JBIC Journal of Biological Inorganic Chemistry (v.19, #6)

A journey into the active center of nitrogenase by Yilin Hu; Markus W. Ribbe (731-736).
Nitrogenase catalyzes the reduction of N2 to NH3, a key step in the global nitrogen cycle. This article describes our journey toward the definition of a complete molecular structure of the active site of nitrogenase, with an emphasis on the discovery of the interstitial carbide and the radical SAM-dependent insertion of this atom into the active FeMo cofactor site of nitrogenase.
Keywords: Nitrogenase; FeMo cofactor; Interstitial carbide; NifB; Radical SAM enzyme

The enzyme nitrogenase catalyzes the six-electron reduction of molecular dinitrogen to ammonium, concomitant with the reduction of protons to yield hydrogen gas. In the MoFe protein component of the nitrogenase system, the unique FeMo cofactor is the active site of catalysis, but its exact mechanism remains under debate. This review focuses on the history of the structure determination of FeMo cofactor, a process that extended over two decades and involved several iterations and corrections that are unusual and unexpected within the established field of X-ray crystallography. However, FeMo cofactor has defied expectations on several occasions, and besides being the largest single iron–sulfur cluster known to bioinorganic chemistry, this [Mo:7Fe:9S:C]:homocitrate moiety is unique in some aspects that have misled researchers several times. We have now arrived at a final and complete description of the atomic structure of FeMo cofactor, and yet many questions regarding various aspects of the catalytic mechanism of this enzyme remain to be answered.
Keywords: Nitrogenase; MoFe protein; FeMo cofactor; X-ray crystallography; Spectroscopy

H-Cluster assembly during maturation of the [FeFe]-hydrogenase by Joan B. Broderick; Amanda S. Byer; Kaitlin S. Duschene; Benjamin R. Duffus; Jeremiah N. Betz; Eric M. Shepard; John W. Peters (747-757).
The organometallic H-cluster at the active site of the [FeFe]-hydrogenase serves as the site of reversible binding and reduction of protons to produce H2. The H-cluster is unique in biology, and consists of a 2Fe subcluster tethered to a typical [4Fe–4S] cluster by a single cysteine ligand. The remaining ligands to the 2Fe subcluster include three carbon monoxides, two cyanides, and a dithiomethylamine. This mini-review will focus on the significant advances in recent years in understanding the pathway for H-cluster biosynthesis, as well as the structures, roles, and mechanisms of the three enzymes directly involved.
Keywords: Hydrogenase; Radical SAM; Metal cluster assembly; HydE; HydG; HydF; Carbon monoxide; Cyanide

Assembly of nonheme Mn/Fe active sites in heterodinuclear metalloproteins by Julia J. Griese; Vivek Srinivas; Martin Högbom (759-774).
The ferritin superfamily contains several protein groups that share a common fold and metal coordinating ligands. The different groups utilize different dinuclear cofactors to perform a diverse set of reactions. Several groups use an oxygen-activating di-iron cluster, while others use di-manganese or heterodinuclear Mn/Fe cofactors. Given the similar primary ligand preferences of Mn and Fe as well as the similarities between the binding sites, the basis for metal specificity in these systems remains enigmatic. Recent data for the heterodinuclear cluster show that the protein scaffold per se is capable of discriminating between Mn and Fe and can assemble the Mn/Fe center in the absence of any potential assembly machineries or metal chaperones. Here we review the current understanding of the assembly of the heterodinuclear cofactor in the two different protein groups in which it has been identified, ribonucleotide reductase R2c proteins and R2-like ligand-binding oxidases. Interestingly, although the two groups form the same metal cluster they appear to employ partly different mechanisms to assemble it. In addition, it seems that both the thermodynamics of metal binding and the kinetics of oxygen activation play a role in achieving metal specificity.
Keywords: Affinity; Chlamydia; Cofactor maturation; Metal selection; Specificity

Mechanisms of iron mineralization in ferritins: one size does not fit all by Justin M. Bradley; Geoffrey R. Moore; Nick E. Le Brun (775-785).
Significant progress has been made in recent years toward understanding the processes by which an iron mineral is deposited within members of the ferritin family of 24mer iron storage proteins, enabled by high-resolution structures together with spectroscopic and kinetic studies. These suggest common characteristics that are shared between ferritins, namely, a highly symmetric arrangement of subunits that provides a protein coat around a central cavity in which the mineral is formed, channels through the coat that facilitate ingress and egress of ions, and catalytic sites, called ferroxidase centers, that drive Fe2+ oxidation. They also reveal significant variations in both structure and mechanism amongst ferritins. Here, we describe three general types of structurally distinct ferroxidase center and the mechanisms of mineralization that they are associated with. The highlighted variation leads us to conclude that there is no universal mechanism by which ferritins function, but instead there exists several distinct mechanisms of ferritin iron mineralization.
Keywords: Iron storage; Ferroxidase; Iron metabolism; Dinuclear iron; Mineralization

