Selective, high-affinity binding of ferric ions by glycine-extended gastrin(17)

Uptake of dietary iron is essential for replenishment of body stores. A role for the hormone gastrin in iron uptake as a chelator of ferric ions in the gastric lumen has been proposed previously [Baldwin, G. S. (1992) Med. Hypotheses 38, 70-74]. Here, spectroscopic evidence of selective, high-affinity binding of ferric ions to progastrin-derived peptides in aqueous solution at low pH is provided. The maximum at 281 nm in the absorption spectrum of glycine-extended gastrin(17) at pH 4.0 increased (2.07 +/- 0.30)-fold in the presence of > or =2 equiv of ferric ions. Titration of glycine-extended gastrin(17) with ferric ions under stoichiometric conditions indicated that the stoichiometry of binding was 2.00 +/- 0.28 mol of Fe(3+)/mol of peptide. Fluorescence quenching experiments yielded values for the stoichiometry and apparent dissociation constant of the ferric ion-glycine-extended gastrin(17) complex at pH 4.0 of 2.39 +/- 0.17 mol of Fe(3+)/mol and 0.62 +/- 0.19 microM, respectively. No interaction was detected with Co(2+), Cu(2+), Mn(2+), or Cr(3+). Electron paramagnetic resonance spectroscopy suggested that the iron ligands were either oxygen or sulfur atoms. Fluorescence quenching experiments with peptides derived from the glycine-extended gastrin(17) sequence indicated that one or more of the five glutamic acid residues were necessary for iron binding. The binding of ferric ions by glycine-extended gastrin(17) at low pH is consistent with a role for progastrin-derived peptides in iron uptake from the lumen of the gastrointestinal tract.     

Structural basis for the observed differential magnetic anisotropic tensorial values in calcium binding proteins

Lanthanide ions (Ln(3+)), which have ionic radii similar to those of Ca(2+), can displace the latter in a calcium binding protein, without affecting its tertiary structure. The paramagnetic Ln(3+) possesses large anisotropic magnetic susceptibilities and produce pseudocontact shifts (PCSs), which have r(-3) dependence. The PCS can be seen for spins as far as 45 A from the paramagnetic ion. They aid in structure refinement of proteins by providing long-range distance constraints. Besides, they can be used to determine the interdomain orientation in multidomain proteins. This is particularly important in the context of a calcium binding protein from Entamoeba histolytica (EhCaBP), which consists of two globular domains connected by a flexible linker region containing 8 residues. As a first step to obtain the interdomain orientation in EhCaBP, a suite of 2D and 3D heteronuclear experiments were recorded on EhCaBP by displacing calcium with Ce(3+), Ho(3+), Er(3+), Tm(3+), Dy(3+), and Yb(3+) ions in separate experiments, and the PCS of (1)H(N) and (15)N spins were measured. Such data have been used in the refinement of the individual domain structures of the protein in parallel with the calculation of the respective magnetic anisotropy tensorial values, which differ substantially (2.1-2.8 times) from what is found in other Ca(2+) binding loops. This study provides a structural basis for such variations in the magnetic anisotropy tensorial values.     

Metal ions binding to NAD-glycohydrolase from the venom of Agkistrodon acutus: regulation of multicatalytic activity

AA-NADase from Agkistrodon acutus venom is a unique multicatalytic enzyme with both NADase and AT(D)Pase activities. Among all identified NADases, only AA-NADase contains Cu(2+) ions that are essential for its multicatalytic activity. In this study, the interactions between divalent metal ions and AA-NADase and the effects of metal ions on its structure and activity have been investigated by equilibrium dialysis, isothermal titration calorimetry, fluorescence, circular dichroism, dynamic light scattering and HPLC. The results show that AA-NADase has two classes of Cu(2+) binding sites, one activator site with high affinity and approximately six inhibitor sites with low affinity. Cu(2+) ions function as a switch for its NADase activity. In addition, AA-NADase has one Mn(2+) binding site, one Zn(2+) binding site, one strong and two weak Co(2+) binding sites, and two strong and six weak Ni(2+) binding sites. Metal ion binding affinities follow the trend Cu(2+) > Ni(2+) > Mn(2+) > Co(2+) > Zn(2+), which accounts for the existence of one Cu(2+) in the purified AA-NADase. Both NADase and ADPase activities of AA-NADase do not have an absolute requirement for Cu(2+), and all tested metal ions activate its NADase and ADPase activities and the activation capacity follows the trend Zn(2+) > Mn(2+) > Cu(2+) ~Co(2+) > Ni(2+). Metal ions serve as regulators for its multicatalytic activity. Although all tested metal ions have no obvious effects on the global structure of AA-NADase, Cu(2+)- and Zn(2+)-induced conformational changes around some Trp residues have been observed. Interestingly, each tested metal ion has a very similar activation of both NADase and ADPase activities, suggesting that the two different activities probably occur at the same site.     

