Mg2+-induced proton release from Escherichia coli ribosome and ribosomal RNA

Escherichia coli ribosome released protons upon addition of Mg2+. The Mg2+-induced proton release was studied by means of the pH-stat technique. The number of protons released from a 70 S ribosome in the Mg2+ concentration range 1-20 mM was about 30 at pH 7 and 7.6, and increased to about 40 at pH 6.5. The rRNA mixture extracted from 70 S ribosome showed proton release of amount and of pH dependence similar to those of the 70 S ribosome but the ribosomal protein mixture released few. This indicates that rRNA is the main source of the protons released from ribosome. The pH titration of rRNA showed that the pKa values of nucleotide bases were downward shifted upon Mg2+ binding. This pKa shift can account for the proton release. The Scatchard plots of proton release from rRNA and ribosome were concave upward, showing that the Mg2+-binding sites leading to proton release were either heterogeneous or had a negative cooperativity. A model assuming heterogeneous Mg2+-binding sites is shown to be unable to explain the proton release. Electrostatic field effect models are proposed in which Mg2+ modulates the electrostatic field of phosphate groups and the potential change induces a shift of the pKa values of bases that leads to the proton release. These models can explain the main features of the proton release.     

Thermodynamics of cation binding to rabbit skeletal muscle calsequestrin. Evidence for distinct Ca(2+)- and Mg(2+)-binding sites

Ca2+ binding to rabbit skeletal calsequestrin was studied at physiological ionic strength by equilibrium flow dialysis, Hummel-Dryer gel filtration and microcalorimetry. 31 Ca(2+)-binding sites with a mean dissociation constant (KD) of 0.79 mM were titrated in the absence, and 23 sites with a KD of 0.88 mM in the presence of 3 mM Mg2+. No cooperativity was observed. For Mg2+ binding, the combination of gel filtration and microcalorimetry yielded a stoichiometry of 26 Mg2+/protein with a KD of 2mM. 1 mM Ca2+ decreased the stoichiometry to 20 Mg2+/protein. Binding of Ca2+ in the absence and presence of 3 mM Mg2+ was accompanied by a release of 2.0 and 2.7 H+/protein, respectively. Mg2+ binding did not lead to a significant proton release suggesting a qualitative difference in the Ca(2+)- and Mg(2+)-binding sites. After correction for proton release, the enthalpy change for Ca2+ binding was very low (-1.5 kJ/protein in the absence, and -15 kJ/protein in the presence of 3 mM Mg2+). The entropy change (+59 J/K.site in the absence and +56 J/K.site in the presence of Mg2+) was therefore virtually the sole driving force for Ca2+ binding. Mg2+ binding is slightly more exothermic (-12.6 kJ/protein), but as for Ca2+, the entropy change (+50 J/K.site) constituted the major driving force of the reaction. A fluorimetric study indicates that the conformation of tryptophan in Mg(2+)-saturated calsequestrin was clearly different from that in the Ca(2+)-saturated protein, but that the (Ca2+ + Mg2+)-saturated protein was not distinct from the Ca(2+)-saturated protein. Thus, in addition to the thermodynamic characterization of the Ca2+/calsequestrin interaction, our data indicate that Ca2+ and Mg2+ do not bind to the same sites on calsequestrin. The data also predict considerable proton fluxes upon Ca(2+)-Mg2+ exchange in vivo.     

Removal of Mn from spinach chloroplasts by sodium cyanide and the binding of Mn2+ to Mn-depleted chloroplasts.

