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Chemical methods for the extraction and refinement of technologically critical rare earth elements (REEs) are energy-intensive, hazardous, and environmentally destructive. Current biobased extraction systems rely on extremophilic organisms and generate many of the same detrimental effects as chemical methodologies. The mesophilic methylotrophic bacterium Methylobacterium extorquens AM1 was previously shown to grow using electronic waste by naturally acquiring REEs to power methanol metabolism. Here we show that growth using electronic waste as a sole REE source is scalable up to 10 L with consistent metal yields without the use of harsh acids or high temperatures. The addition of organic acids increases REE leaching in a nonspecific manner. REE-specific bioleaching can be engineered through the overproduction of REE-binding ligands (called lanthanophores) and pyrroloquinoline quinone. REE bioaccumulation increases with the leachate concentration and is highly specific. REEs are stored intracellularly in polyphosphate granules, and genetic engineering to eliminate exopolyphosphatase activity increases metal accumulation, confirming the link between phosphate metabolism and biological REE use. Finally, we report the innate ability of M. extorquens to grow using other complex REE sources, including pulverized smartphones, demonstrating the flexibility and potential for use as a recovery platform for these critical metals.

Mediator of ERBB2-driven cell motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high-MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.

Introduction: Seeds are the most important carrier of germplasm preservation. However, an irreversible decrease in vigor can occur after the maturation of seeds, denoted as seed aging. Mitochondrion is a crucial organelle in initiation programmed cell death during seed aging. However, the underlying mechanism remains unclear. Methods: Our previous proteome study found that 13 mitochondria proteins underwent carbonylation modification during the aging of Ulmus pumila L. (Up) seeds. This study detected metal binding proteins through immobilized metal affinity chromatography (IMAC), indicating that metal binding proteins in mitochondria are the main targets of carbonization during seed aging. Biochemistry, molecular and cellular biology methods were adopted to detect metal-protein binding, protein modification and subcellular localization. Yeast and Arabidopsis were used to investigate the biological functions in vivo. Results and discussion: In IMAC assay, 12 proteins were identified as Fe²+/Cu²+/Zn²+ binding proteins, including mitochondrial voltage dependent anion channels (VDAC). UpVDAC showed binding abilities to all the three metal ions. His204Ala (H204A) and H219A mutated UpVDAC proteins lost their metal binding ability, and became insensitive to metal-catalyzed oxidation (MCO) induced carbonylation. The overexpression of wild-type UpVDAC made yeast cells more sensitive to oxidative stress, retarded the growth of Arabidopsis seedlings and accelerated the seed aging, while overexpression of mutated UpVDAC weakened these effects of VDAC. These results reveal the relationship between the metal binding ability and carbonylation modification, as well as the probable function of VDAC in regulating cell vitality, seedling growth and seed aging. [ABSTRACT FROM AUTHOR]

New analytical strategies for metal-binding protein facilitate researchers learning about how metals play a significant role in life. Laser ablation–inductively coupled plasma mass spectrometry (LA-ICP-MS) offers many advantages for the metal analysis of biological samples and shows a promising future in protein analysis, but recent advances in LA-ICP-MS-based strategies for identifying metal-binding proteins via endogenous metals remain less updated yet. To present the current status in this field, the main analytical strategies for metal-binding proteins with LA-ICP-MS are reviewed here, including in situ analysis of biospecimens and ex situ analysis with gel electrophoresis. A critical discussion of challenges and future perspectives is also given. Multifarious laser ablation–inductively coupled plasma mass spectrometry (LA-ICP-MS)-based strategies have been developed and applied to investigate the metal-binding proteins in biospecimens in situ or through gel electrophoresis ex situ over the past decades, facilitating researchers disclosing how essential metals are implicated in life or what proteins toxic metals will target. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER), an essential organelle in eukaryotic cells, is widely distributed in myocardial cells. The ER is where secreted protein synthesis, folding, post-translational modification, and transport are all carried out. It is also where calcium homeostasis, lipid synthesis, and other processes that are crucial for normal biological cell functioning are regulated. We are concerned that ER stress (ERS) is widespread in various damaged cells. To protect cells’ function, ERS reduces the accumulation of misfolded proteins by activating the unfolded protein response (UPR) pathway in response to numerous stimulating factors, such as ischemia or hypoxia, metabolic disorders, and inflammation. If these stimulatory factors are not eliminated for a long time, resulting in the persistence of the UPR, it will aggravate cell damage through a series of mechanisms. In the cardiovascular system, it will cause related cardiovascular diseases and seriously endanger human health. Furthermore, there has been a growing number of studies on the antioxidative stress role of metal-binding proteins. We observed that a variety of metal-binding proteins can inhibit ERS and, hence, mitigate myocardial damage.

In recent decades, the scientific community has widely debated the contamination of fish in the Amazon region by mercury species. As the diet of riverside populations in the Amazon region is based mainly on fish, these populations are exposed to mercurial species that can cause serious and irreversible damage to their health. The risks of consuming fish exposed to mercurial species in the Amazon region have motivated toxicological investigations. However, the effect of mercurial species on protein and enzyme levels is still controversial. In this work, analytical and bioanalytical techniques Two-dimensional polyacrylamide gel electrophoresis [2D-PAGE] Graphite Furnace Atomic Absorption Spectrometry [GFAAS], and Mass Spectrometry in Sequence with Electrospray Ionization [ESI–MS/MS] were used to identify proteins associated with mercury (metal-binding protein) in muscle and liver tissues of the fish species Pinirampus pirinampu from the Madeira River, in the Brazilian Amazon. Enzymatic and lipid peroxidation analyses were also used to assess changes related to oxidative stress. Determinations of total mercury by GFAAS indicated higher concentrations in liver tissue (555 ± 19.0 µg kg−1) when compared to muscle tissue (60 ± 2.0 µg kg−1). The fractionation process of tissue proteomes by 2D-PAGE and subsequent mapping of mercury by GFAAS in the protein spots of the gels identified the presence of mercury in three spots of the liver tissue (concentrations in the range of 0.800 to 1.90 mg kg−1). The characterization of protein spots associated with mercury by ESI–MS/MS identified the enzymes triosephosphate isomerase A, adenylate kinase 2 mitochondrial, and glyceraldehyde-3-phosphate dehydrogenase as possible candidates for mercury exposure biomarkers. The muscle tissue did not show protein spots associated with mercury. Enzymatic activity decreased proportionally to the increase in mercury concentrations in the tissues. [ABSTRACT FROM AUTHOR]

Mediator of ERBB2-driven cell motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 ( TFR 2), mitoferrin-2 ( SLC25A28 ), and the global iron response regulator IRP1 ( ACO1 ). These interactions indicate that cells with high-MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells., Competing Interests: ND, EU, MB, FV, AA, PN, MR, MT, SA, JP, HE, AF, FV, OD No competing interests declared, (© 2024, Dolgova, Uhlemann, Boniecki et al.)

