Effects of lipids on the rate-limiting steps in the dark-to-light transition of Photosystem II core complex of Thermostichus vulcanus.

In our earlier works, we have shown that the rate-limiting steps, associated with the dark-to-light transition of Photosystem II (PSII), reflecting the photochemical activity and structural dynamics of the reaction center complex, depend largely on the lipidic environment of the protein matrix. Using chlorophyll-a fluorescence transients (ChlF) elicited by single-turnover saturating flashes, it was shown that the half-waiting time (Δτ 1/2) between consecutive excitations, at which 50% of the fluorescence increment was reached, was considerably larger in isolated PSII complexes of Thermostichus (T.) vulcanus than in the native thylakoid membrane (TM). Further, it was shown that the addition of a TM lipid extract shortened Δτ 1/2 of isolated PSII, indicating that at least a fraction of the 'missing' lipid molecules, replaced by detergent molecules, caused the elongation of Δτ 1/2. Here, we performed systematic experiments to obtain information on the nature of TM lipids that are capable of decreasing Δτ 1/2. Our data show that while all lipid species shorten Δτ 1/2, the negatively charged lipid phosphatidylglycerol appears to be the most efficient species - suggesting its prominent role in determining the structural dynamics of PSII reaction center.

Diagnostic value of [99mTc]Tc-PSMA-I&S-SPECT/CT for the primary staging and restaging of prostate cancer.

Background: A large number of studies have proved that prostate-specific membrane antigen-positron emission tomography/computer tomography (PSMA-PET/CT) provides excellent accuracy in primary staging and restaging of prostate cancer. Less data exist with PSMA-single photon emission computed tomography (SPECT)/CT investigations. Objective: The aim of this study was to evaluate the performance of [99mTc]Tc-PSMA-I&S (for imaging and surgery) in prostate cancer. Design and methods: We retrospectively analysed PSMA-SPECT/CT scans of 20 healthy volunteers and 100 male patients with prostate cancer. All of them had histologically confirmed prostate cancer. In all, 28 patients were examined for primary staging and 72 for biochemical recurrence or progressive disease. Whole body SPECT/CT imaging was carried out 6 h after the intravenous administration of 666 ± 102 MBq [99mTc]Tc-PSMA-I&S. Images were evaluated visually and semi-quantitatively. Results: Patient-based sensitivity, specificity, positive predictive value, negative predictive value and accuracy for primary prostate cancer were 86%, 100%, 100%, 83% and 92%, respectively. For detecting metastases in primary staging, these values were 88%, 100%, 100%, 85% and 93%, respectively. The radiopharmaceutical uptake of primary prostate cancer was significantly higher than in normal prostate. The patient-based sensitivity, specificity, positive predictive value, negative predictive value and accuracy of the method in the visualization of local recurrence were 67%, 100%, 100%, 86% and 89%, and for detecting metastases in restaging were 91%, 92%, 98%, 75% and 91%, respectively. In restaging, detection rates were 37% under prostate-specific antigen level of 1 ng/mL, 74% between 1 and 5 ng/mL and 80% >5 ng/mL. Conclusion: [99mTc]Tc-PSMA-I&S-SPECT/CT can be easily integrated into the routine diagnostic practice, and it provides usable data in primary staging and restaging of prostate cancer. Quantitative assessment of PSMA-SPECT/CT has the potential to be used to differentiate between physiological and pathological intraprostatic tracer uptake.

Etioplasts are more susceptible to salinity stress than chloroplasts and photosynthetically active etio-chloroplasts of wheat (Triticum aestivum L.).

