113Cd-NMR investigation of a cadmium-substituted copper, zinc-containing superoxide dismutase from yeast

113Cd nuclear magnetic resonance spectroscopy has been used to investigate the metal binding sites of cadmium-substituted copper, zinc-containing superoxide dismutase from baker's yeast. NMR signals were obtained for 113Cd(II) at the Cu site as well as for 113Cd(II) at the Zn site. The two subunits in the dimeric enzyme were found to have identical coordination properties towards 113Cd(II) at the Zn site when no copper is coordinated at the Cu site, and when Cu(I) or Cd(II) is coordinated, were found to be very small indicating that 113Cd(II) must be bound to the same number and type of ligands in both cases. Furthermore, the spectra show that the rate of exchange of protein-bound 113Cd(II) and free 113Cd2+ is slow on the NMR time scale also at the Cu site. The present study suggests an explanation for the discrepancy in the literature regarding 113Cd-NMR investigations of bovine superoxide dismutase.     

A spectroscopic characterization of a monomeric analog of copper,zinc superoxide dismutase

A mutated protein of human Cu(II)₂Zn(II)₂ SOD in which residues Phe50 and Gly51 at the dimer interface were substituted by Glu's, thus producing a monomeric species, has been characterized by electronic absorption spectroscopy, EPR, relaxivity and¹H NMR techniques. Such substitutions and/or accompanying remodeling and exposure of the dimer interface to solvent, alter the geometry of the active site: increases in the axiality of the copper chromophore and the Cu-OH₂ distance have been observed. The affinity of both metal binding sites for Co(II) is also altered. The observed NMR parameters of the Co(II) substituted derivative have been interpreted as a function of the decrease of rotational correlation time as a consequence of the lower molecular weight of the mutated protein. Sharper NMR signals are also obtained for the reduced diamagnetic enzyme. Results are consistent with an active site structure similar to that observed for the dimeric analog Thr137Ile characterized elsewhere. An observed proportional decrease in enzymatic activity and affinity for the N₃-anion suggests the importance of electrostatic forces during substrate docking and catalysis.     

Complete 1H, 13C and 15N NMR assignments and secondary structure of the 269-residue serine protease PB92 from Bacillus alcalophilus

Summary The ¹H, ¹³C and ¹⁵N NMR resonances of serine protease PB92 have been assigned using 3D tripleresonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CH_n carbons, all amide (NH and NH₂) nitrogens and 99.2% of the amide and CH_n protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405–411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.Abbreviations 2D/3D/4D two-/three-/four-dimensional - HSQC heteronuclear single-quantum coherence - HMQC heteronuclear multiple-quantum coherence - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - NOE nuclear Overhauser enhancement (connectivity) - NOESY 2D NOE spectroscopy Experiment nomenclature (H(C)CH, etc.) follows the conventions used elsewhere [e.g. Ikura et al. (1990) Biochemistry, 29, 4659–4667].     

Structural Evidence for a Copper-Bound Carbonate Intermediate in the Peroxidase and Dismutase Activities of Superoxide Dismutase

Copper-zinc superoxide dismutase (SOD) is of fundamental importance to our understanding of oxidative damage. Its primary function is catalysing the dismutation of superoxide to O₂ and H₂O₂. SOD also reacts with H₂O₂, leading to the formation of a strong copper-bound oxidant species that can either inactivate the enzyme or oxidise other substrates. In the presence of bicarbonate (or CO₂) and H₂O₂, this peroxidase activity is enhanced and produces the carbonate radical. This freely diffusible reactive oxygen species is proposed as the agent for oxidation of large substrates that are too bulky to enter the active site. Here, we provide direct structural evidence, from a 2.15 Å resolution crystal structure, of (bi)carbonate captured at the active site of reduced SOD, consistent with the view that a bound carbonate intermediate could be formed, producing a diffusible carbonate radical upon reoxidation of copper. The bound carbonate blocks direct access of substrates to Cu(I), suggesting that an adjunct to the accepted mechanism of SOD catalysed dismutation of superoxide operates, with Cu(I) oxidation by superoxide being driven via a proton-coupled electron transfer mechanism involving the bound carbonate rather than the solvent. Carbonate is captured in a different site when SOD is oxidised, being located in the active site channel adjacent to the catalytically important Arg143. This is the probable route of diffusion from the active site following reoxidation of the copper. In this position, the carbonate is poised for re-entry into the active site and binding to the reduced copper.     

