Charge separation in photosystem I. Studies by using methods of ultrafast optical spectroscopy

Wydział FizykiFotosystem I (PSI) to duży białkowo-barwnikowy kompleks wbudowany w błonę tylakoidową, który wykorzystuje energię świetlną do napędzania transbłonowego transportu elektronu. Celem pracy doktorskiej były: (1) weryfikacja istniejących modeli rozdziału ładunku w fotosystemie I, (2) sprawdzenie czy rozdział ładunku jest inicjowany w obu gałęziach przenośników elektronu fotosystemu I czy tylko w jednej z nich, (3) zaproponowanie konkretnego mechanizmu wygaszania wzbudzenia przez zamknięte centra reakcji. Narzędziem badawczym wybranym do rozwiązania wymienionych problemów była ultraszybka spektroskopia optyczna a materiałem badanym – wyizolowane z zielonego glonu Chlamydomonas reinhardtii kompleksy rdzenia fotosystemu I dzikiego szczepu (WT) oraz mutantów punktowych ligandu pierwotnego akceptora elektronu A0. Wyniki przeprowadzonych badań sugerują, że (1) obie gałęzie przenośników elektronu są w jednakowym stopniu zaangażowane w transport elektronu (2) do rozdziału ładunku dochodzi zarówno w otwartych jak i zamkniętych centrach reakcji, (3) rozdział ładunku jest procesem odwracalnym, (4) pierwotnym donorem elektronu jest chlorofil pomocniczy A, a nie jak wcześniej sądzono P700 (5) prawdopodobny mechanizm wygaszania wzbudzenia przez zamknięte centra reakcji polega na rozdziale ładunku, po którym następuje szybka rekombinacja do stanu podstawowego.Photosystem I (PSI) is a large protein-pigment complex embedded in the thylakoid membrane. This complex uses light energy to drive transmembrane electron transfer. The main goals of the presented doctoral thesis were: (1) to verify the existing models of charge separation in photosystem I, (2) to check whether the electron transfer is initiated in one or both electron transfer branches, (3) to reveal the mechanism of antenna excitation quenching by closed RC. The experimental method chosen to solve these problems was the ultrafast optical spectroscopy. The investigated material were complexes of PSI core isolated from green algae Chlamydomonas reinhardtii: wild type (WT) and single site mutants of the axial ligand to the primary electron acceptor A0. The results of experiments suggest that (1) both branches of electron transfer cofactors are equally engaged in electron transfer, (2) the primary charge separation occurs both in open and closed reaction centers, (3) the primary charge separation is a reversible process, (4) the primary electron donor is the accessory chlorophyll A, and not P700 as it was believed earlier (5) the probable mechanism of the excitation quenching in closed RC is primary charge separation followed by fast recombination to the ground state

Comparison of excitation energy transfer in cyanobacterial photosystem I in solution and immobilized on conducting glass

Excitation energy transfer in monomeric and trimeric forms of photosystem I (PSI) from the cyanobacterium Synechocystis sp. PCC 6803 in solution or immobilized on FTO conducting glass was compared using time-resolved fluorescence. Deposition of PSI on glass preserves bi-exponential excitation decay of similar to 4-7 and similar to 21-25 ps lifetimes characteristic of PSI in solution. The faster phase was assigned in part to photochemical quenching (charge separation) of excited bulk chlorophylls and in part to energy transfer from bulk to low-energy (red) chlorophylls. The slower phase was assigned to photochemical quenching of the excitation equilibrated over bulk and red chlorophylls. The main differences between dissolved and immobilized PSI (iPSI) are: (1) the average excitation decay in iPSI is about 11 ps, which is faster by a few ps than for PSI in solution due to significantly faster excitation quenching of bulk chlorophylls by charge separation (similar to 10 ps instead of similar to 15 ps) accompanied by slightly weaker coupling of bulk and red chlorophylls; (2) the number of red chlorophylls in monomeric PSI increases twice-from 3 in solution to 6 after immobilization-as a result of interaction with neighboring monomers and conducting glass; despite the increased number of red chlorophylls, the excitation decay accelerates in iPSI; (3) the number of red chlorophylls in trimeric PSI is 4 (per monomer) and remains unchanged after immobilization; (4) in all the samples under study, the free energy gap between mean red (emission at similar to 710 nm) and mean bulk (emission at similar to 686 nm) emitting states of chlorophylls was estimated at a similar level of 17-27 meV. All these observations indicate that despite slight modifications, dried PSI complexes adsorbed on the FTO surface remain fully functional in terms of excitation energy transfer and primary charge separation that is particularly important in the view of photovoltaic applications of this photosystem