High soil salinity is a global problem in agriculture that directly affects seed germination and the development of the seedlings sown deep in the soil. To study how salinity affected plastid ultrastructure, leaf segments of 11-day-old light- and dark-grown (etiolated) wheat (Triticum aestivum L. cv. Mv Béres) seedlings were floated on Hoagland solution, 600 mM KCl:NaCl (1:1) salt or isosmotic polyethylene glycol solution for 4 h in the dark. Light-grown seedlings were also treated in the light. The same treatments were also performed on etio-chloroplasts of etiolated seedlings greened for different time periods. Salt stress induced slight to strong changes in the relative chlorophyll content, photosynthetic activity, and organization of thylakoid complexes. Measurements of malondialdehyde contents and high-temperature thermoluminescence indicated significantly increased oxidative stress and lipid peroxidation under salt treatment, except for light-grown leaves treated in the dark. In chloroplasts of leaf segments treated in the light, slight shrinkage of grana (determined by transmission electron microscopy and small-angle neutron scattering) was observed, while a swelling of the (pro)thylakoid lumen was observed in etioplasts. Salt-induced swelling disappeared after the onset of photosynthesis after 4 h of greening. Osmotic stress caused no significant alterations in plastid structure and only mild changes in their activities, indicating that the swelling of the (pro)thylakoid lumen and the physiological effects of salinity are rather associated with the ionic component of salt stress. Our data indicate that etioplasts of dark-germinated wheat seedlings are the most sensitive to salt stress, especially at the early stages of their greening.

Prostate-specific membrane antigen-based imaging for stereotactic irradiation of low-volume progressive prostate cancer: a single-center experience.

Introduction: Prostate-specific membrane antigen (PSMA) is a transmembrane protein that may be expressed on the surface of prostate cancer (PC) cells. It enables a more sensitive and specific diagnosis PC, compared to conventional anatomical imaging. Aim: The integration of PSMA-based imaging in the personalized radiotherapy of PC patients and the evaluation of its impact on target volume definition if stereotactic body radiotherapy (SBRT) is planned for locally recurrent or oligometastatic disease. Patients and methods: The data from 363 examinations were analyzed retrospectively. Inclusion criteria were histologically verified PC and clinical data suggesting local recurrence or distant metastasis. Whole-body 99mTc-PSMA-I&S single-photon emission computed tomography (SPECT)/CT or 18F-JK-PSMA-7 positron emission tomography/computer tomography (PET/CT) was carried out, and the evaluation of the scans and biological tumor volume contouring was performed at the Department of Nuclear Medicine. The target volume delineation on topometric CT (TCT) scan was performed at the Department of Oncotherapy. The comparison of the two volumes was performed by image fusion and registration. Results: From 363 PSMA isotope-based examinations, 84 lesions of 64 patients were treated with SBRT. In 50 patients, 70 lesions were examined for intermodality comparison. The target volume defined by the PSMA density was significantly smaller than the tumor size defined by the TCT scan: GTVCT (gross tumor volume on the TCT), 27.58 ± 46.07 cm3; BTVPSMA (biological target volume on the PSMA-based examination), 16.14 ± 29.87 cm3. During geometrical analyses, the Dice similarity coefficient (DSC) was 0.56 ± 0.20 (0.07-0.85). Prostate-specific antigen (PSA) control was performed to evaluate the response: mean pre-radiotherapy (pre-RT) PSA was 16.98 ng/ml ( ± SD: 33.81), and post-RT PSA at 3 months after SBRT was 11.19 ng/ml ( ± SD: 32.85). Three-month post-therapy PSMA-based imaging was performed in 14 cases, in which we observed a decrease or cessation of isotope uptake. Conventional imaging control was performed in 42 cases (65.6% of all cases): 22 (52.4%) complete remissions, 14 (33.3%) partial remissions, four (9.5%) stable diseases, and two (4.8%) progressive diseases were described. Conclusion: PSMA-based imaging is a promising diagnostic method for specifying the stage and detecting the low-volume progression. Our results suggest that PSMA-based hybrid imaging can influence treatment decisions and target volume delineation for SBRT.

New foundations for the physical mechanism of variable chlorophyll a fluorescence. Quantum efficiency versus the light-adapted state of photosystem II.

