Additional Metal Binding Sites Research Articles

Why is binding of a divalent metal cation to a structural motif containing four carboxylate residues not accompanied by a conformational change?

We earlier showed that Torpedo californica acetylcholinesterase (AChE) contains a cluster of four conserved aspartates that can strongly bind divalent cations, which we named the 4D motif. Binding of the divalent metal cations greatly increases its thermal stability. Here we systematically examined all available crystallographic structures of T. californica AChE. Two additional metal-binding sites were identified, both composed of acidic and histidine residues. Relative binding to the 4D and additional sites was studied using metadynamics simulations. It was observed that in crystal structures devoid of metal ions in the 4D site, the conformation of T. californica AChE is almost identical to that in structures in which it is occupied by a divalent metal ion. Closer examination of the 4D motif reveals that three of the four acidic residues form ion pairs with conserved basic residues surrounding them. We named this new motif the 4A/3B motif. Molecular dynamics with quantum potential simulations was used to quantify the 4D motif's binding strength compared with that of the metal-binding site in the protein fXIIIa, which consists of four aspartates, but is devoid of adjacent cationic residues. Whereas fXIIIa's 4D site, in the absence of a metal cation, expanded significantly in the simulation, that of Torpedo AChE displayed only minor periodic changes in size. Furthermore, the energy of metal ion unbinding from the two sites differs by ca. 10 kcal/mol. We identified several other proteins in the PDB that contain the 4A/3B motif, whose conformations are identical in the presence or absence of a metal ion. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at https://proteopedia.org/w/Journal:Protein_Science:4.

Identification of an Additional Metal-Binding Site in Human Dipeptidyl Peptidase III.

Dipeptidyl peptidase III (DPP III, EC 3.4.14.4) is a monozinc metalloexopeptidase that hydrolyzes dipeptides from the N-terminus of peptides consisting of three or more amino acids. Recently, DPP III has attracted great interest from scientists, and numerous studies have been conducted showing that it is involved in the regulation of various physiological processes. Since it is the only metalloenzyme among the dipeptidyl peptidases, we considered it important to study the process of binding and exchange of physiologically relevant metal dications in DPP III. Using fluorimetry, we measured the Kd values for the binding of Zn2+, Cu2+, and Co2+ to the catalytic site, and using isothermal titration calorimetry (ITC), we measured the Kd values for the binding of these metals to an additional binding site. The structure of the catalytic metal's binding site is known from previous studies, and in this work, the affinities for this site were calculated for Zn2+, Cu2+, Co2+, and Mn2+ using the QM approach. The structures of the additional binding sites for the Zn2+ and Cu2+ were also identified, and MD simulations showed that two Cu2+ ions bound to the catalytic and inhibitory sites exchanged less frequently than the Zn2+ ions bound to these sites.

Structural characterization of the Myxococcus xanthus encapsulin and ferritin-like cargo system gives insight into its iron storage mechanism

Encapsulins are bacterial organelle-like cages involved in various aspects of metabolism, especially protection from oxidative stress. They can serve as vehicles for a wide range of medical applications. Encapsulin shell proteins are structurally similar to HK97 bacteriophage capsid protein and their function depends on the encapsulated cargos. The Myxococcus xanthus encapsulin system comprises EncA and three cargos: EncB, EncC, and EncD. EncB and EncC are similar to bacterial ferritins that can oxidize Fe+2 to less toxic Fe+3. We analyzed EncA, EncB, and EncC by cryo-EM and X-ray crystallography. Cryo-EM shows that EncA cages can have T= 3 and T= 1 symmetry and that EncA T= 1 has a unique protomer arrangement. Also, we define EncB and EncC binding sites on EncA. X-ray crystallography of EncB and EncC reveals conformational changes at the ferroxidase center and additional metal binding sites, suggesting a mechanism for Fe oxidation and storage within the encapsulin shell.

Using N-Terminal Coordination of Cu(II) and Ni(II) to Isolate the Coordination Environment of Cu(I) and Cu(II) Bound to His13 and His14 in Amyloid-β(4-16).