Insights into the mechanisms underlying the antiproliferative potential of a Co(II) coordination compound bearing 1,10-phenanthroline-5,6-dione: DNA and protein interaction studies by Daniel V. Luís; Joana Silva; Ana Isabel Tomaz; Rodrigo F. M. de Almeida; Miguel Larguinho; Pedro V. Baptista; Luísa M. D. R. S. Martins; Telma F. S. Silva; Pedro M. Borralho; Cecília M. P. Rodrigues; António S. Rodrigues; Armando J. L. Pombeiro; Alexandra R. Fernandes (787-803).
The very high antiproliferative activity of [Co(Cl)(H2O)(phendione)2][BF4] (phendione is 1,10-phenanthroline-5,6-dione) against three human tumor cell lines (half-maximal inhibitory concentration below 1 μM) and its slight selectivity for the colorectal tumor cell line compared with healthy human fibroblasts led us to explore the mechanisms of action underlying this promising antitumor potential. As previously shown by our group, this complex induces cell cycle arrest in S phase and subsequent cell death by apoptosis and it also reduces the expression of proteins typically upregulated in tumors. In the present work, we demonstrate that [Co(Cl)(phendione)2(H2O)][BF4] (1) does not reduce the viability of nontumorigenic breast epithelial cells by more than 85 % at 1 μM, (2) promotes the upregulation of proapoptotic Bax and cell-cycle-related p21, and (3) induces release of lactate dehydrogenase, which is partially reversed by ursodeoxycholic acid. DNA interaction studies were performed to uncover the genotoxicity of the complex and demonstrate that even though it displays K b (± standard error of the mean) of (3.48 ± 0.03) × 105 M−1 and is able to produce double-strand breaks in a concentration-dependent manner, it does not exert any clastogenic effect ex vivo, ruling out DNA as a major cellular target for the complex. Steady-state and time-resolved fluorescence spectroscopy studies are indicative of a strong and specific interaction of the complex with human serum albumin, involving one binding site, at a distance of approximately 1.5 nm for the Trp214 indole side chain with log K b ~4.7, thus suggesting that this complex can be efficiently transported by albumin in the blood plasma.
Keywords: Cobalt; 1,10-Phenanthroline-5,6-dione; Apoptosis; DNA cleavage; Human serum albumin

Outer-sphere oxidation of Fe(II) in nitrosylmyoglobin by ferricyanide by Jens K. S. Møller; Leif H. Skibsted (805-812).
Nitrosylmyoglobin, MbFe(II)NO, was found to be oxidized by [Fe(CN)6]3− and HClO/ClO, but not by the semistable radical nitrosodisulfonate (anion of Frémy’s salt) or NO2 at ambient temperature in aqueous solution with pH 7.0. The oxidation by HClO/ClO was significantly faster than that by [Fe(CN)6]3−. With excess [Fe(CN)6]3−, MbFe(II)NO was oxidized to metmyoglobin, MbFe(III), in a second-order reaction with k 2 = 1.67 ± 0.10 M−1 s−1 at 288 K without detectable intermediates as determined by stopped-flow spectroscopy. The activation parameters were ΔH  = 43 ± 2 kJ mol−1 and ΔS  = −93 ± 9 J−1 K−1 mol−1. Outer-sphere electron-transfer to [Fe(CN)6]3− was assigned as rate determining rather than NO dissociation from iron(II) followed by electron transfer. Outer-sphere electron transfer from MbFe(II)NO to certain moderate oxidizing agents may thus have a role in labilizing NO association slowly through oxidation of iron(II) to iron(III). In contrast, hypochlorite oxidizes MbFe(II)NO much faster in a complex sequence of processes involving a rate-determining second-order (unidentified) reaction with k 2 = 2.6 ± 0.3 × 103 M−1 s−1 at 288 K and possibly involving protein degradation.
Keywords: Nitrosylmyoglobin; Hypochlorite; Ferricyanide; Nitrite; Dinitrososulfonate; Electron transfer

Selenite-mediated production of superoxide radical anions in A549 cancer cells is accompanied by a selective increase in SOD1 concentration, enhanced apoptosis and Se–Cu bonding by Claire M. Weekley; Gloria Jeong; Michael E. Tierney; Farjaneh Hossain; Aung Min Maw; Anu Shanu; Hugh H. Harris; Paul K. Witting (813-828).
Selenite may exert its cytotoxic effects against cancer cells via the generation of reactive oxygen species (ROS). We investigated sources of, and the cellular response to, superoxide radical anion (O2 ·−) generated in human A549 lung cancer cells after treatment with selenite. A temporal delay was observed between selenite treatment and increases in O2 ·− production and biomarkers of apoptosis/necrosis, indicating that the reduction of selenite by the glutathione reductase/NADPH system (yielding O2 ·−) is a minor contributor to ROS production under these conditions. By contrast, mitochondrial and NADPH oxidase O2 ·− generation were the major contributors. Treatment with a ROS scavenger [poly(ethylene glycol)-conjugated superoxide dismutase (SOD) or sodium 4,5-dihydroxybenzene-1,3-disulfonate] 20 h after the initial selenite treatment inhibited both ROS generation and apoptosis determined at 24 h. In addition, SOD1 was selectively upregulated and its perinuclear cytoplasmic distribution was colocalised with the cellular distribution of selenium. Interestingly, messenger RNA for manganese superoxide dismutase, catalase, inducible haem oxygenase 1 and glutathione peroxidase either remained unchanged or showed a delayed response to selenite treatment. Colocalisation of Cu and Se in these cells (Weekley et al. in J. Am. Chem. Soc. 133:18272–18279, 2011) potentially results from the formation of a Cu–Se species, as indicated by Cu K-edge extended X-ray absorption fine structure spectra. Overall, SOD1 is upregulated in response to selenite-mediated ROS generation, and this likely leads to an accumulation of toxic hydrogen peroxide that is temporally related to decreased cancer cell viability. Increased expression of SOD1 gene/protein coupled with formation of a Cu–Se species may explain the colocalisation of Cu and Se observed in these cells.
Keywords: Selenium; Cell viability; Reactive oxygen species; Synchrotron radiation; Superoxide radical anion

