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.     

Metal ion specificities for folding and cleavage activity in the Schistosoma hammerhead ribozyme

The effects of various metal ions on cleavage activity and global folding have been studied in the extended Schistosoma hammerhead ribozyme. Fluorescence resonance energy transfer was used to probe global folding as a function of various monovalent and divalent metal ions in this ribozyme. The divalent metals ions Ca²⁺, Mg²⁺, Mn²⁺, and Sr²⁺ have a relatively small variation (less than sixfold) in their ability to globally fold the hammerhead ribozyme, which contrasts with the very large difference (>10,000-fold) in apparent rate constants for cleavage for these divalent metal ions in single-turnover kinetic experiments. There is still a very large range (>4600-fold) in the apparent rate constants for cleavage for these divalent metal ions measured in high salt (2 M NaCl) conditions where the ribozyme is globally folded. These results demonstrate that the identity of the divalent metal ion has little effect on global folding of the Schistosoma hammerhead ribozyme, whereas it has a very large effect on the cleavage kinetics. Mechanisms by which the identity of the divalent metal ion can have such a large effect on cleavage activity in the Schistosoma hammerhead ribozyme are discussed.     

Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis

HutP is an RNA-binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis, by binding to cis-acting regulatory sequences on hut mRNA. It requires L-histidine and an Mg²⁺ ion for binding to the specific sequence within the hut mRNA. In the present study, we show that several divalent cations can mediate the HutP–RNA interactions. The best divalent cations were Mn²⁺, Zn²⁺ and Cd²⁺, followed by Mg²⁺, Co²⁺ and Ni²⁺, while Cu²⁺, Yb²⁺ and Hg²⁺ were ineffective. In the HutP–RNA interactions, divalent cations cannot be replaced by monovalent cations, suggesting that a divalent metal ion is required for mediating the protein–RNA interactions. To clarify their importance, we have crystallized HutP in the presence of three different metal ions (Mg²⁺, Mn²⁺ and Ba²⁺), which revealed the importance of the metal ion binding site. Furthermore, these analyses clearly demonstrated how the metal ions cause the structural rearrangements that are required for the hut mRNA recognition.     

Complexation of NADH analogs with divalent metal ions: Dependence on the 3-substituent of 1-benzyl-1,4-dihydropyridines

In light of the critical role of divalent metal ions in the chemistry of coenzyme NADH analogs, complexation of 1-benzyl-3-substituted(X)-1,4-dihydropyridines (1, X=CONH₂; 2, X=CSNH₂; 3, X=COOCH₃; 4, X=COCH₃) with divalent metal ions (Mg²⁺, Zn²⁺, and Co²⁺) in dry acetonitrile was studied spectroscopically and kinetically. Presence of the metal ions causes red-shift of absorption band of NADH analogs and the rate retardation for the reaction between NADH analogs and N-methylacridinium ion. Analysis of the spectroscopic and kinetic data indicates that the NADH analogs form 1 : 1 complexes with the metal ions. The decreasing order of the magnitude of the association constants, K, is 1 2 4 3 for a given metal ion, and Mg²⁺ Zn²⁺ > Co²⁺ for a given NADH analog. The results strongly suggest that the primary binding site for the metal ions is the carbonyl oxygen (or thiocarbonyl sulfur) of the 3-substituent and that the amide nitrogen atom of the 3-substituent of 1 and 2 also ligates the metal ions, forming a bidentate structure and providing extra stability to the complexes of 1 and 2. Inhibition of reaction between NADH analogs and N-methylacridinium ion by the metal ions is attributed to inaccessibility of N-methylacridinium ion to the NADH analogs complexed with metal ions due to electrostatic repulsion.     