Structural consequences of the binding of metal ions (regulatory Ca2+ and catalytic Zn2+) to the metalloenzyme l -alanyl- d -glutamate peptidase of the bacteriophage T5 (Endo T5) and some of its analogues containing single amino acid substitutions in the active center were analyzed by nuclear magnetic resonance (NMR), circular dichroism (CD) and calorimetry. Analyses revealed that the native EndoT5 undergoes strong structural rearrangements as a result of Zn2+ binding. This structural rearrangement resulting in the formation of an active enzyme is completed by the Ca2+ binding. In this case, the NMR spectra uncover the tautomerism of the NH protons of histidine imidazoles responsible for the Zn2+ coordination. For the EndoT5 analogues with point substitutions in the Ca2+-binding site, similar conformational rearrangements are observed upon Zn2+ binding. However, no characteristic changes in the NMR spectra associated with the Ca2+ binding were detected. The roles of the proton exchange in the process of Ca2+-induced activation of the enzymatic activity of EndoT5 is discussed. [ABSTRACT FROM AUTHOR]

Zrt2 is a zinc transporter of the ZIP family. It is predicted to be located in the plasma membrane and it is essential for Candida albicans zinc uptake and growth at acidic pH. Zrt2 from C. albicans is composed of 370 amino acids and contains eight putative transmembrane domains and an extra-membrane disordered loop, corresponding to the amino acid sequence 126–215. This protein region contains at least three possible metal binding motifs: HxHxHxxD (144–153), HxxHxxEHxD (181–193) and the Glu- and Asp- rich sequence DDEEEDxE (161–168). The corresponding model peptides, protected at their termini (Ac-GPHTHSHFGD-NH2, Ac-DDEEEDLE-NH2 and Ac-PSHFAHAQEHQDP-NH2), have been investigated in order to elucidate the thermodynamic and coordination properties of their Zn2+ and Cu2+ complexes, with the further aim to identify the most effective metal binding site among the three fragments. Furthermore, we extended the investigation to the peptides Ac-GPHTHAHFGD-NH2 and Ac-PAHFAHAQEHQDP-NH2, where serine residues have been substituted by alanines in order to check if the presence of a serine residue may favor the displacement of amidic protons by Cu2+. In the native Zrt2 protein, the Ac-GPHTHSHFGD-NH2 region of the Zrt2 loop has the highest metal binding affinity, showing that three alternated histidines separated by only one residue (-HxHxH-) bind Zn2+ and Cu2+ more strongly than the region in which three histidines are separated by two and three His residues (-HxxHxxxH- in Ac-PSHFAHAQEHQDP-NH2). All studied Zrt2 loop fragments have lower affinity towards Zn2+ than the zinc(II) binding site on the Zrt1 transporter; also, all three Zrt2 regions bind Zn2+ and Cu2+ with comparable affinity below pH 5 and, therefore, may equally contribute to the metal acquisition under the most acidic conditions in which the Zrt2 transporter is expressed.

Ribotoxins are fungal proteins that serve as weapons against parasites and insects. They are strongly toxic due to their ability to enter host cells and inactivate ribosomes. Ageritin is the prototype of a new ribotoxin-like protein family present in basidiomycetes. We demonstrate that this enzyme has peculiar binding and enzymatic features. Different from other ribotoxins, its ribonucleolytic activity requires the presence of divalent cations, with a maximum activation in the presence of zinc ions, for which Ageritin exhibits the strongest affinity of binding. We modeled the catalytic metal binding site of Ageritin, made of the putative triad Asp68, Asp70 and His77. This report highlights that Ageritin has the structure and function of an RNase but a Mg2+/Zn2+-dependent mechanism of action, a new finding for ribotoxins. As a zinc-dependent toxin, Ageritin can be classified among the arsenal of zinc-binding proteins involved in fungal virulence. [ABSTRACT FROM AUTHOR]

Highlights • Comprehensive procedure to quickly seek biofactors responsible for metal sorption. • Flagellin of Serratia Se1998 demonstrated a super binding capacity towards Pb. • A potential remediation tool for Pb contamination in aquatic environment. Abstract Metal contaminants present persistent and deleterious threats to environmental ecosystems and human health. Microorganisms can rapidly develop protective mechanisms against metal toxicity, such as metallothionein production. The identification of biological factors related to these protective mechanisms is essential for effective metal remediation. This study presents a robust pathway to rapidly locate and characterize a Pb-binding flagellin in Serratia Se1998, which can bind Pb at a 16:1 Pb: protein ratio. A column gel electrophoresis system hyphenated with inductively coupled plasma mass spectrometry (ICP MS) was constructed to efficiently separate and identify Pb-binding proteins from the whole bacterial proteome. PCR and transgenic assays were used to elucidate the exact sequences and biological function of Pb-binding proteins and heterogeneous expression of Pb-binding flagellin in E. coli could significantly enhance Pb removal from aqueous solution by approximately 45%. This method provides a benchmark procedure to rapidly identify biological factors responsible for metal biosorption. Identification of this unique Pb-binding flagellin highlights that microorganisms can survive high metal stresses due to various complex biological pathways for metal detoxification and remediation. [ABSTRACT FROM AUTHOR]

