV (NUV) laser excitation. By means of a series of thermal stress experiments, and of stress under high levels of optical excitation, we have been able to identify the physical process responsible for the degradation of Eu2+-activated alkaline-earth halophosphate phosphors under typical and extreme operating conditions. In particular, for temperatures equal to or greater than 450 &deg, C the material exhibited a time-dependent drop in the Photo-Luminescence (PL), which was attributed to the thermally induced ionization of the Eu2+ optically active centers. Several analytical techniques, including spatially and spectrally resolved PL, Electron Paramagnetic Resonance (EPR) and X-ray Photo-emission Spectroscopy (XPS) were used to support this hypothesis and to gain insight on the degradation process. By means of further tests, evidence of this degradation process was also found on samples stressed under a relatively low power density of 3 W/mm2 at 405 nm. This indicated that the optically (and thermally) induced ionization of the optically active species is the most critical degradation process for this family of phosphorescent material. The operating limits of a second-generation Eu-doped halophosphate phosphor were also investigated by means of short-term stress under optical excitation. The experimental data showed that a threshold excitation intensity for continuous pumping exists. Above this threshold, decay of the steady-state PL performance and non-recoverable degradation of the material were found to take place. This behavior is a consequence of the extremely harsh excitation regime, mainly due to the thermal management capabilities of the substrate material employed for our experimental purposes rather than from intrinsic properties of the phosphors.

The subtle details of the mechanism of energy flow from carotenoids to chlorophylls in biological light-harvesting complexes are still not fully understood, especially in the ultrafast regime. Here we focus on the antenna complex peridinin–chlorophyll a–protein (PCP), known for its remarkable efficiency of excitation energy transfer from carotenoids—peridinins—to chlorophylls. PCP solutions are studied by means of 2D electronic spectroscopy in different experimental conditions. Together with a global kinetic analysis and multiscale quantum chemical calculations, these data allow us to comprehensively address the contribution of the potential pathways of energy flow in PCP. These data support dominant energy transfer from peridinin S2 to chlorophyll Qy state via an ultrafast coherent mechanism. The coherent superposition of the two states is functional to drive population to the final acceptor state, adding an important piece of information in the quest for connections between coherent phenomena and biological functions., Energy transfer from carotenoids to chlorophylls in light-harvesting is still not fully understood, especially in the ultrafast regime. Here, the authors investigate the coherent dynamics of this process in peridinin-chlorophyll a-protein complex via 2D electronic spectroscopy and quantum chemical calculations.

[FeFe]-hydrogenases catalyze the reversible interconversion of protons to molecular hydrogen (H2) at an active site called H-cluster. The maturation pathway of these enzymes is a complex process involving three proteins, HydE, HydF and HydG. The maturase protein HydF has been suggested to interact with HydE and HydG and to be the transferase that shuttles the complete H-cluster to the hydrogenase; however, the exact molecular mechanism driving this translocation remains unclear. HydF is constituted by three different domains: a N-terminal GTP-binding domain, a dimerization domain and a C-terminal [4Fe4S] cluster-binding domain. To investigate possible conformational changes induced by the GTP binding in the N-terminal domain, we have expressed, in Escherichia coli, a recombinant HydF protein from Thermotoga neapolitana including the GTP-binding domain only. Site-directed mutants were designed in which the native residues were substituted by cysteines and subsequently spin labeled with the nitroxide MTSSL. CW-EPR was used to study the local mobility of the nitroxides at each site, and double spin-labeled mutants have been investigated by PELDOR spectroscopy. We found that the binding of the nucleotide does not induce large conformational effects within the isolated GTP domain, at least at the level of the elements investigated in this work. However, small changes in the distance between spin labels were observed which might reflect diffuse structural rearrangements. We suggest that the variations following the GTP binding could affect the dimer form adopted by the whole HydF protein in solution and, as a consequence, the interactions with the other maturases.

