BioMetals (v.29, #4)

An overview of siderophores for iron acquisition in microorganisms living in the extreme by Luis O. De Serrano; Anne K. Camper; Abigail M. Richards (551-571).
Siderophores are iron-chelating molecules produced by microbes when intracellular iron concentrations are low. Low iron triggers a cascade of gene activation, allowing the cell to survive due to the synthesis of important proteins involved in siderophore synthesis and transport. Generally, siderophores are classified by their functional groups as catecholates, hydroxamates and hydroxycarboxylates. Although other chemical structural modifications and functional groups can be found. The functional groups participate in the iron-chelating process when the ferri-siderophore complex is formed. Classified as acidophiles, alkaliphiles, halophiles, thermophiles, psychrophiles, piezophiles, extremophiles have particular iron requirements depending on the environmental conditions in where they grow. Most of the work done in siderophore production by extremophiles is based in siderophore concentration and/or genomic studies determining the presence of siderophore synthesis and transport genes. Siderophores produced by extremophiles are not well known and more work needs to be done to elucidate chemical structures and their role in microorganism survival and metal cycling in extreme environments.
Keywords: Iron acquisition; Siderophores; Siderophore synthesis and transport; Extremophiles

Iron uptake and transport across physiological barriers by Kari A. Duck; James R. Connor (573-591).
Iron is an essential element for human development. It is a major requirement for cellular processes such as oxygen transport, energy metabolism, neurotransmitter synthesis, and myelin synthesis. Despite its crucial role in these processes, iron in the ferric form can also produce toxic reactive oxygen species. The duality of iron’s function highlights the importance of maintaining a strict balance of iron levels in the body. As a result, organisms have developed elegant mechanisms of iron uptake, transport, and storage. This review will focus on the mechanisms that have evolved at physiological barriers, such as the intestine, the placenta, and the blood–brain barrier (BBB), where iron must be transported. Much has been written about the processes for iron transport across the intestine and the placenta, but less is known about iron transport mechanisms at the BBB. In this review, we compare the established pathways at the intestine and the placenta as well as describe what is currently known about iron transport at the BBB and how brain iron uptake correlates with processes at these other physiological barriers.
Keywords: Iron transport; Gut; Placenta; Blood–brain barrier; HFE

In vitro heme biotransformation by the HupZ enzyme from Group A streptococcus by Ankita J. Sachla; Mahamoudou Ouattara; Elvira Romero; Johnson Agniswamy; Irene T. Weber; Giovanni Gadda; Zehava Eichenbaum (593-609).
In Group A streptococcus (GAS), the metallorepressor MtsR regulates iron homeostasis. Here we describe a new MtsR-repressed gene, which we named hupZ (heme utilization protein). A recombinant HupZ protein was purified bound to heme from Escherichia coli grown in the presence of 5-aminolevulinic acid and iron. HupZ specifically binds heme with stoichiometry of 1:1. The addition of NADPH to heme-bound HupZ (in the presence of cytochrome P450 reductase, NADPH-regeneration system and catalase) triggered progressive decrease of the HupZ Soret band and the appearance of an absorption peak at 660 nm that was resistance to hydrolytic conditions. No spectral changes were observed when ferredoxin and ferredoxin reductase were used as redox partners. Differential spectroscopy with myoglobin or with the ferrous chelator, ferrozine, confirmed that carbon monoxide and free iron are produced during the reaction. ApoHupZ was crystallized as a homodimer with a split β-barrel conformation in each monomer comprising six β strands and three α helices. This structure resembles the split β-barrel domain shared by the members of a recently described family of heme degrading enzymes. However, HupZ is smaller and lacks key residues found in the proteins of the latter group. Phylogenetic analysis places HupZ on a clade separated from those for previously described heme oxygenases. In summary, we have identified a new GAS enzyme-containing split β-barrel and capable of heme biotransformation in vitro; to the best of our knowledge, this is the first enzyme among Streptococcus species with such activity.
Keywords: Heme degradation; Heme metabolism; Enzyme; Spectral analysis

