Imidazolate Bridge Research Articles

Conformational variability of the Cu site in one subunit of bovine CuZn superoxide dismutase: the importance of mobility in the Glu119-Leu142 loop region for catalytic function

The structure of the catalytic site in one subunit of bovine CuZn superoxide dismutase is shown to be highly variable. A series of crystal structures at ∼1.7 Å have been determined using data collected from different crystals. Several conformations are observed for the copper site from one of the subunits. These conformations lie between those expected for the Cu(II) and Cu(I) forms of the enzyme and may represent a slow positional rearrangement of the Cu site during the crystallisation process due to the presence of a trace reductant in the mother liquor. These states perhaps indicate some functionally relevant structural steps that ultimately result in the breakage of the imidazolate bridge between the two metal sites. This behaviour is not observed for the second subunit of the dimeric enzyme, which remains in the five-coordinate, distorted square planar geometry in all cases. We suggest that this asymmetric behaviour may be caused by the lack of mobility for the Glu119-Leu142 loop region in the second subunit caused by crystal contacts. This region forms one wall of the active-site cavity, and its mobility has been suggested, via molecular dynamics studies, to be important for the catalytic mechanism.

Identification of the Cu2+ binding sites in the N-terminal domain of the prion protein by EPR and CD spectroscopy.

Recent evidence indicates that the prion protein (PrP) plays a role in copper metabolism in the central nervous system. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds divalent copper ions (Cu(2+)) in vivo. To elucidate the specific mode and site of binding, we have studied a series of Cu(2+)-peptide complexes composed of 1-, 2-, and 4-octarepeats and several sub-octarepeat peptides, by electron paramagnetic resonance (EPR, conventional X-band and low-frequency S-band) and circular dichroism (CD) spectroscopy. At pH 7.45, two EPR active binding modes are observed where the dominant mode appears to involve coordination of three nitrogens and one oxygen to the copper ion, while in the minor mode two nitrogens and two oxygens coordinate. ESEEM spectra demonstrate that the histidine imidazole contributes one of these nitrogens. The truncated sequence HGGGW gives EPR and CD that are indistinguishable from the dominant binding mode observed for the multi-octarepeat sequences and may therefore comprise the fundamental Cu(2+) binding unit. Both EPR and CD titration experiments demonstrate rigorously a 1:1 Cu(2+)/octarepeat binding stoichiometry regardless of the number of octarepeats in a given peptide sequence. Detailed spin integration of the EPR signals demonstrates that all of the bound Cu(2+) is detected thereby ruling out strong exchange coupling that is often found when there is imidazolate bridging between paramagnetic metal centers. A model consistent with these data is proposed in which Cu(2+) is bound to the nitrogen of the histidine imidazole side chain and to two nitrogens from sequential glycine backbone amides.

Metal-Bound Histidine Modes in UV Resonance Raman Spectra of Cu, Zn Superoxide Dismutase

Ultraviolet resonance Raman [UVRR] spectra of Cu, Zn superoxide dismutase [SOD] contain bands arising from vibrations of metal-bound histidine ligands. Spectra in H2O solution reveal several modes of the His61 side chain, which bridges the Cu2+ and Zn2+ ions as imidazolate. The disappearance of these bands signals disruption of the bridge when the pH is lowered to 3.0, or the Cu2+ is reduced to Cu+. Binding of hydroxide [pH 12] or cyanide to the Cu2+ perturbs the imidazolate modes, reflecting geometry changes induced by these strong-field ligands. In D2O solution several additional bands become enhanced which arise from histidine ligands that have undergone NH/D exchange. Some of these are attributed to Cu-bound ligands and others to Zn-bound ligands, on the basis of selective changes accompanying removal and replacement of the metals. Excitation profiles are similar for these bands, and for the bridging imidazolate bands; they are red-shifted relative to nonligating histidine. The detection of site-speci...

A polymeric, layered bimetallic Mn(ii)Fe(iii) imidazolate network; crystal structure and magnetic properties

The use of a C3 complex bearing three imidazolate groups as a ‘building block’ allowed us to synthesize an infinite corrugated two-dimensional (2D) low-spin Fe(III)–high-spin Mn(II) polymer exhibiting weak ferromagnetic intralayer interaction through imidazolate bridge.