Cobalt substitution studies on bovine erythrocyte superoxide dismutase: evidence for a novel cobalt-superoxide dismutase derivative

Three cobalt derivatives of bovine erythrocyte superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) have been prepared under different pH conditions using a cobalt-thiocyanate complex which has already proved to yield specific substitutions on other copper proteins. The cobalt-protein derivatives have been characterized by optical, circular dichroism and fluorescence spectroscopies. One derivative, referred to as Co2Co2-protein, contains Co(II) ions specifically bound at both Zn(II) and Cu(II) sites. On the basis of their spectroscopic properties, the other two derivatives can be referred as E2Co2- and Co2E2-superoxide dismutase, with cobalt substituting, respectively, at the zinc and the copper sites leaving the contiguous site empty (E). The Co2E2-protein complex represents a novel derivative, since it has never been described in literature. The optical spectrum in the visible region of Co2-Co2-protein well corresponds to the sum of the spectra of the other two derivatives. The circular dichroism spectrum of Co2Co2-derivative, however, is not the sum of individual E2Co2- and Co2E2-proteins, suggesting that the presence of Co(II) in one site strongly affects the geometry of the neighbouring site. Some discrepancies between our spectroscopic data and those reported in literature are discussed. The results obtained from fluorescence experiments indicate that Co(II) ions exert a different quenching effect on the tyrosine emission, depending on whether they are located in the Zn(II) or in the Cu(II) site. The fluorescence quenching can be attributed to a 'heavy atom' and 'paramagnetic ion' effect by Co(II) ions.     

Copper complexes of 1,10-phenanthroline and related compounds as superoxide dismutase mimetics

In a preliminary study we tested CuSO4.5H2O, (Cu(II]2[3,5-diisopropylsalicylate]4.2H2O and a number of copper complexes of substituted 1,10-phenanthrolines for superoxide anion dismutase activity. It appeared that this activity depends on the ligands involved and might be governed by the redox potential of the Cu(I) complex/Cu(II) complex couple. The strong superoxide anion dismutase activity of Cu(II)[DMP]2 complex can be expected considering its high redox potential. Rather surprisingly is the superoxide anion dismutase activity of the Cu(I)[DMP]2 complex since it involves oxidation to Cu(II)[DMP]2 complex. From regression analysis it was established that steric and field effects of the substituents of the investigated phenanthrolines play an important role in SOD activity and therefore it is concluded that complex formation is important for the superoxide dismutase-like activity.     

High-resolution solution structure of siamycin II: Novel amphipathic character of a 21-residue peptide that inhibits HIV fusion

