Asymmetric electrostatic effects on the gating of rat brain sodium channels in planar lipid membranes.

The effects of ionic strength (10-1,000 mM) on the gating of batrachotoxin-activated rat brain sodium channels were studied in neutral and in negatively charged lipid bilayers. In neutral bilayers, increasing the ionic strength of the extracellular solution, shifted the voltage dependence of the open probability (gating curve) of the sodium channel to more positive membrane potentials. On the other hand, increasing the intracellular ionic strength shifted the gating curve to more negative membrane potentials. Ionic strength shifted the voltage dependence of both opening and closing rate constants of the channel in analogous ways to its effects on gating curves. The voltage sensitivities of the rate constants were not affected by ionic strength. The effects of ionic strength on the gating of sodium channels reconstituted in negatively charged bilayers were qualitatively the same as in neutral bilayers. However, important quantitative differences were noticed: in low ionic strength conditions (10-150 mM), the presence of negative charges on the membrane surface induced an extra voltage shift on the gating curve of sodium channels in relation to neutral bilayers. It is concluded that: (a) asymmetric negative surface charge densities in the extracellular (1e-/533A2) and intracellular (1e-/1,231A2) sides of the sodium channel could explain the voltage shifts caused by ionic strength on the gating curve of the channel in neutral bilayers. These surface charges create negative electric fields in both the extracellular and intracellular sides of the channel. Said electric fields interfere with gating charge movements that occur during the opening and closing of sodium channels; (b) the voltage shifts caused by ionic strength on the gating curve of sodium channels can be accounted by voltage shifts in both the opening and closing rate constants; (c) net negative surface charges on the channel's molecule do not affect the intrinsic gating properties of sodium channels but are essential in determining the relative position of the channel's gating curve; (d) provided the ionic strength is below 150 mM, the gating machinery of the sodium channel molecule is able to sense the electric field created by surface changes on the lipid membrane. I propose that during the opening and closing of sodium channels, the gating charges involved in this process are asymmetrically displaced in relation to the plane of the bilayer. Simple electrostatic calculations suggest that gating charge movements are influenced by membrane electrostatic potentials at distances of 48 and 28 A away from the plane of the membrane in the extracellular sides of the channel, respectively.     

Ionic control of enzymic degradation of double-stranded RNA

The pattern of the degradation of various double-stranded polyribonucleotides by several ribonucleases (bovine RNAase A and its cross-linked dimer, bovine seminal RNAase, and pike-whale pancreatic RNAase) has been studied as a function of ionic strength and pH. It appears that (1) there is no direct correlation between the secondary structure of double-stranded RNA and its resistance against enzymatic breakdown, i.e., the stability of the secondary structure of double-helical RNA is not the main variable in the process. (2) The acstivity responses of the enzymes examined to changes of ionic strength and pH suggest that enzymic degradation of double-stranded RNA is mainly controlled by ion concentration, and that the process may fall within the phenomena interpreted by the theory of the ionic control of biochemical reactions advanced by Douzou and Maurel (Douzou, P. and Maurel, P. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1013--1015). (3) The activity curves of the enzyme studied show, at a given pH, a shift toward higher ionic strengths as a function of the basicity of the enzyme protein. This finding explains the already observed correlation between number and/or density of positive charges of a ribonuclease molecule and its ability to attack double-stranded RNA in 0.15 M sodium chloride/0.015 M sodium citrate (SSC). (4) A careful analysis of the influence of ionic strength and pH on the reaction appears to be necessary in order to characterize a ribonuclease which shows activity towards double-stranded RNAs, and to allow a meaningful comparison between different enzymes capable of attacking these substrates.     

