JBIC Journal of Biological Inorganic Chemistry (v.15, #4)

CuA centers and their biosynthetic models in azurin by Masha G. Savelieff; Yi Lu (461-483).
CuA is a binuclear copper center that functions as an electron transfer agent, cycling between a reduced Cu(I)Cu(I) state and an oxidized mixed-valence Cu(+1.5)···Cu(+1.5) state. The copper ions are bridged by two cysteine thiolate ligands and form a copper–copper bond, the first reported of its kind in Nature. Such a “diamond-core” Cu2S(Cys)2 structure allows an unpaired electron to be completely delocalized over the two copper ions and contributes to its highly efficient electron transfer properties. This review provides accounts of how the CuA center was structurally characterized and highlights its salient spectroscopic properties. In the process, it introduces the CuA center in four different systems—native protein systems, soluble protein truncates of native proteins, synthetic models using organic molecules, and biosynthetic models using proteins as ligands—with a greater emphasis on biosynthetic models of CuA, especially on new, deeper insights gained from their studies.
Keywords: Biosynthesis; Electron transfer; Protein engineering; Cytochrome c oxidase

In this B3LYP study, the catalytic mechanisms for the hydrolysis of the three different peptide bonds (Lys28-Gly29, Phe19-Phe20, and His14-Gln15) of Alzheimer amyloid beta (Aβ) peptide by insulin-degrading enzyme (IDE) have been elucidated. For all these peptides, the nature of the substrate was found to influence the structure of the active enzyme–substrate complex. The catalytic mechanism is proposed to proceed through the following three steps: (1) activation of the metal-bound water molecule, (2) formation of the gem-diol intermediate, and (3) cleavage of the peptide bond. With the computed barrier of 14.3, 18.8, and 22.3 kcal/mol for the Lys28-Gly29, Phe19-Phe20, and His14-Gln15 substrates, respectively, the process of water activation was found to be the rate-determining step for all three substrates. The computed energetics show that IDE is the most efficient in hydrolyzing the Lys28-Gly29 (basic polar–neutral nonpolar) peptide bond followed by the Phe19-Phe20 (neutral nonpolar–neutral nonpolar) and His14-Gln15 (basic polar–neutral polar) bonds of the Aβ substrate.
Keywords: Insulin-degrading enzyme; Amyloid beta peptide; Peptide hydrolysis; Metallopeptidase; Density functional theory

Can uranium follow the iron-acquisition pathway? Interaction of uranyl-loaded transferrin with receptor 1 by Miryana Hémadi; Ngûyet-Thanh Ha-Duong; Sophie Plantevin; Claude Vidaud; Jean-Michel El Hage Chahine (497-504).
Transferrin receptor 1 (RD) binds iron-loaded transferrin and allows its internalization in the cytoplasm. Human serum transferrin also forms complexes with metals other than iron, including uranium in the uranyl form (UO2 2+). Can the uranyl-saturated transferrin (TUr2) follow the receptor-mediated iron-acquisition pathway? In cell-free assays, TUr2 interacts with RD in two different steps. The first is fast, direct rate constant, k 1 = (5.2 ± 0.8) × 106 M−1 s−1; reverse rate constant, k −1 = 95 ± 5 s−1; and dissociation constant K 1 = 18 ± 6 μM. The second occurs in the 100-s range and leads to an increase in the stability of the protein–protein adduct, with an average overall dissociation constant K d = 6 ± 2 μM. This kinetic analysis implies in the proposed in vitro model possible but weak competition between TUr2 and the C-lobe of iron-loaded transferrin toward the interaction with R D.
Keywords: Protein–protein interactions; Fast kinetics; Endocytosis; Radioactivity; Toxicity