HYperfine Sublevel CORrElation spectroscopy (HYSCORE) is one of the most widely applied EPR techniques for the investigation of hyperfine couplings in a wide range of spin systems; here, it is applied for the first time to a photoexcited triplet state. The analytical expressions describing the electron spin echo envelope modulations in a HYSCORE experiment on a triplet state coupled to spin ½ nuclei and spin 1 nuclei with weak nuclear quadrupole interactions were derived employing the density matrix formalism and used to determine the characteristic features of this experiment when applied to a triplet state. Experimental HYSCORE spectra recorded on the photoexcited triplet state of a meso-substituted zinc porphyrin are used to investigate the 14N hyperfine interactions in this system. The results are compared to one-dimensional three-pulse Electron Spin Echo Envelope Modulation (ESEEM) experiments and the comparison clearly shows the advantage of the HYSCORE experiment for investigation of the hyperfine couplings of a spin 1 nucleus for triplet states in disordered powder samples. The experimental HYSCORE data are discussed and interpreted in light of the properties of triplet state HYSCORE spectra identified using our theoretical approach.

[FeFe]-hydrogenases contain a complex [4Fe–4S]-2Fe cluster (H-cluster) and are able to efficiently reduce protons to H 2 . Due to their potential exploitation for renewable energy production biotechnologies, significant efforts have been put into understanding the mechanisms driving the H-cluster assembly, which involves three conserved proteins. Among them, HydF works as scaffold upon which the H-cluster precursor is synthesized and carrier to deliver it to the hydrogenase, resulting in its activation. A FeS cluster binding sequence (CxHx 46 - 53 HCxxC) is conserved in all HydF proteins and should in principle provide four ligands to coordinate the Fe atom. However, we found that alternative metal coordination may exist in different HydF proteins and that only the three cysteines are strictly required, whereas the fourth ligand may vary and is, in any case, readily exchangeable. In this work we analyzed by EPR/HYSCORE the FeS cluster proton environment of HydF from Thermotoga neapolitana to determine the possible role of surrounding residues in the non-cysteinyl iron ligation of the protein.

Violaxanthin–chlorophyll a binding protein (VCP) is the major light harvesting complex (LHC) of the Heterokonta Nannochloropsis gaditana. It binds chlorophyll a, violaxanthin and vaucheriaxanthin, the last in the form of 19′ deca/octanoate esters. Photosynthetic apparatus of algae belonging to this group have been poorly characterized in the past, but they are now receiving an increasing interest also because of their possible biotechnological application in biofuel production. In this work, isolated VCP proteins have been studied by means of advanced EPR techniques in order to prove the presence of the photoprotective mechanism based on the triplet–triplet energy transfer (TTET), occurring between chlorophyll and carotenoid molecules. This process has been observed before in several light harvesting complexes belonging to various photosynthetic organisms. We used Optically Detected Magnetic Resonance (ODMR) to identify the triplet states populated by photo-excitation, and describe the optical properties of the chromophores carrying the triplet states. In parallel, time-resolved EPR (TR-EPR) and pulse EPR have been employed to get insight into the TTET mechanism and reveal the structural features of the pigment sites involved in photoprotection. The analysis of the spectroscopic data shows a strong similarity among VCP, FCP from diatoms and LHC-II from higher plants. Although these antenna proteins have differentiated sequences and bind different pigments, results suggest that in all members of the LHC superfamily there is a protein core with a conserved structural organization, represented by two central carotenoids surrounded by five chlorophyll a molecules, which plays a fundamental photoprotective role in Chl triplet quenching through carotenoid triplet formation.