The amyloid-β (Aβ) peptide is a cleavage product of the amyloid precursor protein and has been implicated as a central player in Alzheimer's disease. The N-terminal end of Aβ is variable, and different proportions of these variable-length Aβ peptides are present in healthy individuals and those with the disease. The N-terminally truncated form of Aβ starting at position 4 (Aβ4-x) has a His residue as the third amino acid (His6 using the formal Aβ numbering). The N-terminal sequence Xaa-Xaa-His is known as an amino terminal copper and nickel binding motif (ATCUN), which avidly binds Cu(II). This motif is not present in the commonly studied Aβ1-x peptides. In addition to the ATCUN site, Aβ4-x contains an additional metal binding site located at the tandem His residues (bis-His at His13 and 14) which is also found in other isoforms of Aβ. Using the ATCUN and bis-His motifs, the Aβ4-x peptide is capable of binding multiple metal ions simultaneously. We confirm that Cu(II) bound to this particular ATCUN site is redox silent, but the second Cu(II) site is redox active and can be readily reduced with ascorbate. We have employed surrogate metal ions to block copper coordination at the ATCUN or the tandem His site in order to isolate spectral features of the copper coordination environment for structural characterization using extended X-ray absorption fine structure (EXAFS) spectroscopy. This approach reveals that each copper coordination environment is independent in the Cu2Aβ4-x state. The identification of two functionally different copper binding environments within the Aβ4-x sequence may have important implications for this peptide in vivo.

Effect of Staphylococcus epidermidis on U(VI) sequestration by Al-goethite.

Effect of Staphylococcus epidermidis (S. epidermidis) on U(VI) sequestration by Al-goethite were conducted under different geologic conditions. The batch experiments showed that S. epidermidis significantly enhanced the adsorption rates of U(VI) at pH < 9.0 due to the additional metal binding sites. The maximum adsorption capacities of U(VI) on Al-goethite and Al-goethite +S. epidermidis at pH 4.0 and 310 K were calculated from Langmuir equation to be 13.16 and 47.86 mg/g, respectively. The decreased adsorption of U(VI) on Al-goethite+ S. epidermidis at high carbonate and pH conditions were primarily driven by the electrostatic repulsion between negatively charged U(VI)-carbonate complexes and the negatively charged adsorbents. According to XPS analysis, the adsorbed U(VI) can be reduced to U(IV) by S. epidermidis, whereas inhibited reduction of U(VI) on Al-goethite + S. epidermidis at high pH could be attributed to the complexation of structural Fe(III) with the oxygen-containing functional groups of S. epidermidis. FT-IR analysis further demonstrated that the bonding of structural Fe(III) with functional groups (e.g., carboxyl and phosphate groups) of S. epidermidis. These results herein are important to understand the fate and transport of U(VI) on the mineral-bacteria ternary systems in the near-surface environment.

Insights into structural features of HDAC1 and its selectivity inhibition elucidated by Molecular dynamic simulation and Molecular Docking

Histone deacetylases (HDACs) are a family of proteins whose main function is the removal of acetyl groups from lysine residues located on histone and non-histone substrates, which regulates gene transcription and other activities in cells. HDAC1 dysfunction has been implicated in cancer development and progression; thus, its inhibition has emerged as a new therapeutic strategy. Two additional metal binding sites (Site 1 and Site 2) in HDACs have been described that are primarily occupied by potassium ions, suggesting a possible structural role that affects HDAC activity. In this work, we explored the structural role of potassium ions in Site 1 and Site 2 and how they affect the interactions of compounds with high affinities for HDAC1 (AC1OCG0B, Chlamydocin, Dacinostat and Quisinostat) and SAHA (a pan-inhibitor) using molecular docking and molecular dynamics (MD) simulations in concert with a Molecular-Mechanics-Generalized-Born-Surface-Area (MMGBSA) approach. Four models were generated: one with a potassium ion (K+) in both sites (HDAC1k), a second with K+ only at site 1 (HDAC1ks1), a third with K+ only at site 2 (HDAC1ks2) and a fourth with no K+ (HDAC1wk). We found that the presence or absence of K+ not only impacted the structural flexibility of HDAC1, but also its molecular recognition, consistent with experimental findings. These results could therefore be useful for further structure-based drug design studies addressing new HDAC1 inhibitors.

Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB.

