Electrogenicity at the donor/acceptor sides of cyanobacterial photosystem I

To study electrogenesis the photosystem I particles from Synechococcus elongatus were incorporated into asolectin liposomes, and fast kinetics of laser flash-induced electric potential difference generation has been measured by a direct electrometric method in proteoliposomes absorbed on a phospholipid-impregnated collodion film. The photoelectric response has been found to involve three electrogenic stages associated with (i) iron-sulfur center Fx reduction by the primary electron donor P700, (ii) electron transfer between iron-sulfur centers Fx and FA/FB, and (iii) reduction of photo-oxidized P700+ by reduced cytochrome C553. The relative magnitudes of phases (ii) and (iii) comprised about 20% of phase (i).     

Time-resolved fluorescence and absorption spectroscopy of photosystem I

Picosecond fluorescence and femtosecond transient absorption spectroscopy have been used to investigate the primary energy transfer and trapping processes in a photosystem II deletion mutant from the cyanobacterium Synechocystis sp. PCC 6803, which contains active photosystem I reaction centers with approximately 100 chlorophylls per P700. In all experiments, low levels of excitation were used which avoid annihilation processes. Following 590-nm excitation, at room temperature, spectral equilibration is observed in both fluorescence and absorption measurements and is characterized by a time constant of 4-6 ps. The shape of the spectra associated with the equilibration process indicates that long wavelength pigments (pigments with absorption maxima at longer wavelength than that of the primary electron donor, P700) are present and functional at physiological temperatures in this preparation. The overall decay of excitations in the antenna is characterized by a time constant of 24-28 ps, in both fluorescence and absorption measurements. The 24-28-ps process results in the appearance of absorption changes associated with only P700+ formation. Absorption changes associated with the reduction of the primary electron acceptor were not resolved under the experimental conditions used here.     

Carotenoid-dependent oligomerization of the major chlorophyll a/b light harvesting complex of photosystem II of plants

Under many environmental conditions, plants are exposed to levels of sunlight in excess of those required for photosynthesis. Then, a regulated increase in the rate of nonradiative dissipation of excess excitation energy in the thylakoid membrane correlates with the conversion of the carotenoid violaxanthin into zeaxanthin and provides protection from the damaging effects of excessive irradiation. The hypothesis that these carotenoids specifically control the oligomerization of the light harvesting complexes of photosystem II was tested by investigating the effects of violaxanthin and zeaxanthin on the behavior of the major complex, LHCIIb, on sucrose gradients; it was found that zeaxanthin stimulated the formation of LHCIIb aggregates with reduced chlorophyll fluorescence yield whereas violaxanthin caused the inhibition of such aggregation and an elevation of fluorescence. Measurements of 77 K fluorescence indicated that zeaxanthin was not exerting an additional direct quenching of chlorophyll fluorescence. These effects can explain the physiological control of the light harvesting system by the xanthophyll cycle.     

Effects of photoinhibition on the QA-Fe2+ complex of photosystem II studied by EPR and M?ssbauer spectroscopy

Effects of photoinhibition on the iron-quinone electron acceptor complex of oxygen-evolving photosystem II have been studied using low-temperature EPR and M?ssbauer spectroscopy. Photoinhibition of spinach photosystem II membrane particles at 4 degrees C decreases the EPR signal arising from the interaction of QA- with Fe2+ to 30% in 90 min under our conditions. The free radical EPR signal from QA- induced by cyanide treatment of the iron [Sanakis, Y., et al. (1994) Biochemistry 33, 9922-9928] declines with the same kinetics as the QA-Fe2+ EPR signal. In contrast, Fe2+ is present in about 70% of the centers after 90 min of photoinhibition, as shown by its EPR-detected interaction with NO and by its M?ssbauer absorption. Complete oxidation of this Fe2+ population to Fe3+ by ferricyanide is possible only in the presence of glycolate, which lowers the redox potential of the Fe3+/Fe2+ couple. In a fraction of PSII centers, which reach 30% after 90 min of photoinhibition, the iron cannot be detected. It is concluded that photoinhibition of oxygen-evolving photosystem II affects both QA and Fe2+. However, the photoinhibitory impairment of the QA redox functioning precedes the modification of the non-heme iron. In a considerable portion of the photoinhibited centers, which do not have functional QA, the non-heme iron is still present and redox active, but its redox potential is increased relative to that in the normal centers. This is probably due to a minor modification of the bicarbonate ligation site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spin-lattice relaxation of the phyllosemiquinone radical of photosystem I