Summary The 21-amino acid peptides siamycin II (BMY-29303) and siamycin I (BMY-29304), derived from Streptomyces strains AA3891 and AA6532, respectively, have been found to inhibit HIV-1 fusion and viral replication in cell culture. The primary sequence of siamycin II is CLGIGSCNDFAGCGYAIVCFW. Siamycin I differs by only one amino acid; it has a valine residue at position 4. In both peptides, disulfide bonds link Cys¹ with Cys¹³ and Cys⁷ with Cys¹⁹, and the side chain of Asp⁹ forms an amide bond with the N-terminus. Siamycin II, when dissolved in a 50:50 mixture of DMSO and H₂O, yields NOESY spectra with exceptional numbers of cross peaks for a peptide of this size. We have used 335 NOE distance constraints and 13 dihedral angle constraints to generate an ensemble of 30 siamycin II structures; these have average backbone atom and all heavy atom rmsd values to the mean coordinates of 0.24 and 0.52 Å, respectively. The peptide displays an unusual wedge-shaped structure, with one face being predominantly hydrophobic and the other being predominantly hydrophilic. Chemical shift and NOE data show that the siamycin I structure is essentially identical to siamycin II. These peptides may act by preventing oligomerization of the HIV transmembrane glycoprotein gp41, or by interfering with interactions between gp41 and the envelope glycoprotein gp120, the cell membrane or membrane-bound proteins [Frèchet, D. et al. (1994) Biochemistry, 33, 42–50]. The amphipathic nature of siamycin II and siamycin I suggests that a polar (or apolar) site on the target protein may be masked by the apolar (or polar) face of the peptide upon peptide/protein complexation.Abbreviations ABNR adopted basis Newton Raphson - AIDS acquired immunodeficiency syndrome - CW continuous wave - DMSO dimethylsulfoxide - DQF-COSY two-dimensional double-quantum-filtered correlation spectroscopy - HIV human immunodeficiency virus - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser enhancement spectroscopy - ppm parts per million - P.E.-COSY two-dimensional primitive exclusive correlation spectroscopy - REDAC redundant dihedral angle constraint - rf radio frequency - rmsd root-mean-square difference - SIV simian immunodeficiency virus - sw spectral width - _m mixing time - TOCSY two-dimensional total correlation spectroscopy - TSP trimethylsilyl-2,2,3,3-²H₄-propionate - 2D two-dimensional     

Silibinin induces protective superoxide generation in human breast cancer MCF-7 cells

Abstract

The pharmacological activity of polyphenolic silibinin from milk thistle (Silybum marianum) is primarily due to its antioxidant property. However, this study found that silibinin promoted sustained superoxide (O₂^·–) production that was specifically scavenged by exogenous superoxide dismutase (SOD) in MCF-7 cells, while the activity of endogenous SOD was not changed by silibinin. Previous work proved that silibinin induced MCF-7 cell apoptosis through mitochondrial pathway and this study further proved that O₂^·– generation induced by silibinin was also related to mitochondria. It was found that respiratory chain complexes I, II and III were all involved in silibinin-induced O₂^·– generation. Moreover, it was found that silibinin-induced O₂^·– had protective effect, as exogenous SOD markedly enhanced silibinin-induced apoptosis.     

Crystal structure of the second domain of the human copper chaperone for superoxide dismutase

The human copper chaperone for superoxide dismutase (hCCS) delivers the essential copper ion cofactor to copper,zinc superoxide dismutase (SOD1), a key enzyme in antioxidant defense. Mutations in SOD1 are linked to familial amyotrophic lateral sclerosis (FALS), a fatal neurodegenerative disorder. The molecular mechanisms by which SOD1 is recognized and activated by hCCS are not understood. To better understand this biochemical pathway, we have determined the X-ray structure of the largest domain of hCCS (hCCS Domain II) to 2. 75 A resolution. The overall structure is closely related to that of its target enzyme SOD1, consisting of an eight-stranded beta-barrel and a zinc-binding site formed by two extended loops. The first of these loops provides the ligands to a bound zinc ion, and is analogous to the zinc subloop in SOD1. The second structurally resembles the SOD1 electrostatic channel loop, but lacks many of the residues important for catalysis. Like SOD1 and yCCS, hCCS forms a dimer using a highly conserved interface. In contrast to SOD1, however, the hCCS structure does not contain a copper ion bound in the catalytic site. Notably, the structure reveals a single loop proximal to the dimer interface which is unique to the CCS chaperones.     

