Binding of metal ions toE. coli RNase HI observed by1H−15N heteronuclear 2D NMR

Summary The divalent metal ion binding site and binding constant of ribonuclease HI fromEscherichia coli were investigated by observing chemical shift changes using¹H–¹⁵N heteronuclear NMR. Chemical shift changes were monitored during the titration of the enzyme with salts of the divalent cations. The enzyme was uniformly labeled by¹⁵N, which facilitated the monitoring of the chemical shift change of each cross peak between the backbone amide proton and the amide¹⁵N. The chemical shifts of several amide groups were affected upon the addition of a divalent metal ion: Mg²⁺, Ca²⁺, or Ba²⁺. These amide groups resided close to the active site, consistent with the previous X-ray crystallographic studies. From the titration analysis, a single divalent ion binding site was observed with a weak binding constant (K_D=2–4 mM for the current divalent ions).     

Interactions of DNA with divalent metal ions. II. Proton relaxation enhancement studies

The extent and modes of binding of the divalent metal ions Mn²⁺ and Co²⁺ to DNA and the effects of salt on the binding have been studied by measurements of the effects of these paramagnetic metal ions on the longitudinal and transverse relaxation rates of the protons of the solvent water molecules, a technique that is sensitive to overall binding. The number of water molecules coordinated to the DNA–bound Mn²⁺ and Co²⁺ is found to be between five and six, and the electron spin relaxation times and the electron-nuclear hyperfine constants associated with Mn²⁺ and Co²⁺ are little or not affected by the binding. These observations indicate little disturbance of the hydration sphere of Mn²⁺ and Co²⁺ upon binding to DNA. An average 2–3-fold reduction in the exchange rate of the water of hydration of the bound metal ions and an order-of-magnitude increase in their rotational correlation time are attributed to hydrogen-bond formation with the DNA. The binding constants of Mn²⁺ to DNA, at metal concentrations approaching zero, are found to be inversely proportional to the second power of the salt concentration, in agreement with the predictions of Manning's polyelectrolyte theory. A remarkable quantitative agreement with the polyelectrolyte theory is also obtained for the anticooperativity in the binding of Mn²⁺ to DNA, although the experimental results can be well accounted for by another simple electrostatic model. The various modes of binding of divalent metal ions to DNA are discussed.     

Interactions of DNA with divalent metal ions. I. 31P-nmr studies

³¹P-nmr has been used to investigate the specific interaction of three divalent metal ions, Mg²⁺, Mn²⁺, and Co⁺², with the phosphate groups of DNA. Mg²⁺ is found to have no significant effect on any of the ³¹P-nmr parameters (chemical shift, line-width, T₁, T₂, and NOE) over a concentration range extending from 20 to 160 mM. The two paramagnetic ions, Mn²⁺ and Co²⁺, on the other hand, significantly change the ³¹P relaxation rates even at very low levels. From an analysis of the paramagnetic contributions to the spin–lattice and spin–spin relaxation rates, the effective internuclear metal–phosphorus distances are found to be 4.5 ± 0.5 and 4.1 ± 0.5 Å for Mn²⁺ and Co²⁺, respectively, corresponding to only 15 ± 5% of the total bound Mn²⁺ and Co²⁺ being directly coordinated to the phosphate groups (inner-sphere complexes). This result is independent of any assumptions regarding the location of the remaining metal ions which may be bound either as outer-sphere complexes relative to the phosphate groups or elsewhere on the DNA, possibly to the bases. Studies of the temperature effects on the ³¹P relaxation rates of DNA in the absence and presence of Mn²⁺ and Co²⁺ yielded kinetic and thermodynamic parameters which characterize the association and dissociation of the metal ions from the phosphate groups. A two-step model was used in the analysis of the kinetic data. The lifetimes of the inner-sphere complexes are 3 × 10^?7 and 1.4 × 10^?5 s for Mn²⁺ and Co²⁺, respectively. The rates of formation of the inner-sphere complexes with the phosphate are found to be about two orders of magnitude slower than the rate of the exchange of the water of hydration of the metal ions, suggesting that expulsion of water is not the rate-determining step in the formation of the inner-sphere complexes. Competition experiments demonstrate that the binding of Mg²⁺ ions is 3–4 times weaker than the binding of either Mn²⁺ or Co²⁺. Since the contribution from direct phosphate coordination to the total binding strength of these metal ion complexes is small (～15%), the higher binding strength of Mn²⁺ and Co²⁺ may be attributed either to base binding or to formation of stronger outer-sphere metal–phosphate complexes. At high levels of divalent metal ions, and when the metal ion concentration exceeds the DNA–phosphate concentration, the fraction of inner-sphere phosphate binding increases. In the presence of very high levels of Mg²⁺ (e.g., 3.1M), the inner-sphere ? outer-sphere equilibrium is shifted toward ～100% inner-sphere binding. A comparison of our DNA results and previous results obtained with tRNA indicates that tRNA and DNA have very similar divalent metal ion binding properties. A comparison of the present results with the predictions of polyelectrolyte theories is presented.     