The spin-lattice relaxation time (T1) of the phyllosemiquinone anion radical, A1-, of the photosystem I (PSI) reaction center, were measured between 4.5 and 85 K by electron spin-echo spectroscopy. The selective removal of the iron-sulfur centers, FA, FB, and FX, from PSI allowed the measurement of the intrinsic T1 of the A1- radical. The temperature dependence of the intrinsic (T1)-1 for A1- was found to be approximately T1.3 +/- 0.1. The spin-lattice relaxation of the reduced form of iron-sulfur center FX was also measured at low temperatures, in FA/FB-depleted PSI membranes. It was found that the fast-relaxing FX center enhances the spin-lattice relaxation of the phyllosemiquinone due to dipolar coupling. The effect of the reduced forms of FA/FB on the T1 of the phyllosemiquinone was minor compared to the effect of FX. By analyzing the data with a dipolar model in the light of limitations imposed by other information present in the literature, the distance between the phyllosemiquinone and FX in PSI is estimated to be 14.8 +/- 4 A.     

Spatial relationship of photosystem I, photosystem II, and the light-harvesting complex in chloroplast membranes

We have previously demonstrated (Armond, P. A., C. J. Arntzen, J.-M. Briantais, and C. Vernotte. 1976. Arch. Biochem. Biophys. 175:54-63; and Davis, D. J., P. A. Armond, E. L. Gross, and C. J. Arntzen. 1976. Arch. Biochem. Biophys. 175:64-70) that pea seedlings which were exposed to intermittent illumination contained incompletely developed chloroplasts. These plastids were photosynthetically competent, but did not contain grana. We now demonstrate that the incompletely developed plastids have a smaller photosynthetic unit size; this is primarily due to the absence of a major light-harvesting pigment-protein complex which is present in the mature membranes. Upon exposure of intermittent-light seedlings to continuous white light for periods up to 48 h, a ligh-harvesting chlorophyll-protein complex was inserted into the chloroplast membrane with a concomitant appearance of grana stacks and an increase in photosynthetic unit size. Plastid membranes from plants grown under intermediate light were examined by freeze-fracture electron microscopy. The membrane particles on both the outer (PF) and inner (EF) leaflets of the thylakoid membrane were found to be randomly distributed. The particle density of the PF fracture face was approx. four times that of the EF fracture face. While only small changes in particle density were observed during the greening process under continuous light, major changes in particle size were noted, particularly in the EF particles of stacked regions (EFs) of the chloroplast membrane. Both the changes in particle size and an observed aggregation of the EF particles into the newly stacked regions of the membrane were correlated with the insertion of light-harvesting pigment-protein into the membrane. Evidence is presented for identification of the EF particles as the morphological equivalent of a "complete" photosystem II complex, consisting of a phosochemically active "core" complex surrounded by discrete aggregates of the light-harvesting pigment protein. A model demonstrating the spatial relationships of photosystem I, photosystem II, and the light-harvesting complex in the chloroplast membrane is presented.     