S100 proteins are multifunctional (intra/extra)cellular mostly dimeric calcium-binding proteins engaged into numerous diseases. We have found that monomeric recombinant human S100P protein interacts with intact human serum albumin (HSA) in excess of calcium ions with equilibrium dissociation constant of 25–50 nM, as evidenced by surface plasmon resonance spectroscopy and fluorescent titration by HSA of S100P labelled by fluorescein isothiocyanate. Calcium removal or S100P dimerization abolish the S100P-HSA interaction. The interaction is selective, since S100P does not bind bovine serum albumin and monomeric human S100B lacks interaction with HSA. In vitro glycation of HSA disables its binding to S100P. The revealed selective and highly specific conformation-dependent interaction between S100P and HSA shows that functional properties of monomeric and dimeric forms of S100 proteins are different, and raises concerns on validity of cell-based assays and animal models used for studies of (patho)physiological roles of extracellular S100 proteins. [ABSTRACT FROM AUTHOR]

Metallothioneins are cysteine-rich proteins, which function as (i) metal carriers in basal cell metabolism and (ii) protective metal chelators in conditions of metal excess. Metallothioneins have been characterized from different eukaryotic model and cultivable species. Presently, they are categorized in 15 families but evolutionary relationships between these metallothionein families remain unresolved. Several cysteine-rich protein encoding genes that conferred Cd-tolerance in Cd-sensitive yeast mutants have previously been isolated from soil eukaryotic metatranscriptomes. They were called CRPs for “cysteine-rich proteins”. These proteins, of unknown taxonomic origins, share conserved cysteine motifs and could be considered as metallothioneins. In the present work, we analyzed these CRPs with respect to their amino acid sequence features and their metal-binding abilities towards Cd, Zn and Cu metal ions. Sequence analysis revealed that they share common features with different known metallothionein families, but also exhibit unique specific features. Noticeably, CRPs display two separate cysteine-rich domains which, when expressed separately in yeast, confer Cd-tolerance. The N-terminal domain contains some conserved atypical Cys motifs, such as one CCC and two CXCC ones. Five CRPs were expressed and purified as recombinant proteins and their metal-binding characteristics were studied. All these CRPs chelated Cd(II), Zn(II) and Cu(I), although displaying a better capacity for Zn(II) coordination. All CRPs are able to confer Cd-tolerance, and four of them confer Zn-tolerance in the Zn-sensitive zrc1Δ yeast mutant. We designated these CRPs as environmental metallothioneins belonging to a new formerly undescribed metallothionein family. [ABSTRACT FROM AUTHOR]

Exposure to mercury can interfere with the expression of proteins and enzymes, compromise important pathways, such as apoptosis and glucose metabolism, and even induce the expression of metallothioneins. In this study, analytical techniques were used to determine the concentration of total mercury (THg) in muscle and liver tissue, protein pellets, and spots [using graphite furnace atomic absorption spectrometry (GFAAS)], and molecular techniques were used to identify metalloproteins present in mercury-associated protein spots. Thirty individuals from three different fish species, Cichla sp. (n = 10), Brachyplatystoma filamentosum (n = 10), and Semaprochilodus sp. (n = 10) from the Brazilian Amazon were used. Oxidative stress indicators [such as glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD), a marker of lipid peroxidation (LPO)] and the possible expression of metallothioneins in muscle and liver tissues were investigated. The two piscivorous species, Cichla sp. and B. filamentosum , presented the highest concentrations of mercury in their hepatic tissue, 1219 ± 15.00 and 1044 ± 13.6 μg kg−1, respectively, and in their muscle tissue, 101 ± 1.30 μg kg−1 and 87.4 ± 0.900 μg kg−1, respectively. The non-carnivorous species Semaprochilodus sp. had comparatively low concentrations of mercury in both its hepatic (852 ± 11.1 μg kg−1) and muscle (71.4 ± 0.930 μg kg−1) tissues. The presence of mercury was identified in 24 protein spots using GFAAS; concentrations ranged from 11.5 to 787 μg kg−1, and mass spectrometry identified 21 metal-binding proteins. The activities of GSH-Px, CAT, and SOD, related to oxidative stress, decreased proportionally as tissue Hg concentrations increased, while the levels of LPO markers increased, indicating the presence of stress. Our study results demonstrate possible mercury interference in oxidative stress markers (GSH-Px, CAT, SOD, and LPO), in addition to the identification of 21 metal-binding proteins as possible biomarkers of mercury exposure in fish. [Display omitted] • Metalloproteomic study to assess mercury contamination in Amazon fishes was conducted. • Mercury contamination correlations to oxidative stress in Amazon fishes were assessed. • Twenty-one mercury-associated proteins spots were characterized using LC-MS/MS. • Mercury exposure biomarkers in Amazon fishes were evaluated. • Physiological and functional study of mercury toxicity in fishes was carried out. [ABSTRACT FROM AUTHOR]

Introduction: Seeds are the most important carrier of germplasm preservation. However, an irreversible decrease in vigor can occur after the maturation of seeds, denoted as seed aging. Mitochondrion is a crucial organelle in initiation programmed cell death during seed aging. However, the underlying mechanism remains unclear., Methods: Our previous proteome study found that 13 mitochondria proteins underwent carbonylation modification during the aging of Ulmus pumila L. (Up) seeds. This study detected metal binding proteins through immobilized metal affinity chromatography (IMAC), indicating that metal binding proteins in mitochondria are the main targets of carbonization during seed aging. Biochemistry, molecular and cellular biology methods were adopted to detect metal-protein binding, protein modification and subcellular localization. Yeast and Arabidopsis were used to investigate the biological functions in vivo ., Results and Discussion: In IMAC assay, 12 proteins were identified as Fe 2 +/Cu 2 +/Zn 2 + binding proteins, including mitochondrial voltage dependent anion channels (VDAC). UpVDAC showed binding abilities to all the three metal ions. His204Ala (H204A) and H219A mutated UpVDAC proteins lost their metal binding ability, and became insensitive to metal-catalyzed oxidation (MCO) induced carbonylation. The overexpression of wild-type UpVDAC made yeast cells more sensitive to oxidative stress, retarded the growth of Arabidopsis seedlings and accelerated the seed aging, while overexpression of mutated UpVDAC weakened these effects of VDAC. These results reveal the relationship between the metal binding ability and carbonylation modification, as well as the probable function of VDAC in regulating cell vitality, seedling growth and seed aging., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Liu, Qi, Ye, Kang, Wang, Wang and Xue.)