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons to fix CO2. Although the structure at atomic resolution and the basic photophysical and photochemical functions of PSII are well understood, many important questions remain. The activity of PSII in vitro and in vivo is routinely monitored by recording the induction kinetics of chlorophyll a fluorescence (ChlF). According to the 'mainstream' model, the rise from the minimum level (Fo) to the maximum (Fm) of ChlF of dark-adapted PSII reflects the closure of all functionally active reaction centers, and the Fv/Fm ratio is equated with the maximum photochemical quantum yield of PSII (where Fv=Fm-Fo). However, this model has never been free of controversies. Recent experimental data from a number of studies have confirmed that the first single-turnover saturating flash (STSF), which generates the closed state (PSIIC), produces F1

Fluorescence quenching in thylakoid membranes induced by single-walled carbon nanotubes.

The distinct photochemical and electrochemical properties of single-walled carbon nanotubes (SWCNTs) boosted the research interest in nanomaterial utilization in different in vivo and in vitro photosynthetic biohybrid setups. Aiming to unravel the yet not fully understood energetic interactions between the nanotubes and photosynthetic pigment-protein assemblies in an aqueous milieu, we studied SWCNT effects on the photochemical reactions of isolated thylakoid membranes (TMs), Photosystem II (PSII)-enriched membrane fragments and light-harvesting complexes (LHCII). The SWCNTs induced quenching of the steady-state chlorophyll fluorescence in the TM-biohybrid systems with a corresponding shortening of the average fluorescence lifetimes. The effect was not related to changes in the integrity and macroorganization of the photosynthetic membranes. Moreover, we found no evidence for direct excitation energy exchange between the SWCNTs and pigment-protein complexes, since neither the steady-state nor time-resolved fluorescence of LHCII-biohybrid systems differed from the corresponding controls. The attenuation of the fluorescence signal in the TM-biohybrid systems indicates possible leakage of photosynthetic electrons toward the nanotubes that most probably occurs at the level of the PSII acceptor site. Although it is too early to speculate on the nature of the involved electron donors and intermediate states, the observed energetic interaction could be exploited to increase the photoelectron capture efficiency of natural biohybrid systems for solar energy conversion.

Ultrafast excitation quenching by the oxidized photosystem II reaction center.

Photosystem II (PSII) is the pigment-protein complex driving the photoinduced oxidation of water and reduction of plastoquinone in all oxygenic photosynthetic organisms. Excitations in the antenna chlorophylls are photochemically trapped in the reaction center (RC) producing the chlorophyll-pheophytin radical ion pair P+ Pheo-. When electron donation from water is inhibited, the oxidized RC chlorophyll P+ acts as an excitation quencher, but knowledge on the kinetics of quenching is limited. Here, we used femtosecond transient absorption spectroscopy to compare the excitation dynamics of PSII with neutral and oxidized RC (P+). We find that equilibration in the core antenna has a major lifetime of about 300 fs, irrespective of the RC redox state. Two-dimensional electronic spectroscopy revealed additional slower energy equilibration occurring on timescales of 3-5 ps, concurrent with excitation trapping. The kinetics of PSII with open RC can be described well with previously proposed models according to which the radical pair P+ Pheo- is populated with a main lifetime of about 40 ps, which is primarily determined by energy transfer between the core antenna and the RC chlorophylls. Yet, in PSII with oxidized RC (P+), fast excitation quenching was observed with decay lifetimes as short as 3 ps and an average decay lifetime of about 90 ps, which is shorter than the excited-state lifetime of PSII with open RC. The underlying mechanism of this extremely fast quenching prompts further investigation.

Characterization of the Rate-Limiting Steps in the Dark-To-Light Transitions of Closed Photosystem II: Temperature Dependence and Invariance of Waiting Times during Multiple Light Reactions.

Rate-limiting steps in the dark-to-light transition of Photosystem II (PSII) were discovered by measuring the variable chlorophyll-a fluorescence transients elicited by single-turnover saturating flashes (STSFs). It was shown that in diuron-treated samples: (i) the first STSF, despite fully reducing the QA quinone acceptor molecule, generated only an F1(

Radiation Dosimetry of 99mTc-PSMA I&S: A Single-Center Prospective Study.