Molecular simulations to determine the chelating mechanisms of various metal ions to the His-tag motif: a preliminary study

In the present study, molecular simulations were performed to investigate the chelating mechanisms of various metal ions to the His-tag motifs with various His residues. The chelation mostly involved the i and i+2 His residues for Ni(2+), Zn(2+), Cu(2+), and Co(2+), while the cooperation of 3 His residues was necessary when Fe(3+) was involved in chelation with His-tags having more than 4 His residues. Metal ion was best fitted into the pocket formed by the imidazole nitrogens while it was about equally located among these nitrogen atoms. His-tag6 was found to have little effect on the structural integrity while the target protein contains more than 68 amino acid residues. Ni(2+) interacted with the imidazole nitrogen of His3 in the beginning of chelation, and then entered into the pocket formed by His3 and His5 at 4 ns during the 10 ns molecular dynamics simulations. The fast chelating process resulted in successful application of IMAC techniques in efficient protein purification.     

Fluorescence study of the interaction of Suwannee River fulvic acid with metal ions and Al3+-metal ion competition

In this study emission and synchronous-scan fluorescence spectroscopy have been used to investigate the interaction of the class A (oxygen seeking 'hard acid') metal Al(3+), with Suwannee River fulvic acid (SRFA), as well as competition between Al(3+) and several other metal ions (Ca(2+), Mg(2+), Cu(2+), Pd(2+), La(3+), Tb(3+) and Fe(3+)) for binding sites on SRFA. Of the four metal ions possessing very similar (and relatively low) ionic indices (Ca(2+), Mg(2+), Cu(2+) and Pd(2+)) only the latter two paramagnetic ions significantly quenched SRFA fluorescence emission intensity. Of the four metal ions possessing very similar (and relatively low) covalent indices (Ca(2+), Mg(2+), La(3+) and Tb(3+)) only the last paramagnetic ion (Tb(3+)) significantly quenched SRFA fluorescence. None of these metals was able to significantly compete with SRFA-bound Al(3+).Fe(3+), which differs substantially from all of the other metals examined in this study in that it possesses a relatively high ionic index (but not as high as Al(3+)) and a relatively low covalent index (but not as low as Al(3+)), was able not only to quench SRFA fluorescence but also to compete (at least to some extent) with SRFA-bound Al(3+). Synchronous-scan fluorescence SRFA spectra taken in the absence and presence of Fe(3+) and/or Al(3+) support the view that these two metal ions can compete for sites on SRFA. The results of these fluorescence experiments further confirm the Al(3+), and metal ions that have electronic properties somewhat similar to Al(3+) (such as Fe(3+)) are somewhat unique in their ability to interact strongly with binding sites on fulvic acids.     