Electrostatic interactions during activation of coagulation factor IX via the tissue factor pathway: effect of univalent salts

Interaction between the Gla-domain of coagulation proteins and negatively charged phospholipid membranes is essential for blood coagulation reactions. The interaction is calcium-dependent and mediated both by electrostatic and hydrophobic forces. This report focuses on the electrostatic component of factor IX activation via the extrinsic pathway. Effective charges during the reaction are measured by ionic titration of activity, according to the Debye-Huckel and Gouy-Chapman models. Rates of activation decrease with ionic strength independently of the type of monovalent salt used to control ionic strength. Moreover, the effect of ionic strength decreases at concentrations of charged phospholipid approaching saturation levels, indicating that membrane charges participate directly in the ionic interaction measured. The effective charge on calcium-bound factor IX during activation on phospholipid membranes is 0.95+/-0.1. Possible sites mediating contacts between the Gla-domain and membranes are selected by geometrical criteria in several metal-bound Gla-domain structures. A pocket with a solvent opening-pore of area 24-38 A2 is found in the Gla-domain of factors IX, VII, and prothrombin. The pocket contains atoms with negative partial charges, including carboxylate oxygens from Gla residues, and has a volume of 57-114 A3, sufficient to accommodate additional calcium atoms. These studies demonstrate that electrostatic forces modify the activity coefficient of factor IX during functional interactions and suggest a conserved pocket motif as the contact site between the calcium-bound Gla-domain and charged membranes.     

Chromatin ionic atmosphere analyzed by a mesoscale electrostatic approach

Characterizing the ionic distribution around chromatin is important for understanding the electrostatic forces governing chromatin structure and function. Here we develop an electrostatic model to handle multivalent ions and compute the ionic distribution around a mesoscale chromatin model as a function of conformation, number of nucleosome cores, and ionic strength and species using Poisson-Boltzmann theory. This approach enables us to visualize and measure the complex patterns of counterion condensation around chromatin by examining ionic densities, free energies, shielding charges, and correlations of shielding charges around the nucleosome core and various oligonucleosome conformations. We show that: counterions, especially divalent cations, predominantly condense around the nucleosomal and linker DNA, unburied regions of histone tails, and exposed chromatin surfaces; ionic screening is sensitively influenced by local and global conformations, with a wide ranging net nucleosome core screening charge (56-100e); and screening charge correlations reveal conformational flexibility and interactions among chromatin subunits, especially between the histone tails and parental nucleosome cores. These results provide complementary and detailed views of ionic effects on chromatin structure for modest computational resources. The electrostatic model developed here is applicable to other coarse-grained macromolecular complexes.     

Transient kinetics of electron transfer reactions of flavodoxin: ionic strength dependence of semiquinone oxidation by cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid and computer modeling of reaction complexes

Electron transfer reactions between Clostridum pasteurianum flavodoxin semiquinone and various oxidants [horse heart cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic [horse heart cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid (EDTA)] have been studied as a function of ionic strength by using stopped-flow spectrophotometry. The cytochrome c reaction is complicated by the existence of two cytochrome species which react at different rates and whose relative concentrations are ionic strength dependent. Only the faster of these two reactions is considered here. At low ionic strength, complex formation between cytochrome c and flavodoxin is indicated by a leveling off of the pseudo-first-order rate constant at high cytochrome c concentration. This is not observed for either ferricyanide or ferric EDTA. For cytochrome c, the rate and association constants for complex formation were found to increase with decreasing ionic strength, consistent with negative charges on flavodoxin interacting with the positively charged cytochrome electron transfer site. Both ferricyanide and ferric EDTA are negatively charged oxidants, and the rate data respond to ionic strength changes as would be predicted for reactants of the same charge sign. These results demonstrate that electrostatic interactions involving negatively charged groups are important in orienting flavodoxin with respect to oxidants during electron transfer. We have also carried out computer modeling studies of putative complexes of flavodoxin with cytochrome c and ferricyanide, which relate their structural properties to both the observed kinetic behavior and some more general features of physiological electron transfer processes. The results of this study are consistent with the ionic strength behavior described above.     