99mTc-PSMA I&S is a prostate-specific membrane antigen (PSMA) tracer that can be used for planar and SPECT/CT γ-imaging and radioguided surgery. The primary aim of this study was to estimate the dosimetry of 99mTc-PSMA I&S using a hybrid method (sequential γ-planar imaging and 1 single SPECT/CT) in healthy volunteers. The secondary aim was to depict the tracer biodistribution and tumor-to-background ratios (TBRs) in patients with prostate cancer (PCa). Methods: Dosimetry of 99mTc-PSMA I&S was investigated in 4 healthy volunteers. Whole-body planar imaging was acquired at 1, 2, 3, 6, and 24 h and SPECT/CT at 6 h after tracer injection. Contours of organs were drawn on all acquisitions to determine organ activity at each time point. Absorbed dose was estimated using 2 methods: independent curve-fitting manual method (Levenberg-Marquardt-based algorithm using dose factors from RAdiation Dose Assessment Resource [RADAR] website) and OLINDA/EXM software (version 2.0; HERMES Medical Solutions). Biodistribution of 99mTc-PSMA I&S was assessed in 10 patients with PCa on SPECT/CT images at 6 h. Tumor uptake (SUVmax), and TBR (tumor SUVmax/background organ SUVmean) using muscle (T/M), bladder (T/B), and intestine (T/I) as background organs were determined. Results: The mean injected activity of 99mTc-PSMA I&S was 717 MBq (range: 562-828 MBq). No adverse events related to the injection of 99mTc-PSMA I&S were reported. The average radiation effective dose was 0.0055 mSv/MBq with the RADAR manual method and 0.0052 mSv/MBq with OLINDA/EXM. Total body effective dose ranged between 3.33-4.42 and 3.11-4.23 mSv, respectively. All PCa patients showed high tracer uptake in primary and metastatic lesions with T/M, T/B, and T/I ranging from 5.29-110, 0.11-7.02, and 0.96-16.30, respectively. Conclusion: Effective doses of 99mTc-PSMA I&S were comparable to those known for most of the 99mTc tracers and was lower than for the 68Ga-labeled and 18F-labeled agents. 99mTc-PSMA I&S SPECT/CT showed high TBR in PCa patients. This study can provide required data for translation and approval of 99mTc-PSMA I&S by regulatory agencies.

[Theranostics in 2020: Neuroendocrine tumors]. / Teranosztikumok 2020-ban: Neuroendokrin tumorok.

This article presents the diagnostic and therapeutic nuclear medicine methods for neuroendocrine tumors in accordance with current guidelines. The paper begins with a general characterization of neuroendocrine tumors, followed by a broad introduction to laboratory and imaging diagnostic techniques, and a detailed discussion of peptide receptor radionuclide therapy and meta-iodobenzylguanidine treatment. Finally, the article provides an insight into current research and future developments.

18F-FDG-PET/CT in the evaluation and differential diagnosis of active large-vessel vasculitis. A prospective study. / [18F-FDG-PET/CT-vel szerzett tapasztalataink az aktív nagyérvasculitisek diagnosztikájában és differenciáldiagnózisában. Prospektív vizsgálat.]