The phytotoxic aluminum species (Al3+) is considered as the primary factor limiting crop productivity in over 40 % of world’s arable land that is acidic. We evaluated the responses of two wheat cultivars (Triticum aestivum L.) with differential Al resistance, cv. Yecora E (Al-resistant) and cv. Dio (Al-sensitive), exposed to 0, 37, 74 and 148 μM Al for 14 days in hydroponic culture at pH 4.5. With increasing Al concentration, leaf Ca2+ and Mg2+ content decreased, as well as the effective quantum yield of photosystem II (PSII) photochemistry (Φ PSII ), while a gradual increase in leaf membrane lipid peroxidation, Al accumulation, photoinhibition (estimated as F v /F m ), and PSII excitation pressure (1 − q p ) occurred. However, the Al-resistant cultivar with lower Al accumulation, retained larger concentrations of Ca2+ and Mg2+ in the leaves and kept a larger fraction of the PSII reaction centres (RCs) in an open configuration, i.e. a higher ratio of oxidized to reduced quinone A (QA), than plants of the Al-sensitive cultivar. Four times higher Al concentration in the nutrient solution was required for Al-resistant plants (148 μM Al) than for Al-sensitive (37 μM Al), in order to establish the same closed RCs. Yet, the decline in photosynthetic efficiency in the cultivar Dio was not only due to closure of PSII RCs but also to a decrease in the quantum yield of the open RCs. We suggest that Al3+ toxicity may be mediated by nutrient deficiency and oxidative stress, and that Al-resistance of the wheat cultivar Yecora E, may be due at least partially, from the decreased Al accumulation that resulted to decreased reactive oxygen species (ROS) formation. However, under equal internal Al accumulation (exposure Al concentration: Dio 74 μM, Yecora E 148 μM) that resulted to the same oxidative stress, the reduced PSII excitation pressure and the better PSII functioning of the Al-resistant cultivar was probably due to the larger concentrations of Ca2+ and Mg2+ in the leaves. We propose that the different sensitivities of wheat cultivars to Al3+ toxicity can be correlated to differences in the redox state of QA. Thus, chlorophyll fluorescence measurements can be a promising tool for rapid screening of Al resistance in wheat cultivars.
Keywords: Chlorophyll fluorescence; Nutrient uptake; Oxidative stress; Quantum yield; Redox state; Triticum aestivum

Fluorescence detection of intracellular cadmium with Leadmium Green by Latha M. Malaiyandi; Harsh Sharthiya; Kirk E. Dineley (625-635).
Leadmium Green is a commercially available, small molecule, fluorescent probe advertised as a detector of free intracellular cadmium (Cd2+) and lead (Pb2+). Leadmium Green has been used in various paradigms, such as tracking Cd2+ sequestration in plant cells, heavy metal export in protozoa, and Pb2+ absorption by vascular endothelial cells. However very little information is available regarding its affinity and selectivity for Cd2+, Pb2+, and other metals. We evaluated the in vitro selectivity of Leadmium Green using spectrofluorimetry. Consistent with manufacturer’s claims, Leadmium Green was sensitive to Cd2+ (KD ~600 nM) and also Pb2+ (KD ~9.0 nM) in a concentration-dependent manner, and furthermore proved insensitive to Ca2+, Co2+, Mn2+ and Ni2+. Leadmium Green also responded to Zn2+ with a KD of ~82 nM. Using fluorescence microscopy, we evaluated Leadmium Green in live mouse hippocampal HT22 cells. We demonstrated that Leadmium Green detected ionophore-mediated acute elevations of Cd2+ or Zn2+ in a concentration-dependent manner. However, the maximum fluorescence produced by ionophore-delivered Zn2+ was much less than that produced by Cd2+. When tested in a model of oxidant-induced liberation of endogenous Zn2+, Leadmium Green responded weakly. We conclude that Leadmium Green is an effective probe for monitoring intracellular Cd2+, particularly in models where Cd2+ accumulates rapidly, and when concomitant fluctuations of intracellular Zn2+ are minimal.
Keywords: Zinc; Lead; Heavy metals; Live-cell microscopy; FluoZin-3

Activity of phosphino palladium(II) and platinum(II) complexes against HIV-1 and Mycobacterium tuberculosis by Ntombenhle H. Gama; Afag Y. F. Elkhadir; Bhavna G. Gordhan; Bavesh D. Kana; James Darkwa; Debra Meyer (637-650).
Treatment of human immunodeficiency virus (HIV) is currently complicated by increased prevalence of co-infection with Mycobacterium tuberculosis. The development of drug candidates that offer the simultaneous management of HIV and tuberculosis (TB) would be of great benefit in the holistic treatment of HIV/AIDS, especially in sub-Saharan Africa which has the highest global prevalence of HIV-TB coinfection. Bis(diphenylphosphino)-2-pyridylpalladium(II) chloride (1), bis(diphenylphosphino)-2-pyridylplatinum(II) chloride (2), bis(diphenylphosphino)-2-ethylpyridylpalladium(II) chloride (3) and bis(diphenylphosphino)-2-ethylpyridylplatinum(II) (4) were investigated for the inhibition of HIV-1 through interactions with the viral protease. The complexes were subsequently assessed for biological potency against Mycobacterium tuberculosis H37Rv by determining the minimal inhibitory concentration (MIC) using broth microdilution. Complex (3) showed the most significant and competitive inhibition of HIV-1 protease (p = 0.014 at 100 µM). Further studies on its in vitro effects on whole virus showed reduced viral infectivity by over 80 % at 63 µM (p < 0.05). In addition, the complex inhibited the growth of Mycobacterium tuberculosis at an MIC of 5 µM and was non-toxic to host cells at all active concentrations (assessed by tetrazolium dye and real time cell electronic sensing). In vitro evidence is provided here for the possibility of utilizing a single metal-based compound for the treatment of HIV/AIDS and TB.
Keywords: HIV-1; Mycobacterium tuberculosis ; Palladium complexes; Platinum complexes; Phosphine complexes