Further studies on the contribution of electrostatic and hydrophobic interactions to protein adsorption on dye-ligand adsorbents.

The adsorption equilibria of bovine serum albumin (BSA), gamma-globulin, and lysozyme to three kinds of Cibacron blue 3GA (CB)-modified agarose gels, 6% agarose gel-coated steel heads (6AS), Sepharose CL-6B, and a home-made 4% agarose gel (4AB), were studied. We show that ionic strength has irregular effects on BSA adsorption to the CB-modified affinity gels by affecting the interactions between the negatively charged protein and CB as well as CB and the support matrix. At low salt concentrations, the increase in ionic strength decreases the electrostatic repulsion between negatively charged BSA and the negatively charged gel surfaces, thus resulting in the increase of BSA adsorption. This tendency depends on the pore size of the solid matrix, CB coupling density, and the net negative charges of proteins (or aqueous - phase pH value). Sepharose gel has larger average pore size, so the electrostatic repulsion-effected protein exclusion from the small gel pores is observed only for the affinity adsorbent with high CB coupling density (15.4 micromol/mL) at very low ionic strength (NaCl concentration below 0.05 M in 10 mM Tris-HCl buffer, pH 7.5). However, because CB-6AS and CB-4AB have a smaller pore size, the electrostatic exclusion effect can be found at NaCl concentrations of up to 0.2 M. The electrostatic exclusion effect is even found for CB-6AS with a CB density as low as 2.38 micromol/mL. Moreover, the electrostatic exclusion effect decreases with decreasing aqueous-phase pH due to the decrease of the net negative charges of the protein. For gamma-globulin and lysozyme with higher isoelectric points than BSA, the electrostatic exclusion effect is not observed. At higher ionic strength, protein adsorption to the CB-modified adsorbents decreases with increasing ionic strength. It is concluded that the hydrophobic interaction between CB molecules and the support matrix increases with increasing ionic strength, leading to the decrease of ligand density accessible to proteins, and then the decrease of protein adsorption. Thus, due to the hybrid effect of electrostatic and hydrophobic interactions, in most cases studied there exists a salt concentration to maximize BSA adsorption.     

Nucleotide Binding to Na,K-ATPase: The Role of Electrostatic Interactions.

The contribution of electrostatic forces to the interaction of Na,K-ATPase with adenine nucleotides was investigated by studying the effect of ionic strength on nucleotide binding. At pH 7.0 and 20 degrees C, there was a qualitative correlation between the equilibrium dissociation constant (K(d)) values for ATP, ADP, and MgADP and their total charges. All K(d) values increased with increasing ionic strength. According to the Debye-Hückel theory, this suggests that the nucleotide binding site and its ligands have "effective" charges of opposite signs. However, quantitative analysis of the dependence on ionic strength shows that the product of the effective electrostatic charges on the ligand and the binding site is the same for all nucleotides, and is therefore independent of the total charge of the nucleotide. The data suggest that association of nucleotides with Na,K-ATPase is governed by a partial charge rather than the total charge of the nucleotide. This charge, interacting with positive charges on the protein, is probably the one corresponding to the alpha-phosphate of the nucleotide. Dissociation rate constants measured in complementary transient kinetic experiments were 13 s(-1) for ATP and 27 s(-1) for ADP, independent of the ionic strength in the range 0.1-0.5 M. This implies similar association rate constants for the two nucleotides (about 40 x 10(6) M(-1) s(-1) at I = 0.1 M). The results suggest that long-range Coulombic forces, affecting association rates, are not the main contributors to the observed differences in affinities, and that local interactions, affecting dissociation rates, may play an even greater role.     