Slow binding of metal ions to pigeon liver malic enzyme: a general case

Pigeon liver malic enzyme was inhibited by lutetium ion through a slow-binding process, which resulted in a concave down tracing of the enzyme activity assay. The fast initial rates were independent of lutetium ion concentration, while the slow steady-state rates decreased with increasing Lu(3+) concentration. The observed rate constant for the transition from initial rate to steady-state rate, k(obs), exhibited saturation kinetics as a function of Lu(3+) concentration, suggesting the involvement of an isomerization process between two enzyme forms (R-form and T-form). The binding affinity of Lu(3+) to the R-form is weaker (K(d,Lu) = 14 microM) than that of Mn(2+) (K(m,Mn) = 1.89 microM); however, Lu(3+) has much tighter binding affinity with the T-form ( = 0.83 microM). Lu(3+) was shown to be a competitive inhibitor with respect to Mn(2+), which suggests that Lu(3+) and Mn(2+) are competing for the same metal binding site of the enzyme. These observations are in accordance with the available crystal structure information, which shows a distorted active site region of the Lu(3+)-containing enzyme. Other divalent cations, i.e., Fe(2+), Cu(2+), or Zn(2+), also act as time-dependent slow inhibitors for malic enzyme. The dynamic quenching constants of the intrinsic fluorescence for the metal-free and Lu(3+)-containing enzymes are quite different, indicating the conformational differences between the two enzyme forms. The secondary structure of these two enzyme forms, on the other hand, was not changed. The above results indicated that replacement of the catalytically essential Mn(2+) by other metal ions leads to a slow conformational change of the enzyme and consequently alters the geometry of the active site. The transformed enzyme conformation, however, is unfavorable for catalysis. Both the chemical nature of the metal ion and its correct coordination in the active site are essential for catalysis.     

Engineering [Ln(DPA)3] 3- binding sites in proteins: a widely applicable method for tagging proteins with lanthanide ions

Paramagnetic relaxation enhancements from unpaired electrons observed in nuclear magnetic resonance (NMR) spectra present powerful long-range distance restraints. The most frequently used paramagnetic tags, however, are tethered to the protein via disulfide bonds, requiring proteins with single cysteine residues for covalent attachment. Here we present a straightforward strategy to tag proteins site-specifically with paramagnetic lanthanides without a tether and independent of cysteine residues. It relies on preferential binding of the complex between three dipicolinic acid molecules (DPA) and a lanthanide ion (Ln³⁺), [Ln(DPA)₃]³⁻, to a pair of positively charged amino acids whose charges are not compensated by negatively charged residues nearby. This situation rarely occurs in wild-type proteins, allowing the creation of specific binding sites simply by introduction of positively charged residues that are positioned far from glutamate or aspartate residues. The concept is demonstrated with the hnRNPLL RRM1 domain. In addition, we show that histidine- and arginine-tags present binding sites for [Ln(DPA)₃]³⁻.     

Binding ability of a HHP-tagged protein towards Ni2+ studied by paramagnetic NMR relaxation: The possibility of obtaining long-range structure information

The binding ability of a protein with a metal binding tag towards Ni(2+) was investigated by longitudinal paramagnetic NMR relaxation, and the possibility of obtaining long-range structure information from the paramagnetic relaxation was explored. A protein with a well-defined solution structure (Escherichia coli thioredoxin) was used as the model system, and the peptide His-His-Pro (HHP) fused to the N-terminus of the protein was used as the metal binding tag. It was found that the tag forms a stable dimer complex with the paramagnetic Ni(2+) ion, where each metal ion binds two HHP-tagged protein molecules. However, it was also found that additional sites in the protein compete with the HHP-tag for the binding of the metal ion. These binding sites were identified as the side chain carboxylate groups of the aspartic and glutamic acid residues. Yet, the carboxylate groups bind the Ni(2+) ions considerably weaker than the HHP-tag, and only protons spatially close to the carboxylate sites are affected by the Ni(2+) ions bound to these groups. As for the protons that are unaffected by the carboxylate-bound Ni(2+) ions, it was found that the long-range distances derived from the paramagnetic relaxation enhancements are in good agreement with the solution structure of thioredoxin. Specifically, the obtained long-range paramagnetic distance constraints revealed that the dimer complex is asymmetric with different orientations of the two protein molecules relative to the Ni(2+) ion.     