Introduction: Large-vessel vasculitis has non-specific clinical symptoms, which can delay the diagnosis. Early recognition and treatment of the disease can help to avoid late complications. 18 F-FDG-PET can detect the inflammation of the vessel wall in the early stage of the disease with high sensitivity. CT is used to localize vasculitis. Aim: To examine the performance of 18F-FDG-PET/CT in patients with suspected large-vessel vasculitis, during relapse and remission, focusing on disease activity and extent. Method: 43 patients were evaluated. They were classified according to the clinical questions: steroid-naive suspected vasculitis, suspected vasculitis on steroid treatment, patients with relapse and in remission. We examined 10 cancer patients in control. We carried out visual and quantitative analysis of the 18F-FDG uptake of vessel walls. During quantitative evaluation, we determined standardised uptake values (SUVmax) of vessel wall segments compared to liver. Results: We found active disease in 5 patients examined for primary diagnosis, moreover, in 5 patients with relapse. The disease involved 3 or more vessel segments in fifty percent of the active cases. In the visually active group, the SUVmax was significantly lower in patients on steroid treatment than in steroid-naive cases (1.17 ± 0.11 vs. 1.43 ± 0.29; p = 0.005). We confirmed remission in 2 cases after therapy. In the inactive group, we found other types of inflammatory disorders in 8 cases. Conclusion: 18F-FDG-PET/CT is an effective diagnostic tool for large-vessel vasculitis, and can be used to determine the activity and extent of the disease. Steroid treatment influences the 18F-FDG-uptake of vessel wall. Orv Hetil. 2020; 161(20): 829-838.

Fluorescence-detected linear dichroism imaging in a re-scan confocal microscope equipped with differential polarization attachment.

Confocal laser scanning microscopy is probably the most widely used and one of the most powerful techniques in basic biology, medicine and material sciences that is employed to elucidate the architecture of complex cellular structures and molecular macro-assemblies. It has recently been shown that the information content, signal-to-noise ratio and resolution of such microscopes (LSMs) can be improved significantly by adding different attachments or modifying their design, while retaining their user-friendly features and relatively moderate costs. Differential polarization (DP) attachments, using high-frequency modulation/demodulation circuits, have made LSMs capable of high-precision 2D and 3D mapping of the anisotropy of microscopic samples-without interfering with their 'conventional' fluorescence or transmission imaging (Steinbach et al. in Methods Appl Fluoresc 2:015005, 2014). The resolution and the quality of fluorescence imaging have been enhanced in the recently constructed Re-scan confocal microscopy (RCM) (De Luca et al. in Biomed Opt Express 4:2644-2656, 2013). In this work, we developed the RCM technique further, by adding a DP-attachment modulating the exciting laser beam via a liquid crystal (LC) retarder synchronized with the data acquisition system; by this means, and with the aid of a software, fluorescence-detected linear dichroism (FDLD), characteristic of the anisotropic molecular organization of the sample, could be recorded in parallel with the confocal fluorescence imaging. For demonstration, we show FDLD images of a plant cell wall (Ginkgo biloba) stained with Etzold's staining solution.

Redox transients of P680 associated with the incremental chlorophyll-a fluorescence yield rises elicited by a series of saturating flashes in diuron-treated photosystem II core complex of Thermosynechococcus vulcanus.

Recent chlorophyll-a fluorescence yield measurements, using single-turnover saturating flashes (STSFs), have revealed the involvement of a rate-limiting step in the reactions following the charge separation induced by the first flash. As also shown here, in diuron-inhibited PSII core complexes isolated from Thermosynechococcus vulcanus the fluorescence maximum could only be reached by a train of STSFs. In order to elucidate the origin of the fluorescence yield increments in STSF series, we performed transient absorption measurements at 819 nm, reflecting the photooxidation and re-reduction kinetics of the primary electron donor P680. Upon single flash excitation of the dark-adapted sample, the decay kinetics could be described with lifetimes of 17 ns (∼50%) and 167 ns (∼30%), and a longer-lived component (∼20%). This kinetics are attributed to re-reduction of P680•+ by the donor side of PSII. In contrast, upon second-flash (with Δt between 5 µs and 100 ms) or repetitive excitation, the 819 nm absorption changes decayed with lifetimes of about 2 ns (∼60%) and 10 ns (∼30%), attributed to recombination of the primary radical pair P680•+ Pheo•- , and a small longer-lived component (∼10%). These data confirm that only the first STSF is capable of generating stable charge separation - leading to the reduction of QA ; and thus, the fluorescence yield increments elicited by the consecutive flashes must have a different physical origin. Our double-flash experiments indicate that the rate-limiting steps, detected by chlorophyll-a fluorescence, are not correlated with the turnover of P680.