Cd2+ is highly toxic to Staphylococcus aureus since it blocks dithiols in cytoplasmic 2-oxoglutarate dehydrogenase complex (ODHC) participating in energy conservation process. However, S. aureus 17810R is Cd2+-resistant due to possession of cadA-coded Cd2+ efflux system, recognized here as P-type Cd2+-ATPase. This Cd2+ pump utilizing cellular energy—ATP, ∆μ H + (electrochemical proton potential) and respiratory protons, extrudes Cd2+ from cytoplasm to protect dithiols in ODHC, but the mechanism of Cd2+ extrusion remains unknown. Here we propose that two Cd2+ taken up by strain 17810R via Mn2+ uniporter down membrane potential (∆ψ) generated during glutamate oxidation in 100 mM phosphate buffer (high PiB) are trapped probably by high affinity sites in cytoplasmic domain of Cd2+-ATPase, forming SCdS. This stops Cd2+ transport towards dithiols in ODHC, allowing undisturbed NADH production, its oxidation and energy conservation, while ATP could change orientation of SCdS towards facing transmembrane channel. Now, increased number of Pi-dependent protons pumped electrogenically via respiratory chain and countertransported through the channel down ∆ψ, extrude two trapped cytoplasmic Cd2+, which move to low affinity sites, being then extruded into extracellular space via ∆ψ-dependent Cd2+/H+ exchange. In 1 mM phosphate buffer (low PiB), external Cd2+ competing with decreased number of Pi-dependent protons, binds to ψs of Cd2+-ATPase channel, enters cytoplasm through the channel down ∆ψ via Cd2+/Cd2+ exchange and blocks dithiols in ODHC. However, Mg2+ pretreatment preventing external Cd2+ countertransport through the channel down ∆ψ, allowed undisturbed NADH production, its oxidation and extrusion of two cytoplasmic Cd2+ via Cd2+/H+ exchange, despite low PiB.
Keywords: Cd2+ resistance; Cd2+-ATPase; Energy dependent Cd2+/H+ exchange; Staphylococcus aureus

Parkinson’s disease (PD) is the second most common neurodegenerative disease with gradual loss of dopaminergic neurons. Despite extensive research in the past decades, the etiology of PD remains elusive. Nevertheless, multiple lines of evidence suggest that oxidative stress is one of the common causes in the pathogenesis of PD. It has also been suggested that heavy metal-associated oxidative stress may be implicated in the etiology and pathogenesis of PD. Here we review the roles of redox metals, including iron, copper and cobalt, in PD. Iron is a highly reactive element and deregulation of iron homeostasis is accompanied by concomitant oxidation processes in PD. Copper is a key metal in cell division process, and it has been shown to have an important role in neurodegenerative diseases such as PD. Cobalt induces the generation of reactive oxygen species (ROS) and DNA damage in brain tissues.
Keywords: Parkinson’s disease; Reactive oxygen species; Iron; Copper; Cobalt