Varied metal-binding properties of lipoprotein PsaA in Streptococcus pneumoniae by Nan Li; Xiao-Yan Yang; Zhong Guo; Jing Zhang; Kun Cao; Junlong Han; Gong Zhang; Langxia Liu; Xuesong Sun; Qing-Yu He (829-838).
Streptococcus pneumoniae is a Gram-positive pathogen responsible for pneumonia, otitis media, and meningitis. Manganese and zinc ions are essential for this bacterium, playing regulatory, structural, or catalytic roles as the critical cofactors in the bacterial proteins and metabolic enzymes. Lipoprotein PsaA has been found to mediate Mn2+ and Zn2+ transportation in Streptococcus pneumoniae. In the present work, we conducted a systemic study on the contributions from key amino acids in the metal-binding site of PsaA using various spectroscopic and biochemical methods. Our experimental data indicate that four metal-binding residues contribute unequally to the Mn2+ and Zn2+ binding, and His139 is most important for both the structural stability and metal binding of the protein. PsaA–Mn2+ has a lower thermal stability than PsaA–Zn2+, possibly due to the different coordination preferences of the metals. Kinetics analysis revealed that PsaA–Mn2+ binding is a fast first-order reaction, whereas PsaA–Zn2+ binding is a slow second-order reaction, implying that PsaA kinetically prefers binding Mn2+ to Zn2+. The present results provide complementary information for understanding the mechanisms of metal transport and bacterial virulence via lipoproteins in Streptococcus pneumoniae.
Keywords: Metal transport; Protein folding

Biophysical and morphological studies on the dual interaction of non-octarepeat prion protein peptides with copper and nucleic acids by Juliana A. P. Chaves; Carolina Sanchez-López; Mariana P. B. Gomes; Tháyna Sisnande; Bruno Macedo; Vanessa End de Oliveira; Carolina A. C. Braga; Luciana P. Rangel; Jerson L. Silva; Liliana Quintanar; Yraima Cordeiro (839-851).
Conversion of prion protein (PrP) to an altered conformer, the scrapie PrP (PrPSc), is a critical step in the development of transmissible spongiform encephalopathies. Both Cu(II) and nucleic acid molecules have been implicated in this conversion. Full-length PrP can bind up to six copper ions; four Cu(II) binding sites are located in the octarepeat domain (residues 60–91), and His-96 and His-111 coordinate two additional copper ions. Experimental evidence shows that PrP binds different molecules, resulting in diverse cellular signaling events. However, there is little information about the interaction of macromolecular ligands with Cu(II)-bound PrP. Both RNA and DNA sequences can bind PrP, and this interaction results in reciprocal conformational changes. Here, we investigated the interaction of Cu(II) and nucleic acids with amyloidogenic non-octarepeat PrP peptide models (comprising human PrP residues 106–126 and hamster PrP residues 109–149) that retain His-111 as the copper-anchoring residue. The effect of Cu(II) and DNA or RNA sequences in the aggregation, conformation, and toxicity of PrP domains was investigated at low and neutral pH. Circular dichroism and EPR spectroscopy data indicate that interaction of the PrP peptides with Cu(II) and DNA occurs at pH 7. This dual interaction induces conformational changes in the peptides, modulating their aggregation, and affecting the morphology of the aggregated species, resulting in different cytotoxic effects. These results provide new insights into the role of Cu(II) and nucleic acid sequences in the structural conversion and aggregation of PrP, which are both critical events related to prion pathogenesis.
Keywords: Copper; Prion protein; Nucleic acid; EPR; Toxicity; Spectroscopy

Anticancer activity of structurally related ruthenium(II) cyclopentadienyl complexes by Leonor Côrte-Real; Filipa Mendes; Joana Coimbra; Tânia S. Morais; Ana Isabel Tomaz; Andreia Valente; M. Helena Garcia; Isabel Santos; Manuel Bicho; Fernanda Marques (853-867).
A set of structurally related Ru(η5-C5H5) complexes with bidentate N,N′-heteroaromatic ligands have been evaluated as prospective metallodrugs, with focus on exploring the uptake and cell death mechanisms and potential cellular targets. We have extended these studies to examine the potential of these complexes to target cancer cell metabolism, the energetic-related phenotype of cancer cells. The observations that these complexes can enter cells, probably facilitated by binding to plasma transferrin, and can be retained preferentially at the membranes prompted us to explore possible membrane targets involved in cancer cell metabolism. Most malignant tumors present the Warburg effect, which consists in increasing glycolytic rates with production of lactate, even in the presence of oxygen. The reliance of glycolytic cancer cells on trans-plasma-membrane electron transport (TPMET) systems for their continued survival raises the question of their appropriateness as a target for anticancer drug development strategies. Considering the interesting findings that some anticancer drugs in clinical use are cytotoxic even without entering cells and can inhibit TPMET activity, we investigated whether redox enzyme modulation could be a potential mechanism of action of antitumor ruthenium complexes. The results from this study indicated that ruthenium complexes can inhibit lactate production and TPMET activity in a way dependent on the cancer cell aggressiveness and the concentration of the complex. Combination approaches that target cell metabolism (glycolytic inhibitors) as well as proliferation are needed to successfully cure cancer. This study supports the potential use of some of these ruthenium complexes as adjuvants of glycolytic inhibitors in the treatment of aggressive cancers.A simplified hypothetical model showing the possible relationship between the trans-plasma-membrane electron transport (tPMET) system (ferricyanide reductase), the transferrin receptor, and the Na+/H+ antiporter. This tPMET might be involved in iron uptake and in regulating the NADH-to-NAD+ ratio. As a consequence of tPMET activity, the antiport is probably activated by proton release. (Adapted from Crane et al., 1991; Herst and Berridge, Curr. Mol. Med. 6:895–904, 2006). MET mitochondrial electron transport, TCA tricarboxylic acid
Keywords: Glycolysis; Drug targets; Redox enzymes; Cancer therapy; Ruthenium drugs

