A Brownian dynamics study of the interaction of Phormidium laminosum plastocyanin with Phormidium laminosum cytochrome f

The interaction of Phormidium laminosum plastocyanin (PC) with P. laminosum cytochrome f (cyt f) was studied using Brownian dynamics (BD) simulations. Few complexes and a low rate of electron transfer were observed for wild-type PC. Increasing the positive electrostatic field on PC by the addition of a Zn(2+) ion in the neighborhood of D44 and D45 on PC (as found in crystal structure of plastocyanin) increased the number of complexes formed and the calculated rates of electron transfer as did PC mutations D44A, D45A, E54A, and E57A. Mutations of charged residues on Phormidium PC and Phormidium cyt f were used to map binding sites on both proteins. In both the presence and absence of the Zn(2+) ion, the following residues on PC interact with cyt f: D44, D45, K6, D79, R93, and K100 that lie in a patch just below H92 and Y88 and D10, E17, and E70 located on the upper portion of the PC molecule. In the absence of the Zn(2+) ion, K6 and K35 on the top of the PC molecule also interact with cyt f. Cyt f residues involved in binding PC, in the absence of the Zn(2+) ion, include E165, D187, and D188 that are located on the small domain of cyt f. The orientation of PC in the complexes was quite random in accordance with NMR results. In the presence of the Zn(2+) ion, K53 and E54 in the lower patch of the PC molecule also interact with cyt f and PC interacts with E86, E95, and E123 on the large domain of cyt f. Also, the orientation of PC in the complexes was much more uniform than in the absence of the Zn(2+) ion. The difference may be due to both the larger electrostatic field and the greater asymmetry of the charge distribution on PC observed in the presence of the Zn(2+) ion. Hydrophobic interactions were also observed suggesting a model of cyt f-PC interactions in which electrostatic forces bring the two molecules together but hydrophobic interactions participate in stabilizing the final electron-transfer-active dock.     

Exogenous versus endogenous acceptors in photosystem ii in inhibited chloroplasts

The conditions for steady-state Signal IIf formation in response to single turnover flashes in Tris-treated, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-inhibited chloroplasts have been investigated. DCMU inhibits Signal IIf generation as the photoinactive state, Z P680 Q-A, accumulates. Potassium ferricyanide relieves this inhibition so that Signal IIf can be fully developed on each flash in a flash series. The effectiveness of ferricyanide in stimulating Signal IIf formation is dependent on its concentration, the flash repetition rate, and the salt composition of the chloroplast suspension. There are two models in the literature for Q-A oxidation under these inhibitory conditions: direct oxidation of Q-A by exogenous acceptors like ferricyanide or oxidation of Q-A by an endogenous acceptor, AH, which has a midpoint potential of approximately 400 mV. It is found that the direct exogenous acceptor model accounts well for these data, whereas the AH model does not explain several of these results. The apparent rate constant for the direct oxidation of Q-A by ferricyanide at various concentrations of salt has been calculated from our electron paramagnetic resonance (EPR) data and compared with the corresponding rate constant determined by S. Itoh from fluorescence data (Biochim, Biophys. Acta 504, 324-340, 1978); good agreement is found for the two different experimental approaches.     

Computer simulation of plastocyanin interaction with cytochrome f and photosystem I in cyanobacterium Phormidium laminosum

The paper is devoted to computer simulation of complex formation of protein plastocyanin with transmembrane pigment-protein complex photosystem I and subunit f of cytochrome b ₆ f complex in the cyanobacterium Phormidium laminosum. The computer algorithm considers diffusion and electrostatic interactions of protein molecules. The computer models have shown that electrostatic interactions in the cyanobacterium play a less important role than in higher plants because of different electrostatic potentials created by charged amino acid residues on the protein surfaces.     

Role of electrostatics in the interaction between plastocyanin and photosystem I of the cyanobacterium Phormidium laminosum.