[Initial experiences with 99mTc-PSMA-SPECT/CT in patients with prostate cancer]. / Kezdeti tapasztalatok a 99mTc-PSMA-SPECT/CT-vel prosztatarákos betegekben.

INTRODUCTION: The prostate-specific membrane antigen (PSMA) is a transmembrane protein, that is highly expressed on the surface of prostate cancer cells. In the last few years, several PSMA-specific ligands have been developed, that can be successfully used to detect primary prostate cancer, tumor recurrences and metastases as well. AIM: The goal of our work was to examine the clinical application of a 99mtechnetium-labeled PSMA-radiopharmaceutical as part of the routine diagnostics of prostate cancer. METHOD: We examined 15 male patients with verified prostate adenocarcinoma with suspicion of progression or recurrence of the disease. We performed whole-body PSMA-SPECT/CTs and multiparametric MRIs of the prostate and the pelvic regions within a week. We used 99mTc-mas3-y-nal-k(Sub-KuE) for the PSMA-SPECT scans. The images were visually evaluated by independent observers. The results were compared with the follow-up bone scintigraphies as well. RESULTS: Twenty-two PSMA-positive lesions were found. Nine of them were localized outside, 13 were within the MRI's field of view. From these 13 lesions, 7 matched with the SPECT/CT results and in 5 cases the MRI images showed no abnormalities. In one case, bone metastasis was suspected on the MRI scan but there was no corresponding pathological tracer uptake on the SPECT images. In two patients, none of the examinations showed signs of prostate malignancy. Four patients had PSMA-positive bone metastases. One of them had a matching PSMA/SPECT and bone scintigraphy result and in one case the PSMA examination showed metastasis in contrast to the negative bone scintigraphy. CONCLUSION: PSMA-SPECT/CT with 99mTc-mas3-y-nal-k(Sub-KuE) is a promising diagnostic tool. This technique is capable of visualizing bone metastases and it can detect local recurrences and visceral metastases as well. Orv Hetil. 2018; 159(35): 1433-1440.

Rate-limiting steps in the dark-to-light transition of Photosystem II - revealed by chlorophyll-a fluorescence induction.

Photosystem II (PSII) catalyses the photoinduced oxygen evolution and, by producing reducing equivalents drives, in concert with PSI, the conversion of carbon dioxide to sugars. Our knowledge about the architecture of the reaction centre (RC) complex and the mechanisms of charge separation and stabilisation is well advanced. However, our understanding of the processes associated with the functioning of RC is incomplete: the photochemical activity of PSII is routinely monitored by chlorophyll-a fluorescence induction but the presently available data are not free of controversy. In this work, we examined the nature of gradual fluorescence rise of PSII elicited by trains of single-turnover saturating flashes (STSFs) in the presence of a PSII inhibitor, permitting only one stable charge separation. We show that a substantial part of the fluorescence rise originates from light-induced processes that occur after the stabilisation of charge separation, induced by the first STSF; the temperature-dependent relaxation characteristics suggest the involvement of conformational changes in the additional rise. In experiments using double flashes with variable waiting times (∆τ) between them, we found that no rise could be induced with zero or short ∆τ, the value of which depended on the temperature - revealing a previously unknown rate-limiting step in PSII.

The biophysics of a critical phenomenon: colonization and sedimentation of the photosynthetic bacteria Rubrivivax gelatinosus.