Investigating the role of transferrin in the distribution of iron, manganese, copper, and zinc by Carolina Herrera; Michael A. Pettiglio; Thomas B. Bartnikas (869-877).
The essential role of transferrin in mammalian iron metabolism is firmly established. Integral to our understanding of transferrin, studies in hypotransferrinemic mice, a model of inherited transferrin deficiency, have demonstrated that transferrin is essential for iron delivery for erythropoiesis and in the regulation of expression of hepcidin, a hormone that inhibits macrophage and enterocyte iron efflux. Here we investigate a potential role for transferrin in the distribution of three other physiologic metals, manganese, copper, and zinc. We first assessed metal content in transferrin-rich fractions of wild-type mouse sera and demonstrate that although both iron and manganese cofractionated predominantly with transferrin, the absolute levels of manganese are several orders of magnitude lower than those of iron. We next measured metal content in multiple tissues in wild-type and hypotransferrinemic mice of various ages. Tissue metal imbalances were severe for iron and minimal to moderate for some metals in some tissues in hypotransferrinemic mice. Metal levels measured in a transferrin-replete yet hepcidin-deficient and iron-loaded mouse strain suggested that the observed imbalances in tissue copper, zinc, and manganese levels were not all specific to hypotransferrinemic mice or caused directly by transferrin deficiency. Overall, our results suggest that transferrin does not have a primary role in the distribution of manganese, copper, or zinc to tissues and that the abnormalities observed in tissue manganese levels are not attributable to a direct role for transferrin in manganese metabolism but rather are attributable to an indirect effect of transferrin deficiency on hepcidin expression and/or iron metabolism.
Keywords: Transferrin; Iron; Copper; Zinc; Manganese

Terbium promotes adhesion and osteogenic differentiation of mesenchymal stem cells via activation of the Smad-dependent TGF-β/BMP signaling pathway by Dan-Dan Liu; Kun Ge; Yi Jin; Jing Sun; Shu-Xiang Wang; Meng-Su Yang; Jin-Chao Zhang (879-891).
With its special physical and chemical properties, terbium has been widely used, which has inevitably increased the chance of human exposure to terbium-based compounds. It was reported that terbium mainly deposited in bone after introduction into the human body. Although some studies revealed the effects of terbium on bone cell lines, there have been few reports about the potential effect of terbium on adhesion and differentiation of mesenchymal stem cells (MSCs). In this study, we investigated the effects of terbium on the adhesion and osteogenic and adipogenic differentiation of MSCs and the associated molecular mechanisms. Our data reveal that terbium promoted the osteogenic differentiation in a time-dependent manner and conversely inhibited the adipogenic differentiation of MSCs. Meanwhile, the cell–cell or cell–matrix interaction was enhanced by activating adherent-related key factors, which were evaluated by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Real-time RT-PCR and Western blot analysis were also performed to further detect osteogenic and adipogenic biomarkers of MSCs. The regulation of terbium on differentiation of MSCs led to the interaction between the transforming growth factor β/bone morphogenetic protein and peroxisome-proliferator-activated receptor γ (PPARγ) signaling pathways, resulting in upregulation of the osteogenic master transcription factors, such as Runt-related transcription factor 2, bone morphogenetic protein 2, collagen I, alkaline phosphatase, and osteocalcin, and downregulation of the adipogenic master transcription factors, such as PPARγ2. The results provide novel evidence to elucidate the mechanisms of bone metabolism by terbium and may be helpful for more rational application of terbium-based compounds in the future.The effects of terbium on the osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs) and the associated molecular mechanisms were investigated. The results suggest that terbium promotes the osteogenic differentiation of MSCs via the transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) signaling pathway. ALP alkaline phosphatase, BSP bone sialoprotein, C/EBP CCAAT/enhancer binding protein, Col I collagen I, ERα estrogen receptor α, GDF growth differentiation factor, OCN osteocalcin, PPARγ peroxisome-proliferator-activated receptor γ, Runx2 Runt-related transcription factor 2
Keywords: Terbium; Mesenchymal stem cells; Adhesion; Osteogenic differentiation; Adipogenic differentiation

The class Ib ribonucleotide reductase from Mycobacterium tuberculosis has two active R2F subunits by Marta Hammerstad; Åsmund K. Røhr; Niels H. Andersen; Astrid Gräslund; Martin Högbom; K. Kristoffer Andersson (893-902).
Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication in all living organisms. Class Ib RNRs require either a diiron–tyrosyl radical (Y·) or a dimanganese–Y· cofactor in their R2F subunit to initiate ribonucleotide reduction in the R1 subunit. Mycobacterium tuberculosis, the causative agent of tuberculosis, contains two genes, nrdF1 and nrdF2, encoding the small subunits R2F-1 and R2F-2, respectively, where the latter has been thought to serve as the only active small subunit in the M. tuberculosis class Ib RNR. Here, we present evidence for the presence of an active Fe 2 III –Y· cofactor in the M. tuberculosis RNR R2F-1 small subunit, supported and characterized by UV–vis, X-band electron paramagnetic resonance, and resonance Raman spectroscopy, showing features similar to those for the M. tuberculosis R2F-2–Fe 2 III –Y· cofactor. We also report enzymatic activity of Fe 2 III –R2F-1 when assayed with R1, and suggest that the active M. tuberculosis class Ib RNR can use two different small subunits, R2F-1 and R2F-2, with similar activity.
Keywords: Ribonucleotide reductase; R2; Tyrosyl radical; Mycobacterium tuberculosis ; Iron