Nicotinic acetylcholine receptor channel electrostatics determined by diffusion-enhanced luminescence energy transfer

The electrostatic potentials within the pore of the nicotinic acetylcholine receptor (nAChR) were determined using lanthanide-based diffusion-enhanced fluorescence energy transfer experiments. Freely diffusing Tb3+ -chelates of varying charge constituted a set of energy transfer donors to the acceptor, crystal violet, a noncompetitive antagonist of the nAChR. Energy transfer from a neutral Tb3+ -chelate to nAChR-bound crystal violet was reduced 95% relative to the energy transfer to free crystal violet. This result indicated that crystal violet was strongly shielded from solvent when bound to the nAChR. Comparison of energy transfer from positively and negatively charged chelates indicate negative electrostatic potentials of -25 mV in the channel, measured in low ionic strength, and -10 mV measured in physiological ionic strength. Debye-Hückel analyses of potentials determined at various ionic strengths were consistent with 1-2 negative charges within 8 A of the crystal violet binding site. To complement the energy transfer experiments, the influence of pH and ionic strength on the binding of [3H]phencyclidine were determined. The ionic strength dependence of binding affinity was consistent with -3.3 charges within 8 A of the binding site, according to Debye-Hückel analysis. The pH dependence of binding had an apparent pKa of 7.2, a value indicative of a potential near -170 mV if the titratable residues are constituted of aspartates and glutamates. It is concluded that long-range potentials are small and likely contribute little to selectivity or conductance whereas close interactions are more likely to contribute to electrostatic stabilization of ions and binding of noncompetitive antagonists within the channel.     

Comparison between histones FV and F2a2 of chicken erythrocyte. II. Interaction with homologous DNA.

The conformation and stability of artificial complexes between chicken erythrocyte DNA and homologous histones FV and F2a2 was studied by circular dichroism (CD) and thermal denaturation followed by both absorbance and CD measurements. The complexes are made after a stepwise potassium fluoride gradient dialysis without urea and studied at low ionic strength (10-minus 3 M). 1) No structural changes of the DNA can be detected up to r equals 0.2 with FV and r equals 0.6 for F2a2. With FV at higher values of r the CD spectrum is altered, indicating the organization of DNA and histones in some kind of aggregate. 2) The conformation of histone molecules inside the complexes is not related to the ionic strength of the medium but to an effective ionic environment close to 0.1 M. This ionic strength would also correspond to the melting temperature of histone-covered DNA. 3) From the analysis of the absorbance melting profile the length of DNA covered with an histone molecule can be estimated. A good agreement is found between the negative charge of this piece of DNA and the net positive charge of the histone. 4) Since the CD transition at 227 nm occurs before the second absorbance transition at 280 nm, the DNA is stabilized no longer by native histone but partially or fully denatured histones. The helical regions of the histone molecule are not involved in the binding process, which appears to be almost purely coulombian and most likely related to some structural fit between the pattern of negative charges in the DNA helix and that of positive charges along the peptide chain.     

Electron transfer in hemoproteins. VI. The dependence of the reduction rate of ferricytochrome c by oxymyoglobin on ionic strength

The rate of the redox-reaction between MbO2 and ferri-Cyt c has been investigated in the pH range 5-8 under different ionic strength of the solution. The influence of various anions-phosphate, chloride, sulfate and acetat on the rate of the reaction were also studied. It has been shown that under the low ionic strength, I less than 0.1, all pH-dependence curves have pronounced maximum near pH 6.0. While the ionic strength values increase in this interval the reaction rate falls markedly, the profile of lg k versus square root of I/1 + square root of I is linear. Under high ionic strength values, I greater than 0.1, the reaction rate in MbO2-Cyt c system is only slightly influenced by increasing salt concentrations and by pH changing. The results obtained support the idea that the local interactions of charged groups in " active sites" of MbO2 and Cyt c play the most important role in the mechanism of electron transfer. On the contrary net charges of the molecules have a negligible effect on the rate of the reaction. Compared to anions Cl-, SO42- and CH3COO- which influence the reaction rate in an analogous way, phosphate ions have essential inhibiting effect. This is most likely explained by the specific bonding of the phosphate ions to Cty c in the immediate vicinity from the site of the "active contact" with Mb molecule.     