Manganese and copper were released from spinach chloroplasts by NaCN-treatment, though iron was not affected. The Hill reaction activity was also inhibited by this treatment, but was partially recovered by the addition of either Mn2+ or Cu2+, but not of Fe3+. The interaction of Mn2+ with manganese-depleted chloroplasts by NaCN-treatment was studied using 54Mn2+. A Scatchard plot shows the high and low affinity binding sites of Mn2+ on NaCN-treated chloroplast membrane; high affinity binding being specific for NaCN-treated chloroplast with a binding constant, KH, of 1.9 X 10(5) M-1, and a maximum binding number, NH, of 0.0016 g-atom per mole of chlorophyll. The low binding site was also found on untreated chloroplasts; its binding constant, KL, being 1.2 X 10(4) M-1, and its maximum binding number, NL, of 0.0112 g-atom per mole oc chlorophyll at pH 8.2 NH was proportional to the degree of the removal of Mn by NaCN-treatment and was constant at pH 4--9. NL markedly increased at a high pH with a midpoint of pH 7.9 indicating the exposure of a new, similar binding site. Light illumination partially inhibited the binding of Mn2+. Within 1 min in the dark the binding reaction reached equilibrium in the absence of pyrophosphate, however, 20 min were required to transform into pyrophosphate-resistant form. The pH dependence of the binding of Mn2+ with pKa 7.2 and the ineffectiveness of p-chloromercuribenzoate suggest the possible ligand of Mn2+ is the imidazole nitrogen of the histidine residue.     

Iron as a bound secondary electron donor in modified bacterial reaction centers

The binding and oxidation of ferrous iron were studied in wild-type reaction centers and in mutants that have been modified to be both highly oxidizing and able to bind manganese [Thielges et al. (2005) Biochemistry 44, 7389-7394]. After illumination of wild-type reaction centers, steady-state optical spectroscopy showed that the oxidized bacteriochlorophyll dimer, P+, could oxidize iron but only as a second-order reaction at iron concentrations above 100 microM. In the modified reaction centers, P+ was reduced by iron in the presence of sodium bicarbonate with dissociation constants of approximately 1 microM for two mutants with different metal-binding sites. Transient optical spectroscopy showed that P+ was rapidly reduced with first-order rates of 170 and 275 s-1 for the two mutants. The dependence of the amplitude of this rate on the iron concentration yielded a dissociation constant of approximately 1 microM for both mutants, in agreement with the steady-state determination. The oxidation of bound iron by P+ was confirmed by the observation of a light-induced EPR signal centered at g values of 2.2 and 4.3 and attributed to high-spin Fe3+. Bicarbonate was required at pH 7 for low dissociation constants for both iron and manganese binding. The similarity between iron and manganese binding in these mutants provides insight into general properties of metal-binding sites in proteins.     

Electron spin resonance and magnetic relaxation studies of gadolinium(III) complexes with human transferrin

A human serum transferrin complex was prepared in which Gd(III) was substituted for Fe(III) at the two metal-binding sites. Characteristic changes upon metal binding in both the UV absorption of ligated tyrosines and the solvent proton longitudinal magnetic relaxation rates demonstrated 2/1 metal stoichiometry and pH-dependent binding constants. Binding studies were complicated both by binding of Gd(III) to nonspecific sites on transferrin at pH less than or equal to 7 and by complexation of the Gd(III) by the requisite bicarbonate anion at pH greater than or equal to 6.0. A unique Gd(III) electron spin resonance spectrum, with a prominent signal at g = 4.96, was observed for the specific Gd(III)-transferrin complex. The major features of this spectrum were fit successfully by a model Hamiltonian which utilized crystal field parameters similar to those determined for Fe(III) in transferrin [Aasa, R. (1970) J. Chem. Phys. 52, 3919-3924]. The magnetic field dependence of the solvent proton relaxation rate was measured as a function of both pH and metal ion concentration. An observed biphasic dependence of the relaxation rate on metal concentration is attributed to either sequential metal binding to the two iron-binding sites with different relaxation properties or random binding to two sites that are similar but show conformationally induced changes in relaxation properties as the second metal is bound. The increase in the solvent proton relaxation rate with pH is consistent with a model in which a proton of a second coordination sphere water molecule is hydrogen bonded to a metal ligand which becomes deprotonated at pH 8.5.     

