Reduction kinetics of the four hemes of cytochrome c3 from Desulfovibrio vulgaris by flash photolysis.

The reduction of the tetraheme cytochrome c3 (from Desulfovibrio vulgaris, strains Miyazaki F and Hildenbourough) by flavin semiquinone and reduced methyl viologen follows a monophasic kinetic profile, even though the four hemes do not have equivalent reduction potentials. Rate constants for reduction of the individual hemes are obtained subsequent to incrementally reducing the cytochrome by phototitration. The dependence of each rate constant on the reduction potential difference between the heme and the reductant can be described by outer sphere electron transfer theroy. Thus, the very low reduction potentials of the cytochrome c3 hemes compensate for the very large solvent accessibility of the hemes. The relative rate constants for electron transfer to the four hemes of cytochrome c3 are consistent with the assignments of reduction potential to hemes previously made by Park et al. (Park, J.-S., Kano, K., Niki, S. and Akutsu, H. (1991) FEBS Lett. 285, 149-151) using NMR techniques. The ionic strength dependence of the observed rate constant for reduction by the methyl viologen radical cation indicates that ionic strength substantially alters the structure and/or the heme reduction potentials of the cytochrome. This result is confirmed by reduction with a neutral flavin species (5-deazariboflavin semiquinone) in which the reactivity of the highest potential heme decreases and the reactivity of the lowest potential heme increases at high (500 mM) ionic strength, and by the sensitivity of heme methyl resonances to ionic strength as observed by 1H-NMR. These unusual ionic strength-dependent effects may be due to a combination of structural changes in the cytochrome and alterations of the electrostatic fields at elevated ionic strengths.     

Binding and hydrolysis of ATP by cardiac myosin subfragment 1: effect of solution parameters on transient kinetics

Transient kinetic data of ATP binding and cleavage by cardiac myosin subfragment 1 (S1) were obtained by fluorescence stopped flow and analyzed by using computer modeling based on a consecutive, reversible two-step mechanism: (formula: see text) where M1 and M12 denote myosin species with enhanced fluorescence and K'O = K0/(K0[ATP] + 1). The kinetic constants K0, k12, k23, and k32 and the fractional contributions of M1 and M12 to the total fluorescence are analyzed over a range of systematically varied solution parameters. The initial ATP binding equilibrium (K0), which decreases with increasing pH, is facilitated by a positively charged protein residue with a pK of 7.1. An active-site charge of +1.5 is determined from the ionic strength dependence. The rate constants k12, k23, and k32 also exhibit pK's near neutrality but increase with increasing pH. The majority of the large (-54 kJ/mol) negative free energy of ATP binding occurs upon S1 isomerization, k12, and a large increase in entropy (183 J/kmol at 15 degrees C) is associated with the cleavage step. The equilibrium constant for the cleavage step, K2, is determined as 3.5 at pH 7.0, 15 degrees C, and 200 mM ionic strength. There are no significant changes in fractional contributions to total fluorescence enhancement due to solvent-dependent conformational changes of S1 in these data. When values for the combined rate constants are calculated and compared with those determined by graphical analysis, it is observed that graphical analysis overestimates the binding rate constant (K0k12) by 25% and the hydrolysis rate constant (k23 + k32) by as much as 30%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gel filtration studies on oxyhemerythrin. II. Effect of temperature and ionic strength on the association-dissociation equilibria.

The effects of temperature and ionic strength on the association of oxyhemerythrin have been studied. deltaH degrees and deltaS degrees for association at pH 7.0 are -2.6 kcal and +16.5 eu per mol of monomer. These values suggest that solvent adjacent to the surface of the protein undergoes rearrangement on association. Increasing ionic strength is observed to promote dissociation while decreasing the rate of attainment of equilibrium between monomers and octamers. Qualitatively similar results are observed on lowering the pH from 7.0 to 4.8, thereby linking the effects of increasing ionic strength to those of protonation of specific amino acid residues at the subunit contacts of hemerythrin. The apparent enthalpy of ionization of the amino acid residue controlling dissociation at acidic pH was found to be -1.9 to +2.1 kcal/mol. These values are consistent with a carboxyl group.     