The endoplasmic reticulum (ER), an essential organelle in eukaryotic cells, is widely distributed in myocardial cells. The ER is where secreted protein synthesis, folding, post-translational modification, and transport are all carried out. It is also where calcium homeostasis, lipid synthesis, and other processes that are crucial for normal biological cell functioning are regulated. We are concerned that ER stress (ERS) is widespread in various damaged cells. To protect cells' function, ERS reduces the accumulation of misfolded proteins by activating the unfolded protein response (UPR) pathway in response to numerous stimulating factors, such as ischemia or hypoxia, metabolic disorders, and inflammation. If these stimulatory factors are not eliminated for a long time, resulting in the persistence of the UPR, it will aggravate cell damage through a series of mechanisms. In the cardiovascular system, it will cause related cardiovascular diseases and seriously endanger human health. Furthermore, there has been a growing number of studies on the antioxidative stress role of metal-binding proteins. We observed that a variety of metal-binding proteins can inhibit ERS and, hence, mitigate myocardial damage.

Marine bivalves accumulate high concentrations of potentially toxic heavy metals in their tissues. We report here our investigation of the accumulation patterns of cadmium (Cd), copper (Cu), and lead (Pb) in tissues of the Japanese scallop Mizuhopecten yessoensis after exposure to artificial seawater containing Cd, Cu, or Pb. Our results revealed that accumulation of these heavy metals by this scallop was associated with certain intracellular metal-binding proteins (MBPs). Cd was accumulated at higher concentrations than Cu and Pb, and most of the accumulated metals were detected in the digestive gland. MBPs from the digestive gland were purified and subjected to a preliminary characterization. Three proteins with molecular masses of approximately 28, 37, and 42 kDa, respectively, were isolated by gel-filtration and anion-exchange column chromatography. Characterization of the partial amino acid sequences of these proteins show high sequence similarity to MBPs and ion-transporters. Metalloprotein profiles in the digestive gland indicated that some proteins were upregulated after metal exposure, suggesting that these proteins are involved in mechanisms of metal accumulation and detoxification in M. yessoensis. [ABSTRACT FROM AUTHOR]

[Display omitted] • In vitro biosynthesis of FeSe nanoparticles; • Biosynthesized FeSe nanoparticles for imageable drug-delivery platform; • FeSe nanoparticles with doxorubicin and poly-γ-glutamate (FeSe@DOX@PGA); • Assessment of FeSe@DOX@PGA as drug-delivery system in nude mouse. Biosynthesis of metal nanoparticles is an alternative route to obtain suitable materials for biomedical applications because neither toxic reducing reagents nor surfactants are required for the synthesis, unlike conventional chemical ways. In this study, we demonstrate the in vitro biosynthesis of FeSe nanoparticles using a recombinant fusion protein of Pseudomonas putida metallothionein and Arabidopsis thaliana phytochelatin synthase as the seeding sites under UV irradiation. The resulting FeSe nanoparticles exhibited distinguishable fluorescence properties promising for imageable drug-delivery application. Its demonstration as an imageable drug delivery vehicle is done through poly-γ-glutamic acid (PGA) encapsulation to form the pH-dependent structural changeable shells. Doxorubicin (DOX) was used as a model drug, so that a drug-delivery system of FeSe@DOX@PGA was constructed. It was then administered to mice and assessed for its biodistribution over time. The results suggest that FeSe@DOX@PGA is feasible to be used as an imageable drug-delivery platform. [ABSTRACT FROM AUTHOR]

Three structures of the histidine triad family protein from Entamoeba histolytica, the causative agent of amoebic dysentery, were solved at high resolution within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The structures have sulfate (PDB entry ), AMP (PDB entry ) or GMP (PDB entry ) bound in the active site, with sulfate occupying the same space as the α-phosphate of the two nucleotides. The Cα backbones of the three structures are nearly superimposable, with pairwise r.m.s.d.s ranging from 0.06 to 0.13 Å. [ABSTRACT FROM AUTHOR]

HcgD, a homolog of the ubiquitous Nif3‐like protein family, is found in a gene cluster involved in the biosynthesis of the iron‐guanylylpyridinol (FeGP) cofactor of [Fe]‐hydrogenase. The presented crystal structure and biochemical analyses indicated that HcgD has a dinuclear iron‐center, which provides a pronounced binding site for anionic ligands. HcgD contains a stronger and a weaker bound iron; the latter being removable by chelating reagents preferentially in the oxidized state. Therefore, we propose HcgD as an iron chaperone in FeGP cofactor biosynthesis, which might also stimulate investigations on the functionally unknown but physiologically important eukaryotic Nif3‐like protein family members.HcgD is encoded in the gene cluster involved in FeGP‐cofactor biosynthesis. HcgD is structurally similar to proteins of the ubiquitous Nif3‐like protein family. HcgD has two irons in the conserved binuclear metal‐binding site. One iron can be removed from HcgD by chelating reagents. A hypothesis of the biological role of HcgD is proposed. [ABSTRACT FROM AUTHOR]