Mutational analysis of photosystem I polypeptides. Role of PsaD and the lysyl 106 residue in the reductase activity of the photosystem I

The ADC4 mutant of the cyanobacterium Synechocystis sp. PCC 6803 was studied to determine the structural and functional consequences of the absence of PsaD in photosystem I. Isolated ADC4 membranes were shown to be deficient in ferredoxin-mediated NADP(+) reduction, even though charge separation between P700 and FA/FB occurred with high efficiency. Unlike the wild type, FB became preferentially photoreduced when ADC4 membranes were illuminated at 15 K, and the EPR line shapes were relatively broad. Membrane fragments oriented in two dimensions on thin mylar films showed that the g tensor axes of FA- and FB- were identical in the ADC4 and wild type strains, implying that PsaC is oriented similarly on the reaction center. PsaC and the FA/FB iron-sulfur clusters are lost more readily from the ADC4 membranes after treatment with Triton X-100 or chaotropic agents, implying a stabilizing role for PsaD. The specific role of Lys106 of PsaD, which can be crosslinked to Glu93 of ferredoxin (Lelong et al. (1994) J. Biol. Chem. 269, 10034-10039), was probed by site-directed mutagenesis. Chemical cross-linking and protease treatment experiments did not reveal any drastic alterations in the conformation of the mutant PsaD proteins. The EPR spectra of FA and FB in membranes of the Lys106 mutants were similar to those of the wild type. Membranes of all Lys106 mutants showed wild type rates of flavodoxin reduction and flavodoxin-mediated NADP+ reduction, but had 10-54% decrease in the ferredoxin-mediated NADP+ reduction rates. This implies that Lys106 is a dispensable component of the docking site on the reducing side of photosystem I and an ionic interaction between Lys106 of PsaD and Glu93 of ferredoxin is not essential for electron transfer to ferredoxin. These results demonstrate that PsaD serves distinct roles in modulating the EPR spectral characteristics of FA and FB, in stabilizing PsaC on the reaction center, and in facilitating ferredoxin-mediated NADP+ photoreduction on the reducing side of photosystem I.     

Role of an extrinsic 33 kilodalton protein of photosystem II in the turnover of the reaction center-binding protein D1 during photoinhibition

The reaction center-binding protein D1 of photosystem II (PS II) undergoes rapid turnover under light stress conditions. In the present study, we investigated the role of the extrinsic 33 kDa protein (OEC33) in the early stages of D1 turnover. D1 degradation was measured after strong illumination (1000-5000 microE m-2 S-1) of spinach manganese-depleted, PSII-enriched membrane and core samples in the presence and absence of the OEC33 under aerobic conditions at room temperature. PSII samples lacking the OEC33 were prepared by standard biochemical treatments with Tris or CaCl2/NH2OH while samples retaining the OEC33 were prepared with NH2OH or NaCl/NH2OH. The degradation of D1, monitored by SDS/urea-polyacrylamide gel electrophoresis and Western blotting using specific antibodies against D1, proceeds to a greater extent in NH2OH-treated samples than in Tris-treated samples over a 60 min illumination period. Under the same conditions, significantly more aggregation of D1 occurs in the Tris-treated samples than in the NH2OH-treated samples. The lower level of D1 degradation in Tris-treated samples is not due to secondary proteolysis, as judged from the time course for degradation at 25 degrees C or the degradation pattern at 4 degrees C. Similarly, for NaCl/NH2OH-treated samples, D1 degradation is greater and D1 aggregation less than in CaCl2/NH2OH-treated samples. The effect of the presence of the OEC33 on D1 degradation and aggregation is confirmed by reconstitution experiments in which the isolated OEC33 is restored back to Tris-treated samples. During very strong illumination, significant loss of CP43 also occurs in Tris-treated but not in NH2OH-treated samples. Structural analysis of PS II core complexes by Fourier transform infrared (FT-IR) spectroscopy revealed very little change in the protein secondary structure after 10 min illumination of NH2OH-treated samples while a large 10% decrease of alpha-helix content occurs in Tris-treated samples. On the basis of these results, we suggest that either (1) the OEC33 stabilizes the structural integrity of PS II such that it prevents the photodamaged D1 protein from aggregating with nearby polypeptides and thereby facilitating degradation or (2) the OEC33 specifically stabilizes CP43, a putative D1-specific protease, which normally promotes the efficient degradation of D1.     