Interaction of divalent metal ions with the carboxyl-terminal domain of human voltage-gated proton channel Hv1

The voltage-gated proton channel Hv1 functions as a dimer, in which the intracellular C-terminal domain of the protein is responsible for the dimeric architecture and regulates proton permeability. Although it is well known that divalent metal ions have effect on the proton channel activity, the interaction of divalent metal ions with the channel in detail is not well elucidated. Herein, we investigated the interaction of divalent metal ions with the C-terminal domain of human Hv1 by CD spectra and fluorescence spectroscopy. The divalent metal ions binding induced an obvious conformational change at pH 7 and a pH-sensitive reduction of thermostability in the C-terminal domain. The interactions were further estimated by fluorescence spectroscopy experiments. There are at least two binding sites for divalent metal ions binding to the C-terminal domain of Hv1, either of which is close to His²⁴⁴ or His²⁶⁶ residue. The binding of Zn²⁺ to the two sites both enhanced the fluorescence of the protein at pH 7, whereas the binding of other divalent metal ions to the two sites all resulted fluorescence quenching. The orders of the strength of divalent metal ions binding to the two sites from strong to weak are both Co²⁺, Ca²⁺, Ni²⁺, Mg²⁺, and Mn²⁺. The strength of Ca²⁺, Co²⁺, Mg²⁺, Mn²⁺ and Ni²⁺ binding to the site close to His²⁴⁴ is stronger than that of these divalent metal ions binding to the site close to His²⁶⁶.     

Proton paths in the sarcoplasmic reticulum Ca-ATPase

The sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a) pumps Ca²⁺ and countertransport protons. Proton pathways in the Ca²⁺ bound and Ca²⁺-free states are suggested based on an analysis of crystal structures to which water molecules were added. The pathways are indicated by chains of water molecules that interact favorably with the protein. In the Ca²⁺ bound state Ca₂E1, one of the proposed Ca²⁺ entry paths is suggested to operate additionally or alternatively as proton pathway. In analogs of the ADP-insensitive phosphoenzyme E2P and in the Ca²⁺-free state E2, the proton path leads between transmembrane helices M5 to M8 from the lumenal side of the protein to the Ca²⁺ binding residues Glu-771, Asp-800 and Glu-908. The proton path is different from suggested Ca²⁺ dissociation pathways. We suggest that separate proton and Ca²⁺ pathways enable rapid (partial) neutralization of the empty cation binding sites. For this reason, transient protonation of empty cation binding sites and separate pathways for different ions are advantageous for P-type ATPases in general.     