Photoinduced electron-transfer reaction in a ternary system involving zinc cytochrome c and plastocyanin. Interplay of monopolar and dipolar electrostatic interactions between metalloproteins.

A carbodiimide promotes noninvasive cross-linking between amino groups surrounding the exposed heme edge in zinc cytochrome c and carboxylic groups in the acidic patch in plastocyanin. Eight derivatives of the covalent complex Zncyt/pc(I), which have similar structures but different overall charges because of different numbers and locations of N-acylurea groups, are separated by cation-exchange chromatography. Kinetics of electron transfer from the diprotein complex in the triplet excited state, 3Zncyt/pc(I), to free cupriplastocyanin at pH 7.0 and various ionic strengths is studied by laser flash spectroscopy. This reaction is purely bimolecular for all eight N-acylurea derivatives of the diprotein complex. The overall charges of the derivatives 1 and 2 at pH 7.0 are -2 and 0, respectively; both of them, however, have very large dipole moments of 410-480 D. The rate constants for their reactions with cupriplastocyanin, whose charge at pH 7.0 is -8 and whose dipole moment is 362 D, are determined over the range of ionic strengths from 2.5 mM to 3.00 M. The observed dependence of the rate constants on ionic strength cannot be explained in terms of net charges (monopole-monopole interactions) alone, but it can be fitted quantitatively with a theory that recognizes also monopole-dipole and dipole-dipole interactions [van Leeuwen, J. W. (1983) Biochim. Biophys. Acta 743, 408]. At ionic strengths up to ca. 10 mM monopole-monopole interactions predominate and Br?nsted-Debye-Hückel theory applies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of immobiline membranes for application in a multicompartment electrolyzer for protein purification.

The efficient use of preparative protein purification in a multicompartment electrolyzer with Immobiline membranes depends on the knowledge of membrane characteristics. For that purpose, an experimental investigation of the effects of ionic charges on the membrane characteristics has been carried out through the measurements of membrane swelling and conductance. We also investigated the effects on the electrolyzer behaviour of operating parameters such as the Immobiline concentration and the presence of ion-exchange membranes. Data show that polyacrylamide gel degree of swelling is strongly dependent upon the pH and the ionic strength of the bathing solution as well as on the type and molarity of charges incorporated in the gel. The conductance of supported Immobiline gels in contact with uni-univalent chloride solutions has been measured by means of a mercury cell. The membrane conductance is also influenced by the ionic strength of the equilibrium solution and the presence of weak ionizable groups in the gel matrix. This study has demonstrated the close link between electrochemical and electromechanical properties of Immobiline membranes.     

Ionic control of immobilized enzymes. Kinetics of acid phosphatase bound to plant cell walls.

When an enzyme is bound to an insoluble polyelectrolyte it may acquire novel kinetic properties generated by Donnan effects. It the enzyme is homogeneously distributed within the matrix, a variation of the electrostatic partition coefficient, when substrate concentration is varied, mimics either positive or negative co-operativity. This type of non-hyperbolic behaviour may be distinguished from true co-operativity by an analysis of the Hill plots. If the enzyme is heterogeneously distributed within the polyelectrolyte matrix, an apparent negative co-operativity occurs, even if the electrostatic partition coefficient does not vary when substrate concentration is varied in the bulk phase. If the partition coefficient varies, mixed positive and negative co-operativities may occur. All these effects must be suppressed by raising the ionic strength in the bulk phase. Attraction of cations by fixed negative charges of the polyanionic matrix may be associated with a significant decrease of the local pH. The magnitude of this effect is controlled by the pK of the fixed charges groups of the Donnan phase. The local pH cannot be much lower than the value of this pK. This effect may be considered as a regulatory device of the local pH. Acid phosphatase of sycamore (Acer pseudoplatanus) cell walls is a monomeric enzyme that displays classical Michaelis-Menten kinetics in free solution. However, when bound to small cell-wall fragments or to intact cells, it has an apparent negative co-operativity at low ionic strength. Moreover a slight increase of ionic strength apparently activates the bound enzymes and tends to suppress the apparent co-operativity. At I0.1, or higher, the bound enzyme has a kinetic behavior indistinguishable from that of the purified enzyme in free solution. These results are interpreted in the light of the Donnan theory. Owing to the repulsion of the substrate by the negative charges of cell-wall polygalacturonates, the local substrate concentration in the vicinity of the bound enzyme is smaller than the corresponding concentration in bulk solution. The kinetic results obtained are consistent with the view that there exist at least three populations of bound enzyme with different ionic environments: a first population with enzyme molecules not submitted to electrostatic effects, and two other populations with molecules differently submitted to these effects. The theory allows one to estimate the proportions of enzyme belonging to these populations, as well as the local pH values and the partition coefficients within the cell walls.     