Alleviatory effect of rare earth micro-fertilizer on photosystem II (PSII) photoinhibition in Pseudostellaria heterophylla leaves at photosynthetic midday depression

In order to explore the mitigatory mechanism of rare earths on the midday depression of photosynthesis, this study used fast-phase chlorophyll fluorescence technology combined with an antioxidant enzyme system to analyze the effects of lanthanum and cerium on the photosystem II (PSII) function of Pseudostellaria heterophylla leaves at noon. The results show that the maximum photochemical efficiency (F_v/F_m) and the photosynthetic performance index (PI_ABS) of P. heterophylla leaves during the photosynthetic noon-break are only 0.73 and 0.86, respectively. The leaves of P. heterophylla show obvious photoinhibition. The application of two rare earth fertilizers promotes the light energy absorption, capture, conversion, and electron transfer efficiencies of PSII in the P. heterophylla leaves and alleviates the photoinhibition caused by excess excitation energy. Additionally, the effect of lanthanum is better than that of cerium. In the fertilization groups, P. heterophylla leaves show higher PSII photochemical activity. Fertilization protects the oxygen-evolving complex (OEC) activity of P. heterophylla, thus reducing oxidative damage to the chloroplasts and preventing the dissociation of the thylakoids to maintain the normal physiological functioning of PSII, thereby reducing the degree of photoinhibition. Additionally, fertilization increases the electron transfer ability from Q_A to Q_B on the PSII electron receptor side of P. heterophylla. Correlation analysis shows that the fluorescence parameters V_L, V_K, and V_J are significantly negatively correlated with antioxidant enzyme activity and are significantly positively correlated with malondialdehyde content, relative electric conductivity and reactive oxygen species (ROS), which further indicates that rare earths can enhance the ROS scavenging system in plants and reduce the degree of membrane lipid peroxidation and plasma membrane damage, thereby maintaining the structure and function of the OEC on the donor side of PSII, increasing the electron receptor pool, and contributing to the defense against photoinhibition.     

Energy transfer and charge separation in photosystem I: P700 oxidation upon selective excitation of the long-wavelength antenna chlorophylls of Synechococcus elongatus

Photosystem I of the cyanobacterium Synechococcus elongatus contains two spectral pools of chlorophylls called C-708 and C-719 that absorb at longer wavelengths than the primary electron donor P700. We investigated the relative quantum yields of photochemical charge separation and fluorescence as a function of excitation wavelength and temperature in trimeric and monomeric photosystem I complexes of this cyanobacterium. The monomeric complexes are characterized by a reduced content of the C-719 spectral form. At room temperature, an analysis of the wavelength dependence of P700 oxidation indicated that all absorbed light, even of wavelengths of up to 750 nm, has the same probability of resulting in a stable P700 photooxidation. Upon cooling from 295 K to 5 K, the nonselectively excited steady-state emission increased by 11- and 16-fold in the trimeric and monomeric complexes, respectively, whereas the quantum yield of P700 oxidation decreased 2.2- and 1.7-fold. Fluorescence excitation spectra at 5 K indicate that the fluorescence quantum yield further increases upon scanning of the excitation wavelength from 690 nm to 710 nm, whereas the quantum yield of P700 oxidation decreases significantly upon excitation at wavelengths longer than 700 nm. Based on these findings, we conclude that at 5 K the excited state is not equilibrated over the antenna before charge separation occurs, and that approximately 50% of the excitations reach P700 before they become irreversibly trapped on one of the long-wavelength antenna pigments. Possible spatial organizations of the long-wavelength antenna pigments in the three-dimensional structure of photosystem I are discussed.     