Chemical rescue of H+ delivery in proton transfer mutants of reaction center of photosynthetic bacteria

In the native and most mutant reaction centers of bacterial photosynthesis, the electron transfer is coupled to proton transfer and is rate limiting for the second reduction of Q_B^??→?Q_BH₂. In the presence of divalent metal ions (e.g. Cd²⁺) or in some (“proton transfer”) mutants (L210DN/M17DN or L213DN), the proton delivery to Q_B^? is made rate limiting and the properties of the proton pathway can be directly examined. We found that small weak acids and buffers in large concentrations (up to 1?M) were able to rescue the severely impaired proton transfer capability differently depending on the location of the defects: lesions at the protein surface (proton gate H126H/H128H?+?Cd²⁺), beneath the surface (M17DN?+?Cd²⁺, L210DN/M17DN) or deep inside the protein (L213DN) could be completely, partially or to very small extent recovered, respectively. Small zwitterionic acids (azide/hydrazoic acid) and buffers (tricine) proved to be highly effective rescuers consistent with their enhanced binding affinity and access to any of the proton acceptors (including Q_B^? itself) in the pathway. As a consequence, back titration of the protons at L212Glu could be observed as a pH-dependence of the rate constant of the charge recombination in the presence of azide or formate. Model calculations support the collective influence of the acid cluster on the change of the protonation states upon extension of the cluster with the bound small acid. In proton transfer mutants, the rescuing agents decreased the free energy of activation together with their enthalpic and entropic components. This is in agreement with the hypothesis that they function as protein-penetrating protonophores delivering protons into the chain and select dominating paths out of many alternate routes. We estimate that the proton delivery will be accelerated in one pathway out of 100–200 alternate pathways. The implications for design of the chemical recovery of impaired intra-protein proton transfer pathways in proton transfer mutants are discussed.     

31P-nmr studies of the effect of various metals on substrate binding to yeast enolase

The ³¹P nuclear magnetic resonance (nmr) spectra of product (phosphoenolpyruvate) and substrate (2-phosphoglycerate) binding to 1:1 molar ratios ot yeast enolase were obtained as functions of the level of various metal ions. Levels sufficient to produce substrate and product binding but not catalysis ( 1 equivalent/subunit), produced shifts (with respect to 86% H₃PO₄) to lower shielding of ca. 30 ppm in the case of Co²⁺, 5–8 ppm in the case of Mg²⁺, and 2–3 ppm in the case ofCa²⁺, but virtual obliteration in the case of Mn²⁺. The effects of Mn²⁺ and Co²⁺ are consistent with a close approach of the metal ions to the phosphate groups. The effects of the physiological cofactor and optimum activator Mg²⁺ and the nonactivator Ca²⁺ are interpreted as indicating different degrees of distortion of the R-O-P bond angle in the two metal-enzyme-substrate complexes. Levels of Mg²⁺ sufficient for optimal or near optimal catalysis (2 equivalents/subunit) produce shifts to higher shielding in the ³¹P resonances of both substrate and product. These shifts are intermediate between those in the presence of 1 equivalent/subunit and those of the free ligands. Addition of a second equivalent of Ca²⁺ produces a slight shift to lower shielding of the phosphoenolpyruvate resonance and a small shift to higher shielding in the resonance for 2-phosphoglycerate. Similar levels of Co²⁺ eliminate the resonances for both substrate and product. These effects are interpreted as arising from direct coordination between substrate-dependent metal ion binding and the phosphate esters. Higher levels of Ca²⁺, Mg²⁺, or Co²⁺ or addition of KF, all of which inhibit enzyme activity, have only minor effects on the spectra. The spectrum of inorganic phosphate, a competitive inhibitor, was also examined. KF strongly enhances binding, as does excess Mg²⁺, and the binding is accompanied by a chemical shift to lower shielding of ca 2 ppm. This is not due to formation of a magnesium-fluorophosphate complex, consistent with the findings of other workers.     

Magnetic resonance studies of the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium.

