Dependence of Protein Immobilization and Photocurrent Generation in PSI-FTO Electrodes on the Electrodeposition Parameters.

This study investigates the immobilization of cyanobacterial photosystem I (PSI) from Synechocystis sp. PCC 6803 onto fluorine-doped tin oxide (FTO) conducting glass plates to create photoelectrodes for biohybrid solar cells. The fabrication of these PSI-FTO photoelectrodes is based on two immobilization processes: rapid electrodeposition driven by an external electric field and slower adsorption during solvent evaporation, both influenced by gravitational sedimentation. Deposition and performance of photoelectrodes was investigated by UV-Vis absorption spectroscopy and photocurrent measurements. We investigated the efficiency of PSI immobilization under varying conditions, including solution pH, applied electric field intensity and duration, and electrode polarization, with the goals to control (1) the direction of migration and (2) the orientation of the PSI particles on the substrate surface. Variation in the pH value of the PSI solution alters the surface charge distribution, affecting the net charge and the electric dipole moment of these proteins. Results showed PSI migration to the positively charged electrode at pH 6, 7, and 8, and to the negatively charged electrode at pH 4.4 and 5, suggesting an isoelectric point of PSI between 5 and 6. At acidic pH, the electrophoretic migration was largely hindered by protein aggregation. Notably, photocurrent generation was consistently cathodic and correlated with PSI layer thickness, and no conclusions can be drawn on the orientation of the immobilized proteins. Overall, these findings suggest mediated electron transfer from FTO to PSI by the used electrolyte containing 10 mM sodium ascorbate and 200 µM dichlorophenolindophenol.

Competition between intra-protein charge recombination and electron transfer outside photosystem I complexes used for photovoltaic applications.

Photosystem I (PSI) complexes isolated from three different species were electrodeposited on FTO conducting glass, forming a photoactive multilayer of the photo-electrode, for investigation of intricate electron transfer (ET) properties in such green hybrid nanosystems. The internal quantum efficiency of photo-electrochemical cells (PEC) containing the PSI-based photo-electrodes did not exceed ~ 0.5%. To reveal the reason for such a low efficiency of photocurrent generation, the temporal evolution of the transient concentration of the photo-oxidized primary electron donor, P+, was studied in aqueous suspensions of the PSI complexes by time-resolved absorption spectroscopy. The results of these measurements provided the information on: (1) completeness of charge separation in PSI reaction centers (RCs), (2) dynamics of internal charge recombination, and (3) efficiency of electron transfer from PSI to the electrolyte, which is the reaction competing with the internal charge recombination in the PSI RC. The efficiency of the full charge separation in the PSI complexes used for functionalization of the electrodes was ~ 90%, indicating that incomplete charge separation was not the main reason for the small yield of photocurrents. For the PSI particles isolated from a green alga Chlamydomonas reinhardtii, the probability of ET outside PSI was ~ 30-40%, whereas for their counterparts isolated from a cyanobacterium Synechocystis sp. PCC 6803 and a red alga Cyanidioschyzon merolae, it represented a mere ~ 4%. We conclude from the transient absorption data for the PSI biocatalysts in solution that the observed small photocurrent efficiency of ~ 0.5% for all the PECs analyzed in this study is likely due to: (1) limited efficiency of ET outside PSI, particularly in the case of PECs based on PSI from Synechocystis and C. merolae, and (2) the electrolyte-mediated electric short-circuiting in PSI particles forming the photoactive layer, particularly in the case of the C. reinhardtii PEC.

Electron Transfer in a Bio-Photoelectrode Based on Photosystem I Multilayer Immobilized on the Conducting Glass.

A film of ~40 layers of partially oriented photosystem I (PSI) complexes isolated from the red alga Cyanidioschyzon merolae formed on the conducting glass through electrodeposition was investigated by time-resolved absorption spectroscopy and chronoamperometry. The experiments were performed at a range of electric potentials applied to the film and at different compositions of electrolyte solution being in contact with the film. The amount of immobilized proteins supporting light-induced charge separation (active PSI) ranged from ~10%, in the absence of any reducing agents (redox compounds or low potential), to ~20% when ascorbate and 2,6-dichlorophenolindophenol were added, and to ~35% when the high negative potential was additionally applied. The origin of the large fraction of permanently inactive PSI (65-90%) was unclear. Both reducing agents increased the subpopulation of active PSI complexes, with the neutral P700 primary electron donor, by reducing significant fractions of the photo-oxidized P700 species. The efficiencies of light-induced charge separation in the PSI film (10-35%) did not translate into an equally effective generation of photocurrent, whose internal quantum efficiency reached the maximal value of 0.47% at the lowest potentials. This mismatch indicates that the vast majority of the charge-separated states in multilayered PSI complexes underwent charge recombination.

