Role of electrostatics in the interaction between cytochrome f and plastocyanin of the cyanobacterium Phormidium laminosum

The role of charged residues on the surface of plastocyanin from the cyanobacterium Phormidium laminosum in the reaction with soluble cytochrome f in vitro was studied using site-directed mutagenesis. The charge on each of five residues on the eastern face of plastocyanin was neutralized and/or inverted, and the effect of the mutation on midpoint potentials was determined. The dependence of the overall rate constant of reaction, k(2), on ionic strength was investigated using stopped-flow spectrophotometry. Removing negative charges (D44A or D45A) accelerated the reaction and increased the dependence on ionic strength, whereas removing positive charges slowed it down. Two mutations (K46A, K53A) each almost completely abolished any influence of ionic strength on k(2), and three mutations (R93A, R93Q, R93E) each converted electrostatic attraction into repulsion. At low ionic strength, wild type and all mutants showed an inhibition which might be due to changes in the interaction radius as a consequence of ionic strength dependence of the Debye length or to effects on the rate constant of electron transfer, k(et). The study shows that the electrostatics of the interaction between plastocyanin and cytochrome f of P. laminosum in vitro are not optimized for k(2). Whereas electrostatics are the major contributor to k(2) in plants [Kannt, A., et al. (1996) Biochim. Biophys. Acta 1277, 115-126], this role is taken by nonpolar interactions in the cyanobacterium, leading to a remarkably high rate at infinite ionic strength (3.2 x 10(7) M(-1) s(-1)).     

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 charges on cytochrome f from the cyanobacterium Phormidium laminosum in its interaction with plastocyanin

The role of charge on the surface of cytochrome f from the cyanobacterium Phormidium laminosum in the reaction with plastocyanin was investigated in vitro using site-directed mutagenesis. Charge was neutralized at five acidic residues individually and introduced at a residue close to the interface between the two proteins. The effects on the kinetics of the reaction were measured using stopped-flow spectrophotometry, and the midpoint potentials of the mutant proteins were determined. The dependence of the bimolecular rate constant of reaction, k(2), on ionic strength was determined for the reactions of the cytochrome f mutants with wild-type and mutant forms of plastocyanin. Double mutant cycle analysis was carried out to probe for the presence of specific electrostatic interactions. The effects of mutations on Cyt f were smaller than those seen previously for mutants of plastocyanin [Schlarb-Ridley, B. G. et al. (2002) Biochemistry 41, 3279-3285]. One specific short-range interaction between charged residues of wild-type plastocyanin (Arg93) and wild-type cytochrome f (Asp63) was identified. The kinetic evidence from this study and that of Schlarb-Ridley et al., 2002, appears to conflict with the NMR structure of the P. laminosum complex, which suggests the absence of electrostatic interactions in the final complex [Crowley, P. et al. (2001) J. Am. Chem. Soc. 123, 10444-10453]. The most likely explanation of the apparent paradox is that the overall rate is diffusion controlled and that electrostatics specifically influence the encounter complex and not the reaction complex.     

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.     

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.     

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.     

Relation between interface properties and kinetics of electron transfer in the interaction of cytochrome f and plastocyanin from plants and the cyanobacterium Phormidium laminosum

Cytochrome f and plastocyanin from the cyanobacterium Phormidium laminosum react an order of magnitude faster than their counterparts from chloroplasts when long-range electrostatic interactions have been screened out by high salt concentration [Schlarb-Ridley, B. G., et al. (2002) Biochemistry 41, 3279-3285]. To investigate the relative contributions of the reaction partners to these differences, the reactions of turnip cytochrome f with P. laminosum plastocyanin and P. laminosum cytochrome f with pea plastocyanin were examined. Exchanging one of the plant reaction partners with the corresponding cyanobacterial protein nearly abolished electron transfer at low ionic strength but increased the rate at high ionic strength. This increase was larger for P. laminosum cytochrome f than for P. laminosumplastocyanin. To identify molecular features of P. laminosum cytochrome f that contribute to the increase, the effect of mutations in the N-terminal heme-shielding peptide on the reaction with P. laminosum plastocyanin was determined. Phenylalanine-3 was converted to valine and tryptophan-4 to phenylalanine or leucine. The mutations lowered the rate constant at 0.1 M ionic strength by factors of 0.71 for F4V, 0.42 for W4F, and 0.63 for W4L while introducing little change in the shape of the ionic strength dependence curve. When the N-terminal tetrapeptide (sequence YPFW) was converted into that found in the chloroplast of Chlamydomonas reinhardtii (YPVF), the reaction was slowed further (factor of 0.26). The N-terminal heme-shielding peptide was found to be responsible for 75% of the kinetic differences between cytochrome f from chloroplasts and the cyanobacterium when electrostatic interactions were eliminated.     

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.     