Initiation of plasmid rolling circle replication (RCR) is catalyzed by a plasmid-encoded Rep protein that performs a Tyr- and metal-dependent site-specific cleavage of one DNA strand within the double-strand origin (dso) of replication. The crystal structure of RepB, the initiator protein of the streptococcal plasmid pMV158, constitutes the first example of a Rep protein structure from RCR plasmids. It forms a toroidal homohexameric ring where each RepB protomer consists of two domains: the C-terminal domain involved in oligomerization and the N-terminal domain containing the DNA-binding and endonuclease activities. Binding of Mn2+ to the active site is essential for the catalytic activity of RepB. In this work, we have studied the effects of metal binding on the structure and thermostability of full-length hexameric RepB and each of its separate domains by using different biophysical approaches. The analysis of the temperature-induced changes in RepB shows that the first thermal transition, which occurs at a range of temperatures physiologically relevant for the pMV158 pneumococcal host, represents an irreversible conformational change that affects the secondary and tertiary structure of the protein, which becomes prone to self-associate. This transition, which is also shown to result in loss of DNA binding capacity and catalytic activity of RepB, is confined to its N-terminal domain. Mn2+ protects the protein from undergoing this detrimental conformational change and the observed protection correlates well with the high-affinity binding of the cation to the active site, as substituting one of the metal-ligands at this site impairs both the protein affinity for Mn2+and the Mn2+-driven thermostabilization effect. The level of catalytic activity of the protein, especially in the case of full-length RepB, cannot be explained based only on the high-affinity binding of Mn2+ at the active site and suggests the existence of additional, lower-affinity metal binding site(s), missing in the separate catalytic domain, that must also be saturated for maximal activity. The molecular bases of the thermostabilizing effect of Mn2+ on the N-terminal domain of the protein as well as the potential location of additional metal binding sites in the entire RepB are discussed.

Roles of small laccases from Streptomyces in lignin degradation.

Laccases (EC 1.10.3.2) are multicopper oxidases that can oxidize a range of substrates, including phenols, aromatic amines, and nonphenolic substrates. To investigate the involvement of the small Streptomyces laccases in lignin degradation, we generated acid-precipitable polymeric lignin obtained in the presence of wild-type Streptomyces coelicolor A3(2) (SCWT) and its laccase-less mutant (SCΔLAC) in the presence of Miscanthus x giganteus lignocellulose. The results showed that strain SCΔLAC was inefficient in degrading lignin compared to strain SCWT, thereby supporting the importance of laccase for lignin degradation by S. coelicolor A3(2). We also studied the lignin degradation activity of laccases from S. coelicolor A3(2), Streptomyces lividans TK24, Streptomyces viridosporus T7A, and Amycolatopsis sp. 75iv2 using both lignin model compounds and ethanosolv lignin. All four laccases degraded a phenolic model compound (LM-OH) but were able to oxidize a nonphenolic model compound only in the presence of redox mediators. Their activities are highest at pH 8.0 with a low krel/Kapp for LM-OH, suggesting that the enzymes’ natural substrates must be different in shape or chemical nature. Crystal structures of the laccases from S. viridosporus T7A (SVLAC) and Amycolatopsis sp. 75iv2 were determined both with and without bound substrate. This is the first report of a crystal structure for any laccase bound to a nonphenolic β-O-4 lignin model compound. An additional zinc metal binding site in SVLAC was also identified. The ability to oxidize and/or rearrange ethanosolv lignin provides further evidence of the utility of laccase activity for lignin degradation and/or modification.

A Method for Improving the Optical Properties of a Fluoregenic Di-metal Chelator as a Zn2+Ion Fluorescent Probe by Using a Bridging Substrate