Metal ion enhanced binding of AMD3100 to Asp262 in the CXCR4 receptor

The affinity of AMD3100, a symmetrical nonpeptide antagonist composed of two 1,4,8,11-tetraazacyclotetradecane (cyclam) rings connected through a 1,4-dimethylene(phenylene) linker to the CXCR4 chemokine receptor was increased 7, 36, and 50-fold, respectively, by incorporation of the following: Cu(2+), Zn(2+), or Ni(2+) into the cyclam rings of the compound. The rank order of the transition metal ions correlated with the calculated binding energy between free acetate and the metal ions coordinated in a cyclam ring. Construction of AMD3100 substituted with only a single Cu(2+) or Ni(2+) ion demonstrated that the increase in binding affinity of the metal ion substituted bicyclam is achieved through an enhanced interaction of just one of the ring systems. Mutational analysis of potential metal ion binding residues in the main ligand binding crevice of the CXCR4 receptor showed that although binding of the bicyclam is dependent on both Asp(171) and Asp(262), the enhancing effect of the metal ion was selectively eliminated by substitution of Asp(262) located at the extracellular end of TM-VI. It is concluded that the increased binding affinity of the metal ion substituted AMD3100 is obtained through enhanced interaction of one of the cyclam ring systems with the carboxylate group of Asp(262). It is suggested that this occurs through a strong concomitant interaction of one of the oxygen's directly with the metal ion and the other oxygen to one of the nitrogens of the cyclam ring through a hydrogen bond.     

Kinetics of conformational changes induced by the binding of various metal ions to bovine alpha-lactalbumin.

The kinetic effects of the binding of various metal ions (Ca(2+), Cd(2+), Co(2+), Mg(2+), Mn(2+), Sr(2+) and Zn(2+)) to apo bovine alpha-lactalbumin has been monitored by means of stopped-flow fluorescence spectroscopy. Our results show that the measured rate constant for the binding of metal ions to the Ca(2+)-site increases with increasing binding constant. This is, however, not the case for metal ions binding to the Zn(2+)-site. The binding experiments performed at different temperatures allowed us to calculate the activation energy for the transition from the metal-free to the metal-loaded state of the protein. These values do not depend on the nature of the metal ion but are correlated with the type of binding site. As a result, we were able to demonstrate that Mg(2+), a metal ion which was thought to bind to the Ca(2+)-site, shows the same binding characteristics as Co(2+) and Zn(2+) and therefore most likely interacts with the residues belonging to the Zn(2+)-binding site.     

Characterization of calcium binding properties of lithostathine

The pancreas secretes primarily two types of metabolically important proteins: digestive enzymes and hormones. Lithostathine (LIT) is the only protein excreted from the pancreas that has no known digestive or hormonal activity. Human lithostathine is a 144-amino acid glycoprotein synthesized by the exocrine pancreas that has been implicated in various physiological functions, including inhibition of pancreatic stone formation. To better understand the physiological function of LIT, we expressed the recombinant LIT protein in Escherichia coli and measured its calcium binding properties by equilibrium dialysis and electron paramagnetic resonance (EPR) spectroscopy. Equilibrium dialysis with (45)Ca(2+) showed that LIT binds Ca(2+) with 1:1 stoichiometry. EPR studies using the divalent vanadyl (VO(2+)) ion as a paramagnetic substitute for Ca(2+) also showed that VO(2+) binds to LIT with a metal:protein binding stoichiometry of 1:1 and that VO(2+) competes with Ca(2+) in binding to LIT. Mutations of a cluster of acidic residues on the molecular surface (E30A, D31A, E33A, D37A, D72A, and D73A) resulted in almost complete loss (95-100%) of binding of Ca(2+) and VO(2+), showing that these residues are critical for calcium binding by LIT.     

Adverse effects of alkali and acid on the anticoagulant potency of heparin, evaluated with methyl 2-deoxy-2-sulfamino-alpha-D-glucopyranoside 3-sulfate as a model compound.