Photovoltaic activity of electrodes based on intact photosystem I electrodeposited on bare conducting glass.

We demonstrate photovoltaic activity of electrodes composed of fluorine-doped tin oxide (FTO) conducting glass and a multilayer of trimeric photosystem I (PSI) from cyanobacterium Synechocystis sp. PCC 6803 yielding, at open circuit potential (OCP) of + 100 mV (vs. SHE), internal quantum efficiency of (0.37 ± 0.11)% and photocurrent density of up to (0.5 ± 0.1) µA/cm2. The photocurrent measured for OCP is of cathodic nature meaning that preferentially the electrons are injected from the conducting layer of the FTO glass to the photooxidized PSI primary electron donor, P700+, and further transferred from the photoreduced final electron acceptor of PSI, Fb-, via ascorbate electrolyte to the counter electrode. This observation is consistent with preferential donor-side orientation of PSI on FTO imposed by applied electrodeposition. However, by applying high-positive bias (+ 620 mV) to the PSI-FTO electrode, exceeding redox midpoint potential of P700 (+ 450 mV), the photocurrent reverses its orientation and becomes anodic. This is explained by "switching off" the natural photoactivity of PSI particles (by the electrochemical oxidation of P700 to P700+) and "switching on" the anodic photocurrent from PSI antenna Chls prone to photooxidation at high potentials. The efficient control of the P700 redox state (P700 or P700+) by external bias applied to the PSI-FTO electrodes was evidenced by ultrafast transient absorption spectroscopy. The advantage of the presented system is its structural simplicity together with in situ-proven high intactness of the PSI particles.

Acceleration of the excitation decay in Photosystem I immobilized on glass surface.

Femtosecond transient absorption was used to study excitation decay in monomeric and trimeric cyanobacterial Photosystem I (PSI) being prepared in three states: (1) in aqueous solution, (2) deposited and dried on glass surface (either conducting or non-conducting), and (3) deposited on glass (conducting) surface but being in contact with aqueous solvent. The main goal of this contribution was to determine the reason of the acceleration of the excitation decay in dried PSI deposited on the conducting surface relative to PSI in solution observed previously using time-resolved fluorescence (Szewczyk et al., Photysnth Res 132(2):111-126, 2017). We formulated two alternative working hypotheses: (1) the acceleration results from electron injection from PSI to the conducting surface; (2) the acceleration is caused by dehydration and/or crowding of PSI proteins deposited on the glass substrate. Excitation dynamics of PSI in all three types of samples can be described by three main components of subpicosecond, 3-5, and 20-26 ps lifetimes of different relative contributions in solution than in PSI-substrate systems. The presence of similar kinetic components for all the samples indicates intactness of PSI proteins after their deposition onto the substrates. The kinetic traces for all systems with PSI deposited on substrates are almost identical and they decay significantly faster than the kinetic traces of PSI in solution. We conclude that the accelerated excitation decay in PSI-substrate systems is caused mostly by dense packing of proteins.

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.

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 ~4-7 and ~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 (~10 ps instead of ~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 ~710 nm) and mean bulk (emission at ~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.

Monte Carlo simulations of excitation and electron transfer in grana membranes.

Time-resolved fluorescence measurements on grana membranes with instrumental response function of 3 ps reveal faster excitation dynamics (120 ps) than those reported previously. A possible reason for the faster decay may be a relatively low amount of "extra" LHCII trimers per reaction center of Photosystem II. Monte Carlo modeling of excitation dynamics in C2S2M2 form of PSII-LHCII supercomplexes has been performed using a coarse grained model of this complex, constituting a large majority of proteins in grana membranes. The main factor responsible for the fast fluorescence decay reported in this work was the deep trap constituted by the primary charge separated state in the reaction center (950-1090 cm(-1)). This value is critical for a good fit, whereas typical hopping times between antenna polypeptides (from ~4.5 to ~10.5 ps) and reversible primary charge separation times (from ~4 to ~1.5 ps, respectively) are less critical. Consequently, respective mean migration times of excitation from anywhere in the PSII-LHCII supercomplexes to reaction center range from ~30 to ~80 ps. Thus 1/4-2/3 of the ~120-ps average excitation lifetime is necessary for the diffusion of excitation to reaction center, whereas the remaining time is due to the bottle-neck effect of the trap. Removal of 27% of the Lhcb6 apoprotein pool by mutagenesis of DEG5 gene caused the acceleration of the excitation decay from ~120 to ~100 ps. This effect may be due to the detachment of LHCII-M trimers from PSII-LHCII supercomplexes, accompanied by deepening of the reaction center trap.