Interaction of calcium with bovine plasma protein C

The binding of 45Ca2+ to bovine plasma protein C (PC) and to activated bovine plasma protein C (APC) has been examined by equilibrium ultrafiltration at pH 7.4 and 25 degrees C. Under these conditions, PC possesses 16.0 plus or minus 2.0 equivalent Ca2+ binding sites, of average KD (8.7 plus or minus 1.5) x 10(-4) M, and APC contains 9.0 plus or minus 1.0 equivalent Ca2+ binding sites, with an average KD of (4.3 plus or minus 1.1) x 10(-4) M. Both Mn2+ and Sr2+ were capable of ready displacement of Ca2+ from a Ca2+-PC complex, while Mg2+ was less effective in this regard. The alpha-thrombin-catalyzed activation of PC was inhibited by the presence of Ca2+. A kinetic analysis of this effect demonstrated that it was, in large part, due to an increase in the Km of the reaction. Addition of other divalent cations, e.g. Mn2+, Sr2+, and Mg2+, in place of Ca2+ also resulted in inhibition of the alpha-thrombin-catalyzed activation of PC in a manner which paralleled their ability to displace Ca2+ from a Ca2+-PC complex. On the other hand, the activation of PC by the coagulant protein from Russell's Viper venom was augmented by the presence of Ca2+. Other divalent metal ions, such as Sr2+ and Mn2+, in the absence of Ca2+, also weakly stimulated this reaction. Mg2+ was without notable effect.     

Studies of the pH dependence of the formation of binary and ternary complexes with liver alcohol dehydrogenase

The association of the coenzyme NAD+ to liver alcohol dehydrogenase (LADH) is known to be pH dependent, with the binding being linked to the shift in the pK of some group on the protein from a value of 9-10, in the free enzyme, to 7.5-8 in the LADH-NAD+ binary complex. We have further characterized the nature of this linkage between NAD+ binding and proton dissociation by studying the pH dependence (pH range 6-10) of the proton release, delta n, and enthalpy change, delta Ho(app), for formation of both binary (LADH-NAD+) and ternary (LADH-NAD+-I, where I is pyrazole or trifluoroethanol) complexes. The pH dependence of both delta n and delta Ho(app) is found to be consistent with linkage to a single acid dissociating group, whose pK is perturbed from 9.5 to 8.0 upon NAD+ binding and is further perturbed to approximately 6.0 upon ternary complex formation. The apparent enthalpy change for NAD+ binding is endothermic between pH 7 and pH 10, with a maximum at pH 8.5-9.0. The pH dependence of the delta Ho(app) for both binary and ternary complex formation is consistent with a heat of protonation of -7.5 kcal/mol for the coupled acid dissociating group. The intrinsic enthalpy changes for NAD+ binding and NAD+ plus pyrazole binding to LADH are determined to be approximately 0 and -11.0 kcal/mol, respectively. Enthalpy change data are also presented for the binding of the NAD+ analogues adenosine 5'-diphosphoribose and 3-acetylpyridine adenine dinucleotide.     

Differences in the protein fluorecence of the two iron(III)-binding sites of ovotransferrin.

1. Changes in the tryptophan fluorescence and the visible absorption spectrum resulting from the combination of apo-ovotransferrin with Fe3+, F,E2+, Cu2+, Zn2+, Mn2+, and Cd2+were measured. 2. As expected for a radiationless transfer of electronic excitation energy, only the ions Fe3+, Fe2+and Cu2+, which gave complexes with large extinctions between 300 and 370nm, resulted in large decreases in trytophan fluorescence. 3. The decrease in protein fluorescence was non-linear with increasing occupancy of the Fe3+ -and Cu2+ - binding sites. The decrease in fluorescence on binding of Fe3+ was biphasic and showed that the two metal-binding sites were being occupied sequentially at pH7.4-8.4. The first site reacted with Fe3+ instantaneously, the second was occupied over a minute. 5. The nonidentity of the two sites was also demonstrated by the preparation of a stable hybrid containing both Cu2+ and Zn2+.h Cu2+ and Zn2+     

Metal binding to DNA polymerase I, its large fragment, and two 3',5'-exonuclease mutants of the large fragment