A variety of chemical modifications can induce a reduction in the anticoagulant activity of heparin. Among such modifications are the removal in alkaline solution of the 2-O-sulfonate group of alpha-L-idopyranosyluronic acid 2-sulfate residues (1) and, in a weakly acidic medium, of the N-sulfonate group of residues of 2-deoxy-2-sulfamino-alpha-D-glucopyranose 6-sulfate (2). This study examined the possibility that the losses in anticoagulant potency are related to a concomitant removal of the 3-O-sulfonate group of residues of 2-deoxy-2-sulfamino-alpha-D-glucopyranose 3,6-disulfate (6) in the AT-III binding site. It entailed a synthesis of methyl 2-deoxy-2-sulfamino-alpha-D-glucopyranoside 3-sulfate (7), as a model compound that was subjected to both the strongly alkaline and weakly acidic conditions appropriate for the modification of residues 1 and 2, respectively. The 3-sulfate group of 7 was found to be highly stable in both environments. This indicated that the adverse effects that these conditions have on the anticoagulant properties of heparin are not specifically associated with the 3-sulfate substituent of residues of 6 in the polymer.     

A relationship between 13C-chemical-shift displacements and counterion-condensation theory, in the binding of calcium ion by heparin

Characteristics of the interaction between heparin and calcium ion in the presence of sodium ion have been examined by monitoring the 13C-chemical shift changes as a function of the calcium ion concentration and the total ionic strength. The results indicated that the association between the polyanion and the divalent cation is a delocalized process, as opposed to one involving specific binding. The correspondence found between chemical shift and the number of Ca2+ ions bound per charged group, as derived from the Manning counterion-condensation model, showed that the stoichiometry is not a constant quantity but, rather, varies throughout the titration, and approaches a limiting value of 2 at high dilution. Additional measurements of T1 and line-width were consistent with an intramolecular order-disorder conformational process induced by the binding of calcium ion. Moreover, binding does not occur or is relatively weak with N-desulfated heparin, or chondroitin 4-sulfate and 6-sulfate, each of which possesses fewer sulfate groups than heparin. These differences serve to emphasize the importance of the charge-density parameter in the control of counterion condensation according to the Manning model, and suggest that the spacing between the negatively charged groups is an associated factor.     

Metal binding sites in proteins: identification and characterization by paramagnetic NMR relaxation

A method is presented that allows the identification and quantitative characterization of metal binding sites in proteins using paramagnetic nuclear magnetic resonance spectroscopy. The method relies on the nonselective longitudinal relaxation rates of the amide protons and their dependence on the paramagnetic metal ion concentration and the pH, and on the three-dimensional structure of the protein. The method is demonstrated using Escherichia coli thioredoxin as a model protein and Ni(2+) as the paramagnetic metal ion. Through a least-squares analysis of the relaxation rates, it is found that Ni(2+) binds to a series of specific sites on the surface of thioredoxin. The strongest binding site is found near the N-terminus of the protein, where the metal ion is coordinated to the free NH(2) group of the N-terminal serine residue and the side chain carboxylate group of the aspartic acid residue in position 2. In addition, Ni(2+) binds specifically but more weakly to the surface-exposed side chain carboxylate groups of residues D10, D20, D47, and E85.     

Picogram detection of metal ions by melanin-sensitized piezoelectric sensor

A heavy metal ion sensor was constructed by cross-linking melanin onto the gold electrode of quartz crystal microbalance (QCM). A mercury ion sensitivity of 518+/-37 Hz/ppm was observed, a substantial increase in sensitivity compared to previous reports of 10-50 Hz/ppm with the limit of detection at 5 ppb. Detection of other metal ions including Sn(2+), Ge(4+), Li(+), Zn(2+), Cu(2+), Bi(3+), Co(2+), Al(3+), Ni(2+), Ag(+), and Fe(3+) were also performed. Unexpectedly, binding of Mn(7+), Pb(2+), Cd(2+), and Cr(3+) increased resonant frequencies. The surface profile of melanin thin film upon binding to metal ions was investigated by atomic force microscopy (AFM). Structural change of melanin upon binding to metal ions was characterized by circular dichroism and by infrared spectroscopy. The current study provides the first example of melanin-coated piezoelectric sensor showing high sensitivity and selectivity to metal ions.     

Copper and zinc binding modulates the aggregation and neurotoxic properties of the prion peptide PrP106-126.