Solution structure and metal ion binding sites of the human CPEB3 ribozyme’s P4 domain by Miriam Skilandat; Magdalena Rowinska-Zyrek; Roland K. O. Sigel (903-912).
Three ribozymes are known to occur in humans, the CPEB3 ribozyme, the CoTC ribozyme, and the hammerhead ribozyme. Here, we present the NMR solution structure of a well-conserved motif within the CPEB3 ribozyme, the P4 domain. In addition, we discuss the binding sites and impact of Mg2+ and [Co(NH3)6]3+, a spectroscopic probe for [Mg(H2O)6]2+, on the structure. The well-defined P4 region is a hairpin closed with a UGGU tetraloop that shows a distinct electrostatic surface potential and a characteristic, strongly curved backbone trajectory. The P4 hairpin contains two specific Mg2+ binding sites: one outer-sphere binding site close to the proposed CPEB3 ribozyme active site with potential relevance for maintaining a compact fold of the ribozyme core, and one inner-sphere binding site, probably stabilizing the tetraloop structure. The structure of the tetraloop resembles an RNase III recognition structure, as previously described for an AGUU tetraloop. The detailed knowledge of the P4 domain and its metal ion binding preferences thus brings us closer to understanding the importance of Mg2+ binding for the CPEB3 ribozyme’s fold and function in the cell.
Keywords: NMR; CPEB3; Ribozyme; Mg2+ binding

Pseudoazurin from Sinorhizobium meliloti as an electron donor to copper-containing nitrite reductase: influence of the redox partner on the reduction potentials of the enzyme copper centers by Félix M. Ferroni; Jacopo Marangon; Nicolás I. Neuman; Julio C. Cristaldi; Silvina M. Brambilla; Sergio A. Guerrero; María G. Rivas; Alberto C. Rizzi; Carlos D. Brondino (913-921).
Pseudoazurin (Paz) is the physiological electron donor to copper-containing nitrite reductase (Nir), which catalyzes the reduction of NO2 to NO. The Nir reaction mechanism involves the reduction of the type 1 (T1) copper electron transfer center by the external physiological electron donor, intramolecular electron transfer from the T1 copper center to the T2 copper center, and nitrite reduction at the type 2 (T2) copper catalytic center. We report the cloning, expression, and characterization of Paz from Sinorhizobium meliloti 2011 (SmPaz), the ability of SmPaz to act as an electron donor partner of S. meliloti 2011 Nir (SmNir), and the redox properties of the metal centers involved in the electron transfer chain. Gel filtration chromatography and sodium dodecyl sulfate–polyacrylamide gel electrophoresis together with UV–vis and EPR spectroscopies revealed that as-purified SmPaz is a mononuclear copper-containing protein that has a T1 copper site in a highly distorted tetrahedral geometry. The SmPaz/SmNir interaction investigated electrochemically showed that SmPaz serves as an efficient electron donor to SmNir. The formal reduction potentials of the T1 copper center in SmPaz and the T1 and T2 copper centers in SmNir, evaluated by cyclic voltammetry and by UV-vis- and EPR-mediated potentiometric titrations, are against an efficient Paz T1 center to Nir T1 center to Nir T2 center electron transfer. EPR experiments proved that as a result of the SmPaz/SmNir interaction in the presence of nitrite, the order of the reduction potentials of SmNir reversed, in line with T1 center to T2 center electron transfer being thermodynamically more favorable.
Keywords: Pseudoazurin; Nitrite reductase; Electrochemistry; Redox titration; EPR

Cognate and noncognate metal ion coordination in metal-specific metallothioneins: the Helix pomatia system as a model by Òscar Palacios; Sílvia Pérez-Rafael; Ayelen Pagani; Reinhard Dallinger; Sílvia Atrian; Mercè Capdevila (923-935).
The Helix pomatia metallothionein (MT) system, namely, its two highly specific forms, HpCdMT and HpCuMT, has offered once again an optimum model to study metal–protein specificity. The present work investigates the most unexplored aspect of the coordination behavior of MT polypeptides with respect to either cognate or noncognate metal ions, as opposed to the standard studies of cognate metal ion coordination. To this end, we analyzed the in vivo synthesis of the corresponding complexes with their noncognate metals, and we performed a detailed spectroscopic and spectrometric study of the Zn2+/Cd2+ and Zn2+/Cu+ in vitro replacement reactions on the initial Zn-HpMT species. An HpCuMTAla site-directed mutant, exhibiting differential Cu+-binding abilities in vivo, was also included in this study. We demonstrate that when an MT binds its cognate metal, it yields well-folded complexes of limited stoichiometry, representative of minimal-energy conformations. In contrast, the incorporation of noncognate metal ions is better attributed to an unspecific reaction of cysteinic thiolate groups with metal ions, which is dependent on their concentration in the surrounding milieu, where no minimal-energy structure is reached, and otherwise, the MT peptide acts as a multidentate ligand that will bind metal ions until its capacity has been saturated. Additionally, we suggest that previous binding of an MT polypeptide with its noncognate metal ion (e.g., binding of Zn2+ to the HpCuMT isoform) may preclude the correct folding of the complex with its cognate metal ion.
Keywords: Helix pomatia ; Metallothionein; Cu-specific; Cd-specific

