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Advanced EPR spectroscopy provides new insight into triplet-triplet energy transfer in photosynthesis
Full text linkCarotenoids are essential in photosynthesis. These molecules play several roles in the photosynthetic apparatus: they share with chlorophylls the light-harvesting function, stabilize the pigment-protein complexes and are responsible for the protection of the whole apparatus against the hazardous singlet oxygen. The structural function has been investigated in many different organisms by several biochemical methods: the main finding is that carotenoids are essential for the protein stability. Without carotenoids light-harvesting complexes would not fold and their selective removal from intact proteins often causes unfolding. The light-harvesting function of the carotenoids has been object of extensive investigations. The photo-physics of their singlet states is very complex: it is characterized by the presence of forbidden states, fast dynamics of the excited states and low fluorescence yield. The singlet energy transfer pathways between carotenoids and chlorophylls, which are important for the light-harvesting function, have been also well characterized. Less has been done on the study of the photoprotective role that makes carotenoids essential for the photosynthetic organisms. Carotenoids are involved in photoprotection in many ways and at different stages of the photosynthetic process, in relation to the intensity and duration of the stress conditions. When the sunlight intensity suddenly increases the first photoprotection mechanism activated is the photo-physical triplet-triplet energy transfer from chlorophylls molecules to carotenoids molecules. This interaction scavenges the formation of highly oxidant oxygen species which could be produced by the chlorophyll triplet states, whose production increases under light excess. Carotenoid triplet states are thus very important from a physiological point of view.
In contrast to photosynthetic reaction centres, which have very similar structural architecture, light-harvesting complexes show more variability due to different habitat adaptation of the photosynthetic organisms. This heterogeneity stimulated a comparative investigation on light harvesting complexes in order to point out the common and the different key features adopted by the different organisms for reaching an efficient triplet-triplet energy transfer.
This thesis represents one of the few detailed studies entirely focused on the role of carotenoids molecules in the triplet-triplet energy transfer mechanism in naturally occurring light-harvesting complexes. By means of EPR spectroscopy, the magnetic properties of the carotenoid triplet states have been exploited to characterize the energy transfer pathways in four different light-harvesting complexes: the LHC-II from higher plants, the LHC and the PCPs from dinoflagellates and the chlorosomes from green sulphur bacteria. These antenna proteins have been selected as representative for the light-harvesting strategy variability adopted by different photosynthetic species.
The main results obtained, which have a physiological relevance, can be summarized as follows:
- Among all the pigments present in each protein considered, only specific carotenoids are devoted to the dissipation of chlorophyll triplet energy. It is worth noting that the examined proteins belong to different organisms which are not close in the evolutionary scale. Thus, the presence of a specific triplet energy sink seems to be an important point in the photoprotection mechanism played by triplet-triplet energy transfer.
- EPR spectroscopy does not show any evidence for the existence of triplet excitons involving carotenoids and chlorophylls. Moreover multiple triplet-triplet energy transfers among carotenoids can also be ruled out. Therefore a localized carotenoid triplet state seems to be a common feature for all the pigment-protein complexes.
- The triplet electron density distribution over the conjugate chain of the carotenoids is not heavily influenced by the chemical substitution. It has the same pattern and the same extent even though triplet states belonging to carotenoids with very different molecular structure are considered. This characteristic makes the relative geometry of the chlorophyll/carotenoid couples involved in the triplet-triplet energy transfer not very dependent on the center-to-center distance of the two pigments. Instead the minimum distance between the π-conjugated systems results to be most the important structural requirement.
- Triplet-triplet energy transfer in antenna complexes seems to be properly described by a superexchange mechanism involving a molecular bridge interposed between the chlorophyll/carotenoid couple. This bridge is represented by the fifth ligand of the Mg atom bound to the chlorophyll. The mechanism is supported by ENDOR measurements and DFT calculations: the highest electron density calculated for the carotenoid triplet state is located on the carbon atom closest to the Mg fifth ligand. Thus the bridge likely increases the triplet-triplet energy transfer rate leading to a very efficient quenching.