DNA polymerase I (Pol I) is an enzyme of DNA replication and repair containing three active sites, each requiring divalent metal ions such as Mg2+ or Mn2+ for activity. As determined by EPR and by 1/T1 measurements of water protons, whole Pol I binds Mn2+ at one tight site (KD = 2.5 microM) and approximately 20 weak sites (KD = 600 microM). All bound metal ions retain one or more water ligands as reflected in enhanced paramagnetic effects of Mn2+ on 1/T1 of water protons. The cloned large fragment of Pol I, which lacks the 5',3'-exonuclease domain, retains the tight metal binding site with little or no change in its affinity for Mn2+, but has lost approximately 12 weak sites (n = 8, KD = 1000 microM). The presence of stoichiometric TMP creates a second tight Mn2+ binding site or tightens a weak site 100-fold. dGTP together with TMP creates a third tight Mn2+ binding site or tightens a weak site 166-fold. The D424A (the Asp424 to Ala) 3',5'-exonuclease deficient mutant of the large fragment retains a weakened tight site (KD = 68 microM) and has lost one weak site (n = 7, KD = 3500 microM) in comparison with the wild-type large fragment, and no effect of TMP on metal binding is detected. The D355A, E357A (the Asp355 to Ala, Glu357 to Ala double mutant of the large fragment of Pol I) 3',5'-exonuclease-deficient double mutant has lost the tight metal binding site and four weak metal binding sites. The binding of dGTP to the polymerase active site of the D355A,E357A double mutant creates one tight Mn2+ binding site with a dissociation constant (KD = 3.6 microM), comparable with that found on the wild-type enzyme, which retains one fast exchanging water ligand. Mg2+ competes at this site with a KD of 100 microM. It is concluded that the single tightly bound Mn2+ on Pol I and a weakly bound Mn2+ which is tightened 100-fold by TMP are at the 3',5'-exonuclease active site and are essential for 3',5'-exonuclease activity, but not for polymerase activity. Additional weak Mn2+ binding sites are detected on the 3',5'-exonuclease domain, which may be activating, and on the polymerase domain, which may be inhibitory. The essential divalent metal activator of the polymerase reaction requires the presence of the dNTP substrate for tight metal binding indicating that the bound substrate coordinates the metal.(ABSTRACT TRUNCATED AT 400 WORDS)     

钙调神经磷酸酶B亚基及钙调素的EPR研究

根据顺磁离子Mn~(2+)的取代特性,用EPR方法研究了钙调神经磷酸酶B亚基与其4个Ca~(2+)的结合位点,以及它们亲和力的细微差别。并同时进行了钙调素的对比研究。实验和Scatchard作图表明,B亚基有4个Ca~(2+)结合位点,2个高亲和力结合位点,其解离常数为4×10~(-6)mol/L;2个低亲和力结合位点,解离常数为9×10~(-5)mol/L。钙调素也有2个Ca~(2+)高亲和力结合位点,其解离常数为8×10~(-6)mol/L,2个低亲和力结合位点,解离常数为7×10~(-5)mol/L。钙调神经磷酸酶B亚基和钙调素Mn~(2+)结合位点的EPR研究对B亚基和钙调素在共同调节钙调神经磷酸酶中的作用提供了有用的信息。     

Binding of divalent cations with low density lipoproteins. A study by ESR

The binding of bivalent metal ions Cu2+, Zn2+, Ca2+, Mg2+ to low-density lipoproteins (LDL) was investigated by the ESR technique. The monitoring of ESR spectra of paramagnetic Mn2+ ions in the presence of above-listed cations made it possible to evaluate the dissociation constants of their complexes with LDL. The effective dissociation constant of the complex Mn(2+)-LDL used for calculations was KD = (1.1 +/- 0.4) x 10(-4) M according to literature data. The investigated cations may be classified into two groups: 1) low dissociation constants were characteristic for Cu2+ ions [KD = (1.3 +/- 0.5) x 10(-4) M], which demonstrated a high oxidative ability, and for Zn2+ [KD = (0.95 +/- 0.45) x 10(-4) M] and Mn2+ ions, which could strongly influence the copper-induced LDL oxidation; 2) Ca2+ and Mg2+ were characterized by higher values of KD [(6 +/- 1) x 10(-4) M and (7.5 +/- 1.5) x 10(-4) M, accordingly] and slightly affected the Cu(2+)-induced oxidation of LDL. The results of the present work reinforced our earlier conjecture that cations may influence the process of lipid peroxidation, binding only to particular binding sites on the surface of LDL.     

Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site.     

Binding of thiocyanate and cyanide to manganese(III)-reconstituted horseradish peroxidase: a 15N nuclear magnetic resonance study

Binding of thiocyanate and cyanide ions to Mn(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by relaxation rate measurements (at 50.68 MHz) of 15N resonance of SC15N- and C15N-. At pH = 4.0 the apparent dissociation constant (KD) for thiocyanate and cyanide binding to Mn(III)HRP was deduced to be 156 and 42 mM, respectively. The pH dependence of the 15N line width as well as apparent dissociation constant for thiocyanate and cyanide binding were quantitatively analyzed on the basis of a reaction scheme in which thiocyanate and cyanide in deprotonated form bind to the enzyme in a protonated form. The binding of thiocyanate and cyanide to Mn(III)HRP was found to be facilitated by protonation of an ionizable group on the enzyme [Mn(III)HRP] with a pKa = 4.0. From competitive binding studies it was shown that iodide, thiocyanate and cyanide bind to Mn(III)HRP at the same site; however, the binding site for resorcinol is different. The apparent dissociation constant for iodide binding deduced from competitive binding studies was found to be 117 mM, which agrees very well with the iodide binding to ferric HRP. The binding of thiocyanate and cyanide was shown to be away from the metal center and the distance of the 15N of thiocyanate and cyanide from the paramagnetic manganese ion in Mn(III)HRP was found to be 6.9 and 6.6 A, respectively. Except for cyanide binding, these observations parallel with the iodide and thiocyanate ion binding to native Fe(III)HRP. Water proton relaxivity measurements showed the presence of a coordinated water molecule to Mn(III)HRP with the distance of Mn-H2O being calculated to be 2.6 A. The slow reactivity of H2O2 towards Mn(III)HRP could be attributed to the presence of water at the sixth coordination position of the manganese ion.     

Calculating proton uptake/release and binding free energy taking into account ionization and conformation changes induced by protein-inhibitor association: application to plasmepsin, cathepsin D and endothiapepsin-pepstatin complexes

The protein-inhibitor binding energies of enzymes are often pH dependent, and binding induces either proton uptake or proton release. The proton uptake/release and the binding energy for three complexes with available experimental data were numerically studied: pepstatin-cathepsin D, pepstatin-plasmepsin II and pepstatin-endothiapepsin. Very good agreement with the experimental data was achieved when conformational changes were taken into account. The role of the desolvation energy and the conformational changes was revealed by modeling the complex, the separated molecules in the complex conformation and the free molecules. It was shown that the conformational changes induced by the complex formation are as important for the proton transfer as the loss of solvation energy caused by the burial of interface residues. The residues responsible for the proton transfer were identified and their contribution to the proton uptake/release calculated. These residues were found to be scattered along the whole protein rather than being localized only at the active site. In the case of cathepsin D, these residues were found to be highly conserved among the cathepsin D sequences of other species. It was shown that conformation and ionization changes induced by the complex formation are critical for the correct calculation of the binding energy. Taking into account the electrostatics and the van der Waals (vdW) energies within the Boltzmann distribution of energies and allowing ionization and conformation changes to occur makes the calculated binding energy more realistic and closer to the experimental value. The interplay between electrostatic and vdW forces makes the pH dependence of the binding energy smoother, because the vdW force acts in reaction to the changes of the electrostatic energy. It was found that a small fraction of the ionizable groups remain uncharged in both the free and complexed molecules. The sequence and structural position of these groups aligns well within the three proteases, suggesting that these may have specific role.     