Fluorescent probe based detection methods for metal ions are indispensable tools in many fields, including medical diagnostics, environmental monitoring, living cell studies, and electronics. These methods have multiple advantages over other methods, such as high sensitivity, low cost, ease of application, and versatility. Numerous fluorescent metal ion probes have been designed using many strategies, including fluorogenic metal chelators, fluorescent dye tagged oligonucleotides, catalytic signal amplification, and chemodosimetors. Fluorogenic metal chelators, which consist of a fluorogenic unit (signaling site) covalently linked to chelating moieties (receptor units) with an appropriate spacer, are a general type metal ion probe. The recognition of chelating moieties with metal ions induces a change in the photophysical properties of the fluorescent probe. This is converted into an optical signal expressed as an enhancement or quenching of the fluorophore emission. The recognition can be enhanced to utilize additive reagents that provide additional binding sites for metal ions. For example, 8-aminoquinolino-β-cyclodextrin, developed by Liu et al., exhibited cooperative binding to Zn ion with 1-adamatanoic acid. It also detected Zn ions more efficiently than without 1adamatanoic acid because 1-adamatanoic acid bound to βcyclodextrin to provide additional binding site for Zn ions. This strategy can easily expand to improve the optical properties of metal ion sensors with two metal binding sites. Di-metal complexes may include bridging substrates to complete the metal coordination sphere, and bridging substrates can modulate the properties of the resulting cascade complexes. In particular, bridging substrates provided additional metal ion binding sites and enhanced the binding properties of metal ions to metal ion ligands. Various fluorescent molecules with two metal binding sites have been synthesized as efficient probes for detecting metal ions, such as Zn and Ag. Scaffolds of di-metalic fluoregenic probes have also been devised to detect anions. Most of these molecules can be used as metal ion probes. The optical properties of these molecules can be improved by bridging substrates, which provide additional metal ion binding sites. To validate this concept, (9,10-bis[(2,2-dipicolylamino)methyl]anthracene) (1) was adapted as a Zn ion probe because [Zn2(1)] 4+ efficiently detects phosphate derivatives as potential bridging substrates to provide additional metal binding sites. In various phosphate derivatives, ATP was adapted as a bridging substrate to provide additional metal binding sites because [Zn2(1)] 4+ fluorescence is most enhanced by binding with ATP. Therefore, a combination of 1 and ATP (1/ATP) was prepared as a Zn fluorescent probe. The optical properties of 1/ATP, compared to those of 1, can be improved by providing additional binding sites for metal ions using ATP, as depicted in Scheme 1. To determine the optimal ratio of 1 and ATP, fluorescence titration of ATP was conducted using a 5 μM solution of 1 with 2 equivalents of Zn ions. The fluorescence emission spectra of [Zn2(1)] 4+ in the presence of varied ATP concentrations were evaluated. [Zn2(1)] 4+ fluorescence increased nearly proportionally to ATP concentration and was maximized at one equivalent of ATP (see Supporting information). A sensing system (1/ATP) consiting of 1 (5 μM) and ATP (5 μM) was prepared to evaluate the cooperative binding effect of the bridging substrate by providing additional binding sites for Zn ions. Fluorescence changes of 1/ATP in the presence of various concentrations of Zn were examined. The addition of Zn ions induced greatly enhanced fluorescence. The binding zinc ions within the bis-(2-picolyl)amine in 1 prevents the photo induced electron transfer process

Semi‐Synthesis and Analysis of Chemically Modified Zif268 Zinc‐Finger Domains

Total synthesis of proteins can be challenging despite assembling techniques, such as native chemical ligation (NCL) and expressed protein ligation (EPL). Especially, the combination of recombinant protein expression and chemically addressable solid-phase peptide synthesis (SPPS) is well suited for the redesign of native protein structures. Incorporation of analytical probes and artificial amino acids into full-length natural protein domains, such as the sequence-specific DNA binding zinc-finger motifs, are of interest combining selective DNA recognition and artificial function. The semi-synthesis of the natural 90 amino acid long sequence of the zinc-finger domain of Zif268 is described including various chemically modified constructs. Our approach offers the possibility to exchange any amino acid within the third zinc finger. The realized modifications of the natural sequence include point mutations, attachment of a fluorophore, and the exchange of amino acids at different positions in the zinc finger by artificial amino acids to create additional metal binding sites. The individual constructs were analyzed by circular dichroism (CD) spectroscopy with respect to the integrity of the zinc-finger fold and DNA binding.

Three-dimensional structure of the human copper transporter hCTR1

Copper uptake proteins (CTRs), mediate cellular acquisition of the essential metal copper in all eukaryotes. Here, we report the structure of the human CTR1 protein solved by electron crystallography to an in plane resolution of 7 A. Reminiscent of the design of traditional ion channels, trimeric hCTR1 creates a pore that stretches across the membrane bilayer at the interface between the subunits. Assignment of the helices identifies the second transmembrane helix as the key element lining the pore, and reveals how functionally important residues on this helix could participate in Cu(I)-coordination during transport. Aligned with and sealing both ends of the pore, extracellular and intracellular domains of hCTR1 appear to provide additional metal binding sites. Consistent with the existence of distinct metal binding sites, we demonstrate that hCTR1 stably binds 2 Cu(I)-ions through 3-coordinate Cu-S bonds, and that mutations in one of these putative binding sites results in a change of coordination chemistry.