MetalS3, a database-mining tool for the identification of structurally similar metal sites by Yana Valasatava; Antonio Rosato; Gabriele Cavallaro; Claudia Andreini (937-945).
We have developed a database search tool to identify metal sites having structural similarity to a query metal site structure within the MetalPDB database of minimal functional sites (MFSs) contained in metal-binding biological macromolecules. MFSs describe the local environment around the metal(s) independently of the larger context of the macromolecular structure. Such a local environment has a determinant role in tuning the chemical reactivity of the metal, ultimately contributing to the functional properties of the whole system. The database search tool, which we called MetalS3 (Metal Sites Similarity Search), can be accessed through a Web interface at http://metalweb.cerm.unifi.it/tools/metals3/ . MetalS3 uses a suitably adapted version of an algorithm that we previously developed to systematically compare the structure of the query metal site with each MFS in MetalPDB. For each MFS, the best superposition is kept. All these superpositions are then ranked according to the MetalS3 scoring function and are presented to the user in tabular form. The user can interact with the output Web page to visualize the structural alignment or the sequence alignment derived from it. Options to filter the results are available. Test calculations show that the MetalS3 output correlates well with expectations from protein homology considerations. Furthermore, we describe some usage scenarios that highlight the usefulness of MetalS3 to obtain mechanistic and functional hints regardless of homology.
Keywords: Metalloprotein; Metalloenzyme; Bioinorganic chemistry; Structural biology; Zinc; Iron

Diversity of the metal-transporting P1B-type ATPases by Aaron T. Smith; Kyle P. Smith; Amy C. Rosenzweig (947-960).
The P1B-ATPases are integral membrane proteins that couple ATP hydrolysis to metal cation transport. Widely distributed across all domains of life, these enzymes have been previously shown to transport copper, zinc, cobalt, and other thiophilic heavy metals. Recent data suggest that these enzymes may also be involved in nickel and/or iron transport. Here we have exploited large amounts of genomic data to examine and classify the various P1B-ATPase subfamilies. Specifically, we have combined new methods of data partitioning and network visualization known as Transitivity Clustering and Protein Similarity Networks with existing biochemical data to examine properties such as length, speciation, and metal-binding motifs of the P1B-ATPase subfamily sequences. These data reveal interesting relationships among the enzyme sequences of previously established subfamilies, indicate the presence of two new subfamilies, and suggest the existence of new regulatory elements in certain subfamilies. Taken together, these findings underscore the importance of P1B-ATPases in homeostasis of nearly every biologically relevant transition metal and provide an updated framework for future studies.
Keywords: P-type ATPase; P1B-ATPase; Metal homeostasis; Metal transport; Transitivity clustering

Historical and biochemical aspects of a seventeenth century gold-based aurum vitae recipe by Riccardo Rubbiani; Bettina Wahrig; Ingo Ott (961-965).
The medicinal chemistry and biomedical applications of gold complexes have been intensively studied over the last decades. Some complexes have been used for the treatment of rheumatoid arthritis, and a considerable number of new metallodrug candidates have been developed as new anticancer drugs and anti-infectives. However, the therapeutic use of gold and its complexes goes back to ancient times and was also of great importance for alchemists until the modern age. In this report, we give an overview of the alchemic medicine between the sixteenth and the early eighteenth century and describe the cytotoxicity and thioredoxin reductase (TrxR) inhibition of a typical “aurum vitae” medicine, which was prepared according to a recipe by Bartholomäus Kretschmar from the seventeenth century. “Aurum vitae” consists of a mixture of gold, mercury and antimony complexes and shows the expected cytotoxic and TrxR inhibitory properties providing some rationale for therapeutic effects of this kind of historical medicinal preparation.
Keywords: Alchemy; Antimony; Aurum vitae; Cytotoxicity; Gold

Synthesis, characterization, and evaluation of cis-diphenyl pyridineamine platinum(II) complexes as potential anti-breast cancer agents by Jacqueline Gamboa Varela; Atasi De Chatterjee; Priscilla Guevara; Verenice Ramirez; Alejandro J. Metta-Magaña; Dino Villagrán; Armando Varela-Ramirez; Siddhartha Das; Jose E. Nuñez (967-979).
Although cisplatin is considered as an effective anti-cancer agent, it has shown limitations and may produce toxicity in patients. Therefore, we synthesized two cis-dichlorideplatinum(II) compounds (13 and 14) composed of meta- and para-N,N-diphenyl pyridineamine ligands through a reaction of the amine precursors and PtCl2 with respective yields of 16 and 47 %. We hypothesized that compounds 13 and 14, with lipophilic ligands, should transport efficiently in cancer cells and demonstrate more effectiveness than cisplatin. When tested for biological activity, compounds 13 and 14 were found to inhibit the growth of MCF 7 and MDA-MB-231 cells (IC50s 1 ± 0.4 µM and 1 ± 0.2 µM for 13 and 14, respectively, and IC50 7.5 ± 1.3 µM for compound 13 and 1 ± 0.3 µM for compound 14). Incidentally, these doses were found to be lower than cisplatin doses (IC50 5 ± 0.7 µM for MCF 7 and 10 ± 1.1 µM for MDA-MB-231). Similar to cisplatin, 13 and 14 interacted with DNA and induced apoptosis. However, unlike cisplatin, they blocked the migration of MDA-MB-231 cells suggesting that in addition to apoptotic and DNA-binding capabilities, these compounds are useful in blocking the metastatic migration of breast cancer cells. To delineate the mechanism of action, computer-aided analyses (DFT calculations) were conducted for compound 13. Results indicate that in vivo, the pyridineamine ligands are likely to dissociate from the complex, forming a platinum DNA adduct with anti-proliferative activity. These results suggest that complexes 13 and 14 hold promise as potential anti-cancer agents.
Keywords: Cisplatin; Breast cancer drugs; Platinum(II) complexes; Diphenyl pyridineamine; Apoptosis