The neurodegenerative disease Friedreich ataxia results from a deficiency of frataxin, a mitochondrial protein. Most patients have a GAA expansion in the first intron of both alleles of frataxin gene, whereas a minority of them are heterozygous for the expansion and contain a mutation in the other allele. Frataxin has been claimed to participate in iron homeostasis and biosynthesis of FeS clusters, however its role in both pathways is not unequivocally defined. In this work we combined different advanced spectroscopic analyses to explore the iron-binding properties of human frataxin, as isolated and at the FeS clusters assembly machinery. For the first time we used EPR spectroscopy to address this key issue providing clear evidence of the formation of a complex with a low symmetry coordination of the metal ion. By 2D NMR, we confirmed that iron can be bound in both oxidation states, a controversial issue, and, in addition, we were able to point out a transient interaction of frataxin with a N-terminal 6his-tagged variant of ISCU, the scaffold protein of the FeS clusters assembly machinery. To obtain insights on structure/function relationships relevant to understand the disease molecular mechanism(s), we extended our studies to four clinical frataxin mutants. All variants showed a moderate to strong impairment in their ability to activate the FeS cluster assembly machinery in vitro, while keeping the same iron-binding features of the wild type protein. This supports the multifunctional nature of frataxin and the complex biochemical consequences of its mutations. Fil: Bellanda, Massimo. Università di Padova; Italia Fil: Maso, Lorenzo. Università di Padova; Italia Fil: Doni, Davide. Università di Padova; Italia Fil: Bortolus, M.. Università di Padova; Italia Fil: De Rosa, E.. Università di Padova; Italia Fil: Lunardi, Federica. Università di Padova; Italia Fil: Alfonsi, Arianna. Università di Padova; Italia Fil: Noguera, Martín Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Herrera, Maria Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Santos, Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Carbonera, Donatella. Università di Padova; Italia Fil: Costantini, Paola. Università di Padova; Italia

Although the major light harvesting complexes of diatoms, called FCPs (fucoxanthin chlorophyll a / c binding proteins), are related to the cab proteins of higher plants, the structures of these light harvesting protein complexes are much less characterized. Here, a structural/functional model for the “core” of FCP, based on the sequence homology with LHCII, in which two fucoxanthins replace the central luteins and act as quenchers of the Chl a triplet states, is proposed. Combining the information obtained by time-resolved EPR spectroscopy on the triplet states populated under illumination, with quantum mechanical calculations, we discuss the chlorophyll triplet quenching in terms of the geometry of the chlorophyll–carotenoid pairs participating to the process. The results show that local structural rearrangements occur in FCP, with respect to LHCII, in the photoprotective site.

Advanced electron paramagnetic resonance (EPR) techniques, in combination with Density Functional theory (DFT), have been applied to the comparative study of carotenoid triplet states in two major photosynthetic antenna complexes, the Peridinin-chlorophyll a–protein of dinoflagellates and the light-harvesting complex II of higher plants.Carotenoid triplet states are populated by triplet–triplet energy transfer (TTET) from chlorophyll molecules to photoprotect the system from singlet oxygen formation under light-stress conditions. The TTET process is strongly dependent on the relative arrangement and on the electronic properties of the triplet states involved.The proposed spectroscopic approach exploits the concept of spin conservation during TTET, which leads to recognisable spin polarisation effects in the time-resolved and field-swept echo-detected EPR spectra. The electron spin polarisation produced at the carotenoid acceptor site depends on the initial polarisation of the chlorophyll donor and on the ...

The protein disulfide isomerase (PDI) family comprises a wide set of enzymes mainly involved in thiol- disulfide exchange reactions in the endoplasmic reticulum. Class A PDIs (PDI-A) constitute the smallest members of the family, consisting of a single thioredoxin (TRX) module without any additional domains. To date, their catalytic activity and cellular function are still poorly understood. To gain insight into the role of higher-plant class A PDIs, the biochemical properties of rAtPDI-A, the recombinant form of Arabidopsis thaliana PDI-A, have been investigated. As expressed, rAtPDI-A has only little oxidoreductase activity, but it appears to be capable of binding an iron-sulfur (Fe-S) cluster, most likely a [2Fe-2S] center, at the interface between two protein monomers. A mutational survey of all cysteine residues of rAtPDI-A indicates that only the second and third cysteines of the CXXXCKHC stretch, containing the putative catalytic site CKHC, are primarily involved in cluster coordination. A key role is also played by the lysine residue. Its substitution with glycine, which restores the canonical PDI active site CGHC, does not influence the oxidoreductase activity of the protein, which remains marginal, but strongly affects the binding of the cluster. It is therefore proposed that the unexpected ability of rAtPDI-A to accommodate an Fe-S cluster is due to its very unique CKHC motif, which is conserved in all higher-plant class A PDIs, differentiating them from all other members of the PDI family.