Polybis(4-azabenzimidazolato)-iron(II) and -cobalt(II). 3-D single diamond-like framework materials which exhibit spin canting and ferromagnetic ordering at low temperatures †

The direct reaction between ferrocene and an excess of molten 4-azabenzimidazole yielded amber-green crystals of polybis(4-azabenzimidazolato)iron(II), 1. Single crystal X-ray diffraction studies revealed tetrahedrally coordinated iron ions linked by single imidazolate bridges and forming a 3-D single diamond-like (diamondoid) extended lattice. Magnetization studies show the effects of antiferromagnetic exchange above a critical temperature, Tc = 21 K, and long-range ferromagnetic ordering below this temperature, behavior that classifies 1 as a molecule-based magnet. Magnetic hysteresis studies at 4.8 K yield a coercive field, Hcoer = 80 G and a remnant magnetization, Mrem = 2100 cm3 G mol−1. The isomorphous polybis(4-azabenzimidazolato)cobalt(II), 2, has also been studied. It too is a low temperature molecule-based magnet. For 2, Tc = 11 K and from magnetic hysteresis studies at 10 K, Hcoer = 400 G and Mrem = 22 cm3 G mol−1.

Synthesis and X-Band Spectra of An Imidazolate-Bridged Copper-Zinc Binuclear Complex

The pH-dependence of the EPR spectra of a newly synthesized imidazolate-bridged Cu(II)-Zn(II) complex, [(dien)Cu(im)Zn(dien)]3+ (dien = diethylenetriamine, im = imidazolate anion), has been studied in frozen solution (50% DMSO) to explore the mode of breaking of the imidazolate bridge. The data suggest that the imidazolate bridge in the complex is stable only at high pH (>9) and breaks on the zinc side at lower pH values.

On the Metal-Ion-Coordinating Properties of the Benzimidazolate Residue in Aqueous Solution – Extent of Acidification of Benzimidazole-(N3)H Sites by (N1)-Coordinated Divalent Metal Ions

The stability constants of the 1:1 complexes formed between Mg2+, Mn2+, Co2+, Ni2+ or Cd2+ (= M2+) and the anionic 5(6)-nitrobenzimidazolate [= (NBI–H)−] or 5,6-dinitrobenzimidazolate [= (DNBI–H)−] were determined by potentiometric pH titrations in aqueous solution (25 °C; I = 0.5 M, NaNO3). The acidity constants for the deprotonation of the (N1)H sites in neutral NBI and DNBI (= L) were measured by the same method. The comparison of the stability constants determined for the M(L–H)+ complexes with those calculated from log KMLM versus pKHLHstraight-line plots, which were established recently for neutral benzimidazole-type ligands, reveals that the stabilities of the M(L–H)+ complexes are significantly enhanced, as one might have expected due to the negative charge present on the ligands. Of course, the electron-withdrawing properties of (N3)-bound metal ions facilitate the release of the proton from the (N1)H site in the M(NBI)2+ and M(DNBI)2+ complexes, if compared to the situation in the free ligands. The effect of a metal ion bound to a benzimidazolate or imidazolate residue on the coordination tendency toward a further metal ion (giving rise to an imidazolate bridge) was estimated.

On the Metal-Ion-Coordinating Properties of the Benzimidazolate Residue in Aqueous Solution – Extent of Acidification of Benzimidazole-(N3)H Sites by (N1)-Coordinated Divalent Metal Ions