Pulsed EPR investigations of the Mo(V) centers of the R55Q and R55M variants of sulfite dehydrogenase from Starkeya novella by Trevor D. Rapson; Andrei V. Astashkin; Kayunta Johnson-Winters; Paul V. Bernhardt; Ulrike Kappler; Arnold M. Raitsimring; John H. Enemark (505-514).
Continuous-wave and pulsed electron paramagnetic resonance (EPR) spectroscopy have been used to characterize two variants of bacterial sulfite dehydrogenase (SDH) from Starkeya novella in which the conserved active-site arginine residue (R55) is replaced by a neutral amino acid residue. Substitution by the hydrophobic methionine residue (SDHR55M) has essentially no effect on the pH dependence of the EPR properties of the Mo(V) center, even though the X-ray structure of this variant shows that the methionine residue is rotated away from the Mo center and a sulfate anion is present in the active-site pocket (Bailey et al. in J Biol Chem 284:2053–2063, 2009). For SDHR55M only the high-pH form is observed, and samples prepared in H2 17O-enriched buffer show essentially the same 17O hyperfine interaction and nuclear quadrupole interaction parameters as SDHWT enzyme. However, the pH dependence of the EPR spectra of SDHR55Q, in which the positively charged arginine is replaced by the neutral hydrophilic glutamine, differs significantly from that of SDHWT. For SDHR55Q the blocked form with bound sulfate is generated at low pH, as verified by 33S couplings observed upon reduction with 33S-labeled sulfite. This observation of bound sulfate for SDHR55Q supports our previous hypothesis that sulfite-oxidizing enzymes can exhibit multiple pathways for electron transfer and product release (Emesh et al. in Biochemistry 48:2156–2163, 2009). At pH ≥ 8 the high-pH form dominates for SDHR55Q.
Keywords: Sulfite-oxidizing enzyme; Electron spin echo envelope modulation; Sulfur metabolism; 17O labeling

Biological assays and noncovalent interactions of pyridine-2-carbaldehyde thiosemicarbazonecopper(II) drugs with [poly(dA–dT)]2, [poly(dG–dC)]2, and calf thymus DNA by Rebeca Ruiz; Begoña García; Javier Garcia-Tojal; Natalia Busto; Saturnino Ibeas; José M. Leal; Célia Martins; Jorge Gaspar; Joaquín Borrás; Rubén Gil-García; Marta González-Álvarez (515-532).
The interaction of the Cu(II) drugs CuL(NO3) and CuL′(NO3) (HL is pyridine-2-carbaldehyde thiosemicarbazone and HL′ is pyridine-2-carbaldehyde 4N-methylthiosemicarbazone, in water named [CuL]+ and [CuL′]+) with [poly(dA–dT)]2, [poly(dG–dC)]2, and calf thymus (CT) DNA has been probed in aqueous solution at pH 6.0, I = 0.1 M, and T = 25 °C by absorbance, fluorescence, circular dichroism, and viscosity measurements. The results reveal that these drugs act as groove binders with [poly(dA–dT)]2, with a site size n = 6–7, whereas they act as external binders with [poly(dG–dC)]2 and/or CT-DNA, thus establishing overall electrostatic interaction with n = 1. The binding constants with [CuL′]+ were slightly larger than with [CuL]+. The title compounds display some cleavage activity in the presence of thiols, bringing about the rupture of the DNA strands by the reactive oxygen species formed by reoxidation of Cu(I) to Cu(II); this feature was not observed in the absence of thiols. Mutagenic assays performed both in the presence and in the absence of S9 mix, probed by the Ames test on TA 98, TA 100, and TA 102, were negative. Weak genotoxic activity was detected for [CuL]+ and [CuL′]+, with a significative dose–response effect for [CuL′]+, which was shown to be more cytotoxic in the Ames test and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assays. Methylation of the terminal NH2 group enhances the antiproliferative activity of the pyridine-2-carbaldehyde thiosemicarbazones.
Keywords: Pyridine-2-carbaldehyde thiosemicarbazone; Copper(II); DNA; Thermodynamic properties; Cytotoxicity