The interactions between photosystem I and five charge mutants of plastocyanin from the cyanobacterium Phormidium laminosum were investigated in vitro. The dependence of the overall rate constant of reaction, k2, on ionic strength was investigated using laser flash photolysis. The rate constant of the wild-type reaction increased with ionic strength, indicating repulsion between the reaction partners. Removing a negative charge on plastocyanin (D44A) accelerated the reaction and made it independent of ionic strength; removing a positive charge adjacent to D44 (K53A) had little effect. Neutralizing and inverting the charge on R93 slowed the reaction down and increased the repulsion. Specific effects of MgCl2 were observed for mutants K53A, R93Q and R93E. Thermodynamic analysis of the transition state revealed positive activation entropies, suggesting partial desolvation of the interface in the transition state. In comparison with plants, plastocyanin and photosystem I of Phormidium laminosum react slowly at low ionic strength, whereas the two systems have similar rates in the range of physiological salt concentrations. We conclude that in P. laminosum, in contrast with plants in vitro, hydrophobic interactions are more important than electrostatics for the reactions of plastocyanin, both with photosystem I (this paper) and with cytochrome f[Schlarb-Ridley, B.G., Bendall, D.S. & Howe, C.J. (2002) Biochemistry41, 3279-3285]. We discuss the implications of this conclusion for the divergent evolution of cyanobacterial and plant plastocyanins.     

Regulation of nitrate assimilation in the cyanobacterium Phormidium laminosum

The intracellular ratio of 2-oxoglutarate to glutamine has been analyzed under nutritional conditions leading to different activity levels of nitrate-assimilating enzymes in Phormidium laminosum (Agardh) Gom. This non-N₂-fixing cyanobacterium adapted to the available nitrogen source by modifying its nitrate reductase (NR; EC 1.7.7.2), nitrite reductase (NiR; EC 1.7.7.1) and glutamine synthetase (GS; EC 6.3.1.2) activities. The 2-oxoglutarate/glutamine ratio was similar in cells adapted to grow with nitrate or ammonium. However, metabolic conditions that increased this ratio [i.e., nitrogen starvation or l-methionine-d,l-sulfoximine (MSX) treatment] corresponded to high activity levels of NR, NiR, GS (except in MSX-treated cells) and glutamate synthase (GOGAT; EC 1.4.7.1). By contrast, metabolic conditions that diminished this ratio (i.e., addition of ammonium to nitrate-growing cells or addition of nitrate or ammonium to nitrogen-starved cells) resulted in low activity levels. The variation in the 2-oxoglutarate/glutamine ratio preceded the changes in enzyme activities. These results suggest that changes in the 2-oxoglutarate/glutamine ratio could be the signal that triggers the adaptation of P. laminosum cells to variations in the available nitrogen source, as occurs in enterobacteria.Abbreviations Chl chlorophyll - GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - GS glutamine synthetase (EC 6.3.1.2) - MSX l-methionine-d,l-sulfoximine - NiR nitrite reductase (EC 1.7.7.1) - NR nitrate reductase (EC 1.7.7.2) - TP total protein This work has been partially supported by grants from the Spanish Ministry of Education and Science (DGICYT PB88-0300 and PB92-0464) and the University of the Basque Country (042.310-EC203/94). M.I.T. was the recipient of a fellowship from the Basque Government.     

Photosynthetic electron transport in a cell-free preparation from the thermophilic blue-green alga Phormidium laminosum.

1. A cell-free preparation of membrane fragments was prepared from the thermophilic blue-green alga Phormidium laminosum by lysozyme treatment of the cells followed by osmotic shock to lyse the spheroplasts. The membrane fragments showed high rates of photosynthetic electron transport and O2 evolution (180-250 mumol of O2/h per mg of chlorophyll a with 2,6-dimethyl-1,4-benzoquinone as electron acceptor). O2-evolution activity was stable provided that cations (e.g. 10mM-Mg2+ or 100mM-Na+) or glycerol (25%, v/v) were present in the suspending medium. 2. The components of the electron-transport chain in P. laminosum were similar to those of other blue-green algae: the cells contained Pigment P700, plastocyanin, soluble high-potential cytochrome c-553, soluble low-potential cytochrome c-54 and membrane-bound cytochromes f, b-563 and b-559 (both low- and high-potential forms). The amounts and midpoint potentials of the membrane-bound cytochromes were similar to those in higher-plant chloroplasts. 3. Although O2 evolution in P. laminosum spheroplasts was resistant to high temperatures, thermal stability was not retained in the cell-free preparation. However, in contrast with higher plants, O2 evolution in P. laminosum membrane fragments was remarkably resistant to the non-ionic detergent Triton X-100.     