The stability constants of the 1:1 complexes formed between Mg2+, Mn2+, Co2+, Ni2+ or Cd2+ (= M2+) and the anionic 5(6)-nitrobenzimidazolate [= (NBI–H)−] or 5,6-dinitrobenzimidazolate [= (DNBI–H)−] were determined by potentiometric pH titrations in aqueous solution (25 °C; I = 0.5 M, NaNO3). The acidity constants for the deprotonation of the (N1)H sites in neutral NBI and DNBI (= L) were measured by the same method. The comparison of the stability constants determined for the M(L–H)+ complexes with those calculated from log KMLM versus pKHLHstraight-line plots, which were established recently for neutral benzimidazole-type ligands, reveals that the stabilities of the M(L–H)+ complexes are significantly enhanced, as one might have expected due to the negative charge present on the ligands. Of course, the electron-withdrawing properties of (N3)-bound metal ions facilitate the release of the proton from the (N1)H site in the M(NBI)2+ and M(DNBI)2+ complexes, if compared to the situation in the free ligands. The effect of a metal ion bound to a benzimidazolate or imidazolate residue on the coordination tendency toward a further metal ion (giving rise to an imidazolate bridge) was estimated.

Characterization of the Spectroscopic Properties of the Cu,Co Cluster in a Prokaryotic Superoxide Dismutase

A Cu,Co derivative of the Cu,ZnSOD fromPhotobacterium leiognathi,in which cobalt has been selectively substituted for zinc, has been prepared and spectroscopically investigated. The derivative shows three bands in the visible region at 530, 566, and 600 nm when copper is in the oxidized state. Reduction or depletion of the copper ion produce a shift of the band absorbing at 600 to 590 nm because of the detachment from copper of the imidazolate bridging the two metals when copper is in the oxidized state. Numerous isotropically shifted1H NMR lines are observed when copper is oxidized, confirming the presence of the imidazolate bridge between the two metals. Comparison of the optical and the NMR spectra with those observed for the eukaryotic enzyme reveals the occurrence of slight but unambiguous differences diagnostic of a different degree of distortion of the metal cluster between the prokaryotic and eukaryotic enzymes.

Crystallographic structures of bovine copper-zinc superoxide dismutase reveal asymmetry in two subunits: functionally important three and five coordinate copper sites captured in the same crystal

A key feature of the generally accepted catalytic mechanism of CuZn superoxide dismutase (CuZnSOD) is the breakage of the imidazolate bridge between copper and zinc and the loss of a coordinated water molecule from copper on reduction from Cu(II) to Cu(I). Crystal structures exist for the enzyme from a number of sources in the oxidised, five coordinate copper form. For the reduced form two structures from different sources have been determined only recently but provide contradictory results. We present crystal structures of bovine CuZnSOD (BSOD) in two different space groups. The structure of the P2 12 12 1 form (pBSOD), at 1.65 Å resolution clearly shows one subunit with Cu in the five coordinate, oxidised form, and the other with Cu in the three coordinate form expected for the reduced state. This mixed state of pBSOD is confirmed by XANES data of these crystals. The pBSOD structure has thus captured each subunit in one of the two oxidation state conformations and thus provides direct crystallographic evidence for the superoxide dismutase mechanism involving the breakage of the imidazole bridge between Cu and Zn. A shift in the position of copper in subunit A poises the catalytic centre to undergo the first stage of catalysis via dissociation of Cu from His61 with a concomittant movement of the coordinated water molecule towards His61, which rotates by ∼20°, enabling it to form a hydrogen bond to the water molecule. The Cu-Zn separation in the reduced site is increased by ∼0.5 Å. In contrast the 2.3 Å resolution structure in space group C222 1 (cBSOD) shows both of the Cu atoms to be in the five coordinate, oxidised form but in this space group the whole of subunit A is significantly more disordered than subunit B. An examination of published structures of “oxidised” SODs, shows a trend towards longer Cu-Zn and Cu-His61 separations in subunit A, which together with the structures reported here indicate a potential functional asymmetry between the subunits of CuZnSODs. We also suggest that the increased separation between Cu and Zn is a precursor to breakage of His61.

A structure-based mechanism for copper-zinc superoxide dismutase.