Characterizing metalloendonuclease mixed metal complexes by global kinetic analysis by Charulata B. Prasannan; Fuqian Xie; Cynthia M. Dupureur (533-545).
To test the role of a secondary metal ion in a two metal ion metallonuclease mechanism, some groups have introduced a nonsupportive metal ion [usually Ca(II)] in cleavage reactions. Stimulation of Mg(II)- or Mn(II)-supported activity has been taken as evidence that the second metal ion is regulatory. However, this activity has yet to be dissected to determine what processes and species contribute to this observation. Here, we test global kinetic analysis as an approach to this problem. Taking advantage of the various binding and cleavage constants established for PvuII endonuclease, we apply cleavage data obtained under a range of Mg(II) and Ca(II) concentrations to a number of kinetic models which specify A and B sites for both metal ions and various active species. The data are best fit and simulated with models which feature Ca(II) being held more strongly in the B (or secondary) site. This mixed metal enzyme species is the only one which forms appreciably and exhibits a cleavage rate constant similar to that observed when there is only one Mg(II) per active site (approximately 0.01 s−1). Thus, in the case of PvuII endonuclease, Ca(II) does not stimulate cleavage. However, a simulated increase in activity at moderate Ca(II) concentrations can be rationalized with a cleavage rate constant for the mixed species similar to that when two Mg(II) ions are present in the active site. This provides an important insight into the underlying basis for the Ca(II)-stimulated activity observed for some metallonucleases that is not accessible by any other means.
Keywords: Structure–function relationship; Enzyme kinetics; Nucleic acid; Thermodynamics; Binding affinity

In the present study, we demonstrated that gadolinium-containing particles formed in cell culture medium acted as a biologically active entity to mediate cell cycle progression in NIH3T3 cells. The particles were observed to accumulate at the cell surface by scanning electron microscopy. Energy-dispersive X-ray analysis was undertaken and confirmed that gadolinium was incorporated in the agglomerated particles. Moreover, the smaller gadolinium particles exhibited a stronger cell-cycle-promoting effect than the larger ones, but they shared the common signaling pathways. Both extracellular signal regulated kinase and phosphatidylinositol 3-kinase signaling pathways were activated by gadolinium-containing particles and may account for their proliferation-promoting effect on NIH3T3 cells. Furthermore, the study showed that the free gadolinium ion released from gadolinium-containing particles may be responsible for the proliferation effect. This study will be helpful to clarify the biological effect of the insoluble species formed from Gd3+ as well as other multivalent metal ions under physiological conditions and will help to improve their medical applications.
Keywords: Gadolinium-containing bioparticles; Cell cycle progression; Extracellular-signal-regulated protein kinase; Phosphatidylinositol 3-kinase; Nephrogenic systemic fibrosis

A systematic investigation of multiheme c-type cytochromes in prokaryotes by Shailesh Sharma; Gabriele Cavallaro; Antonio Rosato (559-571).
Multiheme c-type cytochromes (MHCs) are metalloproteins that can play various biochemical roles, including enzymatic activity and electron transfer. As electron transfer proteins, the presence of multiple heme cofactors in the vicinity allows electrons to rapidly travel relatively long distances. MHCs are often characterized by relatively low structural complexity, with the heme cofactors being largely responsible for maintaining the structure in place, owing to the protein–heme covalent linkages. In this work, we analyzed an extensive ensemble of 594 complete prokaryotic proteomes, amounting to more than 1.9 million sequences, to characterize their content in MHCs. We identified 1,659 MHCs in 258 organisms. The presence of MHCs was found to correlate quite well with the capability of an organism to synthesize or take up heme. For two organisms, the presence of MHCs in the proteome could be taken as a hint to the presence of divergent heme uptake pathways. The most common numbers of heme-binding motifs in a sequence were four (25%) and two (23%), followed by five (13%) and ten (9.8%). The average protein-to-heme ratio was relatively similar for all MHCs, except diheme proteins, regardless of the number of motifs at around 60 ± 30. The latter ratio could in favorable cases be a useful indicator for functional assignments of novel MHCs. Finally, we showed that the amount of structural information currently available for MHCs is limited with respect to the diversity of this broad class of metalloproteins. Experimental efforts in the structural investigation of MHCs are thus warranted.
Keywords: Cytochrome c ; Metalloproteome; Heme; Electron transfer