Phosphate uptake by phosphorus-starved cells of the cyanobacterium Phormidium laminosum

Phosphorus(P)-starved cells of the cyanobacterium Phormidium laminosum have been investigated in relation to their phosphate uptake characteristics. P-deficient cells showed much higher phosphate uptake rates from ultrapure water supplemented with this anion than P-sufficient ones. After 9 days of starvation in P-free medium, the total cellular P content of P-deficient cells was approximately five times lower than that of cells grown in the presence of phosphate. Phosphate uptake by P-deficient cells occurred in both light and dark under aerobic conditions. In anaerobiosis, light was required for uptake, suggesting that the necessary energy could be derived from the respiratory electron transport chain. Phosphate uptake in P-deficient cells was sensitive to vanadate, suggesting the involvement of a plasma membrane ATPase.     

EPR characteristics of oxygen-evolving photosytem II particles from Phormidium laminosum

The EPR characteristics of oxygen evolving particles prepared from Phormidium laminosum are described. These particles are enriched in Photosystem II allowing EPR investigation of signals which were previously small or masked by those from Photosystem I in other preparations. EPR signals from a Signal II species and high potential cytochrome beta-559 appear as they are photooxidised at cryogenic temperatures by Photosystem II. The Signal II species is a donor close to the Photosystem II reaction centre and may represent part of the charge accumulation system of water oxidation. An EPR signal from an iron-sulphur centre which may represent an unidentified component of photosynthetic electron transport is also described. The properties of the oxygen evolving particles show that the preparation is superior to chloroplasts or unfractionated alga membranes for the study of Photosystem II with a functional water oxidation system.     

Changes in non-core regions stabilise plastocyanin from the thermophilic cyanobacterium Phormidium laminosum

We report a theoretical investigation on the different stabilities of two plastocyanins. The first one belongs to the thermophilic cyanobacterium Phormidium laminosum and the second one belongs to its mesophilic relative Synechocystis sp. These proteins share the same topology and secondary-structure elements; however, the melting temperatures of their oxidised species differ by approximately 15 K. Long-time-scale molecular dynamics simulations, performed at different temperatures, show that the thermophilic protein optimises a set of intramolecular interactions (interstrand hydrogen bonding, salt bridging and hydrophobic clustering) within the region that comprises the strands β₅ and β₆, loop L₅ and the helix. This region exhibits most of the differences in the primary sequence between the two proteins and, in addition, it is involved in the interaction with known physiological partners. Further work is in progress to unveil the specific structural features responsible for the different thermal stability of the two proteins.     

Carotenoid composition in the cyanobacterium Phormidium laminosum. Effect of nitrogen starvation

When pigments of the non-N2-fixing cyanobacterium Phormidium laminosum were carefully extracted and analyzed in a completely O2-free atmosphere, by either high performance liquid chromatography (HPLC) or thin layer chromatography (TLC), the presence of only two carotenoids (namely, beta-carotene and nostoxanthin) was detected. However, exposure of pigments to an air atmosphere during their manipulation led to the rapid appearance in the organic extracts of at least three additional carotenoids (identified as caloxanthin, zeaxanthin and beta-cryptoxanthin). This fact could explain the presence in cyanobacteria of such hydroxylated derivatives of beta-carotene widely reported in the literature. Nitrogen starvation also resulted in an important decrease on the relative beta-carotene/nostoxanthin content of cells, suggesting that this nutritional condition affects thylakoid membranes more drastically than cytoplasmic membranes.     