Universality of energy and electron transfer processes in photosystem I

Femtosecond transient absorption spectroscopy has been used to investigate the photoinduced energy and electron transfer processes in photosystem I (PS I) particles from cyanobacteria, green algae, and higher plants. At room temperature, the kinetics observed in all three species are very similar: Following 590 nm excitation, an equilibration process(es) with a 3.7-7.5 ps lifetime was observed, followed by a 19-24 ps process that is associated with trapping. In all three species long-wavelength pigments (pigments that absorb at longer wavelengths than the primary electron donor) were observed. The difference spectrum associated with reduction of the primary electron acceptor [Ao(-)-Ao) difference spectrum] was obtained for all three species. The (Ao(-)-Ao) difference spectra obtained from measurements using detergent-isolated PS I particles from spinach and Chlamydomonas reinhardtii are similar but clearly membrane fragments. In all three species the reduced primary electron acceptor (Ao(-)) is reoxidized extremely rapidly, in about 20 ps. The difference spectrum associated with Ao reduction appears to contain contributions from more than a single chlorophyll pigment.     

Photosystem I is an early target of photoinhibition in barley illuminated at chilling temperatures

Light-induced damage to photosystem I (PSI) was studied during low-light illumination of barley (Hordeum vulgare L.) at chilling temperatures. A 4-h illumination period induced a significant inactivation of PSI electron transport activity. Flash-induced P700 absorption decay measurements revealed progressive damage to (a) the iron-sulfur clusters FA and FB, (b) the iron-sulfur clusters FA, FB, and FX, and (c) the phylloquinone A1 and the chlorophyll AO or P700 of the PSI electron acceptor chain. Light-induced PSI damage was also evidenced by partial degradation of the PSI-A and PSI-B proteins and was correlated with the appearance of smaller proteins. Aggravated photodamage was observed upon illumination of barley leaves infiltrated with KCN, which inhibits Cu,Zn-superoxide dismutase and ascorbate peroxidase. This indicates that the photodamage of PSI in barley observed during low-light illumination at chilling temperatures arises because the defense against active oxygen species by active oxygen-scavenging enzymes is insufficient at these specific conditions. The data obtained demonstrate that photoinhibition of PSI at chilling temperatures is an important phenomenon in a cold-tolerant plant species.     

Temperature dependence of forward and reverse electron transfer from A1-, the reduced secondary electron acceptor in photosystem I

Electron-transfer reactions following the formation of P700(+)A1- have been studied in isolated Photosystem I complexes from Synechococcus elongatus between 300 and 5 K by flash absorption spectroscopy. (1) In the range from 300 to 200 K, A1- is reoxidized by electron transfer to the iron-sulfur cluster FX. The rate slows down with decreasing temperature, corresponding to an activation energy of 220 +/- 20 meV in this temperature range. Analyzing the temperature dependence of the rate in terms of nonadiabatic electron-transfer theory, one obtains a reorganization energy of about 1 eV and an edge-to-edge distance between A1 and FX of about 8 A assuming the same distance dependence of the electron-transfer rate as in purple bacterial reaction centers. (2) At temperatures below 150 K, different fractions of PS I complexes attributed to frozen conformational substates can be distinguished. A detailed analysis at 77 K gave the following results: (a) In about 45%, flash-induced electron transfer is limited to the formation and decay of the secondary pair P700(+)A1-. The charge recombination occurs with a t1/2 of about 170 micros. (b) In about 20%, the state P700(+)FX- is formed and recombines with complex kinetics (t1/2 = 5-100 ms). (c) In about 35%, irreversible formation of P700(+)FA- or P700(+)FB- is possible. (3) The transition from efficient forward electron transfer at higher temperatures to heterogeneous photochemistry at low temperatures has been investigated in different glass-forming solvents. The yield of forward electron transfer to the iron-sulfur clusters decreases in a narrow temperature interval. The temperature of the half-maximal effect varies between different solvents and appears to be correlated with their liquid to glass transition. It is proposed that reorganization processes in the surroundings of the reactants which are required for the stabilization of the charge-separated state become arrested near the glass transition. This freezing of protein motions and/or solvent reorganization may affect electron-transfer reactions through changes in the free-energy gap and the reorganization energy. (4) The rate of charge recombination between P700(+) and A1- increases slightly (about 1.5-fold) when the temperature is decreased from 300 to 5 K. This charge recombination characterized by a large driving force is much less influenced by the solvent properties than the forward electron-transfer steps from A1- to FX and FA/B.     