Vanadium as a chemoprotectant: effect of vanadium(III)-l-cysteine complex against cyclophosphamide-induced hepatotoxicity and genotoxicity in Swiss albino mice by Abhishek Basu; Arin Bhattacharjee; Somnath Singha Roy; Prosenjit Ghosh; Pramita Chakraborty; Ila Das; Sudin Bhattacharya (981-996).
Vanadium is an essential micronutrient for living systems and has antioxidant and genoprotective property. In the present study, the protective role of an organovanadium compound vanadium(III)-l-cysteine (VC-III) was evaluated against hepatotoxicity and genotoxicity induced by cyclophosphamide (CP) (25 mg/kg b.w., i.p.) in Swiss albino mice. Treatment with VC-III (1 mg/kg b.w., p.o.) mitigated CP-induced hepatic injury as indicated by reduction in activities of alanine transaminase, aspartate transaminase, alkaline phosphatase by 1.57-, 1.58- and 1.32-fold in concomitant treatment schedule and by 1.83-, 1.77- and 1.45-fold in pretreatment schedule, respectively, and confirmed by histopathological evidences. Parallel to these changes, VC-III ameliorated CP-induced oxidative stress in liver by 1.46-, 1.26-, 1.32- and 1.42-fold in concomitant treatment group and by 1.95-, 1.40-, 1.46- and 1.73-fold in pretreatment group at the level of H2O2, superoxide, nitric oxide and lipid peroxidation, respectively. VC-III also enhanced activities of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase and glutathione (reduced) level in mice liver by 1.46-, 1.37-, 1.29-, 1.44- and 1.45-fold in concomitant treatment schedule and by 1.64-, 1.65-, 1.42-, 1.49- and 1.57-fold in pretreatment schedule, respectively. In addition, the organovanadium compound could efficiently attenuate CP-induced chromosomal aberrations, DNA fragmentation and apoptosis in bone marrow cells and DNA damage in lymphocytes by 1.49-, 1.43-, 1.48- and 1.59-fold in concomitant treatment group and by 1.76-, 1.92-, 1.99- and 2.15-fold in pretreatment group, respectively. Thus, the present study showed that VC-III could exert protection against CP-induced hepatotoxicity and genotoxicity.
Keywords: Organovanadium; Cyclophosphamide; Oxidative stress; Genotoxicity; Comet assay

Characterising the atypical 5′-CG DNA sequence specificity of 9-aminoacridine carboxamide Pt complexes by Hieronimus W. Kava; Anne M. Galea; Farhana Md. Jamil; Yue Feng; Vincent Murray (997-1007).
In this study, the DNA sequence specificity of four DNA-targeted 9-aminoacridine carboxamide Pt complexes was compared with cisplatin, using two specially constructed plasmid templates. One plasmid contained 5′-CG and 5′-GA insert sequences while the other plasmid contained a G-rich transferrin receptor gene promoter insert sequence. The damage profiles of each compound on the different DNA templates were quantified via a polymerase stop assay with fluorescently labelled primers and capillary electrophoresis. With the plasmid that contained 5′-CG and 5′-GA dinucleotides, the four 9-aminoacridine carboxamide Pt complexes produced distinctly different damage profiles as compared with cisplatin. These 9-aminoacridine complexes had greatly increased levels of DNA damage at CG and GA dinucleotides as compared with cisplatin. It was shown that the presence of a CG or GA dinucleotide was sufficient to reveal the altered DNA sequence selectivity of the 9-aminoacridine carboxamide Pt analogues. The DNA sequence specificity of the Pt complexes was also found to be similarly altered utilising the transferrin receptor DNA sequence.
Keywords: 9-Aminoacridine; Anticancer drug; Cisplatin; Cisplatin analogues; DNA sequence specificity

Electrostatic effects control the stability and iron release kinetics of ovotransferrin by Sandeep Kumar; Deepak Sharma; Rajesh Kumar; Rajesh Kumar (1009-1024).
The contribution of electrostatic interactions to the stability of ovotransferrin-Fe3+ (oTf-Fe3+) complex has been assessed by equilibrium experiments that measure iron retention level of diferric-ovotransferrin (Fe2oTf) as a function of pH and urea in the presence of salts (NaCl, Na2SO4, NaBr, NaNO3) and sucrose at 25 °C. As [salt] is increased, the pH-midpoint for iron release increases monoexponentially and plateau at ~0.4(±0.05) M NaCl/NaBr/NaNO3 or ~0.15(±0.03) M Na2SO4. However, at pH 7.4, the urea-midpoints for iron release (based on fluorescence emission at 340 nm) and for unfolding of Fe2oTf and apo-ovotransferrin (based on ellipticity values at 222 and 282 nm) decrease at low salt concentrations [≤0.1(±0.02) M Na2SO4 or ≤0.35(±0.15) M NaCl], but increase at higher salt concentrations. Furthermore, Na2SO4 has a greater effect than NaCl in increasing the urea-midpoints for iron release and unfolding. These results indicate that at low salt concentrations, the electrostatic effects destabilize the oTf-Fe3+ complex and also decrease the structural stability of the proteins. In contrast, at higher concentrations, salt ions behave according to Hofmeister series. At pH 5.6, as [salt] is increased, the rate constants for reductive iron release (Fe2+ release) and urea denaturation-induced iron release (Fe3+ release) from the N-lobe of oTf (FeNoTf) increase monoexponentially and plateau at ~0.4(±0.1) M NaNO3/NaCl or ~0.2(±0.05) M Na2SO4. These results suggest that the anion-binding-induced conformational change as well as the electrostatic screening of surface Coulombic interactions plays important role in accelerating the iron release from FeNoTf under endosomal pH conditions.
Keywords: Iron release; Electrostatic interactions; Anion-binding; Conformational change; Structural stability