Interaction of thiocyanate with horseradish peroxidase. 1H and 15N nuclear magnetic resonance studies

Interaction of thiocyanate with horseradish peroxidase (HRP) was investigated by relaxation rate measurements (at 50.68 MHz) of the 15N resonance of thiocyanate nitrogen and by following the hyperfine shifted ring methyl proton resonances (at 500 MHz) of the heme group of SCN-.HRP solutions. At pH 4.0, the apparent dissociation constant (KD) for thiocyanate binding to HRP was deduced to be 158 mM from the relaxation rate measurements. Chemical shift changes of 1- and 8-ring methyl proton resonances in the presence of various amounts of thiocyanate at pH 4.0 yielded KD values of 166 and 136 mM, respectively. From the pH dependence of KD and the 15N resonance line width, it was observed that thiocyanate binds to HRP only under acidic conditions (pH less than 6). The binding was found to be facilitated by protonation of an acid group on the enzyme with pKa 4.0. The pH dependence of the 15N line width as well as the apparent dissociation constant were quantitatively analyzed on the basis of a reaction scheme in which thiocyanate in deprotonated ionic form binds to the enzyme in protonated acidic form. The KD for thiocyanate binding to HRP was also evaluated in the presence of an excess of exogenous substrates such as resorcinol, cyanide, and iodide ions. It was found that the presence of cyanide (which binds to heme iron at the sixth coordination position) and resorcinol did not have any effect on the binding of thiocyanate, indicating that the binding site of the thiocyanate ion is located away from the ferric center as well as from the aromatic donor binding site. The KD in the presence of iodide, however, showed that iodide competes with thiocyanate for binding at the same site. The distance of the bound thiocyanate ion from the ferric center was deduced from the 15N relaxation time measurements and was found to be a 6.8 A. From the distance as well as the change in the chemical shifts and line width of 1- and 8-methyl proton resonances, it is suggested that the binding site of thiocyanate may be located near heme, placed symmetrically with respect to 1- and 8-methyl groups of the heme of HRP. Similarity in the modes of binding of iodide and thiocyanate suggests that the oxidation of thiocyanate ion by H2O2 may also proceed via the two-electron transfer pathway under acidic conditions, as is the case for iodide.     

Retardation of proton transfer caused by binding of the transition metal ion to the bacterial reaction center is due to pKa shifts of key protonatable residues

Transition metal ions bind to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides and slow the light-induced electron and proton transfer to the secondary quinone, Q(B). We studied the properties of the metal ion-RC complex by measuring the pH dependence of the dissociation constant and the stoichiometry of proton release upon ligand formation. We investigated the mechanism of inhibition by measuring the stoichiometry and kinetics of flash-induced proton binding, the transfer of (first and second) electrons to Q(B), and the rate of steady-state turnover of the RC in the absence and presence of Cd(2+) and Ni(2+) on a wide pH range. The following results were obtained. (1) The complexation of transition metal ions Cd(2+) and Ni(2+) with the bacterial RC showed strong pH dependence. This observation was explained by different (pH-dependent) states of the metal-ligand cluster: the complex formation was strong when the ligand (Asp and His residues) was deprotonated and was much weaker if the ligand was partly (or fully) protonated. A direct consequence of the model was the pH-dependent proton release upon complexation. (2) The retardation of transfer of electrons and protons to Q(B) was also strongly pH-dependent. The effect was large in the neutral pH range and decreased toward the acidic and alkaline pH values. (3) Steady-state turnover measurements indicated that the rate of the second proton transfer was much less inhibited than that of the first one, which became the rate-limiting step in continuous turnover of the RC. (4) Sodium azide partly recovered the proton transfer rate. The effect is not due to removal of the bound metal ion by azide but probably by formation of a proton-transporting azide network similarly as water molecules may build up proton pathways. (5) We argue that the inhibition comes mainly from pK(a) shifts of key protonatable residues that control the proton transfer along the H-bond network to Q(B). The electrostatic interaction between the metal ion and these residues may result in acidic pK(a) shifts between 1.5 and 2.0 that account for the observed retardation of the electron and proton transfer.     