The effects of solvent viscosity on the kinetic parameters of myosin and heavy meromyosin ATPase

The effects of different solvent viscosities on the kinetic parameters of ATP hydrolysis by myosin and heavy meromyosin (HMM) were investigated at high and low ionic strength (i.e., 0.53 and 0.08 M KCl where myosin is polymerized into thick filament). The solvent viscosity was adjusted by the addition of appropriate amounts of sucrose. The maximum rate constants (V _m ) for both myosin and HMM decreased monotonically with increasing solvent viscosity at either ionic strength. The Michaelis constants (K _m ) for soluble myosin and HMM became minima at a viscosity nearly twice that of the solvent without sucrose, then increased abruptly with increasing solvent viscosity. On the other hand,K _m of polymerized myosin at the low ionic strength decreased monotonically with increasing solvent viscosity. These experimental results are discussed with special reference to Kramers' kinetic theory of a chemically reacting system in viscous media.     

Electrostatic influence of local cysteine environments on disulfide exchange kinetics

The ionic strength dependence of the bimolecular rate constant for reaction of the negative disulfide 5,5'-dithiobis (2-nitrobenzoic acid) with cysteines in fragments of naturally occurring proteins was determined by stopped-flow spectroscopy. The Debye-Hückel relationship was applied to determine the effective charge at the cysteine and thereby determine the extent to which nearby neighbors in the primary sequence influence the kinetics. Corrections for the secondary salt effect on cysteine pKs were determined by direct spectrometric pH titration of sulfhydryl groups or by observation of the ionic strength dependence of kinetics of cysteine reaction with the neutral disulfide 2,2'-dithiodipyridine. Quantitative expressions was verified by model studies with N-acetyl-cystein. At ionic strengths equal to or greater than 20 mM, the net charge at the polypeptide cysteine site is the sum of the single negative charge of the thiolate anion and the charges of the amino acids immediately preceding and following the cysteine in the primary sequence. At lower ionic strengths, more distant residues influence kinetics. At pH 7.0, 23 degree C, and an ionic strength of 20 mM, rate constants for reaction of the negative disulfide with a cysteine having two positive neighbors, one positive and one neutral neighbor, or two neutral neighbors are 132000, 3350, and 367 s-1 M-1, respectively. This corresponds to a contribution to the activation energy of 0.65- 1.1 kcal/mol per ion pair involved in collision between the cysteine and disulfide regions. The results permit the estimation that cysteine local environments may provide a means of achieving a 10(6)-fold range in rate constants in disulfide exchange reactions in random-coil proteins. This range may prove useful in developing strategies for directing disulfide pairing in synthetic proteins.     

The effects of ionic strength on the self-association of human spectrin.

The self-association of human spectrin has been studied by means of sedimentation equilibrium in the analytical ultracentrifuge at pH 7.5 and over a range of ionic strength from 0.009 to 1.0 M. Increasing ionic strength above 0.1 M reduces the equilibrium constants for all of the measurable steps in the self-association reaction. These results support the concept of charge-charge interactions stabilizing the tetramer and higher oligomers with respect to the heterodimer. In addition, increasing ionic strength brought about a dissociation of the heterodimer to component polypeptide chains. Dissociation to the heterodimers is also enhanced with a decrease in ionic strength below 0.05 M. This low ionic strength-dependent dissociation is consistent with generalised electrostatic repulsion; however, this effect also correlates with some loss of alpha-helical content as revealed by circular dichroism. The secondary, tertiary and quaternary structures may all be partially disrupted by electrostatic free energy at low ionic strength.     