Together with the ‘functional’ results, this thesis demonstrates also how triplet state EPR techniques, applied to the study of the molecules involved in triplet-triplet energy transfer, can be used to derive structural information if the crystallographic structure of the light-harvesting protein is not known. As a matter of fact, EPR spectra can be conveniently used to extract some experimental constraints useful in the modelling of protein structure, since their shapes strictly depend on the relative geometry of the couples involved in the triplet-triplet energy transfer. In photosynthesis this is particularly relevant since many proteins involved in the process are large, embedded in the membrane and bound many cofactors. All these characteristics are severe limitations in using x-ray diffraction or NMRI caroteni sono assi portanti del processo fotosintetico. A queste molecole sono affidati molteplici ruoli: condividono con le clorofille la funzione di raccolta dell’energia luminosa, stabilizzano i complessi proteina-pigmento e sono responsabili della protezione dell’intero apparato fotosintetico contro l’azione ossidante dell’ossigeno di singoletto. La funzione strutturale è stata studiata mediante diverse tecniche biochimiche in molteplici organismi: è stato dimostrato che i carotenoidi sono essenziali per la stabilità strutturale dei complessi proteici. Senza carotenoidi i complessi antenna non sono in grado di assemblarsi e la loro rimozione selettiva da proteine intatte ne causa la denaturazione. La funzione di raccolta della luce è stata estensivamente studiata tramite spettroscopie ottiche. La foto-fisica degli stati di singoletto nei caroteni è molto complessa e caratterizzata dalla presenza di transizioni proibite, dalla dinamica veloce tra stati eccitati e dalla bassa resa di fluorescenza. Anche i cammini di trasferimento di energia di singoletto tra caroteni e clorofille, importanti per la funzione di raccolta della luce, sono stati ampiamente caratterizzati. Molto meno è stato fatto riguardo al ruolo fotoprotettivo che rende i carotenoidi essenziali per gli organismi fotosintetici. I carotenoidi sono implicati nella fotoprotezione in moti modi e in diversi gradi in relazione alla durata e all’intensità delle condizioni di stress. Quando l’intensità della luce solare aumenta improvvisamente il primo meccanismo di fotoprotezione attivato è il trasferimento di energia tripletto-tripletto dalle molecole di clorofilla verso le molecole di carotenoide: questo trasferimento di energia previene la formazione di specie all’ossigeno altamente ossidanti che potrebbero essere prodotte dalla clorofilla in stato di tripletto. Lo stato di tripletto di carotenoide è quindi fisiologicamente importante.
A differenza dei centri di reazione fotosintetici, che condividono tutti un’architettura strutturale molto simile, i complessi antenna esibiscono una più spiccata variabilità dovuta ai differenti adattamenti ambientali. Questa eterogeneità ha stimolato un’indagine comparativa al fine di evidenziare le caratteristiche fondamentali comuni o particolari adottate dai diversi organismi per rendere il trasferimento di energia di tripletto efficiente.
Questa tesi rappresenta uno dei pochi studi dettagliati completamente focalizzati sul ruolo delle molecole carotenoidi nel meccanismo di trasferimento di energia tripletto-ripletto in complessi antenna naturali. Attraverso la spettroscopia EPR, si sono sfruttate le proprietà magnetiche degli stati di tripletto dei caroteni per caratterizzare i cammini di trasferimento di energia in quattro diversi complessi antenna: la proteina LHC-II caratteristica delle piante superiori, le proteine LHC e PCP caratteristiche dei dinoflagellati ed i clorosomi caratteristici dei batteri verdi sulfurei. Queste proteine sono state selezionate come campione rappresentativo della variabilità nelle strategie di fotoprotezione adottate dai diversi organismi fotosintetici.
I risultati ottenuti con rilevanza fisiologica possono essere schematizzati come segue:
- Tra tutti i pigmenti presenti in ogni proteina considerata, solo specifici carotenoidi sono deputati alla dissipazione dell’energia di tripletto delle clorofille. Vale la pena notare che le proteine considerate appartengono ad organismi distanti nella scala evolutiva. Sembra perciò che la presenza di specifiche ‘trappole di energia’ sia centrale nella fotoprotezione giocata dai trasferimenti di energia tripletto-tripletto
- I dati EPR non mostrano alcuna evidenza circa la formazione di eccitoni di tripletto che coinvolgano caroteni e clorofille. Inoltre, permettono di escludere la presenza trasferimenti multipli di energia di tripletto tra carotenoidi. Perciò, la presenza di uno stato di tripletto di carotenoide localizzato sembra essere una caratteristica comune a tutti i complessi antenna considerati.