Growth inhibitory effects of the Diruthenium-Ibuprofen compound, [Ru2Cl(Ibp)4], in human glioma cells in vitro and in the rat C6 orthotopic glioma in vivo by Marcel Benadiba; Iguatinã de M. Costa; Rodrigo L. S. R. Santos; Fernanda Oliveira Serachi; Denise de Oliveira Silva; Alison Colquhoun (1025-1035).
The Diruthenium-Ibuprofen compound [Ru2Cl(Ibp)4] (or RuIbp) is known to cause significant inhibition of C6 rat glioma cell proliferation in vitro. RuIbp increased the expression of cell cycle-related proteins such as p21 and p27 and the pro-apoptotic protein Bax, as well as causing a reduction in mitochondrial membrane potential and a modest increase in apoptosis in vitro. The present study extended these findings by (i) investigating the effects of RuIbp on human glioma cell line proliferation in vitro and (ii) investigating the acute and chronic toxicology of the compound in normal Wistar rats. The compound was then tested for its anti-tumour properties by either chronic 14 days intra-peritoneal (IP) administration or chronic Alzet osmotic pump infusion, in the rat C6 orthotopic glioma model in vivo. The IP injection of RuIbp caused a 41 % inhibition of tumour area without significant toxic effects but with an increase in blood neutrophils and monocytes and a decrease in blood lymphocytes. In an attempt to reduce this effect RuIbp was administered by Alzet osmotic pump infusion directly into the tumour at a dose of 15 mg/kg with an infusion rate of 0.5 µL/h for 14 days. The direct infusion of RuIbp caused a 45 % inhibition of tumour area without alterations in differential blood leukocyte counts. These results prove the efficacy of RuIbp in human glioma cell lines in vitro and in an in vivo glioma model and point to its potential as an inhibitor of tumour growth in vivo.
Keywords: Diruthenium; Ibuprofen; Glioma; Cancer; Osmotic pump

T versus D in the MTCXXC motif of copper transport proteins plays a role in directional metal transport by Moritz S. Niemiec; Artur P. G. Dingeldein; Pernilla Wittung-Stafshede (1037-1047).
To avoid toxicity and control levels of metal ions, organisms have developed specific metal transport systems. In humans, the cytoplasmic Cu chaperone Atox1 delivers Cu to metal-binding domains of ATP7A/B in the Golgi, for incorporation into Cu-dependent proteins. The Cu-binding motif in Atox1, as well as in target Cu-binding domains of ATP7A/B, consists of a MX1CXXC motif where X1 = T. The same motif, with X1 = D, is found in metal-binding domains of bacterial zinc transporters, such as ZntA. The Asp is proposed to stabilize divalent over monovalent metals in the binding site, although metal selectivity in vivo appears predominantly governed by protein–protein interactions. To probe the role of T versus D at the X1 position for Cu transfer in vitro, we created MDCXXC variants of Atox1 and the fourth metal-binding domain of ATP7B, WD4. We find that the mutants bind Cu like the wild-type proteins, but when mixed, in contrast to the wild-type pair, the mutant pair favors Cu-dependent hetero-dimers over directional Cu transport from Atox1 to WD4. Notably, both wild-type and mutant proteins can bind Zn in the absence of competing reducing agents. In presence of zinc, hetero-complexes are strongly favored for both protein pairs. We propose that T is conserved in this motif of Cu-transport proteins to promote directional metal transfer toward ATP7B, without formation of energetic sinks. The ability of both Atox1 and WD4 to bind zinc ions may not be a problem in vivo due to the presence of specific transport chains for Cu and Zn ions.
Keywords: Atox1; Wilson disease protein; Metal transport; Size exclusion chromatography; Calorimetry

Simultaneous observation of the metabolism of cisplatin and NAMI-A in human plasma in vitro by SEC-ICP-AES by Melani Sooriyaarachchi; Jason L. Wedding; Hugh H. Harris; Jürgen Gailer (1049-1053).
Single drug-based cancer therapies are frequently associated with the development of drug resistance. To overcome this problem, combination therapy with two or more anticancer drugs is a promising strategy, but clinical studies are logistically challenging and costly. Intermediary in vitro studies, however, can provide critical insight to decide whether one should proceed to in vivo studies. To this end, cisplatin and the Ru-based anticancer drug NAMI-A were added to human plasma and the size distribution of Pt-containing and Ru-containing entities was determined over a 2 h period. The results revealed a dramatically different rate of plasma protein binding for each drug and/or their hydrolysis products. Both drugs bound to the same apparent plasma proteins, but crucially they did not adversely affect each other’s metabolism. Therefore, combination therapy of patients with these metallodrugs should be further assessed in clinical studies in order to systematically develop an effective combination therapy protocol to prevent the resurgence of cancer.
Keywords: Cisplatin; NAMI-A; Combination therapy; Plasma; Size-exclusion chromatography