Interaction of various types of photosystem I complexes with exogenous electron acceptors

Interaction of photosystem I (PS I) complexes from cyanobacteria Synechocystis sp. PCC 6803 containing various quinones in the A₁-site (phylloquinone PhQ in the wild-type strain (WT), and plastoquinone PQ or 2,3-dichloronaphthoquinone Cl ₂ NQ in the menB deletion strain) and different numbers of Fe₄S₄ clusters (intact WT and F_X-core complexes depleted of F_A/F_B centers) with external acceptors has been studied. The efficiency of interaction was estimated by measuring the light-induced absorption changes at 820 nm due to the reduction of the special pair of chlorophylls (P₇₀₀ ⁺) by an external acceptor(s). It was shown that externally added Cl ₂ NQ is able to effectively accept electrons from the terminal iron-sulfur clusters of PS I. Moreover, the efficiency of Cl ₂ NQ as external acceptor was higher than the efficiency of the commonly used artificial electron acceptor, methylviologen (MV) for both the intact WT PS I and for the F_X-core complexes. The comparison of the efficiency of MV interaction with different types of PS I complexes revealed gradual decrease in the following order: intact WT?>?menB?>?F_X-core. The effect of MV on the recombination kinetics in menB complexes of PS I with Cl ₂ NQ in the A₁-site differed significantly from all other PS I samples. The obtained effects are considered in terms of kinetic efficiency of electron acceptors in relation to thermodynamic and structural characteristics of PS I complexes.     

Changes in nitrogen source modify distribution of excitation energy in the cyanobacterium Phormidium laminosum

In an attempt to clarify the interactions between the available nitrogen source and the photosystems in cyanobacteria, O₂ exchange and fluorescence emission were monitored in spheroplasts and intact cells of the non N₂-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl₁) growing on different nitrogen sources or in the absence of nitrogen. Short-term (time scale of seconds to minutes), NH⁺₄ addition to NO⁻₃-growing or N-starved cells and, to a minor extent, NO⁻₃addition to N-starved cells, induced state 2 transitions both in light and dark. Long term (time scale of days), the fluorescence yield of PSII relative to that of PSII at 77 K was higher in NO⁻₃- than in NH⁺₄ growing cells, and even higher in N-starved cells. In the dark, the plastoquinone pool was more reduced in NH⁺₄- than in NO⁻₃-growing cells. Both PSII and PSI activities and the degree of linking between both photosystems were affected in the long term, so that non-cyclic electron transport decreased in parallel to the ferredoxin requirement to assimilate each nitrogen source. Results indicate that nitrogen metabolism exerts short- and long-term control over the photosynthetic apparatus, which acclimates to the energy requirement of the available nitrogen source.     

A thermal unfolding study of plastocyanin from the thermophilic cyanobacterium Phormidium laminosum

The thermal unfolding of the plastocyanin from Phormidium laminosum, a thermophilic cyanobacterium, is herein described. The main objective of this work is to identify structural factors responsible for the higher stability observed in proteins from thermophilic organisms. With the aid of fluorescence spectroscopy, EPR, and NMR, the factors influencing the unfolding process of the protein were investigated, and procedures for its study have been standardized. The different spectroscopic techniques used provided consistent results showing that the thermal unfolding of plastocyanin is irreversible under all the conditions investigated and that this irreversibility does not appear to be related to the presence of oxygen. The oxidized plastocyanin species has proven to be more stable than the reduced one, with respect to both the required temperature for protein unfolding (up to a 9 degrees C difference between the two forms) and the kinetics of the process. The behavior of this plastocyanin contrasts with that of other cupredoxins whose unfolding had previously been studied. The unfolding pH dependence and kinetic studies indicate a process with a tight control around the physiological pH in which plastocyanin plays its redox role and the protein's isoelectric point (5.2), suggesting a close compromise between function and stability.