- La distribuzione della densità elettronica dello stato di tripletto lungo la catena coniugata della molecola di carotenoide non risulta essere particolarmente influenzata dalla presenza di sostituenti chimici: presenta la stesso andamento e la stessa estensione anche tra carotenoidi con strutture chimiche molto differenti. Questa evidenza rende la geometria relativa della coppia clorofilla/carotenoide coinvolta nel trasferimento di energia non particolarmente dipendente dalla distanza tra i centri dei sistemi coniugati delle due molecole. Mentre sembra che la minima distanza tra i sistemi coniugati sia il requisito strutturale fondamentale.
- Il trasferimento di energia tripletto-tripletto nei complessi antenna sembra essere correttamente descritto dal meccanismo di super-scambio che coinvolge un ponte molecolare intercalato tra la coppia clorofilla/carotenoide. Questo ponte è rappresentato dal quinto legando dell’atomo di Mg legato al centro della molecola di clorofilla. Gli esperimenti ENDOR ed i calcoli DFT condotti supportano questa conclusione: la densità elettronica più alta riscontrata appartiene all’atomo di carbonio più vicino al quinto legando dell’atomo di Mg. Verosimilmente il ponte molecolare aumenta la velocità di trasferimento di energia garantendo un processo molto efficiente.
Parallelamente a questi conclusioni ‘funzionali’, questa tesi dimostra anche come le tecniche EPR, applicate allo studio di molecole coinvolte nei trasferimenti di energia tripletto-tripletto, possano essere utilizzate per ricavare informazioni strutturali quando la struttura cristallografica non sia nota. Infatti, poiché la loro forma spettrale dipende direttamente dalla geometria della coppia coinvolta nel trasferimento di energia di tripletto, gli spettri EPR possono essere opportunamente impiegati per estrarre vincoli sperimentali da impiegare nella modellizzazione di strutture proteiche.
In ambito fotosintetico questo risultato è particolarmente rilevante dal momento che molte proteine coinvolte nel processo sono grandi, di membrana e legano molti cofattori. Tutte queste caratteristiche costituiscono importanti limitazioni nell’utilizzo della diffrazione ai raggi x o delle tecniche NMR
Photo‐Induced Radicals in Carbon Nitride and their Magnetic Signature
Full text linkAs a metal-free semiconductor, carbon nitride is a promising material for sustainable photocatalysis. From the large number of studies, it seems apparent that the photocatalytic activity is related to the number and type of defects present in the structure. Many defects are paramagnetic and photoresponsive and, for this reason, Electron Paramagnetic Resonance (EPR) spectroscopy is a powerful method to derive fundamental information on the structure – local, extended and electronic – of such defects which in turn impact the optical, magnetic and chemical properties of a material. This review aims at critically discussing the interpretation of EPR data of native and photoinduced radical defects in carbon nitride research highlighting strengths and limitations of this spectroscopic techniqu
Unravelling electronic and structural requisites of triplet–triplet energy transfer by advanced electron paramagnetic resonance and density functional theory
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Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
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Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
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From electron spin to relaxivity: a multidisciplinary perspective on first-row transition metal-based MRI probes
No full text: The electron spin is a key enabler of some of the most advanced current technologies. A prime example is the development of MRI contrast agents, where precisely engineered electron spin properties are utilized to enhance the capabilities of one of the most powerful diagnostic tools in the medical science. Clinically approved contrast agents are based on paramagnetic gadolinium(iii) complexes. However, to alleviate health and environmental concerns, as well as for specialized applications, alternatives are sought after. Due to their rich chemistry, abundance and low toxicity first-row paramagnetic transition metal ions are emerging as an appealing alternative. A large experimental effort is needed to engineer the new generation of contrast agents. The primary source of information comes from Nuclear Magnetic Relaxation Dispersion (NMRD) profiles. While fitting these profiles can, in principle, yield all the structural and dynamic parameters that influence relaxation, the underlying theoretical models demonstrate a significant challenge. The parameters affect the NMRD profiles in highly coupled, non-separable ways, meaning that a simple, unconstrained fit often results in a non-unique solution. Consequently, the independent experimental determination of some, and preferably most, of these parameters offers a considerable advantage in obtaining reliable and physically meaningful information. This perspective outlines an integrated approach that exploits Electron Paramagnetic Resonance (EPR) spectroscopy for the accurate determination of key molecular parameters. Specifically, EPR is used to quantify the rotational correlation time, the closest proton-metal distance, and the electron spin density at the proton. This methodology is particularly relevant for contrast agents based on first-row transition metal ions. We discuss the contribution of EPR in a complementary context with well-established techniques such as NMR and DFT
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