The component analysis of tryptophan fluorescence spectra of melittin during its oligomerization

The oligomerization of melittin with increasing ionic strength and protein concentration was investigated using the methods of decomposition of its tryptophan fluorescence spectra into "elementary" log-normal components. At high ionic strength (up to 2 M KCl), the emission spectra of tetrameric melittin are well described as the sum of two log-normal components, suggesting the presence of tryptophan residues in two sorts of environment with greatly differing polarity. Measurements of fluorescence spectra by iodide showed that these two spectral components possess different Stern-Volmer constants, that is, the tryptophans emitting them have different solvent accessibility, which does not correlate with the crystallographic structure of tetrameric melittin. Moreover, in the oligomerization transition induced by ionic strength, the tetrameric intermediate is formed, which has log-normal spectral components with relative contributions differing from those in 2 M KCl.     

Use of local electrostatic environments of cysteines to enhance formation of a desired species in a reversible disulfide exchange reaction

The role of electrostatic factors has been evaluated for the reversible disulfide exchange reaction between N-acetylcysteine (A) and a peptide fragment (B) comprising residues 85-114 of Kunitz soybean trypsin inhibitor. In A, the sulfhydryl group has a negative carboxyl neighbor on the cysteine itself. In B, the only charged group within five residues of the single cysteine at position 86 is the positive N-terminal amino group on residue 85. The concentrations of the monomers A and B and of the disulfides AA, AB and BB have been determined as a function of time in kinetic experiments at pH 7, 23 degrees C and ionic strengths of 20 mM and 1 M. At both ionic strengths the sulfhydryl acid dissociation constants Ka have been determined for A and B, as well as the four rate constants for the disulfide exchange reaction. The electrostatic effects are small in magnitude but occur in expected directions. Local cysteine environments enhance formation of the mixed disulfide (AB), having a favorable configuration of adjacent unlike charges and generate decreases in the AA and BB disulfides joining regions of identical charge. These experiments represent an initial step towards use of intrinsic protein functional groups to direct formation of specific disulfides in a synthetic protein.     

The solvent effects on the kinetics of bacterial formate dehydrogenase reaction

The increase in concentration of organic cosolvents results in a 2-2.5-fold increase of the maximal reaction rate and a decrease of Michaelis constant for formate of NAD(+)-dependent formate dehydrogenase from methylotrophic bacteria Pseudomonas sp. 101. These parameters, however, are not affected with the increase of ionic strength. For the logarithm of both Vmax and Km a linear function of the reciprocal of solvent dielectric permittivity was found. The decrease of Km is possibly due to the dielectric screening effect on the substrate binding energy. The increase in Vmax is explained by a model based on a solvent-dependent electrostatic image force, acting on the charges moved in the course of the catalytic step of the enzyme reaction.     

Golgi β-glucuronidase of androgen-stimulated mouse kidney

The equilibrium constant of the phosphoglyceromutase reaction was determined over a range of pH (5.4-7.9), in solutions of different ionic strength (0.06-0.3) and in the presence of Mg(2+), at 30 degrees C and at 20 degrees C. The values obtained (8.65-11.65) differ substantially from previously published values. The third acid dissociation constants were redetermined for 2- and 3-phosphoglycerate, and in contrast with previous reports the pK values (7.03 and 6.97 respectively at zero ionic strength) were closely similar. The Mg(2+)-binding constants were measured spectrophotometrically and the values, 286mm(-1) and 255mm(-1) for 2- and 3-phosphoglycerate at pH7 and ionic strength 0.02, were also very similar. From the relative lack of effect of temperature, pH and ionic strength it is concluded that the equilibrium constant differs from unity largely because of entropic factors. At low ionic strength, in the neutral region, the pH-dependence can be attributed to the small difference in the acid dissociation constants, but the difference in dissociation constants does not explain the pH-dependence in the acid region or at high ionic strength. Within physiological ranges of pH, Mg(2+) concentration and ionic strength there will be little variation in equilibrium constant.     