Implications of the effects of viscosity, macromolecular crowding, and temperature for the transient interaction between cytochrome f and plastocyanin from the cyanobacterium Phormidium laminosum

The reaction between cytochrome f and plastocyanin is a central feature of the photosynthetic electron-transport system of all oxygenic organisms. We have studied the reaction in solution to understand how the very weak binding between the two proteins from Phormidium laminosum can nevertheless lead to fast rates of electron transfer. In a previous publication [Schlarb-Ridley, B. G., et al. (2003) Biochemistry 42, 4057-4063], we suggested that the reaction is diffusion-controlled because of a strong effect of viscosity of the medium. The effects of viscosity and temperature have now been examined in detail. High molecular mass viscogens (Ficoll 70 and Dextran 70), which might mimic in vivo conditions, had little effect up to a relative viscosity of 4. Low molecular mass viscogens (ethane diol, glycerol, and sucrose) strongly decreased the bimolecular rate constant (k(2)) over a similar viscosity range. The effects correlated well with the viscosities of the solutions of the three reagents but not with their dielectric constants or molalities. A power law dependence of k(2) on viscosity suggested that k(2) depends on two viscosity-sensitive reactions in series, while the reverse reactions are little affected by viscosity. The results were incompatible with diffusion control of the overall reaction. Determination of the effect of temperature on k(2) gave an activation enthalpy, DeltaH(++) = 45 kJ mol(-)(1), which is also incompatible with diffusion control. The results were interpreted in terms of a model in which the stable form of the protein-protein complex requires further thermal activation to be competent for electron transfer.     

Characterization of photosystem ii electron acceptors in Phormidium laminosum

Chlorophyll a fluorescence has been used to monitor the redox state of the primary electron acceptor of photosystem II (PS II) in the blue-green alga Phormidium laminosum during equilibrium titrations. The shape of induction curves measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) have been analyzed. The induction curves were very similar in unfractionated thylakoid membranes and PS II particles. In both, the fast (alpha) phase was sigmoidal, and was followed by a slow (beta) exponential tail. Thus, the structural organization and complexity of the particles (J. M. Bowes and P. Horton, Biochim, Biophys. Acta 680, 127-133 (1982), as indicated by the occurrence of energy transfer between alpha centers and presence of beta centers, must preexist in the membranes. Redox titration of the initial level of fluorescence indicated the presence of a single quencher QH in the unfractionated thylakoids, midpoint potential: Em7.0 approximately -35 mV (n = 1). Thus, the occurrence of a single acceptor is characteristic for P. laminosum and the absence of a low potential acceptor in PS II particles (J.M. Bowes, P. Horton, and D.S. Bendall, FEBS Lett. 135, 261-264 (1981] was not the result of its removal during their preparation. The midpoint potential of Q varied by -60 mV/pH unit in PS II particles and membrane fragments, with a pK at pH greater than 8.5 (particles) and at pH 7.5 (fragments). In PS II particles, DCMU raised the pK by approximately 0.5 pH units. It is argued that the pH dependence of Q is conferred by protonation of a protein which accompanies its reduction rather than protonation of the semiquinone Q X itself.     

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.     

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.     

Cloning and sequence analysis of a signal peptidase I from the thermophilic cyanobacterium Phormidium laminosum

Type I signal peptidases are a widespread family of enzymes which remove the presequences from proteins translocated across cell membranes, including thylakoid and cytoplasmic membranes of cyanobacteria and thylakoid membranes of chloroplasts. We have cloned and sequenced a signal peptidase gene from the thermophilic cyanobacterium Phormidium laminosum which is believed to encode an enzyme common to both membrane systems. The deduced amino acid sequence is 203 residues long and although the overall similarity among signal peptidases is rather low there are a number of identifiable conserved regions present. The P. laminosum enzyme is predicted to have a single transmembrane domain, in contrast to other Gram-negative bacterial sequences, but similar to other type I signal peptidases.     

Thermal unfolding of plastocyanin from the mesophilic cyanobacterium Synechocystis sp. PCC 6803 and comparison with its thermophilic counterpart from Phormidium laminosum

The thermal unfolding of plastocyanin from the mesophilic cyanobacterium Synechocystis is described herein, and the results are compared with those obtained for the homologous thermophilic protein from Phormidium laminosum. The thermal unfolding is irreversible under all the conditions that were investigated. Plastocyanin from the thermophilic organism, both in the native state and in the apoprotein form, proved to be more thermostable than its mesophilic counterpart under all experimental conditions. Synechocystis reduced plastocyanin has been shown to be more stable than the oxidized species, both with respect to the required temperature for protein unfolding and with respect to the kinetics of the process. This behavior contrasts with that observed for Phormidium plastocyanin, in which the oxidized form is the more stable one. The unfolding pH dependence and kinetic studies indicate that around physiological pH, the most kinetically stable form is also the one more resistant to temperature variations, suggesting a close compromise between function and stability. Molecular dynamics simulations suggest that Phormidium and Synechocystis plastocyanins follow different unfolding pathways that affect different protein areas and which could be responsible for the observed dissimilar thermal resistance.     

Expression of plastocyanin and cytochrome f of the cyanobacterium Phormidium laminosum in Escherichia coli and Paracoccus denitrificans and the role of leader peptides.

The gene for plastocyanin from the cyanobacterium Phormidium laminosum was successfully expressed in Escherichia coli. Expression of the gene for cytochrome f resulted in the production of holocytochrome f in the periplasmic space of E. coli, but the yield was low. Expression in Paracoccus denitrificans yielded no holoprotein. When the region encoding the cytochrome f leader sequence was replaced with more typical bacterial leader sequences (those from the P. laminosum plastocyanin gene and the Paracoccus versutus cytochrome c-550 gene), much higher yields were consistently obtained in both species. Overexpressed proteins were compared to those isolated from P. laminosum and found to be identical in mass, isoelectric point, redox midpoint potential and (for plastocyanin) 1H-NMR spectrum.