A Charge Reversal Differentiates (p)ppGpp Synthesis by Monofunctional and Bifunctional Rel Proteins

A major regulatory mechanism evolved by microorganisms to combat stress is the regulation mediated by (p)ppGpp (the stringent response molecule), synthesized and hydrolyzed by Rel proteins. These are divided into bifunctional and monofunctional proteins based on the presence or absence of the hydrolysis activity. Although these proteins require Mg(2+) for (p)ppGpp synthesis, high Mg(2+) was shown to inhibit this reaction in bifunctional Rel proteins from Mycobacterium tuberculosis and Streptococcus equisimilis. This is not a characteristic feature in enzymes that use a dual metal ion mechanism, such as DNA polymerases that are known to carry out a similar pyrophosphate transfer reaction. Comparison of polymerase Polbeta and Rel(Seq) structures that share a common fold led to the proposal that the latter would follow a single metal ion mechanism. Surprisingly, in contrast to bifunctional Rel, we did not find inhibition of guanosine 5'-triphosphate, 3'-diphosphate (pppGpp) synthesis at higher Mg(2+) in the monofunctional RelA from Escherichia coli. We show that a charge reversal in a conserved motif in the synthesis domains explains this contrast; an RXKD motif in the bifunctional proteins is reversed to an EXDD motif. The differential response of these proteins to Mg(2+) could also be noticed in fluorescent nucleotide binding and circular dichroism experiments. In mutants where the motifs were reversed, the differential effect could also be reversed. We infer that although a catalytic Mg(2+) is common to both bifunctional and monofunctional proteins, the latter would utilize an additional metal binding site formed by EXDD. This work, for the first time, brings out differences in (p)ppGpp synthesis by the two classes of Rel proteins.

Construction of an additional metal-binding site in human metallothionein-2

We have constructed a new metal-binding site in the human metallothionein-2 (hMT-2), using the protein as a scaffold to investigate the structure and function of metal-binding. Potential metal-binding sites were designed within hMT-2 on the basis of structures generated by homology modeling. Amino acid residues D11, C13, C26 and S28 in the beta-domain of hMT-2 (hMT-2beta) were found, by computer search, to form a potential tetrahedral Cys4 metal-binding site. Six mutant proteins were constructed with the following amino acid substitutions: D11C, S28C and D11C/S28C in hMT-2 and the same mutations in hMT-2beta, respectively. These single-mutant and double-mutant proteins bound one gram atom of cadmium or zinc ions per gram molecule of protein more than the corresponding wild-type proteins. The circular dichroism spectra suggested that the structures of the single-mutant proteins that bound Cd or Zn were similar to that of the D11C/S28C double-mutant proteins. To evaluate the metal-binding affinity of the mutant proteins, we performed pH titrations of wild-type and mutant proteins. The stability with changes in pH of all the mutant proteins was higher than that of the wild-type proteins, and that of the double-mutant D11C/S28C protein was highest. Consequently, it appears that we were able to create novel proteins that bound metal ions at high density and with high affinity.

Structural Insights into the Mechanism of Nuclease A, a ββα Metal Nuclease from Anabaena