The abnormal form of the prion protein (PrP) is believed to be responsible for the transmissible spongiform encephalopathies. A peptide encompassing residues 106-126 of human PrP (PrP106-126) is neurotoxic in vitro due its adoption of an amyloidogenic fibril structure. The Alzheimer's disease amyloid beta peptide (Abeta) also undergoes fibrillogenesis to become neurotoxic. Abeta aggregation and toxicity is highly sensitive to copper, zinc, or iron ions. We show that PrP106-126 aggregation, as assessed by turbidometry, is abolished in Chelex-100-treated buffer. ICP-MS analysis showed that the Chelex-100 treatment had reduced Cu(2+) and Zn(2+) levels approximately 3-fold. Restoring Cu(2+) and Zn(2+) to their original levels restored aggregation. Circular dichroism showed that the Chelex-100 treatment reduced the aggregated beta-sheet content of the peptide. Electron paramagnetic resonance spectroscopy identified a 2N1S1O coordination to the Cu(2+) atom, suggesting histidine 111 and methionine 109 or 112 are involved. Nuclear magnetic resonance confirmed Cu(2+) and Zn(2+) binding to His-111 and weaker binding to Met-112. An N-terminally acetylated PrP106-126 peptide did not bind Cu(2+), implicating the free amino group in metal binding. Mutagenesis of either His-111, Met-109, or Met-112 abolished PrP106-126 neurotoxicity and its ability to form fibrils. Therefore, Cu(2+) and/or Zn(2+) binding is critical for PrP106-126 aggregation and neurotoxicity.     

1H NMR spectroscopic studies on the interactions between human plasma antithrombin III and defined low molecular weight heparin fragments.

The effects of length and composition upon the antithrombin-binding properties of heparin have been investigated for two series of structurally related heparin oligosaccharides. Each series consists of a tetrasaccharide, hexasaccharide, and octasaccharide heparin fragment composed of alternating hexuronic acid (either iduronate 2-sulfate or glucuronate) and glucosamine 6,N-disulfate residues. These two series represent dominant structural motifs in intact heparin and differ from each other by the presence of a glucuronic acid in one series in place of an iduronate 2-sulfate residue penultimate to the reducing end of the fragment. Perturbations to the 1H resonances in the NMR spectrum of antithrombin upon binding of the two series of heparin fragments are compared to those generated by intact heparin binding, as well as to the effects of binding of a synthetic high-affinity pentasaccharide. All of the heparin fragments examined appear to bind to antithrombin at the same site. Three of the heparin fragments (hexasaccharide-2, octasaccharide-2, and octasaccharide-1) produce almost identical perturbations in the antithrombin 1H NMR spectrum compared to binding of intact heparin, including perturbations of resonances from tryptophan 49. This indicates that neither the glucuronic acid nor the trisulfated glucosamine residue (structural elements known to be part of the high-affinity heparin motif) are necessary for the majority of the conformational changes induced upon heparin fragment binding to antithrombin. However, the low anticoagulant activity of these fragments indicates that the changes in protein conformation upon fragment binding, as manifested by these 1H resonance perturbations, are not sufficient for catalytic activation of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory copper binding site on the spinach cytochrome b6f complex: implications for Qo site catalysis