The binding of Mn²⁺ to the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium was examined by electron paramagnetic resonance studies. Two types of binding sites were observed: one to two tight sites with a dissociation constant of 3–5 μm and five to six weaker sites with a dissociation constant of 40–70 μm. The activator constant for Mn²⁺ was found to be 9 μm for the glutamine-linked anthranilate synthetase activity and 4 μm for the phosphoribosyltransferase activity. These values are both in the range of the dissociation constant for the tight sites. Water proton relaxation rate measurements showed that the binary enhancement values for both classes of sites were equivalent, ?_b = 10.7 ± 2.0. The addition of chorismate to the Mn²⁺-enzyme complexes when predominantly the tight Mn²⁺ sites were occupied resulted in a large decrease in the observed enhancement (?_T = 2.0). Addition of 5-phosphoribosyl-1-pyrophosphate to the enzyme-Mn²⁺ complexes caused large decreases in the water proton relaxation rate (?_T = 1.5) when tight or tight plus weaker Mn²⁺ sites were occupied. No changes in the water proton relaxation rate were observed when glutamine, pyruvate, or anthranilate were added; a small decrease was observed when enzyme-Mn²⁺ was titrated with tryptophan. Tryptophan significantly altered the effect of the binding of chorismate but not of 5-phosphoribosyl-1-pyrophosphate. The effect of tryptophan on the water proton relaxation rate of a Mn²⁺-enzyme-chorismate complex using a variant enzyme complex which is tryptophan hypersensitive (P. D. Robison, and H. R. Levy, 1976, Biochim. Biophys. Acta. 445, 475–485) occurred at lower concentrations than for the normal enzyme complex. The uncomplexed anthranilate synthetase subunit was titrated with Mn²⁺ and found to have one to two binding sites with a dissociation constant of 300 ± 100 μm. This dissociation constant is much larger than the activator constant for Mn²⁺ for uncomplexed anthranilate synthetase which was determined to be 4 μm. These results indicate that the Mn²⁺-binding sites on anthranilate synthetase are altered when the enzyme complex is formed and that both chorismate and 5-phosphoribosyl-1-pyrophosphate interact closely with enzyme-bound Mn²⁺ or cause a large effect upon its environment.     

Interaction of Calcium Ion with Bovine Caseins

In order to clarify the interaction of calcium ion with casein, the volume change associated with the interaction was measured by dilatometric procedures. When CaCl₂ was added to the casein solutions at neutral pH, a volume increase occurred and reached a constant saturated value of about 700 ml per 10⁶ g protein with increasing CaCl₂ concentrations for whole-, α_s- and β-casein solutions, but there was no volume change for κ-casein solution. On the other hand, the binding of calcium ion to the casein fractions was determined by a gel filtration procedure at pH 6.0 to 9.0. The number of Ca²⁺ ions bound to the caseins increased with the CaCl₂ concentration and pH value, and the relative order of binding capacities for the caseins was: α_s-casein > whole-casein > β-casein > κ-casein.

It was found that the volume changes obtained by the dilatometry were smaller than the calculated volume increases based on the assumption that these are caused by the binding of Ca²⁺ ion to the caseins. Therefore it is necessary to introduce another factor which reduces the volume increase due to the Ca²⁺ ion binding in order to reasonably explain the measured volume changes. At present it is presumed that there occurs the unfolding of peptide chain of casein molecule on Ca²⁺ ion binding, which has been known to decrease the volume of the protein solution.     

Conformational and kinetic features of the morphine-Mn(II) interaction as probed by nuclear paramagnetic resonance investigations

The nature of binding between manganese ions and morphine was studied using Fourier transform proton nuclear magnetic resonance techniques. Proton relaxation times in the presence of Mn(II) ions were determined together with their temperature dependence. Slow exchange conditions were observed for the NCH₃ group, while fast exchange conditions applied for all the other protons. The rotational correlation time of the complex was approximated by that of the free morphine molecule, as measured by selective and nonselective proton relaxation rate measurements. The distances between the metal ion and proton nuclei of morphine were evaluated on the basis of an association constant, measured from water proton spin-lattice relaxation rate binding studies. The results indicate that the metal binds directly to the two oxydryls with K_ass = 9.7 × 10^?3.The rate constant for the interaction of Mn(II) with the opiate is 2.25 × 10⁴ sec^?1 at 27°C, as determined from the temperature dependence of longitudinal relaxation rate of the NCH₃ group.     