Ion Permeation and Block of P2X2 Purinoceptors: Single Channel Recordings

P2X₂ purinoceptors are cation-selective channels activated by ATP and its analogues. Using single channel measurements we studied the channel's selectivity for the alkali metal ions and organic monovalent cations NMDG⁺, Tris⁺, TMA⁺, and TEA⁺. The selectivity sequence for currents carried by alkali metal ions is: K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺, which is Eisenman sequence IV. This is different from the mobility sequence of the ions in free solution suggesting there is weak interaction between the ions and the channel interior. The relative conductance for alkali ions increases linearly in relation to the Stokes radius. The organic ions NMDG⁺, Tris⁺, TMA⁺ and TEA⁺ were virtually impermeant. The divalent ions (Mn²⁺, Mg²⁺, Ca²⁺ and Ba²⁺) induced a fast block visible as a reduction in amplitude of the unitary currents. Using a single-site binding model, the divalent ions exhibited an equilibrium affinity sequence of Mn²⁺ > Mg²⁺ > Ca²⁺ > Ba²⁺. Received: 3 May 1999/Revised: 23 August 1999     

Inhibitory Effects of the Divalent Metal Ions on Biomethanation by Isolated Mesophilic Methanogen in AC21 Medium in Presence or Absence of Juices from Water Hyacinth

The effect of divalent metal ions on biomethanation was studied using pre-reduced methanogenic AC21 medium in absence or in presence of leaf or root extracts of Eichornia grassipes (water hyacinth) in vitro. The metal ions like Cu²⁺, Fe²⁺, Ni²⁺, and Co²⁺ were found to be effective in increasing methane production in vitro at low concentrations, but toxic inhibitory effects were observed at relatively high concentrations. Zn²⁺ ion was inhibitory in character with exceptions. The bioavailability of metal ions in AC21 medium alone or with juices from leaf or root extracts of plant (water hyacinth in the present case) was limited due to complex formation of metal ions with different constituents in the medium, precipitation, etc. However, the results of the microscopic experiments (in vials) with metal ions for methane production by isolated methanogens in the methanogenic medium should not be compared with the conversion of biomass to methane in biodigesters. In such cases, not only a large number of methanogenic bacteria of different strains but also a consortium of bacteria of different specificities is usually involved. Though the biomass may contain sufficient concentrations of metal ions, it is advisable to add metal ions like Cu²⁺, Fe²⁺ , Zn ²⁺ ,Co²⁺, etc. in the biomass, but the concentrations of the metal ions should be limited to 10–20 µM of the biomass to make methane generation successful.     

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.     

Effects of divalent metal ions on the calcium pump and membrane phosphorylation in human red cells

In inside-out red cell membrane vesicles ATP-dependent calcium transport is activated by the divalent metal ions Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Fe²⁺. This activation is based on the formation of Me²⁺-ATP complexes which can serve as energy-donor substrates for the calcium pump, and probably, satisfy the requirement for free Me²⁺ in this transport process. Higher Me²⁺ concentrations inhibit calcium transport with various efficiencies. Mn²⁺ directly competes with Ca²⁺ at the transport site, while other divalent metal ions investigated have no such effect. The formation of the hydroxylamine-sensitive phosphorylated intermediate (EP) of the red cell membrane calcium pump from [γ-³²P]ATP is induced by Ca²⁺ while rapid dephosphorylation requires the presence of Mg²⁺. At higher concentrations Mn²⁺ and Ni²⁺ inhibit predominantly the formation of EP, while Co²⁺ and Fe²⁺ block dephosphorylation. The possible sites and nature of the divalent metal interactions with the red cell calcium pump are discussed. Hydroxylamine-insensitive membrane phosphorylation in inside-out vesicles from [γ-³²P]ATP is significantly stimulated by Mn²⁺ and Co²⁺, as compared to that produced by Mg²⁺, Fe²⁺ and Ni²⁺. Part of this labelling is found in phospholipids, especially in phosphatidylinositol. The results presented for the metal dependency of protein and lipid phosphorylation in red cell membranes may help in the characterization of ATP consumptions directly related to the calcium pump and those involved in various regulatory processes.     