Gene expression of selenoproteins can be regulated by selenoprotein K silencing in chicken myoblasts by Ruifeng Fan; Haidong Yao; Xia Zhao; Changyu Cao; Tianshu Yang; Yilin Luan; Ziwei Zhang; Shiwen Xu (679-689).
The aim of the present study was to clarify the effect of Selenoprotein K (Selk) silencing on the mRNA expression of 25 selenoproteins in chicken myoblasts. The specific small interfering RNA (siRNA) for Selk gene was designed and transfected into chicken myoblasts. Post-transfection mRNA expression of 25 selenoproteins was determined at various time periods i.e., 24, 48 and 72 h. Moreover, based on the results of expression of 25 selenoproteins, correlation analysis and principal component analysis (PCA) were used for further analysis. The results showed that the designed siRNA effectively inhibited Selk expression (decreased by 20, 29 and 43 % on 24, 48 and 72 h, respectively) and the mRNA expression levels of the 23 selenoproteins were influenced by silencing Selk differently (P < 0.05). Time-dependent pattern of mRNA expression after siRNA treatment in three groups were found similar: one group including Gpx1, Gpx2, Gpx3, Gpx4, Txnrd1, Txnrd2, Txnrd3, Sepw1, Selh, Sepp1, Selo and Sepx1, another group including Sepn1, Sels, Selt, Selm and Sep15 and other group including Dio2 and Dio3. The results of correlation analysis showed that Gpx1, Gpx2, Gpx3, Gpx4, Dio1, Dio3, Sepn1, Sels, Sepw1, Selt, Selh, Sep15, Seli and Selu had a positive correlation with Selk, while Dio2 and Sepp1 had a negative correlation with Selk. PCA data also indicated that Txnrd1, Txnrd2, Dio2, Selpb, Sepp1and Selo may play special roles in response to Selk silencing. In summary, these results indicated that different selenoproteins possess and exhibits distinct responses to silencing of Selk in chicken myoblasts.
Keywords: Selenoprotein K; RNA interference; Chicken myoblasts; Selenoproteins

Functional link between ferroxidase activity of ceruloplasmin and protective effect of apo-lactoferrin: studying rats kept on a silver chloride diet by Valeria A. Kostevich; Alexey V. Sokolov; Stanislav O. Kozlov; Anna Yu. Vlasenko; Nikolay N. Kolmakov; Elena T. Zakharova; Vadim B. Vasilyev (691-704).
Strongly pronounced argyrosis caused by adding AgCl to the feed of laboratory rats efficiently mimics the deficiency of ceruloplasmin (CP) ferroxidase activity. Bringing the concentration of AgCl in the feedstuff of lactating rats to 250 mg % and keeping their progeny (Ag-rats) for 3 months on the same silver-containing feed provided the serum iron content 1.4 times lower than that in the control group. Besides, the ferroxidase activity of CP dropped to zero. In CP purified from sera of Ag-rats two copper ions were substituted with two silver ions. Using rat models of both post-hemorrhagic and hemolytic anemia we showed that the deficiency of CP ferroxidase activity in Ag-rats affects the iron content in serum, though does not prevent the recovery of hemoglobin level accompanied by exhaustion of iron caches in liver and spleen. When apo-lactoferrin (apo-LF) was administered to Ag-rats suffering from either post-hemorrhagic or hemolytic anemia, both hemoglobin and serum iron were restored more rapidly than in the control animals. In independent experiments Ag-rats were compared with those fed on regular diet and the former displayed a prolonged 3-day stabilization of hypoxia-inducible factors 1 and 2 alpha (HIF-1a and HIF-2a) along with an increased serum concentration of erythropoietin. Introduction to Ag-rats of active CP separately or together with apo-LF reduced that effect to 1 day only. It is concluded that saturation of apo-LF with iron, provided by active CP, can strongly affect its protective capacity.
Keywords: Iron metabolism; Ferroxidase; Ceruloplasmin; Lactoferrin; Transferrin; Erythropoietin; Post-hemorrhagic anemia; Hemolytic anemia; Hypoxia-inducible factor

Glutaredoxins are a family of small molecular weight proteins that have a central role in cellular redox regulation. Human GRX1 (hGRX1) has also been shown to play an integral role in copper homeostasis by regulating the redox activity of the metalated sites of copper chaperones such as ATOX1 and SOD1, and the copper efflux proteins ATP7A and ATP7B. To further elucidate the role of hGRX1 in copper homeostasis, we examined the impact of RNA interference-mediated knockdown of CG6852, a putative Drosophila orthologue of hGRX1. CG6852 shares ~41 % amino acid identity with hGRX1 and key functional domains including the metal-binding CXXC motif are conserved between the two proteins. Knockdown of CG6852 in the adult midline caused a thoracic cleft and reduced scutellum, phenotypes that were exacerbated by additional knockdown of copper uptake transporters Ctr1A and Ctr1B. Knockdown of CG6852 in the adult eye enhanced a copper-deficiency phenotype caused by Ctr1A knockdown while ubiquitous knockdown of CG6852 resulted a mild systemic copper deficiency. Therefore we conclude that CG6852 is a putative orthologue of hGRX1 and may play an important role in Drosophila copper homeostasis.
Keywords: Drosophila ; hGRX1; Copper; CTR1; ATP7