Unraveling Geometrical Site Confinement in Highly Efficient Iron‐Doped Electrocatalysts toward Oxygen Evolution Reaction

Introduction of iron in various catalytic systems has served a crucial function to significantly enhance the catalytic activity toward oxygen evolution reaction (OER), but the relationship between material properties and catalysis is still elusive. In this study, by regulating the distinctive geometric sites in spinel, Fe occupies the octahedral sites (Fe³⁺_(Oh)) and confines Co to the tetrahedral site (Co²⁺_(Td)), resulting in a strikingly high activity (η_{j = 10 mA cm}^?2 = 229 mV and η_{j = 100 mA cm}^?2 = 281 mV). Further enrichment of Fe ions would occupy the tetrahedral sites to decline the amount of Co²⁺_(Td) and deteriorate the OER activity. It is also found that similar tafel slope and peak frequency in Bode plot of electrochemical impedance spectroscopy indicate that Co²⁺_(Td) ions are primarily in charge of water oxidation catalytic center. By means of electrochemical techniques and in situ X‐ray absorption spectroscopy, it is proposed that Fe³⁺_(Oh) ions mainly confine cobalt ions to the tetrahedral site to restrain the multipath transfer of cobalt ions during the dynamic structural transformation between spinel and oxyhydroxide, continuously activating the catalytic behavior of Co²⁺_(Td) ions. This material‐related insight provides an indication for the design of highly efficient OER electrocatalysts.     

Luminescent sensor for Cd2+, Hg2+ and Ag+ in water based on a sulphur‐containing receptor: quantitative binding–softness relationship

A water‐soluble, high‐output fluorescent sensor, based on a lumazine ligand with a thiophene substituent for Cd²⁺, Hg²⁺ and Ag⁺ metal ions, is reported. The sensor displays fluorescence enhancement upon Cd²⁺ binding (log  β = 2.79 ± 0.08) and fluorescence quenching by chelating with Ag⁺ and Hg²⁺ (log β = 4.31 ± 0.15 and 5.42 ± 0.1, respectively). The mechanism of quenching is static and occurs by formation of a ground‐state non‐fluorescent complex followed by rapid intersystem crossing. The value of the Stern–Volmer quenching rate constant (k_q) by Ag⁺ ions is close to 6.71 × 10¹² mol/L/s at 298 K. The thermodynamic parameters (ΔG, ΔH and ΔS) were also evaluated and indicated that the complexation process is spontaneous, exothermic and entropically favourable. The quantitative linear relationship between the softness values of Klopman (σ_K) or Ahrland (σ_A) and the experimental binding constants (β) being in the order of Hg²⁺ > Ag⁺ > Cd²⁺ suggests that soft–soft interactions are the key for the observed sensitivity and selectivity in the presence of other metal ions, such as: Pb²⁺, Ni²⁺, Mn²⁺, Cu²⁺, Co²⁺, Zn²⁺ and Mg²⁺ ions. Copyright © 2008 John Wiley & Sons, Ltd.     

Characterization of mercury(II)-induced inhibition of photochemistry in the reaction center of photosynthetic bacteria

Mercuric contamination of aqueous cultures results in impairment of viability of photosynthetic bacteria primarily by inhibition of the photochemistry of the reaction center (RC) protein. Isolated reaction centers (RCs) from Rhodobacter sphaeroides were exposed to Hg²⁺ ions up to saturation concentration (~?10³ [Hg²⁺]/[RC]) and the gradual time- and concentration-dependent loss of the photochemical activity was monitored. The vast majority of Hg²⁺ ions (about 500 [Hg²⁺]/[RC]) had low affinity for the RC [binding constant K_b?~?5 mM^?1] and only a few (~?1 [Hg²⁺]/[RC]) exhibited strong binding (K_b?~?50 μM^?1). Neither type of binding site had specific and harmful effects on the photochemistry of the RC. The primary charge separation was preserved even at saturation mercury(II) concentration, but essential further steps of stabilization and utilization were blocked already in the 5 < [Hg²⁺]/[RC]?<?50 range whose locations were revealed. (1) The proton gate at the cytoplasmic site had the highest affinity for Hg²⁺ binding (K_b?~?0.2 μM^?1) and blocked the proton uptake. (2) Reduced affinity (K_b?~?0.05 μM^?1) was measured for the mercury(II)-binding site close to the secondary quinone that resulted in inhibition of the interquinone electron transfer. (3) A similar affinity was observed close to the bacteriochlorophyll dimer causing slight energetic changes as evidenced by a?~?30 nm blue shift of the red absorption band, a 47 meV increase in the redox midpoint potential, and a?~?20 meV drop in free energy gap of the primary charge pair. The primary quinone was not perturbed upon mercury(II) treatment. Although the Hg²⁺ ions attack the RC in large number, the exertion of the harmful effect on photochemistry is not through mass action but rather a couple of well-defined targets. Bound to these sites, the Hg²⁺ ions can destroy H-bond structures, inhibit protein dynamics, block conformational gating mechanisms, and modify electrostatic profiles essential for electron and proton transfer.     