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 (6-8ps, 25-30ps, 166-314ps). 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 12-15ps 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.

Transport of NaYF4:Er3+, Yb3+ up-converting nanoparticles into HeLa cells.

An effective, simple and practically useful method to incorporate fluorescent nanoparticles inside live biological cells was developed. The internalization time and concentration dependence of a frequently used liposomal transfection factor (Lipofectamine 2000) was studied. A user friendly, one-step technique to obtain water and organic solvent soluble Er(3+) and Yb(3+) doped NaYF4 nanoparticles coated with polyvinylpyrrolidone was obtained. Structural analysis of the nanoparticles confirmed the formation of nanocrystals of the desired sizes and spectral properties. The internalization of NaYF4 nanoparticles in HeLa cervical cancer cells was determined at different nanoparticle concentrations and for incubation periods from 3 to 24 h. The images revealed a redistribution of nanoparticles inside the cell, which increases with incubation time and concentration levels, and depends on the presence of the transfection factor. The study identifies, for the first time, factors responsible for an effective endocytosis of the up-converting nanoparticles to HeLa cells. Thus, the method could be applied to investigate a wide range of future 'smart' theranostic agents. Nanoparticles incorporated into the liposomes appear to be very promising fluorescent probes for imaging real-time cellular dynamics.

Time-resolved absorption measurements quantify the competition of energy and electron transfer between quantum dots and cytochrome c.

We applied transient absorption spectroscopy to study the early photodynamics in a system composed of CdTe quantum dots (QDs) and cytochrome c (Cyt c) protein. In the QDs and Cyt c mixtures, about 25 % of the excited QD electrons quickly relax (∼23 ps) to the ground state and roughly 75 % decay on slower time scale - mostly due to quenching by Cyt c. On the basis of the assumed model, we estimated the contribution of electron transfer and other mechanisms to this quenching. The primary quenching mechanism is probably energy transfer but electron transfer makes a significant contribution (∼8 %), resulting in photoreduction of Cyt c. The lifetime of one fraction of reduced Cyt c (35-90 %) is âˆ¼ 1 ms and the lifetime of the remaining fraction was longer than the âˆ¼ 50-ms time window of the experiment. We speculate that, in the former fraction, the back electron transfer from the reduced Cyt c to QDs occurs and the latter fraction of Cyt c is stably reduced.

Treatment with Intravenous Methylprednisolone in Patients with Graves' Orbitopathy Significantly Affects Adrenal Function: Assessment of Serum, Salivary Cortisol and Serum Dehydroepiandrosterone Sulfate.

Treatment of active, moderate-to-severe Graves' orbitopathy (GO) is the administration of intravenous methylprednisolone (IVMP). IVMP may be followed by additional therapy with oral prednisone. The aim of this study was to analyze the impact of IVMP on adrenal function by evaluation of serum, salivary cortisol and serum dehydroepiandrosterone sulfate (DHEA-S). Fourteen patients received IVMP treatment (cumulative dose of 4.5 g in 12 weekly infusions) followed by oral prednisone (for three months). All patients showed normal adrenal function before the 12th IVMP pulse and one patient was diagnosed with secondary adrenal insufficiency (AI) after prednisone treatment. DHEA-S was significantly lower before the 12th IVMP pulse and after oral prednisone (p = 0.015 and p = 0.00002, respectively) in comparison to evaluation before therapy. DHEA-S levels were below the reference range in one and three patients before the 12th IVMP pulse and after prednisone therapy, respectively. We observed decreased serum (p = 0.05) and salivary (p = 0.011) cortisol levels after oral prednisone therapy in comparison to evaluation before therapy. Treatment with IVMP in a cumulative dose of 4.5 g affects adrenal function, causing more severe impairment of DHEA-S secretion than that of cortisol but does not cause secondary AI. Additional therapy with oral glucocorticoids after IVMP can cause secondary AI.