Abstract: Ascidians are well known to accumulate extremely high levels of vanadium in their blood cells. Several key proteins related to vanadium accumulation and physiological function have been isolated from vanadium-rich ascidians. Of these, vanadium(IV)-binding protein-129 (VBP-129) is a unique protein that has been identified from the blood plasma of an ascidian Ascidia sydneiensis samea, but its metal binding domains are not known. In this study, several deletion and point mutants of VBP-129 were generated, and their metal binding abilities were assessed by immobilized metal ion affinity chromatography (IMAC) and electron spin resonance spectroscopy (ESR). The internal partial protein, VBP-Int41, did not bind to VIV, but the two constructs, VBP-N52 and VBP-Int55, added with additional 11 or 14 neighboring amino acids bound to VIV. Mutations for cysteine-47 and lysine-50 in VBP-Int55 diminished VIV-binding in VBP-Int55, suggesting that these amino acid residues play important roles in binding VIV. ESR titration analysis revealed that VBP-129, VBP-N52 and VBP-Int55 could bind to 6, 3 and 2 VIV ions, respectively. ESR spectrum analysis indicated a N2O2 coordination geometry, which is similar to vanabins. The cysteines may contribute to the maintenance of the three-dimensional structure that is necessary for binding VIV ions. VBP-129 did not have a VV-reductase activity, as expected from its tissue localization in blood plasma. This study provided the evidences that VBP-129 possesses VIV-binding domains that make a similar coordination to VIV as those by vanabins but VBP-129 acts solely as a VIV-chaperon in blood plasma. [Copyright &y& Elsevier]

Abstract: Background: Vanabins are a unique protein family of vanadium-binding proteins with nine disulfide bonds. Possible binding sites for VO2+ in Vanabin2 from a vanadium-rich ascidian Ascidia sydneiensis samea have been detected by nuclear magnetic resonance study, but the metal selectivity and metal-binding ability of each site was not examined. Methods: In order to reveal functional contribution of each binding site, we prepared several mutants of Vanabin2 by in vitro site-directed mutagenesis and analyzed their metal selectivity and affinity by immobilized metal-ion affinity chromatography and Hummel Dreyer method. Results: Mutation at K10/R60 (site 1) markedly reduced the affinity for VO2+. Mutation at K24/K38/R41/R42 (site 2) decreased the maximum binding number, but only slightly increased the overall affinity for VO2+. Secondary structure of both mutants was the same as that of the wild type as assessed by circular dichroism spectroscopy. Mutation in disulfide bonds near the site 1 did not affect its high affinity binding capacity, while those near the site 2 decreased the overall affinity for VO2+. General significance: These results suggested that the site 1 is a high affinity binding site for VO2+, while the site 2 composes a moderate affinity site for multiple VO2+. [Copyright &y& Elsevier]

Zrt2 is a zinc transporter of the ZIP family. It is predicted to be located in the plasma membrane and it is essential for Candida albicans zinc uptake and growth at acidic pH. Zrt2 from C. albicans is composed of 370 amino acids and contains eight putative transmembrane domains and an extra-membrane disordered loop, corresponding to the amino acid sequence 126–215. This protein region contains at least three possible metal binding motifs: HxHxHxxD (144–153), HxxHxxEHxD (181–193) and the Glu- and Asp- rich sequence DDEEEDxE (161–168). The corresponding model peptides, protected at their termini (Ac-GPHTHSHFGD-NH2, Ac-DDEEEDLE-NH2 and Ac-PSHFAHAQEHQDP-NH2), have been investigated in order to elucidate the thermodynamic and coordination properties of their Zn2+ and Cu2+ complexes, with the further aim to identify the most effective metal binding site among the three fragments. Furthermore, we extended the investigation to the peptides Ac-GPHTHAHFGD-NH2 and Ac-PAHFAHAQEHQDP-NH2, where serine residues have been substituted by alanines in order to check if the presence of a serine residue may favor the displacement of amidic protons by Cu2+. In the native Zrt2 protein, the Ac-GPHTHSHFGD-NH2 region of the Zrt2 loop has the highest metal binding affinity, showing that three alternated histidines separated by only one residue (-HxHxH-) bind Zn2+ and Cu2+ more strongly than the region in which three histidines are separated by two and three His residues (-HxxHxxxH- in Ac-PSHFAHAQEHQDP-NH2). All studied Zrt2 loop fragments have lower affinity towards Zn2+ than the zinc(II) binding site on the Zrt1 transporter; also, all three Zrt2 regions bind Zn2+ and Cu2+ with comparable affinity below pH 5 and, therefore, may equally contribute to the metal acquisition under the most acidic conditions in which the Zrt2 transporter is expressed. [ABSTRACT FROM AUTHOR]

Abstract: Escherichia coli hosts able to over-express metal-binding proteins (MerP) originating from Gram-positive (Bacillus cereus RC607) and Gram-negative (Pseudomonas sp. K-62) bacterial strains were used to adsorb Ni2+, Zn2+ and Cr3+ in aqueous solutions. The initial adsorption rate and adsorption capacity were determined to evaluate the performance of the biosorbents. With the expression of MerP protein, the metal adsorption capacity of the recombinant strains for Ni2+, Zn2+ and Cr3+ significantly improved. The cells carrying Gram-positive merP gene (GB) adsorbed Zn2+ and Cr3+ at a capacity of 22.3 and 0.98mmol/gbiomass, which is 121% and 72% higher, respectively, over that of the MerP-free host cells. Adsorption capacity of the cells carrying Gram-negative merP gene (GP) also increased 144% and 126% for Zn2+ and Cr3+, respectively. Both recombinant strains also exhibited 24% and 5% enhancement in adsorption of Ni2+ for GB and GP, respectively. The initial adsorption rate of the recombinant biosorbents was also higher than that of the MerP-free host, suggesting an increased metal-binding affinity with MerP expression. Severe cell damage on GB biosorbent was observed after Cr3+ adsorption, probably due to the metal toxicity effect on the cells. [Copyright &y& Elsevier]

Abstract: The blood cells of ascidians accumulate extremely high levels of the transition metal vanadium. We previously isolated four vanadium-binding proteins (Vanabins 1–4) and a homologous protein (VanabinP) from the vanadium-rich ascidian Ascidia sydneiensis samea. In the present study, we identified cDNAs encoding five different Vanabin2-related proteins in A. sydneiensis samea blood cells. It was notable that the sequences of the encoded proteins vary from that of Vanabin2 at up to 14 specific positions, while both the polypeptide length and the 18 cysteine residues were completely conserved. The most divergent protein, named 14MT, differed from Vanabin2 at all 14 positions. Using immobilized metal-ion affinity chromatography, we found that Vanabin2 and 14MT have the same metal-ion selectivity, but the overall affinity of 14MT for VO2+ is higher than that of Vanabin2. Binding number for VO2+ ions was the same between Vanabin2 and 14MT as assessed by gel filtration. These results suggested that sequence variations were under strict evolutionary constraints and high-affinity binding sites for VO2+ are conserved among Vanabin2 variants. [Copyright &y& Elsevier]