4,5-Bis(diphenylphosphinoyl)-1,2,3-triazole ligand: Studies on metal complex formations in liquid-liquid distribution systems

The formation reactions of hydrophobic metal complexes of divalent typical element and transition metal ions with a novel chelating ligand containing N and O donor atoms, 4,5-bis(diphenylphosphinoyl)-1,2,3-triazole (L^TH), were investigated by the liquid-liquid distribution method carried out on metal ions between chloroform and aqueous solutions. The liquid-liquid distribution reaction formulae of metal ions via the formation of hydrophobic metal complexes were revealed, along with their equilibrium constants. Three types of hydrophobic mononuclear and binuclear metal complexes distributed into chloroform solutions were found, namely, ML₂ (M = Mg²⁺, Zn²⁺, Pb²⁺; L = L^T−), ML₂(HL) (M = Cd²⁺, Mn²⁺), and M₂L₃(OH) (M = Co²⁺, Ni²⁺, Cu²⁺). Linear free energy relationships were found between the equilibrium constants of the liquid-liquid distribution reactions and the stability constants of 1:1 complexes consisting of a divalent metal ion and a glycinate. These relationships suggest the chelate formation of N,O-coordination with a heterocyclic five-membered ring in the metal complexes with L^TH.     

Queuine-containing isoacceptor of tyrosine tRNA in Drosophila melanogaster: Alteration of levels by divalent cations

Dietary cadmium causes the queuine-containing, Q(+), isoacceptors to increase relative to the guanine-containing, Q(?), ones of tRNA^Tyr, tRNA^His and tRNA^Asp of Drosophila melanogaster. Of the other divalent cations examined, Sr²⁺, Ni²⁺, Cu²⁺, Zn²⁺ and Hg²⁺, only Hg²⁺ failed to cause an increase in Q(+)tRNA^Tyr. For these results, all pre-adult stages of the organism were spent on media containing the divalent ions. Adult flies that had developed on a normal diet also responsed to divalent ions; Hg²⁺ as well as Cd²⁺, Sr²⁺ and Zn²⁺ caused an increase in Q(+)tRNA^Tyr in 4 days. Using adult flies, the rate of the response was measured; when placed on a Cd²⁺-containing diet, they formed significantly more Q(+)tRNA^Tyr within 24 h as compared to adults on a normal diet. Whether the queuine is derived from the diet or from de novo synthesis is yet to be determined. Since the metal ions represent a range of values in the ‘hard-soft’ classification, different sites of reaction are expected, yet for Drosophila a common result is an alteration in the ratio of Q(+) and Q(?) isoacceptors of these tRNAs. The transition to Q(+)tRNA may be an early indication of the metabolic imbalances resulting from the presence of the divalent cation.     

Resolution of an ATP-metal chelate from metal-free ATP by reverse-phase high-performance liquid chromatography

The modulation of many enzymatic reactions involved in the metabolism of nucleotide phosphates such as ATP often require divalent metal ions. In the present study reverse-phase high-performance liquid chromatography (HPLC) was used to study the chelation of divalent metal ions, such as Mn²⁺, Mg²⁺, and Ca²⁺, by ATP. The results of our study using radiolabeled [⁴⁵Ca] showed that the metal-ATP chelate formed in solution was retained longer than the metal-free ATP due to the nonpolar groups on the column packing. Recovery of the two forms of ATP showed that the [⁴⁵Ca] coeluted exclusively with the ATP-metal chelate. Other experiments showed that the retention time of the chelated form of the ATP was unaffected by eluent flow rate, but was affected by eluant pH and methanol concentration. The amount of ATP in the chelated form was found to be dependent on the amount of the metal in solution and that under appropriate conditions, i.e., with 0.1 m CaCl₂ in the mobile phase, on the divalent cation as well. Thus, we found that in terms of effectiveness in chelate formation, the metal ions were Ca²⁺ > Mg²⁺ > Mn²⁺. Recovery of the chelate and its reanalysis by HPLC revealed that the complex had dissociated. The chelate could be reformed by restoring the metal concentration to its original value and dissociated again by the addition of EDTA. The resolution of the ATP in a metal chelated form from the ATP in an unchelated form is discussed in terms of the stability of these chelates and the role of the hydrophobic groups of the column packing used in the reverse-phase HPLC in enhancement of this stability.     