A reaction cycle is proposed for the mechanism of copper-zinc superoxide dismutase (CuZnSOD) that involves inner sphere electron transfer from superoxide to Cu(II) in one portion of the cycle and outer sphere electron transfer from Cu(I) to superoxide in the other portion of the cycle. This mechanism is based on three yeast CuZnSOD structures determined by X-ray crystallography together with many other observations. The new structures reported here are (1) wild type under 15 atm of oxygen pressure, (2) wild type in the presence of azide, and (3) the His48Cys mutant. Final R-values for the three structures are respectively 20.0%, 17.3%, and 20.9%. Comparison of these three new structures to the wild-type yeast Cu(I)ZnSOD model, which has a broken imidazolate bridge, reveals the following: (i) The protein backbones (the "SOD rack") remain essentially unchanged. (ii) A pressure of 15 atm of oxygen causes a displacement of the copper ion 0.37 A from its Cu(I) position in the trigonal plane formed by His46, His48, and His120. The displacement is perpendicular to this plane and toward the NE2 atom of His63 and is accompanied by elongated copper electron density in the direction of the displacement suggestive of two copper positions in the crystal. The copper geometry remains three coordinate, but the His48-Cu bond distance increases by 0.18 A. (iii) Azide binding also causes a displacement of the copper toward His63 such that it moves 1.28 A from the wild-type Cu(I) position, but unlike the effect of 15 atm of oxygen, there is no two-state character. The geometry becomes five-coordinate square pyramidal, and the His63 imidazolate bridge re-forms. The His48-Cu distance increases by 0.70 A, suggesting that His48 becomes an axial ligand. (iv) The His63 imidazole ring tilts upon 15 atm of oxygen treatment and azide binding. Its NE2 atom moves toward the trigonal plane by 0.28 and 0.66 A, respectively, in these structures. (v) The replacement of His48 by Cys, which does not bind copper, results in a five-coordinate square pyramidal, bridge-intact copper geometry with a novel chloride ligand. Combining results from these and other CuZnSOD crystal structures, we offer the outlines of a structure-based cyclic mechanism.

E.P.R. study of an imidazolate-bridged Cu-Zn complex of glycylglycine

An imidazolate-bridged heterometallic binuclear copper–zinc complex Na[(glygly)Cu–im–Zn(glygly)], where H 2glygly is glycylglycine and imH is imidazole, has been synthesized. X-band e.p.r. spectra of the complex at different pH values in frozen solution (50% DMSO, 77 K) suggest that the imidazolate bridged complex is stable in the pH range 7–10. At lower pH (<7) the imidazolate bridge breaks on the copper side. At higher pH (>10), the e.p.r. changes suggest either hydroxylation of the copper centre or breaking of the imidazolate bridge on the zinc side.

The synthesis, structure and SOD-like behaviors of a μ-imidazolato-dicopper(II) complex with a binucleating hexaazamacrocycle

The synthesis, crystal structure and magnetic properties of the imidazolate-bridged dinuclear copper(II) complex [LCu 2(Im)(DMSO) 2](ClO 4) 3(DMSO), where ImH is imidazole, and L is 3, 6, 9, 17, 20, 23-hexaazatricyclo[23.3.1.1 11,13]triaconta-1(29), 11(30), 12, 14, 25(26), 27-hexaene, have been studied. Single crystal X-ray diffraction determination reveals the distorted tetragonal pyramid geometries of the imidazolate bridged dicopper(II) center are incorporated within the binucleating macrocycle. Both copper atoms are five-coordinated by four basal nitrogen atoms (three from the macrocycle and one from the imidazolate) and one oxygen atom from the DMSO molecule. Two coordinated DMSO molecules are situated at the same side of the macrocycle. The Cu–Cu separation in the complex is 5.93 Å. Magnetic measurements and ESR spectroscopy studies exibit an antiferromagnetic exchange interaction with a coupling constant of J= − 26.94 cm −1. Investigations on the pH-dependent ESR of the title compound in frozen 50% aqueous DMSO solution confirm the existence of the bridged cation [LCu 2(Im)] 3+ as a major species in solution mainly in the range 5.2⩽pH⩽11.5. The imidazolate bridge is broken in the pH range of 5.2–3.3, which is close to that for the native enzyme.