Interaction of divalent metal ions with human serum albumin

ESR study was carried out of the interaction between Zn2+, Cu2+, Ca2+, Mg2+ ions and human serum albumin (HSA) in the presence of Mn2+ ions which depends on pH. Competitive binding of these ions with "manganese-binding" sites of albumin was shown to depend on pH. An analysis of concentration dependence of binding these ions with human serum albumin confirmed earlier supposition about the nature of the binding sites of Mn2+ ions with HSA.     

Proton release during successive oxidation steps of the photosynthetic water oxidation process: stoichiometries and pH dependence.

Flash-induced absorption changes of pH-indicating dyes were investigated in photosystem II enriched membrane fragments, in order to retrieve the individual contributions to proton release of the successive transitions of the Kok cycle. These stoichiometric coefficients were found to be, in general, noninteger and to vary as a function of pH. Proton release on the S0----S1 step decreases from 1.75 at pH 5.5 to 1 at pH 8, while, on S1----S2 the stoichiometry increases from 0 to 0.5 in the same pH range and remains close to 1 for S2----S3. These findings are analyzed in terms of pK shifts of neighboring amino acid residues caused by electrostatic interactions with the redox centers involved in the two first transitions. The electrochromic shift of a chlorophyll, associated with the S transitions, responding to local electrostatic effects was investigated under similar conditions. The pH dependence of this signal upon the successive transitions was found correlated with the titration of the proton release stoichiometries, expressing the electrostatic balance between the oxidation and deprotonation processes.     

Binding of beta-scorpion toxin: a physicochemical study

The binding to rat brain synaptosomes of a beta-scorpion toxin, i.e., toxin II of Centruroides suffusus suffusus (Css II), was studied as a function of pH, temperature, and concentration of some monovalent and divalent cations. At 10 degrees C and pH 6.0, the specific binding of 125I-labeled Css II corresponds to a single class of noninteracting high-affinity binding sites (KD = 0.18 nM) with a capacity (4.2 pmol/mg of protein) that is almost identical with that generally accepted for saxitoxin. The equilibrium dissociation constant of beta-scorpion toxin is pH independent, but the maximum binding capacity is reduced with increasing pH. Li+, guanidinium, Ca2+, Mg2+, and Mn2+ modified the apparent KD of the 125I-labeled Css II toxin. The equilibrium dissociation constant varies markedly with the temperature. The van't Hoff plot of the data is curvilinear, corresponding to a standard free-energy change associated with an entropy-driven process. The association rate constant also varies considerably with the temperature whereas the Arrhenius plot is linear between 1 and 30 degrees C. The energy of activation determined from these data is 17.6 kcal/mol. These results support the hypothesis that a cluster of nonpolar amino acid residues present on one face of the molecule is involved in the toxin-receptor interaction.     

Influence of pH on the Mn2+ activation of and binding to yeast enolase: a functional study.

The influence of pH on the activation of yeast enolase by Mn2+ was measured by steady-state kinetics. The pH influence on the binding of Mn2+ to apoenolase and the enolase-substrate complex was measured by EPR spectroscopy. At pH values above 6.6, activation by Mn2+ is fit by Michaelis-Menten kinetics, but at higher concentrations of Mn2+, inhibition is observed. Under conditions analogous to the kinetic studies, the enzyme binds two Mn2+ per dimer with a Kd in the micromolar range. In the presence of the substrate 2-phosphoglycerate, three thermodynamically distinct cation binding sites per monomer are detected and the binding constants are determined by a fit to the data. As the pH decreases, the reaction velocity decreases and the cation inhibition becomes minimal. Under these conditions, only two Mn2+ binding sites per monomer are observed; the third site must be the inhibitory site. The velocity and kinetic constants are minimally affected by buffer except at pH 5.8 with PIPES. Under these conditions, the velocity is only about 40% that observed with other buffers and only a single binding site for Mn2+ per monomer is detected in the presence or absence of substrate. A direct role in the catalytic mechanism by the second cation is called to question. The binding constant for Mn2+ at site I is independent of pH over the range from 7.5 to 5.2, and the binding at site II increases only slightly over this same pH range. These results indicate that the cation sites at positions I and II contain ligands that are pH independent over this range.(ABSTRACT TRUNCATED AT 250 WORDS)