Exploring the biochemical mechanisms of cytotoxic gold compounds: a proteomic study by Francesca Magherini; Alessandra Modesti; Luca Bini; Michele Puglia; Ida Landini; Stefania Nobili; Enrico Mini; Maria Agostina Cinellu; Chiara Gabbiani; Luigi Messori (573-582).
We have recently shown that a group of structurally diverse gold compounds are highly cytotoxic toward a panel of 36 human tumor cell lines through a variety of biochemical mechanisms. A classic proteomic approach is exploited here to gain deeper insight into those mechanisms. This investigation is focused on Auoxo6, a novel binuclear gold(III) complex, and auranofin, a clinically established gold(I) antiarthritic drug. First, the 72-h cytotoxicity profiles of Auoxo6 and auranofin were determined against A2780 human ovarian carcinoma cells. Subsequently, protein extraction from gold-treated A2780 cells sensitive to cisplatin and 2D gel electrophoresis separation were carried out according to established procedures. Notably, both metallodrugs caused relatively modest changes in protein expression in comparison with controls as only 11 out of approximately 1,300 monitored spots showed appreciable quantitative changes. Very remarkably, six altered proteins were in common between the two treatments. Eight altered proteins were identified by mass spectrometry; among them was ezrin, a protein associated with the cytoskeleton and involved in apoptosis. Interestingly, two altered proteins, i.e., peroxiredoxins 1 and 6, are known to play crucial roles in the cell redox metabolism. Increased cleavage of heterogeneous ribonucleoprotein H was also evidenced, consistent with caspase 3 activation. Overall, the results of the present proteomic study point out that the mode of action of Auoxo6 is strictly related to that of auranofin, that the induced changes in protein expression are limited and selective, that both gold compounds trigger caspase 3 activation and apoptosis, and that a few affected proteins are primarily involved in cell redox homeostasis.
Keywords: Proteomic; Gold compound

Functional characterization of iron-substituted neural zinc finger factor 1: metal and DNA binding by Angelique N. Besold; Seung Jae Lee; Sarah L. J. Michel; Niall Lue Sue; Holly J. Cymet (583-590).
Neural zinc finger factor 1 (NZF-1) is a nonclassical zinc finger protein involved in neuronal development. NZF-1 contains multiple copies of a unique CCHHC zinc-binding domain that recognize a promoter element in the β-retinoic acid receptor gene termed β-retinoic acid receptor element (β-RARE). Previous studies have established that a two-domain fragment of NZF-1 bound with zinc is sufficient for specific DNA binding. Proper functioning of the nervous system relies heavily on iron and misregulation of this highly redox active metal has serious consequences. Several classes of zinc finger proteins have been shown to bind other metal ions, including iron. To determine if ferrous iron can coordinate to the metal-binding sites of NZF-1 and assess the functional consequences of such coordination, a fragment of NZF-1 that contains two zinc-binding domains, NZF-1 double finger (NZF-1-DF), was prepared. UV–vis spectroscopy experiments demonstrated that Fe(II) is capable of binding to NZF-1-DF. Upon reconstitution with either Fe(II) or Zn(II), NZF-1-DF binds selectively and tightly (nanomolar affinity) to its target β-RARE DNA sequence, whereas apo-NZF-1-DF does not bind to DNA and instead aggregates.
Keywords: Neural zinc finger factor 1; Zinc finger; Iron binding; DNA; Fluorescence anisotropy

Design, synthesis, and evaluation of cyclofenil derivatives for potential SPECT imaging agents by Hua Zhu; Liliang Huang; Yuanqing Zhang; Xiaoping Xu; Yanhong Sun; Yu-Mei Shen (591-599).
To develop technetium- and rhenium-labeled nonsteroidal estrogen imaging agents for estrogen receptor (ER) positive breast tumors, two groups of rhenium and technetium cyclofenil derivatives were synthesized and characterized. The binding affinities of the rhenium complexes for ERs were determined. The tricarbonyl rhenium complex showed the highest binding affinity for ERs (81.2 for ERβ, 16.5 for ERα). Tricarbonyl technetium cyclofenil complexes were obtained in high radiochemical purity and radiochemical yields. The results of studies of their octanol/water partition and in vitro stability are presented. These results demonstrate that these radiolabeled cyclofenil derivatives may be considered as potential breast cancer imaging agents.
Keywords: Cyclofenil; Tricarbonyl rhenium; Radiolabeling; Nonsteroidal; Estrogen receptor