Abstract: Human Dicer contains two RNase III domains (RNase IIIa and RNase IIIb) that are responsible for the production of short interfering RNAs and microRNAs. These small RNAs induce gene silencing known as RNA interference. Here, we report the crystal structure of the C-terminal RNase III domain (RNase IIIb) of human Dicer at 2.0 Å resolution. The structure revealed that the RNase IIIb domain can form a tightly associated homodimer, which is similar to the dimers of the bacterial RNase III domains and the two RNase III domains of Giardia Dicer. Biochemical analysis showed that the RNase IIIb homodimer can cleave double-stranded RNAs (dsRNAs), and generate short dsRNAs with 2 nt 3′ overhang, which is characteristic of RNase III products. The RNase IIIb domain contained two magnesium ions per monomer around the active site. The distance between two Mg-1 ions is approximately 20.6 Å, almost identical with those observed in bacterial RNase III enzymes and Giardia Dicer, while the locations of two Mg-2 ions were not conserved at all. We presume that Mg-1 ions act as catalysts for dsRNA cleavage, while Mg-2 ions are involved in RNA binding. [Copyright &y& Elsevier]

Abstract: Several species of ascidians, the so-called tunicates, accumulate extremely high levels of vanadium ions in their blood cells. We previously identified a family of vanadium-binding proteins, named Vanabins, from blood cells and blood plasma of a vanadium-rich ascidian, Ascidia sydneiensis samea. The 3-dimensional structure of Vanabin2, the predominant vanadium-binding protein in blood cells, has been revealed, and the vanadium-binding properties of Vanabin2 have been studied in detail. Here, we used Far Western blotting to identify a novel protein that interacts with Vanabin2 from a blood cell cDNA library. The protein, named Vanabin-interacting protein 1 (VIP1), was localized in the cytoplasm of signet ring cells and giant cells. Using a two-hybrid method, we revealed that VIP1 interacted with Vanabins 1, 2, 3, and 4 but not with Vanabin P. The N-terminal domain of VIP1 was shown to be important for the interaction. Further, Vanabin1 was found to interact with all of the other Vanabins. These results suggest that VIP1 and Vanabin1 act as metal chaperones or target proteins in vanadocytes. [Copyright &y& Elsevier]

Virion infectivity factor (Vif) is an accessory protein encoded by HIV-1 and is critical for viral infection of the host CD4+ T cell population. Vif induces ubiquitination and subsequent degradation of Apo3G, a cytosolic cytidine deaminase that otherwise targets the retroviral genome. Interaction of Vif with the cellular Cullin5-based E3 ubiquitin ligase requires a conserved BC box and upstream residues that are part of the conserved H-(Xaa)5-C-(Xaa)17–18-C-(Xaa)3–5-H (HCCH) motif. The HCCH motif is involved in stabilizing the Vif-Cullin 5 interaction, but the exact role of the conserved His and Cys residues remains elusive. In this report, we find that full-length HIV-1 Vif. as well as a HCCH peptide, is capable of binding to zinc with high specificity. Zinc binding induces a conformational change that leads to the formation of large protein aggregates. EDTA reversed aggregation and regenerated the apoprotein conformation. Cysteine modification studies with the HCCH peptide suggest that C114 is critical for stabilizing the fold of the apopeptide, and that C133 is located in a solvent-exposed region with no definite secondary structure. Selective alkylation of C133 reduced metal-binding specificity of the HCCH peptide, allowing cobalt to bind with rates comparable to that with zinc. This study demonstrates that the HCCH motif of HIV-1 Vif is a unique metal-binding domain capable of mediating protein-protein interactions in the presence of zinc and adds to a growing list of examples in which metal ion binding induces protein misfolding and/or aggregation. [ABSTRACT FROM AUTHOR]

Abstract: Vanadium-binding proteins, or Vanabins, have recently been isolated from the vanadium-rich ascidian, Ascidia sydneiensis samea. Recent reports indicate that Vanabin2 binds twenty V(IV) ions at pH 7.5, and that it has a novel bow-shaped conformation. However, the role of Vanabin2 in vanadium accumulation by the ascidian has not yet been determined. In the present study, the effects of acidic pH on selective metal binding to Vanabin2 and on the secondary structure of Vanabin2 were examined. Vanabin2 selectively bound to V(IV), Fe(III), and Cu(II) ions under acidic conditions. In contrast, Co(II), Ni(II), and Zn(II) ions were bound at pH 6.5 but not at pH 4.5. Changes in pH had no detectable effect on the secondary structure of Vanabin2 under acidic conditions, as determined by circular dichroism spectroscopy, and little variation in the dissociation constant for V(IV) ions was observed in the pH range 4.5–7.5, suggesting that the binding state of the ligands is not affected by acidification. Taken together, these results suggest that the reason for metal ion dissociation upon acidification is attributable not to a change in secondary structure but, rather, that it is caused by protonation of the amino acid ligands that complex with V(IV) ions. [Copyright &y& Elsevier]

Abstract: This review describes recent tendencies for metal-binding and metalloprotein analysis, emphasizing metal quantification in proteins through X-ray, atomic absorption, mass spectrometric techniques, and others. Hyphenated techniques such as capillary electrophoresis–synchrotron radiation X-ray fluorescence (CE–SRXRF), laser ablation–inductively coupled plasma-mass spectrometry (LA–ICP-MS), matrix-assisted laser desorption/ionisation–time-of-flight mass spectrometry (MALDI–TOF-MS), etc. are also presented. As protein separation techniques electrophoresis (mainly sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE), capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) are indicated, due to their inherent sensitivity, resolution and/or easy implementation. Latest challenges in metallomics are also commented. [Copyright &y& Elsevier]