Metal-ion-dependent hydrophobic-interaction chromatography of α-lactalbumins

α-Lactalbumins from bovine, human, goat, sheep, and horse milk bind to phenyl-Sepharose in the presence of EDTA and can be eluted by addition of Ca²⁺ (0.001–100 m ). This property has been utilized to purify these proteins in a one-step purification from milk whey. α-Lactalbumin purified in this manner has the same ultraviolet and proton nuclear magnetic resonance spectra as that purified by other methods. Using binding to phenyl-Sepharose as an assay, the conformation of bovine α-lactalbumin upon the addition of several metal ions that are known to interact with this protein was investigated. Lanthanides, Mn²⁺, Mg²⁺, and Cd²⁺ can substitute for Ca²⁺, whereas Zn²⁺, Al³⁺, and Co²⁺ cannot. Surprisingly, whereas lower concentrations of La³⁺, Mn²⁺, and Cd²⁺ (1 m and less) caused elution from the hydrophobic support, higher concentrations (10 m ) were ineffective. These observations can be rationalized assuming the presence of two distinct metal-ion binding sites with different specificities.     

Role of water-bridged interactions in metal ion coupled protein allostery

Allosteric communication between distant parts of proteins controls many cellular functions, in which metal ions are widely utilized as effectors to trigger the allosteric cascade. Due to the involvement of strong coordination interactions, the energy landscape dictating the metal ion binding is intrinsically rugged. How metal ions achieve fast binding by overcoming the landscape ruggedness and thereby efficiently mediate protein allostery is elusive. By performing molecular dynamics simulations for the Ca²⁺ binding mediated allostery of the calmodulin (CaM) domains, each containing two Ca²⁺ binding helix-loop-helix motifs (EF-hands), we revealed the key role of water-bridged interactions in Ca²⁺ binding and protein allostery. The bridging water molecules between Ca²⁺ and binding residue reduces the ruggedness of ligand exchange landscape by acting as a lubricant, facilitating the Ca²⁺ coupled protein allostery. Calcium-induced rotation of the helices in the EF-hands, with the hydrophobic core serving as the pivot, leads to exposure of hydrophobic sites for target binding. Intriguingly, despite being structurally similar, the response of the two symmetrically arranged EF-hands upon Ca²⁺ binding is asymmetric. Breakage of symmetry is needed for efficient allosteric communication between the EF-hands. The key roles that water molecules play in driving allosteric transitions are likely to be general in other metal ion mediated protein allostery.     

Effect of Ca2+ on the self assembly of a nonionic peptide aggregate

Summary Conductometry, circular dichroism and fluorescence spectroscopy are the techniques employed to investigate the effect of added calcium ions and other monovalent and divalent metal ions on aqueous solutions of nonionic peptide aggregates, Boc-Leu-Asn-OEt (1). It is observed that among all the metal ions studied, Ca²⁺ ions facilitate the aggregation of the peptide. The interior dielectric constant of the micelles (ε) was found to depend upon the proportion of Ca²⁺ complexed peptide with the peptide mononers in the micelles. When Ca²⁺ ion becomes 1/4th of the peptide concentration, there is a structural transition leading to drastic change in the interior of the micro dielectric constant (ɛ _m).     

Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli

ZnuA is the periplasmic Zn²⁺-binding protein associated with the high-affinity ATP-binding cassette ZnuABC transporter from Escherichia coli. Although several structures of ZnuA and its homologs have been determined, details regarding metal ion stoichiometry, affinity, and specificity as well as the mechanism of metal uptake and transfer remain unclear. The crystal structures of E. coli ZnuA (Eco-ZnuA) in the apo, Zn²⁺-bound, and Co²⁺-bound forms have been determined. ZnZnuA binds at least two metal ions. The first, observed previously in other structures, is coordinated tetrahedrally by Glu59, His60, His143, and His207. Replacement of Zn²⁺ with Co²⁺ results in almost identical coordination geometry at this site. The second metal binding site involves His224 and several yet to be identified residues from the His-rich loop that is unique to Zn²⁺ periplasmic metal binding receptors. Electron paramagnetic resonance and X-ray absorption spectroscopic data on CoZnuA provide additional insight into possible residues involved in this second site. The second site is also detected by metal analysis and circular dichroism (CD) titrations. Eco-ZnuA binds Zn²⁺ (estimated K _d < 20 nM), Co²⁺, Ni²⁺, Cu²⁺, Cu⁺, and Cd²⁺, but not Mn²⁺. Finally, conformational changes upon metal binding observed in the crystal structures together with fluorescence and CD data indicate that only Zn²⁺ substantially stabilizes ZnuA and might facilitate recognition of ZnuB and subsequent metal transfer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Subcellular distribution and chemical forms of Co<Superscript>2+</Superscript> in three barley genotypes under different Co<Superscript>2+</Superscript> levels

The toxicity of many heavy metals in plants is closely associated with its subcellular distribution and chemical forms. The subcellular distribution and chemical forms of cobalt (Co²⁺) were investigated using 3 barley genotypes differing in Co²⁺ toxicity resistance, namely Yan66 (resistant), Ea 52 (sensitive), and Humai 4 (moderate), under two Co²⁺ levels (25 and 100 µM). Higher Co²⁺ level in cultural solution significantly increased Co²⁺ accumulation in all subcellular fractions, with vacuole and cell wall having higher concentration. In comparison with 25 µM Co²⁺, 100 µM Co²⁺ treatment caused significant increase of Co²⁺ concentration in the forms of F-NaCl (extracted with 1 M NaCl), F-Ac (extracted with 2% HAc), F-HCl (extracted by 0.6 M HCl), and F-residue (residue forms) in both shoots and roots. There was a significant difference among genotypes in Co²⁺ subcellular distribution and chemical forms, with Ea52 accumulating more Co²⁺ in organelles and Yan66 accumulating more Co²⁺ in vacuole and cell wall. Moreover, the inorganic form of Co²⁺ extracted with 80% ethanol (F-ethanol) and water-soluble form (F-H₂O) were significantly increased in Ea52, while Yan66 accumulated more Co²⁺ in the forms of low-bioavailable molecules (F-NaCl, F-HAc, and F-HCl). The results suggest that the vacuolar sequestration and cell wall deposition of Co²⁺ is a key resistant mechanism for genotype Yan66.     

Each Phaseolus vulgaris isolectin exists in five electrophoretic forms related to the number of metal ions bound per molecule

Analysis of the pH 8.3 electrophoresis of the Phaseolus vulgaris seed lectins suggests that each of the five isolectins exists in five different forms. Incubations of the lectins in the presence of EDTA or of metal ions such as Mn²⁺, Mg²⁺, Co²⁺ and Ni²⁺ shows that their different electrophoretic behaviours are related to the binding of metal ions to the molecules.Determinations of the isolectin metal content by atomic absorption measurements demonstrate that at pH 8.3 their five electrophoretic forms are related to the number of subunits (0, 1, 2, 3 or 4) which contain metal ions in the tetrameric molecules.