Heterogeneous behavior of metalloproteins toward metal ion binding and selectivity: insights from molecular dynamics studies

About one-third of the existing proteins require metal ions as cofactors for their catalytic activities and structural complexities. While many of them bind only to a specific metal, others bind to multiple (different) metal ions. However, the exact mechanism of their metal preference has not been deduced to clarity. In this study, we used molecular dynamics (MD) simulations to investigate whether a cognate metal (bound to the structure) can be replaced with other similar metal ions. We have chosen seven different proteins (phospholipase A₂, sucrose phosphatase, pyrazinamidase, cysteine dioxygenase (CDO), plastocyanin, monoclonal anti-CD4 antibody Q425, and synaptotagmin 1 C₂B domain) bound to seven different divalent metal ions (Ca²⁺, Mg²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Ba²⁺, and Sr²⁺, respectively). In total, 49 MD simulations each of 50 ns were performed and each trajectory was analyzed independently. Results demonstrate that in some cases, cognate metal ions can be exchanged with similar metal ions. On the contrary, some proteins show binding affinity specifically to their cognate metal ions. Surprisingly, two proteins CDO and plastocyanin which are known to bind Fe²⁺ and Cu²⁺, respectively, do not exhibit binding affinity to any metal ion. Furthermore, the study reveals that in some cases, the active site topology remains rigid even without cognate metals, whereas, some require them for their active site stability. Thus, it will be interesting to experimentally verify the accuracy of these observations obtained computationally. Moreover, the study can help in designing novel active sites for proteins to sequester metal ions particularly of toxic nature.     

Identification of brain ecto-apyrase as a phosphoprotein

The divalent cation requirements of NOS activity in bovine retina homogenate supernatant were investigated. Supernatants were assayed under standard conditions (in mM: EDTA 0.45, Ca²⁺ 0.25, Mg²⁺ 4.0). In order to investigate the enzyme's dependence on divalent cations, the tissue homogenate was depleted of di- and trivalent cations by passing it over a cation-exchange column (Chelex 100). Surprisingly, NOS activity was 50-100% higher in this preparation. However, addition of either EDTA (33 M) or EGTA (1 mM) almost fully inhibited NOS activity, suggesting a requirement for residual divalent metal cation(s). Phenanthroline or iminodiacetic acid at low concentrations had little effect on activity, suggesting no requirement for Fe²⁺, Zn²⁺ or Cu²⁺. Ca²⁺ had a moderate stimulatory effect, with an optimum activity around 0.01 mM. Mg²⁺ or Mn²⁺ had little effect at concentrations < 0.25 mM. However, in the presence of EDTA, Mn²⁺ or Ca²⁺ markedly stimulated NOS activity with the optimum at 0.1 mM. At high concentrations (> 0.1-0.2 mM), all divalent cations tested (Ba²⁺, Zn²⁺, Co²⁺, Mn²⁺, Mg²⁺, Ca²⁺), as well as La³⁺, dose-dependently inhibited NOS activity. We propose that retinal NOS requires low concentrations of naturally occurring divalent metal ions, most probably Ca²⁺, for optimal activity and is inhibited by high di- and trivalent metal concentrations, probably by competition with Ca²⁺.     

Effect of the chelation of metal cation on the antioxidant activity of chondroitin sulfates

The antioxidant potencies of chondroitin sulfates (CSs) from shark cartilage, salmon cartilage, bovine trachea, and porcine intestinal mucosa were compared by three representative methods for the measurement of the antioxidant activity; DPPH radical scavenging activity, superoxide radical scavenging activity, and hydroxyl radical scavenging activity. CSs from salmon cartilage and bovine trachea showed higher potency in comparison with CSs from shark cartilage and porcine intestinal mucosa. Next, CS from salmon cartilage chelating with Ca²⁺, Mg²⁺, Mn²⁺, or Zn²⁺ were prepared, and their antioxidant potencies were compared. CS chelating with Ca²⁺ or Mg²⁺ ions showed rather decreased DPPH radical scavenging activity in comparison with CS of H⁺ form. In contrast, CS chelating with Ca²⁺ or Mg²⁺ ion showed remarkably enhanced superoxide radical scavenging activity than CS of H⁺ or Na⁺ form. Moreover, CS chelating with divalent metal ions, Ca²⁺, Mg²⁺, Mn²⁺, or Zn²⁺, showed noticeably higher hydroxyl radical scavenging activity than CS of H⁺ or Na⁺ form. The present results revealed that the scavenging activities of, at least, superoxide radical and hydroxyl radical were enhanced by the chelation with divalent metal ions.     