Effects of hydrogen sulfide on the heme coordination structure and catalytic activity of the globin-coupled oxygen sensor AfGcHK by Veronika Fojtikova; Martina Bartosova; Petr Man; Martin Stranava; Toru Shimizu; Marketa Martinkova (715-729).
AfGcHK is a globin-coupled histidine kinase that is one component of a two-component signal transduction system. The catalytic activity of this heme-based oxygen sensor is due to its C-terminal kinase domain and is strongly stimulated by the binding of O2 or CO to the heme Fe(II) complex in the N-terminal oxygen sensing domain. Hydrogen sulfide (H2S) is an important gaseous signaling molecule and can serve as a heme axial ligand, but its interactions with heme-based oxygen sensors have not been studied as extensively as those of O2, CO, and NO. To address this knowledge gap, we investigated the effects of H2S binding on the heme coordination structure and catalytic activity of wild-type AfGcHK and mutants in which residues at the putative O2-binding site (Tyr45) or the heme distal side (Leu68) were substituted. Adding Na2S to the initial OH-bound 6-coordinate Fe(III) low-spin complexes transformed them into SH-bound 6-coordinate Fe(III) low-spin complexes. The Leu68 mutants also formed a small proportion of verdoheme under these conditions. Conversely, when the heme-based oxygen sensor EcDOS was treated with Na2S, the initially formed Fe(III)–SH heme complex was quickly converted into Fe(II) and Fe(II)–O2 complexes. Interestingly, the autophosphorylation activity of the heme Fe(III)–SH complex was not significantly different from the maximal enzyme activity of AfGcHK (containing the heme Fe(III)–OH complex), whereas in the case of EcDOS the changes in coordination caused by Na2S treatment led to remarkable increases in catalytic activity.
Keywords: Hydrogen sulfide; Heme-based oxygen sensor; Autophosphorylation; Histidine kinase; Intramolecular catalytic regulation; Two-component signal transduction

Reduced LINE-1 methylation is associated with arsenic-induced genotoxic stress in children by Apurba K. Bandyopadhyay; Somnath Paul; Shanta Adak; Ashok K. Giri (731-741).
Early life exposure to arsenic has profound effect towards development of arsenic induced toxic outcomes. Some districts in the state of West Bengal, India are highly affected by arsenic, mainly through ground water. In children, not much of the toxic outcomes like dermatological lesions are observed but it is thought that the exposure leads to transient alteration in their biological processes that leads to various deleterious health effects later on. We evaluated the global methylation status by analyzing the LINE-1 methylation profile in children from arsenic exposed region between the age group 5–15 years along with the cytogenetic stress induced by arsenic as measured by lymphocyte micronucleus (MN) frequency. A total of 52 arsenic exposed and 32 unexposed children were analyzed. Whole blood DNA was used to measure the LINE-1 methylation by qRT-MSP. We found a significant association of MN-frequency in exposed individuals with highly depleted LINE-1 methylation compared to the exposed individuals with near baseline (which was comparable to unexposed control) methylation index as well as with those with the hypermethylated LINE-1 promoters. From our results, we interpret that LINE-1 methylation index may serve as a potent global epigenetic mark to detect the degree of arsenic genotoxicity at a very early age. We propose that this may be utilized to determine the extent of toxic influence exerted by arsenic, from a very early age.
Keywords: Arsenic; Early exposure; LINE-1 methylation; Cytogenetic stress; Epigenetic alteration

The characterization of anti-T. cruzi activity relationships between ferrocenyl, cyrhetrenyl complexes and ROS release by César Echeverría; Valentina Romero; Rodrigo Arancibia; Hugo Klahn; Ignacio Montorfano; Ricardo Armisen; Vincenzo Borgna; Felipe Simon; Rodrigo Ramirez-Tagle (743-749).
Trypanosoma cruzi (T. cruzi) is the parasite that causes Chagas disease. Nifurtimox is the most used drug against the T. cruzi, this drug increases intermediaries nitro group, being mainly responsible for the high toxicity component, for this reason it is important to study new organic compounds and thus improve therapeutic strategies against Chagas disease. The electronic effects of ferrocenyl and cyrhetrenyl fragments were investigated by DFT calculation. A close correlation was found between HOMO–LUMO gap of nitro radical NO 2 with the experimental reduction potential found for nitro group and IC50 of two forms the T. cruzi (epimastigote and trypomastigote). The IC50 on human hepatoma cells is higher for both compounds compared to IC50 demonstrated in the two forms the T. cruzi, and additionally show reactive oxygen species release. The information obtained in this paper could generate two new drugs with anti-T. cruzi activity, but additional studies are needed.
Keywords: Trypanosoma cruzi ; Reactive oxygen species; Anti-chagasic; DFT