Viscosity as an additional physico-chemical parameter for studying chromatin structure

The viscosity values of chromatin in higher order structures, which range from 0.1 to 0.4 dl/g, are considerably lower than those of isolated DNA. These low values are consistent with other physico-chemical parameters, such as sedimentation and diffusion coefficients. When deducing molecular mass and compact shape of chromatin molecules in solvents of nearly physiological ionic strength, all these parameters are in general agreement. A decrease in ionic strength increases viscosity and decreases s-value. Both effects are consistent with a chromatin model postulating a very compact quaternary structure which unravels in low ionic environment to an unfolded but not completely extended tertiary structure.     

Kinetics of reduction of high redox potential ferredoxins by the semiquinones of Clostridium pasteurianum flavodoxin and exogenous flavin mononucleotide. Electrostatic and redox potential effects

We have measured the ionic strength dependence of the rate constants for the electron-transfer reactions of flavin mononucleotide (FMN) and flavodoxin semiquinones with 10 high redox potential ferredoxins (HiPIP's). The rate constants were extrapolated to infinite ionic strength by using a theoretical model of electrostatic interactions developed in our laboratory. In all cases, the sign of the electrostatic interaction was the same as the protein net charge, but the magnitudes were much smaller. The results are consistent with a model in which the electrical charges are approximately uniformly distributed over the HiPIP surface and in which there are both short- and long-range electrostatic interactions. An electrostatic field calculation for Chromatium vinosum HiPIP is consistent with this. The presumed site of electron transfer includes that region of the protein surface to which the iron-sulfur cluster is nearest and appears to be relatively hydrophobic. The principal short-range electrostatic interaction would involve the negative charge on the iron-sulfur cluster. For some net negatively charged proteins, this effect is magnified, and for net positively charged HiPIP's, it is counterbalanced. The rate constants extrapolated to infinite ionic strength can be correlated with redox potential differences between the reactants, as has previously been shown for cytochrome-flavin semiquinone reactions. Both electrostatic and redox potential effects are magnified for the flavodoxin semiquinone as compared to the FMN semiquinone-HiPIP reactions. This was also observed previously for the flavin semiquinone-cytochrome reactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Design and characterization of the anion-sensitive coiled-coil peptide.

As a model for analyzing the role of charge repulsion in proteins and its shielding by the solvent, we designed a peptide of 27 amino acid residues that formed a homodimeric coiled-coil. The interface between the coils consisted of hydrophobic Leu and Val residues, and 10 Lys residues per monomer were incorporated into the positions exposed to solvent. During the preparation of a disulfide-linked dimer in which the two peptides were linked in parallel by the two disulfide bonds located at the N and C terminals, a cyclic monomer with an intramolecular disulfide bond was also obtained. On the basis of CD and 1H-NMR, the conformational stabilities of these isomers and several reference peptides were examined. Whereas all these peptides were unfolded in the absence of salt at pH 4.7 and 20 degrees C, the addition of NaClO4 cooperatively stabilized the alpha-helical conformation. The crosslinking of the peptides by disulfide bonds significantly decreased the midpoint salt concentration of the transition. The 1H-NMR spectra in the presence of NaClO4 suggested that, whereas the disulfide-bonded dimer assumed a native-like conformation, the cyclic monomer assumed a molten globule-like conformation with disordered side chains. However, the cyclic monomer exhibited cooperative transitions against temperature and Gdn-HCl that were only slightly less cooperative than those of the disulfide-bonded parallel dimer. These results indicate that the charge repulsion critically destabilizes the native-like state as well as the molten globule-like state, and that the solvent-dependent charge repulsion may be useful for controlling the conformation of designed peptides.     

Acetylcholinesterase: diffusional encounter rate constants for dumbbell models of ligand.