In response to environmental changes, the photosynthetic bacterium Rubrivivax gelatinosus (Rvx.) can switch from a planktonic lifestyle to a phototrophic biofilm. Like in critical phenomena, the colonization and sedimentation of the cells is abrupt and hard to predict causally, and the underlying biophysics of the mechanisms involved is not known. Herein, we report basic experimental observations and quantitative explanations as keys to understanding microbial turnover of aggregates. (1) The moment of sedimentation can be controlled by the height of the tube of cultivation, by the concentrations of externally added Ficoll (a highly branched polymer) and/or of internally produced polysaccharides (constituents of the biofilm). (2) The observed translational diffusion coefficient of the planktonic bacteria is the sum of diffusion coefficients coming from random Brownian and twitching movements of the bacteria and amounts to 14 (µm)2/s. (3) This value drops hyperbolically with the association number of the cell aggregates and with the concentration of the exopolysaccharides in the biofilm. In the experiments described herein, their effects could be separated. (4) The critical conditions of colonization and sinking of the cells will be achieved if the height of the tube meets the scale height that is proportional to the ratio of the diffusion coefficient and the net mass of the bacterium. The decisive role of the web-like structure of a biofilm, the organization of bacteria from loose cooperativity to solid aggregation, and the possible importance of similar controls in other phototrophic microorganisms are discussed.

Photoprotection in intact cells of photosynthetic bacteria: quenching of bacteriochlorophyll fluorescence by carotenoid triplets.

Upon high light excitation in photosynthetic bacteria, various triplet states of pigments can accumulate leading to harmful effects. Here, the generation and lifetime of flash-induced carotenoid triplets (3Car) have been studied by observation of the quenching of bacteriochlorophyll (BChl) fluorescence in different strains of photosynthetic bacteria including Rvx. gelatinosus (anaerobic and semianaerobic), Rsp. rubrum, Thio. roseopersicina, Rba. sphaeroides 2.4.1 and carotenoid- and cytochrome-deficient mutants Rba. sphaeroides Ga, R-26, and cycA, respectively. The following results were obtained: (1) 3Car quenching is observed during and not exclusively after the photochemical rise of the fluorescence yield of BChl indicating that the charge separation in the reaction center (RC) and the carotenoid triplet formation are not consecutive but parallel processes. (2) The photoprotective function of 3Car is not limited to the RC only and can be described by a model in which the carotenoids are distributed in the lake of the BChl pigments. (3) The observed lifetime of 3Car in intact cells is the weighted average of the lifetimes of the carotenoids with various numbers of conjugated double bonds in the bacterial strain. (4) The lifetime of 3Car measured in the light is significantly shorter (1-2 µs) than that measured in the dark (2-10 µs). The difference reveals the importance of the dynamics of 3Car before relaxation. The results will be discussed not only in terms of energy levels of the 3Car but also in terms of the kinetics of transitions among different sublevels in the excited triplet state of the carotenoid.

Characterization of mercury(II)-induced inhibition of photochemistry in the reaction center of photosynthetic bacteria.

Mercuric contamination of aqueous cultures results in impairment of viability of photosynthetic bacteria primarily by inhibition of the photochemistry of the reaction center (RC) protein. Isolated reaction centers (RCs) from Rhodobacter sphaeroides were exposed to Hg2+ ions up to saturation concentration (~ 103 [Hg2+]/[RC]) and the gradual time- and concentration-dependent loss of the photochemical activity was monitored. The vast majority of Hg2+ ions (about 500 [Hg2+]/[RC]) had low affinity for the RC [binding constant Kb ~ 5 mM-1] and only a few (~ 1 [Hg2+]/[RC]) exhibited strong binding (Kb ~ 50 µM-1). Neither type of binding site had specific and harmful effects on the photochemistry of the RC. The primary charge separation was preserved even at saturation mercury(II) concentration, but essential further steps of stabilization and utilization were blocked already in the 5 < [Hg2+]/[RC] < 50 range whose locations were revealed. (1) The proton gate at the cytoplasmic site had the highest affinity for Hg2+ binding (Kb ~ 0.2 µM-1) and blocked the proton uptake. (2) Reduced affinity (Kb ~ 0.05 µM-1) was measured for the mercury(II)-binding site close to the secondary quinone that resulted in inhibition of the interquinone electron transfer. (3) A similar affinity was observed close to the bacteriochlorophyll dimer causing slight energetic changes as evidenced by a ~ 30 nm blue shift of the red absorption band, a 47 meV increase in the redox midpoint potential, and a ~ 20 meV drop in free energy gap of the primary charge pair. The primary quinone was not perturbed upon mercury(II) treatment. Although the Hg2+ ions attack the RC in large number, the exertion of the harmful effect on photochemistry is not through mass action but rather a couple of well-defined targets. Bound to these sites, the Hg2+ ions can destroy H-bond structures, inhibit protein dynamics, block conformational gating mechanisms, and modify electrostatic profiles essential for electron and proton transfer.