香蒲对不同浓度Pb~(2+)胁迫的生理应答及其细胞超微结构变化

通过室内水培试验,研究了不同浓度Pb2+(0、0.25、0.50、1.00和2.00mmol·L-1)胁迫对东方香蒲根和叶中Pb含量、叶绿素含量、丙二醛(MDA)含量、抗氧化酶(SOD、CAT和POD)活性以及亚细胞结构的影响。结果显示:(1)随着外源Pb2+浓度的增加,Pb在香蒲根和叶中的积累量均显著高于对照,且Pb在根中的含量明显高于叶中,并与外源Pb2+浓度呈显著正相关关系。(2)香蒲叶片中的叶绿素a和叶绿素b含量随着外源Pb2+浓度的增加呈先升后降趋势,均在处理浓度为0.50mmol·L-1时达到峰值。(3)胁迫处理叶片的MDA含量与对照相比变化不显著,但根中MDA含量呈显著下降趋势。(4)叶片中SOD活性在1.00mmol·L-1 Pb2+处理时达到峰值,然后下降,但始终高于对照,CAT和POD活性则均低于对照组;根中SOD活性除1.00mmol·L-1 Pb2+处理组外均显著低于对照组,CAT和POD活性分别在0.25和0.50mmol·L-1 Pb2+处理时达到峰值,然后随处理Pb2+浓度升高而下降。(5)电镜观察发现,Pb2+胁迫使香蒲叶细胞中叶绿体被膜破裂,类囊体膨胀、破损;根和叶细胞中的线粒体被膜均破裂、内腔空泡化,细胞核核膜破损、核仁消失、染色质凝集。研究表明,Pb2+胁迫致使东方香蒲根、叶生理代谢失衡,亚细胞结构出现不可逆的损伤,这为从分子水平研究Pb2+作用的具体机理以及香蒲在重金属污染修复中的应用提供了依据。     

An epr signal from the half-reduced type 3 copper pair in Rhus vernicifera laccase

A new type of Cu(II) epr signals have been produced in native and type 2 copper depleted Rhus vernicifera laccase. They are shown to originate from one of the type 3 copper ions that are epr silent in the resting enzyme. The new epr signals show high rhombicity in g tensor and are similar to those observed in other proteins, such as superoxide dismutase and half-met hemocyanin. The half-reduced type 3 copper pair is formed by reduction with an electron from type 1 Cu(I) but only after a reoxidation of the copper pair, either by peroxide or dioxygen. It is suggested that the half-reduction of the type 3 copper pair only occurs in molecules where type 2 copper ion is either reduced or absent.     

X-ray absorption edge spectroscopy of Co(II)-binding sites of copper- and zinc-containing proteins

X-ray absorption near-edge spectroscopy (XANES) of Co(II) in three derivatives of superoxide dismutase, namely [Cu(II)-Co(II)], [Cu(I)-Co(II)] and [...-Co(II)], suggests a tetrahedral coordination of the metal for all compounds. Significant differences, detected in the spectrum of the [Cu(II)-Co(II)] derivative as compared to the other species, indicate that a conformational change and/or a different charge of the imidazole bridging the two metal sites in superoxide dismutase occur in coincidence with the change of copper valence. The XANES spectra of the cobalt derivatives of alcohol dehydrogenase, carbonic anhydrase and stellacyanin show features that can be accounted for by an increasing degree of covalency in the metal first sphere of coordination, in the following order: alcohol dehydrogenase greater than stellacyanin greater than superoxide dismutase greater than or equal to carbonic anhydrase.     

Superoxide dismutase, a study of the electronic properties of the copper and zinc by X-ray absorption spectroscopy

The x-ray absorption for copper and zinc in oxidized and reduced superoxide dismutase, as well as in various model compounds, was studied. Upon reduction of the protein, the added electron affects the copper site almost exclusively, while the zinc remains virtually unchanged. Reduction decreases the charge on the copper atom [toward Cu(I)] and changes the configuration of the copper site so that it becomes less symmetric. An analysis of the copper absorption observed with the oxidized enzyme and a comparison with that for Cu(II)(imid)4 suggests that the copper is not simply ligated to four imidazoles. The addition of H2O2 to superoxide dismutase reduces the copper to Cu(I), while oxygen addition to the peroxide-reduced protein restores the copper to Cu(II).     

The Zn-Site in Bovine Copper,Zinc Superoxide Dismutase Studied by 111Cd Pac

The active site in bovine copper, zinc superoxide dismutase (Cu₂. Zn₂ SOD) has been studied by ¹¹¹Cd time differential Perturbed Angular Correlation (PAC) on enzyme with Zn²⁺ replaced by excited 'Cd²⁺. The PAC spectra obtained for both the oxidized and the reduced form of Cu₂Cd₂SOD show no asymmetry between the two Zn-sites in the dimeric enzyme. The spectv further reveal that a significant change has taken place at the Zn-site in the reduced form compared to the oxidized form.