For some enzymes, virtually every substrate molecule that encounters the entrance to the active site proceeds to reaction, at low substrate concentrations. Such diffusion-limited enzymes display high apparent bimolecular rate constants ((kcat/KM)), which depend strongly upon solvent viscosity. Some experimental studies provide evidence that acetylcholinesterase falls into this category. Interestingly, the asymmetric charge distribution of acetylcholinesterase, apparent from the crystallographic structure, suggests that its electrostatic field accelerates the encounter of its cationic substrate, acetylcholine, with the entrance to the active site. Here we report simulations of the diffusion of substrate in the electrostatic field of acetylcholinesterase. We find that the field indeed guides the substrate to the mouth of the active site. The computed encounter rate constants depend upon the particular relative geometries of substrate and enzyme that are considered to represent successful encounters. With loose reaction criteria, the computed rates exceed those measured experimentally, but the rate constants vary appropriately with ionic strength. Although more restrictive reaction criteria lower the computed rates, they also lead to unrealistic variation of the rate constants with ionic strength. That these simulations do not agree well with experiment suggests that the simple diffusion model is incomplete. Structural fluctuations in the enzyme or events after the encounter may well contribute to rate limitation.     

Effects of solvent and ionic medium on the kinetics of axial ligand substitution in vitamin B12. Part II. the reaction between aquocobalamin and thiourea in dioxane-water and acetonitrile-water mixtures

The rate constants for the reaction of aquocobalamin with thiourea were measured as a function of ionic strength, pH and solvent composition in dioxane-water and acetonitrile-water mixtures. With the help of solubility measurements a complete quantitative analysis of solvent effects on the reaction profile could be made. The transfer Gibbs energy of the initial state strongly depends on solvent composition. Because the transition state and the final state closely follow the initial state, this is not reflected in the rate constants.For the acetonitrile-water mixtures the transfer enthalpy and transfer entropy were determined and were found to exhibit the familiar compensation effect.It is concluded that, when the solvent changes, vitamin B₁₂ creates its own micro-environment around the active metal site, so that the reactivity is effectively solvent independent. The mechanism of activation is dissociative.     

Pharmacological and kinetic analysis of K channel gating currents

We have measured gating currents from the squid giant axon using solutions that preserve functional K channels and with experimental conditions that minimize Na channel contributions to these currents. Two pharmacological agents were used to identify a component of gating current that is associated with K channels. Low concentrations of internal Zn2+ that considerably slow K channel ionic currents with no effect on Na channel currents altered the component of gating current associated with K channels. At low concentrations (10-50 microM) the small, organic, dipolar molecule phloretin has several reported specific effects on K channels: it reduces K channel conductance, shifts the relationship between channel conductance and membrane voltage (Vm) to more positive potentials, and reduces the voltage dependence of the conductance-Vm relation. The K channel gating charge movements were altered in an analogous manner by 10 microM phloretin. We also measured the dominant time constants of the K channel ionic and gating currents. These time constants were similar over part of the accessible voltage range, but at potentials between -40 and 0 mV the gating current time constants were two to three times faster than the corresponding ionic current values. These features of K channel function can be reproduced by a simple kinetic model in which the channel is considered to consist of two, two-state, nonidentical subunits.     

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.     

Axial pertubations on the electronic and magnetic properties of ferric porphyrins: II. Solvent effects on the proton NMR spectra of low-spin cyano complexes

The proton NMR spectra of a series of low-spin bis-cyano ferric complexes of tetraarylporphyrins and octaethylporphyrin in a variety of solvents have been recorded and analyzed. The hyperfine shifts are shown to be very sensitive to the solvent, experiencing an overall downfield bias as the solvent hydroge-bonding donor strength increased. The characteristic pattern of the contact and dipolar shifts for the meso-aryl group in tetraarylporphyrin complexes are shown to permit a quantitative separation of the dipolar and contact contributions to the hyperfine shift. The separated components indicate that increased solvent hydrogen bonding strength significantly decreases the magnetic anisotropy of the iron and diminishes porphyrin → iron π bonding. The changes in anisotropy with solvent are shown to be consistent with the coordinated cyanide acting as a proton acceptor. Although similar effects are found to be absent in bis-imidazole complexes, a downfield bias of half the magnitude of the bis-cyano complexes is observed in mixed cyano/imidazole complexes. Hence, the heme hyperfine shifts in cyano-metmyoglobins and -hemoglobins may serve as probes for the protonation of the distal histidyl imidazole.