Nuclease A (NucA) is a nonspecific endonuclease from Anabaena sp. capable of degrading single- and double-stranded DNA and RNA in the presence of divalent metal ions. We have determined the structure of the Δ(2-24),D121A mutant of NucA in the presence of Zn2+ and Mn2+ (PDB code 1ZM8). The mutations were introduced to remove the N-terminal signal peptide and to reduce the activity of the nonspecific nuclease, thereby reducing its toxicity to the Escherichia coli expression system. NucA contains a ββα metal finger motif and a hydrated Mn2+ ion at the active site. Unexpectedly, NucA was found to contain additional metal binding sites ∼26 A apart from the catalytic metal binding site. A structural comparison between NucA and the closest analog for which structural data exist, the Serratia nuclease, indicates several interesting differences. First, NucA is a monomer rather than a dimer. Second, there is an unexpected structural homology between the N-terminal segments despite a poorly conserved sequence, which in Serratia includes a cysteine bridge thought to play a regulatory role. In addition, although a sequence alignment had suggested that NucA lacks a proposed catalytic residue corresponding to Arg57 in Serratia, the structure determined here indicates that Arg93 in NucA is positioned to fulfill this role. Based on comparison with DNA-bound nuclease structures of the ββα metal finger nuclease family and available mutational data on NucA, we propose that His124 acts as a catalytic base, and Arg93 participates in the catalysis possibly through stabilization of the transition state.

Copper induces increased beta-sheet content in the scrapie-susceptible ovine prion protein PrPVRQ compared with the resistant allelic variant PrPARR.

Prion diseases are characterized by conformational change in the copper-binding protein PrP (prion protein). Polymorphisms in ovine PrP at amino acid residues 136, 154 and 171 are associated with variation in susceptibility to scrapie. PrPVRQ [PrP(Val136/Arg154/Gln171)] or PrPARQ [PrP(Ala136/Arg154/Gln171)] animals show susceptibility to scrapie, whereas those that express Ala136/Arg154/Arg171 (PrPARR) show resistance. Results are presented here that show PrPVRQ and PrPARR display different conformational responses to metal-ion interaction. At 37 degrees C copper induced different levels of b-sheet content in the allelic variants of ovine full-length prion protein (amino acid 25-232). PrPVRQ showed a significant increase in b-sheet content when exposed to copper at 37 degrees C, whereas PrPARR remained relatively unchanged. The conversion of a-helical PrPVRQ to b-sheet form was shown by CD spectroscopy and the decreased binding of C-terminal specific monoclonal anti-PrP antibodies. This conversion to an increased b-sheet form did not occur with truncated PrPVRQ (amino acids 89-233), which demonstrates that additional metal-binding sites outside of the N-terminus may not overtly influence the overall structure of ovine PrP. Despite the difference in b-sheet content, both the scrapie-susceptible and -resistant allelic forms of ovine PrP acquired resistance to proteinase K digestion following exposure to copper at 37 degrees C, suggesting the potential for disease-associated PrPARR to accumulate in vivo. Our present study demonstrates that allelic variants of ovine PrP differ in their structure and response to the interaction with copper. These observations will contribute to a better understanding of the mechanism of susceptibility and resistance to prion disease.

Monomeric Cu,Zn-superoxide Dismutase Is a Common Misfolding Intermediate in the Oxidation Models of Sporadic and Familial Amyotrophic Lateral Sclerosis

Proteinacious intracellular aggregates in motor neurons are a key feature of both sporadic and familial amyotrophic lateral sclerosis (ALS). These inclusion bodies are often immunoreactive for Cu,Zn-superoxide dismutase (SOD1) and are implicated in the pathology of ALS. On the basis of this and a similar clinical presentation of symptoms in the familial (fALS) and sporadic forms of ALS, we sought to investigate the possibility that there exists a common disease-related aggregation pathway for fALS-associated mutant SODs and wild type SOD1. We have previously shown that oxidation of fALS-associated mutant SODs produces aggregates that have the same morphological, structural, and tinctorial features as those found in SOD1 inclusion bodies in ALS. Here, we show that oxidative damage of wild type SOD at physiological concentrations ( approximately 40 microm) results in destabilization and aggregation in vitro. Oxidation of either mutant or wild type SOD1 causes the enzyme to dissociate to monomers prior to aggregation. Only small changes in secondary and tertiary structure are associated with monomer formation. These results indicate a common aggregation prone monomeric intermediate for wild type and fALS-associated mutant SODs and provides a link between sporadic and familial ALS.