Semi-empirical calculations based on the Angular Overlap Model (AOM) and coordinates from the crystal structure of the native enzyme are in agreement with the experimental PAC data of the oxidized enzyme. The results indicate that Cd²⁺ coordinates in the same manner as Zn²⁺ and that the crystal structure of SOD is valid for the enzyme in solution. The PAC spectrum of the reduced enzyme can be explained by extending the AOM calculations to the enzyme in the reduced form and assuming that the imidazol ring of His61 is no longer bridging the copper and cadmium ions in the reduced state.     

Resonance assignment and structural analysis of acid denatured E. coli [U-15N]-glutaredoxin 3

Virtually complete sequence specific ¹H and ¹⁵N resonance assignments are presented for acid denatured reduced E. coli glutaredoxin 3. The sequential resonance assignments of the backbone rely on the combined use of 3D F₁-decoupled ROESY-¹⁵N-HSQC and 3D ¹⁵N-HSQC-(TOCSY-NOESY)-¹⁵N-HSQC using a single uniformly ¹⁵N labelled protein sample. The sidechain resonances were assigned from a 3D TOCSY-¹⁵N-HSQC and a homonouclear TOCSY spectrum. The presented assignment strategy works in the absence of chemical exchange peaks with signals from the native conformation and without ¹³C/¹⁵N double labelling. Chemical shifts, ³J(H, NH) coupling constants and NOEs indicate extensive conformational averaging of both backbone and side chains in agreement with a random coil conformation. The only secondary structure element persisting at pH 3.5 appears to be a short helical segment comprising residues 37 to 40.Abbreviations HSQC heteronuclear single quantum coherence - NMR nuclear magnetic resonance - NOE nuclear Overhauser effect - NOESY two-dimensional NOE spectroscopy - ROE nuclear Overhauser effect in the rotating frame - ROESY two-dimensional ROE spectroscopy - TOCSY total correlation spectroscopy - TPPI time proportional phase incrementation Correspondence to: G. Otting     

Copper uptake and intracellular distribution in the human intestinal Caco-2 cell line

The apical uptake of ⁶⁴CuCl₂ was investigated in human differentiated intestinal Caco-2 cells grown on permeable supports. At pH 6.0 in the apical compartment, the uptake of copper was linear over the first 6 min and between 10 and 80 M CuCl₂ exhibited non-saturable transport kinetics. In addition, copper uptake was energy-independent, affected by the valency state of copper, preferring Cu(II) over Cu(I), and not influenced by high (10 mM) extracellular calcium. The intracellular distribution of copper was investigated by FPLC at different times of uptake (`pulse') and of `chase'. Intracellular copper initially bound predominantly to low molecular weight components (i.e., glutathione), and subsequently shifted to higher molecular weight components such as metallothionein and Cu,Zn superoxide dismutase.     

Multiple protein domains contribute to the action of the copper chaperone for superoxide dismutase.

The copper chaperone for superoxide dismutase (SOD1) inserts the catalytic metal cofactor into SOD1 by an unknown mechanism. We demonstrate here that this process involves the cooperation of three distinct regions of the copper chaperone for SOD1 (CCS): an amino-terminal Domain I homologous to the Atx1p metallochaperone, a central portion (Domain II) homologous to SOD1, and a short carboxyl-terminal peptide unique to CCS molecules (Domain III). These regions fold into distinct polypeptide domains as revealed through proteolysis protection studies. The biological roles of the yeast CCS domains were examined in yeast cells. Surprisingly, Domain I was found to be necessary only under conditions of strict copper limitation. Domain I and Atx1p were not interchangeable in vivo, underscoring the specificity of the corresponding metallochaperones. A putative copper site in Domain II was found to be irrelevant to yeast CCS activity, but SOD1 activation invariably required a CXC in Domain III that binds copper. Copper binding to purified yeast CCS induced allosteric conformational changes in Domain III and also enhanced homodimer formation of the polypeptide. Our results are consistent with a model whereby Domain I recruits cellular copper, Domain II facilitates target recognition, and Domain III, perhaps in concert with Domain I, mediates copper insertion into apo-SOD1.     