Stoichiometry and kinetics of mercury uptake by photosynthetic bacteria.

Mercury adsorption on the cell surface and intracellular uptake by bacteria represent the key first step in the production and accumulation of highly toxic mercury in living organisms. In this work, the biophysical characteristics of mercury bioaccumulation are studied in intact cells of photosynthetic bacteria by use of analytical (dithizone) assay and physiological photosynthetic markers (pigment content, fluorescence induction, and membrane potential) to determine the amount of mercury ions bound to the cell surface and taken up by the cell. It is shown that the Hg(II) uptake mechanism (1) has two kinetically distinguishable components, (2) includes co-opted influx through heavy metal transporters since the slow component is inhibited by Ca2+ channel blockers, (3) shows complex pH dependence demonstrating the competition of ligand binding of Hg(II) ions with H+ ions (low pH) and high tendency of complex formation of Hg(II) with hydroxyl ions (high pH), and (4) is not a passive but an energy-dependent process as evidenced by light activation and inhibition by protonophore. Photosynthetic bacteria can accumulate Hg(II) in amounts much (about 105) greater than their own masses by well-defined strong and weak binding sites with equilibrium binding constants in the range of 1 (µM)-1 and 1 (mM)-1, respectively. The strong binding sites are attributed to sulfhydryl groups as the uptake is blocked by use of sulfhydryl modifying agents and their number is much (two orders of magnitude) smaller than the number of weak binding sites. Biofilms developed by some bacteria (e.g., Rvx. gelatinosus) increase the mercury binding capacity further by a factor of about five. Photosynthetic bacteria in the light act as a sponge of Hg(II) and can be potentially used for biomonitoring and bioremediation of mercury-contaminated aqueous cultures.

Induction and anisotropy of fluorescence of reaction center from photosynthetic bacterium Rhodobacter sphaeroides.

Submillisecond dark-light changes of the yield (induction) and anisotropy of fluorescence under laser diode excitation were measured in the photosynthetic reaction center of the purple bacterium Rhodobacter sphaeroides. Narrow band (1-2 nm) laser diodes emitting at 808 and 865 nm were used to selectively excite the accessory bacteriochlorophyll (B, 800 nm) or the upper excitonic state of the bacteriochlorophyll dimer (P-, 810 nm) and the lower excitonic state of the dimer (P+, 865 nm), respectively. The fluorescence spectrum of the wild type showed two bands centered at 850 nm (B) and 910 nm (P-). While the monotonous decay of the fluorescence yield at 910 nm tracked the light-induced oxidation of the dimer, the kinetics of the fluorescence yield at 850 nm showed an initial rise before a decrease. The anisotropy of the fluorescence excited at 865 nm (P-) was very close to the limiting value (0.4) across the whole spectral range. The excitation of both B and P- at 808 nm resulted in wavelength-dependent depolarization of the fluorescence from 0.35 to 0.24 in the wild type and from 0.30 to 0.24 in the reaction center of triple mutant (L131LH-M160LH-M197FH). The additivity law of the anisotropies of the fluorescence species accounts for the wavelength dependence of the anisotropy. The measured fluorescence yields and anisotropies are interpreted in terms of very fast energy transfer from (1)B* to (1)P- (either directly or indirectly by internal conversion from (1)P+) and to the oxidized dimer.