Oligonucleotides are potent antioxidants acting primarily through metal ion chelation by Eyal Zobel; Eylon Yavin; Hugo E. Gottlieb; Meirav Segal; Bilha Fischer (601-620).
We report on a rather unknown feature of oligonucleotides, namely, their potent antioxidant activity. Previously, we showed that nucleotides are potent antioxidants in FeII/CuI/II–H2O2 systems. Here, we explored the potential of 2′-deoxyoligonucleotides as inhibitors of the FeII/CuI/II-induced ·OH formation from H2O2. The oligonucleotides [d(A)5,7,20; d(T)20; (2′-OMe-A)5] proved to be highly potent antioxidants with IC50 values of 5–17 or 48–85 μM in inhibiting FeII/CuI- or CuII-induced H2O2 decomposition, respectively, thus representing a 40–215-fold increase in potency as compared with Trolox, a standard antioxidant. The antioxidant activity is only weakly dependent on the oligonucleotides’ length or base identity. We analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry and 1H-NMR spectroscopy the composition of the d(A)5 solution exposed to the aforementioned oxidative conditions for 4 min or 24 h. We concluded that the primary (rapid) inhibition mechanism by oligonucleotides is metal ion chelation and the secondary (slow) mechanism is radical scavenging. We characterized the CuI–d(A)5 and CuII–d(A)7 complexes by 1H-NMR and 31P-NMR or frozen-solution ESR spectroscopy, respectively. CuI is probably coordinated to d(A)5 via N1 and N7 of two adenine residues and possibly also via two phosphate/bridging water molecules. The ESR data suggest CuII chelation through two nitrogen atoms of the adenine bases and two oxygen atoms (phosphates or water molecules). We conclude that oligonucleotides at micromolar concentrations prevent FeII/CuI/II-induced oxidative damage, primarily through metal ion chelation. Furthermore, we propose the use of a short, metabolically stable oligonucleotide, (2′-OMe-A)5, as a highly potent and relatively long lived (t 1/2 ~ 20 h) antioxidant.
Keywords: Oligonucleotides; EPR spectroscopy; Antioxidants; CuI/II ; FeII

Tissue-specific interplay between copper uptake and efflux in Drosophila by Tim Binks; Jessica Charlotte Lye; James Camakaris; Richard Burke (621-628).
The vinegar fly Drosophila melanogaster is proving to be an excellent system to study the in vivo regulation of the essential metal copper. The Ctr1A/B and DmATP7 copper transport proteins have well-established roles in Drosophila copper uptake and efflux, respectively. Both Ctr1A and DmATP7 are essential genes, whereas Ctr1B mutants are viable but die in excess or depleted copper conditions. Less is known about the tissue-specific requirements for these three genes and how they interact to maintain copper homeostasis in different cell types. Here, we use targeted overexpression and suppression of each gene to examine these questions in vivo. We find that in the epidermal cells that form the adult thoracic and abdominal cuticle, Ctr1A plays a major role in copper uptake, whereas Ctr1B plays only a minor supporting role and DmATP7, as previously shown, is essential for transfer of copper to the trans-Golgi network. We also find that the copper chaperone dSco1 appears necessary for supplying the mitochondria with copper in these tissues. In contrast, in the developing Drosophila eye, DmATP7 appears to be non-essential unless copper levels in these cells are artificially elevated. Again, Ctr1A is the main copper uptake gene in the eye, but when ectopically expressed, Ctr1B has greater phenotypic effects than Ctr1A. Furthermore, Ctr1A and Ctr1B show a dramatic synergistic interaction, indicating their relationship is more complicated than a simply additive one and that they may in fact act cooperatively for optimal copper import.
Keywords: Copper homeostasis; Drosophila ; DmATP7; Ctr1; Copper