Polycation induced actin bundles

Three polycations, polylysine, the polyamine spermine and the polycationic protein lysozyme were used to study the formation, structure, ionic strength sensitivity and dissociation of polycation-induced actin bundles. Bundles form fast, simultaneously with the polymerization of MgATP-G-actins, upon the addition of polycations to solutions of actins at low ionic strength conditions. This indicates that nuclei and/or nascent filaments bundle due to attractive, electrostatic effect of polycations and the neutralization of repulsive interactions of negative charges on actin. The attractive forces between the filaments are strong, as shown by the low (in nanomolar range) critical concentration of their bundling at low ionic strength. These bundles are sensitive to ionic strength and disassemble partially in 100 mM NaCl, but both the dissociation and ionic strength sensitivity can be countered by higher polycation concentrations. Cys374 residues of actin monomers residing on neighboring filaments in the bundles can be cross-linked by the short span (5.4 Å) MTS-1 (1,1-Methanedyl Bismethanethiosulfonate) cross-linker, which indicates a tight packing of filaments in the bundles. The interfilament cross-links, which connect monomers located on oppositely oriented filaments, prevent disassembly of bundles at high ionic strength. Cofilin and the polysaccharide polyanion heparin disassemble lysozyme induced actin bundles more effectively than the polylysine-induced bundles. The actin-lysozyme bundles are pathologically significant as both proteins are found in the pulmonary airways of cystic fibrosis patients. Their bundles contribute to the formation of viscous mucus, which is the main cause of breathing difficulties and eventual death in this disorder.     

Cooperative, excluded-site binding and its dynamics for the interaction of gene 5 protein with polynucleotides

The binding of gene 5 protein to various single-stranded polynucleotides is investigated by fluorescence titrations and stopped-flow measurements. The association state of gene 5 protein itself is analyzed by equilibrium sedimentation: the monomer-dimer equilibrium found in the micromolar concentration range is described by a stability constant of 8 X 10(5) M-1. The fluorescence quenching upon binding to polynucleotides, studied over a broad concentration range and analyzed in terms of a cooperative excluded-site binding model, provides binding constants for "isolated" and for "cooperative" sites. The cooperativity for various ribo- and deoxyribopolymers is between 400 and 800 and is virtually independent of the ionic strength. The binding to isolated sites is strongly dependent upon the ionic strength; analysis in terms of polyelectrolyte theory indicates the compensation of 4 +/- 0.5 charges upon complex formation. The number of nucleotide residues covered by one protein molecule is also found to be 4 +/- 0.5 units. The affinity of gene 5 protein for polynucleotides increases in the series poly(C) less than poly(dA) less than poly(A) less than poly(U) much less than poly(dT); the binding constant for poly(dT) is roughly a factor of 1000 higher than that for the other polymers. Model studies with Lys-Tyr-Lys and Lys-Trp-Lys suggest that the preferential interaction with poly(dT) is not simply due to enhanced stacking interactions between the aromatic amino acids and the thymine residues. Stopped-flow reaction curves obtained by mixing of gene 5 protein with poly(dT) in the micromolar concentration range show three relaxation processes with time constants between 1 ms and 1 s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrostatic effects and calcium ion concentration as modulators of acid phosphatase bound to plant cell walls