Effect of the P700 pre-oxidation and point mutations near A0 on the reversibility of the primary charge separation in Photosystem I from Chlamydomonas reinhardtii

AbstractTime-resolved fluorescence studies with a 3-ps temporal resolution were performed in order to: (1) test the recent model of the reversible primary charge separation in Photosystem I (Müller et al., 2003; Holwzwarth et al., 2005, 2006), and (2) to reconcile this model with a mechanism of excitation energy quenching by closed Photosystem I (with P700 pre-oxidized to P700+). For these purposes, we performed experiments using Photosystem I core samples isolated from Chlamydomonas reinhardtii wild type, and two mutants in which the methionine axial ligand to primary electron acceptor, A0, has been change to either histidine or serine. The temporal evolution of fluorescence spectra was recorded for each preparation under conditions where the “primary electron donor,” P700, was either neutral or chemically pre-oxidized to P700+. For all the preparations under study, and under neutral and oxidizing conditions, we observed multiexponential fluorescence decay with the major phases of ∼7 ps and ∼25 ps. The relative amplitudes and, to a minor extent the lifetimes, of these two phases were modulated by the redox state of P700 and by the mutations near A0: both pre-oxidation of P700 and mutations caused slight deceleration of the excited state decay. These results are consistent with a model in which P700 is not the primary electron donor, but rather a secondary electron donor, with the primary charge separation event occurring between the accessory chlorophyll, A, and A0. We assign the faster phase to the equilibration process between the excited state of the antenna/reaction center ensemble and the primary radical pair, and the slower phase to the secondary electron transfer reaction. The pre-oxidation of P700 shifts the equilibrium between the excited state and the primary radical pair towards the excited state. This shift is proposed to be induced by the presence of the positive charge on P700+. The same charge is proposed to be responsible for the fast A+A0−→AA0 charge recombination to the ground state and, in consequence, excitation quenching in closed reaction centers. Mutations of the A0 axial ligand shift the equilibrium in the same direction as pre-oxidation of P700 due to the up-shift of the free energy level of the state A+A0−

Uphill energy transfer in photosystem I from Chlamydomonas reinhardtii. Time-resolved fluorescence measurements at 77 K

Energetic properties of chlorophylls in photosynthetic complexes are strongly modulated by their interaction with the protein matrix and by inter-pigment coupling. This spectral tuning is especially striking in photosystem I (PSI) complexes that contain low-energy chlorophylls emitting above 700 nm. Such low-energy chlorophylls have been observed in cyanobacterial PSI, algal and plant PSI–LHCI complexes, and individual light-harvesting complex I (LHCI) proteins. However, there has been no direct evidence of their presence in algal PSI core complexes lacking LHCI. In order to determine the lowest-energy states of chlorophylls and their dynamics in algal PSI antenna systems, we performed time-resolved fluorescence measurements at 77 K for PSI core and PSI–LHCI complexes isolated from the green alga Chlamydomonas reinhardtii. The pool of low-energy chlorophylls observed in PSI cores is generally smaller and less red-shifted than that observed in PSI–LHCI complexes. Excitation energy equilibration between bulk and low-energy chlorophylls in the PSI–LHCI complexes at 77 K leads to population of excited states that are less red-shifted (by ~ 12 nm) than at room temperature. On the other hand, analysis of the detection wavelength dependence of the effective trapping time of bulk excitations in the PSI core at 77 K provided evidence for an energy threshold at ~ 675 nm, above which trapping slows down. Based on these observations, we postulate that excitation energy transfer from bulk to low-energy chlorophylls and from bulk to reaction center chlorophylls are thermally activated uphill processes that likely occur via higher excitonic states of energy accepting chlorophylls