A Novel Series of Heterotrinuclear Complexes Involving Imidazolate Bridging Schiff's Base Ligands. Synthesis, Crystal Structure, and Magnetic Properties

Mononuclear copper(II) complexes with an unsymmetrical tridentate Schiff's base involving two imidazole moieties, [Cu(HL)(H2O)](ClO4)2 (1) and [Cu(L)(hfac)] (3), have been prepared and characterized, where HL stands for the 1:1 condensation product of 2-imidazolecarboxaldehyde and histamine. Under basic conditions, 1 easily undergoes a self-assembly process affording an insoluble imidazolate-bridged polymeric species {[Cu(L)(H2O)](ClO4)}n (2). By using 1 and 3 as “ligand complexes” toward M(hfac)2 (hfac = hexafluoroacetylacetonate) a series of imidazolate-bridged heterometal trinuclear complexes with the formula [Cu(L)(hfac)M(hfac)2Cu(hfac)(L)] (M = ZnII, CuII, NiII, MnII) (4−7) can be prepared. The structures of these complexes have been solved by X-ray crystallographic methods. All the trinuclear complexes are isostructural and consist of trinuclear molecules with a V-shaped conformation, which are formed by two CuL(hfac) “ligands” bonded through imidazolate bridges to the central metal in cis-position. The ligand environment about the central metal atom can be described as a distorted octahedron, with the nitrogen atoms from the imidazolate groups and two oxygen atoms of the hfac ligands in the equatorial plane and the remaining two oxygen atoms from the hfac ligands filling the axial positions at longer distances. The coordination geometry of the copper(II) ion is very close to a square pyramid with one oxygen atom from the hfac in a more distant axial position. The magnetic susceptibility data (2−300 K) revealed antiferromagnetic interactions between copper(II) ions and the central metal. The magnetic susceptibility data were quantitatively analyzed using the theoretical expressions deduced from the spin Hamiltonian for a symmetrical three-spin system H = −JCuM(SCu1·SM + SM·SCu2) to give the coupling parameters JCuCu = −60.6 cm-1, JCuNi = −21.6 cm-1, and JCuMn = −3.2 cm-1. These magnetic behaviors are discussed on the basis of the localized-orbital model of exchange interactions.

Does the Imidazole Bridge Break During Catalysis in Cu, Zn Superoxide Dismutase ? Judgement from XAFS and Crystallography

The binuclear (Cu, Zn) site of superoxide dismutase is bridged by an imidazole ligand which is thought to dissociate from Cu upon reduction, forming the mechanistic basis of this important enzyme. Conflicting crystallographic structures of the reduced enzyme have been reported in the last 12 months. High resolution XAFS data have been collected for both oxidised and reduced forms of the enzyme using the same buffer systems employed in the crystallographic studies. These data and their comprehensive analysis based on fast curved wave theory with multiple scattering, conclusively show that upon reduction of the enzyme very little change takes place at the Zn site but that Cu becomes three coordinate, thus confirming the XAFS results of Blackburn et al. some 13 years ago.

Reactions of the cis-diamminediaquaplatinum (II) cation with histidine and related molecules

The reaction of cis-[Pt(NH 3) 2(H 2O) 2] 2+ ( 1) with histidine (H 3his +) at pH 2–3 gave initially complexes with histidine bound through carboxylate only, then, after standing, the complex containing an amine nitrogen (N A), carboxylate oxygen-chelate ring [Pt(NH 3) 2(H 2-his- N A , O)] 2+. Increasing the pH to 8–9 caused loss of one imidazole proton, followed by isomerization to the species with a imidazole N(3), N A-chelate ring, [Pt(NH 3) 2(Hhis- N A ,N(3))]. From the variation of NMR parameters with pH, p K a for loss of the last imidazole proton was determined (11.2 ± 0.1). Histidine methyl ester and histidinamide each reacted slowly with 1 at pH 5.5 to give the N A , N(3)-chelate complex. With N-(histidyl)glycine the initial complexes at pH 5 contained the ligand bound only through carboxylate, but a N A , N(3)-chelate complex then formed. With an excess of 1, a second diammineplatinum moiety was bound, initially through the free carboxylate, then chelated by carboxylate and peptide nitrogen. With N-acetylhistidine and N-( β-alanyl)histidine at pH 4–5, the initial complexes also contained carboxylate-bound ligands, then a chelate ring was formed involving carboxylate and the deprotonated amide or peptide nitrogen, N A. With N-(glycyl)histidine, more complex reactions involving the terminal nitrogen atom also occurred. In alkaline solution, these N A , O-chelate complexes reacted slowly to form a dinuclear complex with one ligand bound to one Pt atom through N A and N(3), and to the second platinum through N(1) of bridging imidazolate. The second ligand was bound monodentate to the second platinum through N A.