The isolated cytochrome (cyt) b(6)f complex from spinach is inhibited by Cu(2+) with a K(D) of about 1 microM at pH 7.6 in the presence of 1.6 microM decyl-plastoquinol (C(10)-PQH(2)) as a substrate. Inhibition was competitive with respect to C(10)-PQH(2) but noncompetitive with respect to horse heart cyt c or plastocyanin (PC). Inhibition was also pH-sensitive, with an apparent pK at about 7, above which inhibition was stronger, suggesting that binding occurred at or near a protonatable amino acid residue. Equilibrium binding titrations revealed ca. 1.4 tight Cu(2+) binding sites with a K(D) of about 0.5 microM and multiple (>8) weak (K(D) > 50 microM) binding sites per complex. Pulsed electron paramagnetic resonance (EPR) techniques were used to identify probable binding sites for inhibitory Cu(2+). A distinct enhancement of the relaxation time constant for the EPR signal from bound Cu(2+) was observed when the cyt f was paramagnetic. The magnitude and temperature-dependence of this relaxation enhancement were consistent with a dipole interaction between Cu(2+) and the cyt f (Fe(3+)) heme at a distance of between 30 and 54 A, depending upon the relative orientations of Cu(2+) and cyt f heme g-tensors. Two-pulse electron spin-echo envelope modulation (ESEEM) and 4-pulse 2-dimensional hyperfine sublevel correlation (2D HYSCORE) measurements of Cu(2+) bound to isolated cyt b(6)f complex indicated the presence of a weakly coupled nitrogen nucleus. The nuclear quadrupole interaction (NQI) and the hyperfine interaction (HFI) parameters identified one Cu(2+) ligand as an imidazole nitrogen of a His residue, and electron-nuclear double resonance (ENDOR) confirmed the presence of a directly coordinated nitrogen. A model of the 3-dimensional structure of the cytochrome b(6)f complex was constructed on the basis of sequences and structural similarities with the mitochondrial cyt bc(1) complex, for which X-ray structures have been solved. This model indicated three possible His residues as ligands to inhibitory Cu(2+). Two of these are located on the "Rieske" iron-sulfur protein protein (ISP) while the third is found on the cyt f protein. None of these potential ligands appear to interact directly with the quinol oxidase (Q(o)) binding pocket. A model is thus proposed wherein Cu(2+) interferes with the interaction of the ISP protein with the Q(o) site, preventing the binding and subsequent oxidation of plastoquinonol. Implications for the involvement of ISP "domain movement" in Q(o) site catalysis are discussed.     

Metal binding to heparin monosaccharides: D-glucosamine-6-sulphate, D-glucuronic acid, and L-iduronic acid

In order to ascertain which residues in heparin may be responsible for its metal binding capacities we have investigated metal binding to some of its component monosaccharides by 1H and 13C NMR. The diamagnetic Zn ion and the paramagnetic Ni ion were used as probes. 4-Methylumbelliferyl-2-deoxy-2-acetamido-6-O-sulpho-D-glucosamine was used as a model for O-sulphates. Only weak interactions with the sulphate group were found. The 4C1 ring conformation of sodium methyl-beta-D-glucopyranosiduronate was not perturbed by binding to its carboxylate and little evidence exists for chelation. By contrast, the ring conformation of the sodium methyl-alpha-L-idopyranosiduronate is affected by the addition of Zn greater than Pb greater than Cd greater than Ca much greater than K ions. The sodium salt is suggested to be an equilibrium mixture of the 2SO and 1C4 ring conformations. Cation binding to the carboxylate group shifts this equilibrium towards the 1C4 conformation and suggests additional binding to O5 or, less likely, O4. This effect appears to be electrostatic in nature, as excess Na and protonation produce similar shifts. Lead complexation is different from the other ions and suggests some covalent character. The control of the ring conformation of iduronic acid by metal ions may have biological implications for the action of heparin and heparin-like compounds.     

Probing the location of the substrate binding site of ascorbate oxidase near type 1 copper: an investigation through spectroscopic, inhibition and docking studies

The present investigation addresses the problem of the binding mode of phenolic inhibitors and the substrate ascorbate to the active site of ascorbate oxidase. The results from both types of compounds indicate that the binding site is located in a pocket near the type 1 copper center. This information is of general interest for blue multicopper oxidases. Docking calculations performed on the ascorbate oxidase-ascorbate complex show that binding of the substrate occurs in a pocket near type 1 Cu, and is stabilized by at least five hydrogen bonding interactions with protein residues, one of which involves the His512 Cu ligand. Similar docking studies show that the isomeric fluorophenols, which act as competitive inhibitors toward ascorbate, bind to the enzyme in a manner similar to ascorbate. The docking calculations are supported by 19F NMR relaxation measurements performed on fluorophenols in the presence of the enzyme, which show that the bound inhibitors undergo enhanced relaxation by the paramagnetic effect of a nearby Cu center. Unambiguous support to the location of the inhibitor close to type 1 Cu was obtained by comparative relaxation measurements of the fluorophenols in the presence of the ascorbate oxidase derivative where a Zn atom selectively replaces the paramagnetic type 2 Cu. The latter experiments show that contribution to relaxation of the bound inhibitors by the type 2 Cu site is negligible.