Remodeling of excitation energy transfer in extremophilic red algal PSI-LHCI complex during light adaptation.

Photosynthetic PSI-LHCI complexes from an extremophilic red alga C. merolae grown under varying light regimes are characterized by decreasing size of LHCI antenna with increasing illumination intensity [1]. In this study we applied time-resolved fluorescence spectroscopy to characterize the kinetics of energy transfer processes in three types of PSI-LHCI supercomplexes isolated from the low (LL), medium (ML) and extreme high light (EHL) conditions. We show that the average rate of fluorescence decay is not correlated with the size of LHCI antenna and is twice faster in complexes isolated from ML-grown cells (~25-30 ps) than from both LL- and EHL-exposed cells (~50-55 ps). The difference is mainly due to a contribution of a long ~100-ps decay component detected only for the latter two PSI samples. We propose that the lack of this phase in ML complexes is caused by perfect coupling of this antenna to PSI core and lack of low-energy chlorophylls in LHCI. On the other hand, the presence of the slow, ~100-ps, fluorescence decay component in LL and EHL complexes may be due to the weak coupling between PSI core and LHCI antenna complex, and due to the presence of particularly low-energy or red chlorophylls in LHCI. Our study has revealed the remarkable functional flexibility of light harvesting strategies that have evolved in the extremophilic red algae in response to harsh or limiting light conditions involving accumulation of low energy chlorophylls that exert two distinct functions: as energy traps or as far-red absorbing light harvesting antenna, respectively.

On the nature of uncoupled chlorophylls in the extremophilic photosystem I-light harvesting I supercomplex.

Photosystem I core-light-harvesting antenna supercomplexes (PSI-LHCI) were isolated from the extremophilic red alga Cyanidioschyzon merolae and studied by three fluorescence techniques in order to characterize chlorophylls (Chls) energetically uncoupled from the PSI reaction center (RC). Such Chls are observed in virtually all optical experiments of any PSI core and PSI-LHCI supercomplex preparations across various species and may influence the operation of PSI-based solar cells and other biohybrid systems. However, the nature of the uncoupled Chls (uChls) has never been explored deeply before. In this work, the amount of uChls was controlled by stirring the solution of C. merolae PSI-LHCI supercomplex samples at elevated temperature (~303 K) and was found to increase from <2% in control samples up to 47% in solutions stirred for 3.5 h. The fluorescence spectrum of uChls was found to be blue-shifted by ~20 nm (to ~680 nm) relative to the fluorescence band from Chls that are well coupled to PSI RC. This effect indicates that mechanical stirring leads to disappearance of some red Chls (emitting at above ~700 nm) that are present in the intact LHCI antenna associated with the PSI core. Comparative diffusion studies of control and stirred samples by fluorescence correlation spectroscopy together with biochemical analysis by SDS-PAGE and BN-PAGE indicate that energetically uncoupled Lhcr subunits are likely to be still physically attached to the PSI core, albeit with altered three-dimensional organization due to the mechanical stress.

Excitation energy transfer and charge separation are affected in Arabidopsis thaliana mutants lacking light-harvesting chlorophyll a/b binding protein Lhcb3.

The composition of LHCII trimers as well as excitation energy transfer and charge separation in grana cores of Arabidopsis thaliana mutant lacking chlorophyll a/b binding protein Lhcb3 have been investigated and compared to those in wild-type plants. In grana cores of lhcb3 plants we observed increased amounts of Lhcb1 and Lhcb2 apoproteins per PSII core. The additional copies of Lhcb1 and Lhcb2 are expected to substitute for Lhcb3 in LHCII trimers M as well as in the LHCII "extra" pool, which was found to be modestly enlarged as a result of the absence of Lhcb3. Time-resolved fluorescence measurements reveal a deceleration of the fast phase of excitation dynamics in grana cores of the mutant by ~15 ps, whereas the average fluorescence lifetime is not significantly altered. Monte Carlo modeling predicts a slowing down of the mean hopping time and an increased stabilization of the primary charge separation in the mutant. Thus our data imply that absence of apoprotein Lhcb3 results in detectable differences in excitation energy transfer and charge separation.