In metal-polluted environments, high concentrations of metals such as zinc, cadmium, copper, and lead are present. Many studies have been conducted to determine uptake and binding of the nonessential, toxic metal cadmium. Effects of essential metals on this uptake and binding remain unclear. The aim of the present study was to determine the effect of zinc on cadmium kinetics and metallothionein induction in the springtail Orchesella cincta. Exposure experiments were conducted with cadmium only, zinc only, and cadmium 1 zinc. Metallothionein was isolated to determine zinc binding to or induction of this cadmiumbinding protein. In animals exposed to cadmium only and to cadmium 1 zinc, metallothionein was induced. In animals exposed to zinc only, no metallothionein was induced, and zinc did not bind to the metallothionein induced by cadmium. Cadmium uptake rate was significantly lower in animals exposed to cadmium 1 zinc than in animals exposed to cadmium only. No significant pattern of uptake and elimination of zinc was observed. Zinc concentrations in the animals were not significantly different for the different exposures, demonstrating that this element is regulated by O. cincta. These results suggest a protective role of zinc in the uptake of cadmium but no interference of zinc with the internal cadmium distribution and elimination. [ABSTRACT FROM AUTHOR]

About 90 years ago, Henze discovered high levels of vanadium in the blood (coelomic) cells of an ascidian collected from the Bay of Naples. His discovery attracted the interdisciplinary attention of chemists, physiologists, and biochemists. Two decades ago, we quantified the vanadium levels in several ascidian tissues definitively using neutron-activation analysis and revealed that some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, corresponding to about 107 times that found in seawater. Vanadium accumulated is reduced to the +3 oxidation state via the +4 oxidation state and stored in vacuoles of vanadocytes (vanadium-containing blood cells) where high levels of protons and sulfate are also contained. To investigate this unusual phenomenon, we isolated several proteins and genes that are expressed in vanadocytes. To date, three types of vanadium-binding protein, designated as Vanabins, have been isolated, with molecular masses of 12.5, 15, and 16 kDa, along with the cDNAs encoding these proteins. In addition, four types of enzyme related to the pentose phosphate pathway that produces NADPH were revealed to be located in vanadocytes. The pentose phosphate pathway participates in the reduction of V(V) to V(IV). The cDNA for each of the vacuolar-type H+&z.sbnd;ATPase (V&z.sbnd;ATPase) A, B, C, and D subunits, which are located on the vacuolar membranes of vanadocytes, has been isolated and analyzed. V&z.sbnd;ATPase generates a proton-motive force, and is thought to provide the energy for vanadium accumulation. To clarify the entire mechanism involved in the accumulation and reduction, much more genes and proteins expressed in the blood cells need to be systematically identified. Thus, we have performed an expressed sequence tag (EST) analysis of blood cells and have established the functional assay system to elucidate the functions of genes and proteins obtained from ascidian blood cells. [Copyright &y& Elsevier]

The Asian periwinkle, Littorina brevicula, is highly resistant to a wide range of heavy metal concentrations and its metal-binding protein(s) are induced in the presence of cadmium (Cd) and zinc (Zn). In this study, we isolated and characterized a novel copper-binding protein (Cu-BP). Following purification by Sephacryl S-100 chromatography, Cu-BP contained an equal amount of Zn in non-exposed physiological conditions. However, Zn is replaced by Cu at the binding site upon addition of excess Cu (100 μM CuCl2) to the cytosol or after a long period (60 days) of exposure of the periwinkles to the metal ion (150 μg/l CuCl2). The ligand was further purified by DEAE-Sepharose anion-exchange chromatography and C18 reverse-phase HPLC. The molecular weight of the purified protein was determined as 11.38 kDa by MALDI-TOF MS analyses. This Cu-BP is distinct from common mollusk metallothionein (MT) in that it contains significantly lower number of Cys (8 residues) and high levels of aromatic amino acids, Tyr and Phe. The protein additionally contains His and Met, which are absent in the MT-like Cd-BP of L. brevicula. The finding that Cu-BP in the Asian periwinkle is distinct from MT-like Cd-BP suggests that the timely expression of specific metal-binding proteins allows added protection against each heavy metal in severely polluted conditions. [Copyright &y& Elsevier]

Metallothioneins (MTs) are a class of metal-binding proteins characterized by a high cysteine content (up to 30% of the amino acid residues), low molecular weight, and lack of aromatic amino acid residues. Remarkable metal-binding properties have been reported both in vivo and in vitro by a number of different analytical techniques. Chemical and spectroscopic studies have shown that an unusually wide range of metals bind to MT. 113Cd and 1H-NMR techniques have been used successfully to determine the structures in a number of different proteins. Together with X-ray diffraction results, analyses of these NMR data have established that in mammalian Cd7–MT and Zn7–MT, the metals are tetrahedrally coordinated in two isolated domains with stoichiometries of M4S11 and M3S9. Recently, Stillman, collaborators, and coworkers have characterized the Zn(II), Cd(II), Cu(I), Ag(I), Au(I) and Hg(II) containing forms of MTs from mammalian, yeast, and recombinant human sources using a number of different spectroscopic techniques. Optical spectroscopy, and in particular circular dichroism and luminescence, have provided rich details of a complicated metal binding chemistry, whether metals are added directly to the metal free, apo-MT, or to the Zn-containing protein. Electrospray ionization mass spectrometry is a powerful technique for the characterization of proteins in general directly from solution. For MTs, both from mammalian and recombinant sources, use of this technique allows study of the details of metal binding to the protein as a function of metal loading and pH. Kinetic studies allow determination of the metal binding mechanism when Cu(I) binds to both the metal-free and Zn(II) protein. Scanning tunneling microscopy is a surface analysis technique capable of producing nanoscale images with atomic resolution and is being applied to obtain images of MT on Au(111) surfaces. Molecular modeling, together with XANES, XAFS, and structural information from 2D-1H-NMR data allow the determination of 3D structures of a range of MTs. Finally, molecular dynamics (MD) calculations have been carried out to investigate the motion of the metal-containing and metal free peptides, with special interest in the region of the metal binding site. [Copyright &y& Elsevier]