[FeFe]-hydrogenases catalyse the reduction of protons to hydrogen at a complex 2Fe[4Fe4S] center called H-cluster. The assembly of this active site is a multistep process involving three proteins, HydE, HydF and HydG. According to the current models, HydF has the key double role of scaffold, upon which the final H-cluster precursor is assembled, and carrier to transfer it to the target hydrogenase. The X-ray structure of HydF indicates that the protein is a homodimer with both monomers carrying two functional domains: a C-terminal FeS cluster-binding domain, where the precursor is assembled, and a N-terminal GTPase domain, whose exact contribution to cluster biogenesis and hydrogenase activation is still elusive. We previously obtained several hints suggesting that the binding of GTP to HydF could be involved in the interactions of this scaffold protein with the other maturases and with the hydrogenase itself. In this work, by means of site directed spin labeling coupled to EPR/PELDOR spectroscopy, we explored the conformational changes induced in a recombinant HydF protein by GTP binding, and provide the first clue that the HydF GTPase domain could be involved in the H-cluster assembly working as a molecular switch similarly to other known small GTPases.

Water-Soluble Chlorophyll Proteins (WSCPs) from Brassicaceae are non-photosynthetic proteins which tetramerize upon binding four chlorophyll (Chl) molecules. The bound Chls are highly photostable, despite the lack of bound carotenoids known, in Chl-containing photosynthetic proteins, to act as singlet oxygen and Chl triplet (3Chl) quenchers. Although the physiological function of WSCPs is still unclear, it is likely to be related to their biochemical stability and their resistance to photodegradation. To get insight into the origin of this photostability, the properties of the 3Chl generated in WSCPs upon illumination were investigated. We found that, unlike the excited singlet states, which are excitonic states, the triplet state is localized on a single Chl molecule. Moreover, the lifetime of the 3Chl generated in WSCPs is comparable to that observed in other Chl-containing systems and is reduced in presence of oxygen. In contrast to previous observations, we found that WSCP actually photosensitizes singlet oxygen with an efficiency comparable to that of Chl in organic solvent. We demonstrated that the observed resistance to photooxidation depends on the conformation of the phytyl moieties, which in WSCP are interposed between the rings of Chl dimers, hindering the access of singlet oxygen to the oxidizable sites of the pigments.

In photosynthetic reaction centers, reduction of the secondary acceptors leads to triplet charge recombination of the primary radical pair (RP). This process is spin selective and in a magnetic field it populates only the T0 state of the donor triplet state. As a result, the triplet state of the donor has a distinctive spin polarization pattern that can be measured by transient electron paramagnetic resonance (TREPR) spectroscopy. In heliobacterial reaction centers (HbRCs), the primary donor, P800, is composed of two bacteriochlorophyll g′ molecules and its triplet state has not been studied as extensively as those of other reaction centers. Here, we present TREPR and optically detected magnetic resonance (ODMR) data of 3P800 and show that although it can be detected by ODMR it is not observed in the TREPR data. We demonstrate that the absence of the TREPR spectrum is a result of the fact that the zero-field splitting (ZFS) tensor of 3P800 is maximally rhombic, which results in complete cancelation of the absorptive and emissive polarization in randomly oriented samples.

We present a novel pulsed electron paramagnetic resonance (EPR) spectroscopic ruler to test the performance of a recently developed spin-labeling method based on the photoexcited triplet state (S=1). Four-pulse electron double resonance (PELDOR) experiments are carried out on a series of helical peptides, labeled at the N-terminal end with the porphyrin moiety, which can be excited to the triplet state, and with the nitroxide at various sequence positions, spanning distances in the range 1.8–8 nm. The PELDOR traces provide accurate distance measurements for all the ruler series, showing deep envelope modulations at frequencies varying in a progressive way according to the increasing distance between the spin labels. The upper limit is evaluated and found to be around 8 nm. The PELDOR-derived distances are in excellent agreement with theoretical predictions. We demonstrate that high sensitivity is acquired using the triplet state as a spin label by comparison with Cu(II)–porphyrin analogues. The new labeling approach has a high potential for measuring nanometer distances in more complex biological systems due to the properties of the porphyrin triplet state.