A study on reduction potentials of porcine and bovine superoxide dismutase and dissociation of bridging imidazolate

A study on reduction potentials of porcine and bovine superoxide dismutase and dissociation of bridging imidazolate

The crystal and molecular structure of μ-imidazolatobis [bis (1–10 phenanthroline)copper(II)] nitrate

[Cu 2(phen) 4(im)](NO 3) 3·3H 2O·2CH 3OH crystallizes in the triclinic space group P 1(#2) with a = 13.803(10) A ̊ , b = 16.844(8) A ̊ , c = 13.395(9) A ̊ , α = 111.1(2)°, β = 116.8(1)°, γ = 89.6(1)° and Z = 2 . The structure consists of the dimeric [Cu 2(phen) 4(im)] +3 ion with the imidazolate ion bridging the two Cu(phen) 2 moieties. The lattice contains three water molecules and two methanol molecules of solvation as well as the nitrate anions. The two [CuN 4(im)] chromophores are distorted square pyramidal with one of the cis phenanthroline ligands asymmetrically coordinated to the axial (Cu-Nphen = 2.232(9) Å and 2.24(1) Å) and equatorial (Cu-Nphen = 2.016(9) Å and 2.020(9) Å) sites. The remaining equatorial sites are occupied by the second phen ligand on each Cu and the bridging imidazolate ion. The relationship between the structure and the antiferromagnetic behavior of the cation is discussed.

Unusual trigonal-planar copper configuration revealed in the atomic structure of yeast copper-zinc superoxide dismutase.

The three-dimensional structure of yeast copper-zinc superoxide dismutase (CuZnSOD) has been determined in a new crystal form in space group R32 and refined against X-ray diffraction data using difference Fourier and restrained crystallographic refinement techniques. The unexpected result is that the copper ion has moved approximately 1 angstrom from its position in previously reported CuZnSOD models, the copper-imidazolate bridge is broken, and a roughly trigonal planar ligand geometry characteristic of Cu(I) rather than Cu(II) is revealed. Final R values for the two nearly identical room temperature structures are 18.6% for all 19 149 reflections in the 10.0-1.7 angstrom resolution range and 18. 2% for 17 682 reflections (F > 2 sigma) in the 10.0-1.73 angstrom resolution range. A third structure has been determined using X-ray data collected at -180 degrees C. The final R value for this structure is 19.0% (R(free) = 22.9%) for all 24 356 reflections in the 10.0-1.55 angstrom resolution range. Virtually no change in the positions of the ligands to the zinc center is observed in these models. The origin of the broken bridge and altered Cu-ligand geometry is discussed.

New Type 2 Copper-Cysteinate Proteins. Copper Site Histidine-to-Cysteine Mutants of Yeast Copper-Zinc Superoxide Dismutase.

Preparation and characterization of two new site-directed mutant copper-zinc superoxide dismutase proteins from Saccharomyces cerevisiae, i.e., His46Cys (H46C) and His120Cys (H120C), in which individual histidyl ligands in the copper-binding site were replaced by cysteine, are reported here. These two mutant CuZnSOD proteins may be described as type 2 (or normal) rather than type 1 (or blue) copper-cysteinate proteins and are characterized by their yellow rather than blue color, resulting from intense copper-to-sulfur charge transfer bands around 400 nm, their type 2 EPR spectra, with large rather than small nuclear hyperfine interactions, and their characteristic type 2 d-d electronic absorption spectra. An interesting difference between these two copper site His-to-Cys mutations is that the imidazolate bridge between the two metal sites that is characteristic of the wild-type protein remains intact in the case of the H46C mutant but is not present in the case of the H120C mutant.