Reactions of plastocyanin and cytochrome 553 with photosystem I of Scenedesmus

Chloroplast material active in photosynthetic electron transport has been isolated from Scenedesmus acutus (strain 270/3a). During homogenization, part of cytochrome 553 was solubilized, and part of it remained firmly bound to the membrane. A direct correlation between membrane cytochrome 553 and electron transport rates could not be found. Sonification removes plastocyanin, but leaves bound cytochrome 553 in the membrane. Photooxidation of the latter is dependent on added plastocyanin. In contrast to higher plant chloroplasts, added soluble cytochrome 553 was photooxidized by 707 nm light without plastocyanin present. Reduced plastocyanin or cytochrome 553 stimulated electron transport by Photosystem I when supplied together or separately. These reactions and cytochrome 553 photooxidation were not sensitive to preincubation of chloroplasts with KCN, indicating that both redox proteins can donate their electrons directly to the Photosystem I reaction center. Scenedesmus cytochrome 553 was about as active as plastocyanin from the same alga, whereas the corresponding protein from the alga Bumilleriopsis was without effect on electron transport rates. It is suggested that besides the reaction sequence cytochrome 553 leads to plastocyanin leads to Photosystem I reaction center, a second pathway cytochrome 553 leads to Photosystem I reaction center may operate additionally.     

Long-term effects of selective decontamination on antimicrobial resistance

OBJECTIVE: To determine whether selective decontamination of the digestive tract exerts any long-term effects on antimicrobial resistance patterns. DESIGN: A surveillance and interventional study comparing the antimicrobial sensitivity patterns of clinically important bacterial isolates the year before a 2-yr, double-blind, randomized, controlled study of selective decontamination of the digestive tract, and for the year thereafter when no use of the regimen was made. SETTING: A ten-bed respiratory intensive care unit (ICU) in a 1,200-bed teaching hospital. PATIENTS: All 1,528 patients admitted to the ICU over the 4-yr study period were included. There were 406 patients admitted in the year before the study of decontamination of the digestive tract (65% medical, 23% surgical, and 12% trauma), of whom 76% required mechanical ventilation. There were 719 patients admitted during the 2-yr study of selective decontamination (55% medical, 28% surgical, and 17% trauma), of whom 79.6% required mechanical ventilation. There were 403 patients admitted in the subsequent year (61% medical, 25% surgical, and 14% trauma), of whom 76.9% required mechanical ventilation. INTERVENTIONS: We performed daily clinical monitoring to detect nosocomial infection, with microbiological investigation when clinically indicated, as well as twice-weekly routine microbiological surveillance sampling. Antimicrobial susceptibility testing using standard laboratory methods was also performed. Selective decontamination of the digestive tract included parenteral cefotaxime and oral and enteral polymyxin E, amphotericin B, and tobramycin. MEASUREMENTS AND MAIN RESULTS: The occurrence rate of nosocomial infection was 20.6%, 16.6%, and 25.3%, respectively, in the three study periods. In the year after selective decontamination, there was an increase in the occurrence rate of infection (p = .005), with an-associated increase in infections caused by the Enterobacteriaceae, while a reduction in the level of resistance to the third-generation cephalosporins were found (p = .07). There was a progressive increase in the occurrence rate of infections caused by Acinetobacter species (p = .05). Only 11 infections over the 4 yrs were caused by Enterococcus species. Staphylococcal infections were uncommon (5.7% of admissions), and the level of methicillin resistance did not change. No increase in aminoglycoside resistance occurred. CONCLUSION: No long-term effects on antimicrobial resistance or the spectrum of nosocomial pathogens could be attributed to the use of selective decontamination of the digestive tract over a 2-yr period in a respiratory ICU admitting all categories of patients.     

Electrophysical properties of granulated films based on borides of rare-earth elements. I. Temperature dependence of resistance

Translated from Poroshkovaya Metallurgiya, No. 4(304), pp. 66–71, April, 1988.