At 'low' ionic strength, acid phosphatase bound to plant cell walls exhibits an apparent negative co-operativity, whereas it displays classic Michaelis-Menten kinetics in free solution. Conversely, at 'high' ionic strength, the bound enzyme and the soluble enzyme behave identically. This apparent negative co-operativity is explained by the existence of an electrostatic partition of the charged substrate by the fixed negative charges of the cell wall. Raising the ionic strength suppresses these electrostatic repulsion effects. Calcium may be removed from the cell walls by acid treatment and the acid phosphatase is apparently strongly inhibited. This inhibition occurs together with an increased apparent negative co-operativity of the enzyme. Incubating cell wall fragments previously depleted of calcium with CaCl2 restores the initial behaviour of the enzyme. Calcium, which tightly binds to cell wall pectic compounds, has by itself no effect on the enzyme in free solution. It affects the net charge of the cell wall and therefore the amplitude of electrostatic repulsion effects. Non-linear least-square fitting methods make it possible to estimate the density of fixed negative charges as well as the electrostatic partition coefficient, for both the 'native' and 'calcium-deprived' cell wall fragments. It may be shown directly that calcium loading and unloading in the cell wall controls the electrostatic effects, by monitoring proton extrusion from cell wall fragments upon raising the ionic strength. Proton outflux in the bulk phase is considerably enhanced upon removal of calcium from the cell walls. The main conclusion is that loading and unloading of calcium during cell elongation and division may regulate the activity of cell wall enzymes.     

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)).     

Interaction of cytochrome c with the phosphorprotein phosvitin.

Candida krusei cytochrome c forms a molecular complex with phosphorprotein phosvitin in weakly alkaline solution of low ionic strength. At most, about 22 molecules of cytochrome c bind to a phosvitin molecule. The complex at the binding ratio below about 11 (half of the maximum ratio) as a much higher binding strength. Several lines of evidence indicate that the marked difference in the binding strength is due to the difference in negative charges on phosvitin molecule concerned in the binding of a cytochrome c molecule. The phosvitin-bound cytochrome c seems to have a preferred orientation with the front surface of the molecule containing the exposed heme edge in contact with the phosvitin molecule.     

Electrostatic effects on the kinetics of oxidation-reduction reactions of c-type cytochromes.

The kinetics of the oxidation-reduction reactions between horse heart cytochrome c, Euglena gracilis cytochrome c552, and ions (ascorbate, ferricyanide, and ferrocyanide) was investigated as a function of ionic strength at pH 7, 25 degrees C. The ionic strength was varied between 0.002 and 0.02 M. Data were analyzed according to four different functions of ionic strength. Results showed that the Kirkwood-Tanford smeared charge model holds well for the calculation of the activity coefficients and that the whole charges of these proteins are reflected in the rates of their reactions. Chemical modifications or changes in the pH that altered the charge of the proteins affected the primary salt effects as predicted by the smeared charge model.     

Kinetics of renaturation of denatured DNA. I. Spectrophotometric results

The kinetics of renaturation of heat- or formamide-denatured DNA have been studied by following the change of optical density at a constant temperature. Solvents of different ionic strength and various DNA samples have been used. At the lower ionic strengths studied, the reaction follows second-order kinetics, substantiating the hypothesis that strands of native DNA separate upon denaturation and recombine during renaturation. As the ionic strength is increased at a constant temperature, the reaction deviates from simple second-order behavior. This appears to be the result of the inhibition to rewinding caused by short helical segments in the denatured DNA which are more stable at the higher ionic strenth.