A Mass Spectrometric Study of Metal Binding to Osteocalcin

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry was used to investigate Ca 2+, Mg 2+, and La 3+ binding to bovine bone osteocalcin (OCN). OCN was shown to bind 3 mol Ca 2+ per mol protein. There was also evidence for the presence of four additional metal binding sites. Ca 2+ increased the formation of the OCN dimer. Mg 2+ bound to OCN to the same extent as Ca 2+ but did not induce the dimerization of OCN. La 3+ bound to a lesser extent than either Ca 2+ or Mg 2+ to OCN and, like Mg 2+, did not influence dimerization. Each Gla residue of OCN participates in Ca 2+ binding, whereas Mg 2+ binding may occur preferentially at sites other than Gla residues. This implies that the different natures of Ca 2+- and Mg 2+-containing OCN complexes influence the tendency of OCN to form a dimer.

Silver co-ordination compounds with aliphatic nitriles containing amino nitrogen as H bond donor or additional metal binding site: formation of a ring, a chain, a polymeric sheet and a 3D polymeric channeled structure

Three co-ordination polymers and a discrete 20-membered macrometallacycle were prepared by crystallization of silver salts with the conformationally flexible, polyfunctional ligands N-(2-cyanoethyl)glycine (ceg), 3,3′-iminodipropionitrile (idpn) and tris(2-cyanoethyl)amine (tca). Single-crystal X-ray analyses of [Ag2(tca)2](ClO4)2 (1), [Ag(idpn)]ClO4 (2), [Ag(ceg)]CF3SO3 (3) and [Ag(ceg)]ClO4·0.5H2O (4) revealed a bimetallic macrocycle (1), a 2D sheet structure with 4.82 topology (2), a 3D network structure with rhomboid channels (3) and a 1D chain structure (4). The rhomboid channels in 3 are generated by 36-membered macrometallacyclic subunits consisting of four Ag atoms and four ceg ligands with ceg binding through the carboxylate group and the nitrile nitrogen. The amino nitrogen of ceg is protonated and serves as a hydrogen bond donor for the triflate anions that are incorporated into the macrocycles, thus stabilizing the channeled structure.

Expression, Purification, and Characterization of Recombinant Forms of Membrane-Bound Cytochromec-550nm fromBacillus subtilis

Bacillus subtilisexpresses a cytochromec-550nm that participates in respiratory electron transfer and is an integral membrane protein. Analysis of theB. subtiliscytochromec-550nm amino acid sequence predicts a single N-terminal transmembrane helix attached to a water-soluble heme binding domain [C. von Wachenfeldt and L. Hederstedt (1990)J. Biol. Chem.265, 13939–13948]. We have purified cytochromec-550nm from wild-typeB. subtilisandB. subtilistransformed with the shuttle vector pHP13 containing the gene forB. subtiliscytochromec-550nm (cccA). InB. subtilistransformed with pHP13/cccA there is better than eightfold more membrane-bound cytochromec-550nm than in wild-typeB. subtilis. The overexpressed cytochromec-550nm can be purified by chromatography on hydroxylapatite and Q-Sepharose media. A six-histidine tag has been added to the C-terminus of cytochromec-550nm fromB. subtilisas a further aid for purification. This strain produces cytochromec-550nm to a level fourfold greater than wild type and allows for one-step purification using metal affinity chromatography. UV-Vis spectroscopy detects no change in the heme C spectrum due to the addition of six histidines. Neither form ofB. subtiliscytochromec-550nm is stable in its reduced state in aerated buffer, unless EDTA is added. The two forms, wild-type and his-tagged, of cytochromeschave similar midpoint redox potentials of 195 and 185 mV, respectively, and are equally good substrates forB. subtiliscytochromecoxidase. We conclude that the addition of the histidine tag eases the purification of cytochromec-550nm fromB. subtilisplasma membranes and that the additional metal binding site does not compromise the stability or functional properties of the protein.

Effect of calcium and magnesium ions on radiation-induced inactivation of plant peroxidases

Radiation-induced inactivation of soybean, horseradish, peanut, and tobacco peroxidases in the presence and in the absence of calcium and magnesium salts involves two steps which differ in the character of the effect of the metal cation. The effect of calcium cations is determined by the total charge of the enzyme molecule under experimental conditions. In general, calcium stabilizes the peroxidases, although in the case of anionic soybean and tobacco peroxidases it destabilizes the enzymes at the first inactivation step, probably due to absorption on their surface and changes in the native conformation. The effect of magnesium ions is determined by the specific features of the enzyme structure, and the data obtained allow us to suggest the existence of additional metal-binding sites in tobacco and peanut peroxidases.