Surface potential of phosphatidylserine monolayers. I. Divalent ion binding effect

Surface potentials of phosphatidylserine monolayers have been measured in the presence of different divalent ion concentrations in order to determine the way in which divalent ions bind to the membrane surface. The association constants for divalent ions (Mg²⁺, Ca²⁺ and Mn²⁺) with the phosphatidylserine membrane have been obtained from the experimental data and simple ion binding theory. The order of divalent ion binding to the membrane is Mn²⁺ > Ca²⁺ > Mg²⁺. However, none of the divalent ions used completely neutralized the negative charge of phosphatidylserine even at relatively high concentrations. The amounts of the divalent ions bound depended upon the concentration of the monovalent ions present in the subphase. It is suggested that the amounts of bound ions obtained from the use of radioisotope tracer methods may include a considerable contribution from the excess free ions in the double layer region of the phosphatidylserine membrane.     

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).     

Metal‐dependent assembly of a protein nano‐cage

Short, alpha‐helical coiled coils provide a simple, modular method to direct the assembly of proteins into higher order structures. We previously demonstrated that by genetically fusing de novo–designed coiled coils of the appropriate oligomerization state to a natural trimeric protein, we could direct the assembly of this protein into various geometrical cages. Here, we have extended this approach by appending a coiled coil designed to trimerize in response to binding divalent transition metal ions and thereby achieve metal ion‐dependent assembly of a tetrahedral protein cage. Ni²⁺, Co²⁺, Cu²⁺, and Zn²⁺ ions were evaluated, with Ni²⁺ proving the most effective at mediating protein assembly. Characterization of the assembled protein indicated that the metal ion–protein complex formed discrete globular structures of the diameter expected for a complex containing 12 copies of the protein monomer. Protein assembly could be reversed by removing metal ions with ethylenediaminetetraacetic acid or under mildly acidic conditions.     

Cation-induced conformational changes in tRNA molecules

The uv absorption spectra and melting profiles of an initially ion-free solution of E. coli unfractionated tRNA are significantly modified by the addition of either Na⁺, Mg²⁺, or Mn²⁺ or of other first-series transition-metal ions such as Ni²⁺, Co²⁺, and Zn²⁺. The main effect of the addition of all monovalent or divalent cations examined is an increase of the ordered and stacking stabilized tRNA structure, as revealed by a drop in the absorption near 260 nm, as well as in the 4-TU absorption region. Sharp differences have, however, been detected in the 290–305-nm range in the presence of the various ions studied. When transition-metal ions were added to a tRNA solution, an absorption peak appeared at 294 nm. This effect is interpreted as a perturbation of the electronic structure of the bases due to direct binding of metal ions to the bases. An analysis of the variation in the spectrum as a function of metal concentration and of the thermal melting reversibility in the presence of various metal ions supports the conclusion that while all ions investigated are involved in binding to the phosphate groups of tRNA, transition-metal ions are also able to bind directly to the bases.     

A comparative study on intrinsic fluorescence of BSA and lysozyme proteins in presence of different divalent ions from their solution and thin film conformations

Optical emission behaviours of lysozyme and bovine serum albumin, from bulk and thin film geometry, were studied in the presence of three different divalent ions (Mg²⁺, Ca²⁺ or Ba²⁺) using different spectroscopic [steady‐state fluorescence, UV–Vis and Fourier transform infra‐red (FTIR)] techniques. Additionally, protein thin films on silicon surfaces were prepared and morphological studies were carried out using atomic force microscopy. Dynamic quenching was mainly identified for both proteins in the presence of Mg²⁺, Ca²⁺ and Ba²⁺ ions. The molecular conformation of the proteins was modified in thin films compared with that in solution, consequently quenching efficiencies also varied. ATR‐FTIR studies confirmed the conformational changes of proteins in the presence of all divalent ions. All metal ions used were divalent in nature and belonged to the same group of the periodic table but, depending on their individual characteristics such as electron affinity, ionic radius, etc., the magnitude of the protein and hydrated ion interaction varied and accordingly the quenching efficiency was modified. Quenching was maximum for Ca²⁺ ions, followed by the other two ions. Our study clearly illustrates the geometry‐dependent physical and biological functions of proteins.