Pharmacokinetics of levodopa and 3-O-methyldopa in parkinsonian patients treated with levodopa and ropinirole and in patients with motor complications

Parkinson’s disease (PD) is a progressive, neurodegenerative disorder primarily affecting dopaminergic neuronal systems, with impaired motor function as a consequence. The most effective treatment for PD remains the administration of oral levodopa (LD). Long-term LD treatment is frequently associated with motor fluctuations and dyskinesias, which exert a serious impact on a patient’s quality of life. The aim of our study was to determine the pharmacokinetics of LD: used as monotherapy or in combination with ropinirole, in patients with advanced PD. Furthermore, an effect of ropinirole on the pharmacokinetics of 3-OMD (a major LD metabolite) was assessed. We also investigated the correlation between the pharmacokinetic parameters of LD and 3-OMD and the occurrence of motor complications. Twenty-seven patients with idiopathic PD participated in the study. Thirteen patients received both LD and ropinirole, and fourteen administered LD monotherapy. Among 27 patients, twelve experienced fluctuations and/or dyskinesias, whereas fifteen were free of motor complications. Inter- and intra-individual variation in the LD and 3-OMD concentrations were observed. There were no significant differences in the LD and 3-OMD concentrations between the patients treated with a combined therapy of LD and ropinirole, and LD monotherapy. There were no significant differences in the LD concentrations in patients with and without motor complications; however, plasma 3-OMD levels were significantly higher in patients with motor complications. A linear one-compartment pharmacokinetic model with the first-order absorption was adopted for LD and 3-OMD. Only mean exit (residence) time for 3-OMD was significantly shorter in patients treated with ropinirole. Lag time, V/F, CL/F and t(max) of LD had significantly lower values in patients with motor complications. On the other hand, AUC were significantly higher in these patients, both for LD and 3-OMD. 3-OMD C(max) was significantly higher in patients with motor complications as well. Our results showed that ropinirole does not influence LD or 3-OMD concentrations. Higher 3-OMD levels play a role in inducing motor complications during long-term levodopa therapy

Excitation dynamics in Photosystem i from Chlamydomonas reinhardtii. Comparative studies of isolated complexes and whole cells

Identical time-resolved fluorescence measurements with ∼ 3.5-ps resolution were performed for three types of PSI preparations from the green alga, Chlamydomonas reinhardtii: isolated PSI cores, isolated PSI-LHCI complexes and PSI-LHCI complexes in whole living cells. Fluorescence decay in these types of PSI preparations has been previously investigated but never under the same experimental conditions. As a result we present consistent picture of excitation dynamics in algal PSI. Temporal evolution of fluorescence spectra can be generally described by three decay components with similar lifetimes in all samples (6-8 ps, 25-30 ps, 166-314 ps). In the PSI cores, the fluorescence decay is dominated by the two fastest components (∼ 90%), which can be assigned to excitation energy trapping in the reaction center by reversible primary charge separation. Excitation dynamics in the PSI-LHCI preparations is more complex because of the energy transfer between the LHCI antenna system and the core. The average trapping time of excitations created in the well coupled LHCI antenna system is about 12-15 ps longer than excitations formed in the PSI core antenna. Excitation dynamics in PSI-LHCI complexes in whole living cells is very similar to that observed in isolated complexes. Our data support the view that chlorophylls responsible for the long-wavelength emission are located mostly in LHCI. We also compared in detail our results with the literature data obtained for plant PSI