Decomposition of reactive oxygen species by copper(II) bis(1-pyrazolyl)methane complexes

Two bis(1-pyrazolyl)alkane ligands, bis(3,5-dimethyl-1-pyrazolyl)methane and bis(4-iodo-3,5-dimethyl-1-pyrazolyl)methane, and their copper(II) complexes, bis(3,5-dimethyl-1-pyrazolyl)methanedinitratocopper(II) [CuL₁(NO₃)₂] and bis(4-iodo-3,5-dimethyl-1-pyrazolyl)methanedinitratocopper(II) [CuL₂(NO₃)₂]·2H₂O, were prepared. Physiochemical properties of the copper(II) complexes were studied by spectroscopic (UV–vis, IR, EPR) techniques and cyclic voltammetry. Spectroscopic analysis revealed a 1:1 stoichiometry of ligand:copper(II) ion and a bindentate coordination mode for the nitrate ions in both of the complexes. According to experimental and theoretical ab initio data, the copper(II) ion is located in an octahedral hexacoordinated environment. Both complexes were able to catalyze the dismutation of superoxide anion () (pH 7.5) and decomposition of H₂O₂ (pH 7.5) and peroxynitrite (pH 10.9). In addition, both complexes exhibited superoxide dismutase (SOD) like activity toward extracellular and intracellular reactive oxygen species produced by activated human neutrophils in whole blood. Thus, these complexes represent useful SOD mimetics with a broad range of antioxidant activity toward a variety of reactive oxidants.     

Determination of the Superoxide Dismutase-Like Activity of Cimetidine-Cu(II) Complexes

Using the direct method of pulse radiolysis to determine the superoxide dismutase like activity of copper(II) cimetidine complexes, it was found that the reaction rate constant with O^?₂, k_cat, was (8.5 ± 0.5) × 10⁸ M^?1s^?1 independent of the cimetidine concentrations present in excess of 50–200 μM over the metal. The results suggest that either the 1:1 ligand to metal complex does not catalyze O^?₂ dismutation at a comparable rate to that of the 2:1 complex, or that the stability constant of the last species is much higher than that determined earlier by Kimura el al.,¹ and only the 2:1 species is present in the solutions. With the indirect methods of cytochrome c and NBT for determining the ability of these complexes to catalyze O^?₂ dismutation, these compounds exhibited a much lower SOD activity. and k_cat was determined to be (5.0 ± 0.3) × 10⁶ and (7.± 0.4) × 10¹ M^?1s^?1. respectively using the two assays.     

X-ray absorption investigation of a unique protein domain able to bind both copper(I) and copper(II) at adjacent sites of the N-terminus of Haemophilus ducreyi Cu,Zn superoxide dismutase

The N-terminal metal binding extension of the Cu,Zn superoxide dismutase from Haemophilus ducreyi is constituted by a histidine-rich region followed by a methione-rich sequence which shows high similarity with protein motifs involved in the binding of Cu(I). X-ray absorption spectroscopy experiments selectively carried out with peptides corresponding to the two metal binding regions indicate that both sequences can bind either Cu(II) or Cu(I). However, competition experiments demonstrate that Cu(II) is preferred by histidine residues belonging to the first half of the motif, while the methionine-rich region preferentially binds Cu(I) via the interaction with three methionine sulfur atoms. Moreover, we have observed that the rate of copper transfer from the peptides to the active site of a copper-free form of the Cu,Zn superoxide dismutase mutant lacking the N-terminal extension depends on the copper oxidation state and on the residues involved in metal binding, histidine residues being critically important for the efficient transfer. Differences in the enzyme reactivation rates in the presence of mixtures of the two peptides when compared to those obtained with the single peptides suggest that the two halves of the N-terminal domain functionally interact during the process of copper transfer, possibly through subtle modifications of the copper coordination environment.