Two small multimeric histidine-rich proteins, AgNt84 and Ag164, encoded by two nodule-specific cDNAs isolated from nodule cDNA libraries of the actinorhizal host plant Alnus glutinosa, represent a new class of plant metal binding proteins. This paper reports the characterization of the purified in vitro-expressed proteins by size exclusion chromatography, circular dichroism, equilibrium dialysis, metal affinity chromatography coupled with mass spectrometry, and nuclear magnetic resonance spectroscopy. These analyses reveal that each polypeptide is capable of binding multiple atoms of Zn2+, Ni2+, Co2+, Cu2+, Cd2+ and Hg2+. A reversible shift in histidine Cε1 and Cδ2 protons in NMR analysis occurred during titration of this protein with ZnCl2 strongly suggesting that histidine residues are responsible for metal binding. AgNt84 and Ag164 are not related to metal binding metallothioneins and phytochelatins and represent a new class of plant metal binding proteins that we propose to call metallohistins. Possible biological roles in symbioses for AgNt84 and Ag164, and their potential for use in bioremediation are discussed. [ABSTRACT FROM AUTHOR]

Bacteria are commonly used for bioremediation of heavy metal pollution and strategies to improve their performance in this respect are desirable. In this study, an Escherichia coli strain was engineered to express a common metallothionein-α domain. The metallothionein-α domain was over-expressed in the cytoplasm of E. coli as a fusion to the carboxyl terminal of maltose binding protein. The fusion protein was highly soluble in the cytoplasm of E. coli. When grown in the presence of cadmium, cells expressing the metallothionein-α fusion protein showed increased viability compared with control cells. Cells expressing the metallothionein-α also demonstrated increased accumulation of cadmium. [ABSTRACT FROM AUTHOR]

Ion uptake, transport, and sequestration are essential to meet the nutritional requirements for plant growth and development. Furthermore, regulation of these processes is critical for plants to tolerate toxic levels of ions. The examination of isoprenylated proteins encoded by Arabidopsis thaliana and Glycine max cDNAs revealed a unique family of proteins containing putative metal-binding motifs (the core sequence is M/LXCXXC). Here, we describe this new class of proteins, which are capable of being isoprenylated and binding transition metal ions. Members of this family contain consensus isoprenylation (CaaX) sites, which we demonstrate are efficiently isoprenylated in vitro. ATFP3, a representative of the Arabidopsis family, was expressed in Escherichia coli and examined for metal-binding activity in vitro. Analysis of the interaction of ATFP3 with metal-chelating columns (IMAC) suggested that it binds to Cu2+, Ni2+, or Zn2+. To test whether proteins with these characteristics are present in other plant species, tobacco BY2 cells were labeled in vivo with [14C]mevalonate and the resulting mevalonate-labeled proteins were tested for metal-binding activity. Several soluble, isoprenylated proteins which bound copper-IMAC columns were revealed. Consistent with a wide-spread distribution of these proteins in plants, their presence was observed in Arabidopsis, soybean, and tobacco. [ABSTRACT FROM AUTHOR]

Metal-binding proteins were investigated in livers of bream caught in the River Elbe from Steti (Czech Republic) to Hamburg (Federal Republic of Germany). A major zinc and copper binding protein fraction with a low molecular weight of 10 000 to 12 000 Da and with properties similar to mammalian hepatic metallothionein was isolated from bream livers using gel filtration chromatography. Two protein isoforms could be separated by reversed phase-high performance liquid chromatography (RP-HPLC), however, mercury was associated with only one isoform. The possibility of different detoxification potentials of the isoforms is discussed. Maximal concentrations of metal-binding protein were detected in samples from Dresden. If metal-binding proteins are to be included in a biological monitoring study, further investigations are required. [ABSTRACT FROM AUTHOR]

The kinetics of binding of Cu (II), Tb (III) and Fe(III) to ovotransferrin have been investigated using the stopped-flow technique. Rate constants for the second-order reaction, k, were determined by monitoring the absorbance change upon formation of the metal-transferrin complex in time range of milliseconds to seconds. The N and C sites appeared to bind a particular metal ion with the same rate; thus, average formation rate constants k (average) were 2.4 × 10 M s and 8.3 × 10 M S for Cu (II) and Tb (III) respectively. Site preference ( N site for Cu (II) and C site for Tb (III)) is then mainly due to the difference in dissociation rate constant for the metals. Fe (III) binding from Fe-nitrilotriacetate complex to apo-ovotransferrin was found to be more rapid, giving an average formation rate constant k (average) of 5 × 10 M s, which was followed by a slow increase in absorbance at 465 nm. This slow process has an apparent rate constant in the range 3 s to 0.5 s, depending upon the degree of Fe (III) saturation. The variation in the rate of the second phase is thought to reflect the difference in the rate of a conformational change for monoferric and diferric ovotransferrins. Monoferric ovotransferrin changes its conformation more rapidly (3.4s) than diferric ovotransferrin (0.52 s). A further absorbance decrease was observed over a period of several minutes; this could be assigned to release of NTA from the complex, as suggested by Honda et al. (1980). [ABSTRACT FROM AUTHOR]

High concentrations of mercury found in soils, sediments, fish, and humans of the Amazon region have gained prominence in scientific studies during the last decade. However, studies related to the elucidation of mercury toxicity mechanisms in ichthyofauna at the molecular and metallomic levels that seek to elucidate physiological and functional aspects, as well as the search for biomarkers of mercury exposure, are still sparse. In the search for these answers, the present study analyzed the hepatic tissue proteome of the Arapaima gigas (pirarucu) fish species collected in the Jirau hydroelectric power plant reservoir in the state of Rondônia state, Brazil, in order to identify mercury-related metal-binding proteins and to elucidate their physiological and functional aspects. The proteomic profile of the hepatic tissue of Arapaima gigas was obtained by two-dimensional electrophoresis (2D-PAGE) and the presence of mercury was mapped in the protein SPOTS by graphite furnace atomic absorption spectrometry(GFAAS). Mercury was detected in 18 protein SPOTS with concentrations ranging from 0.13 ± 0.003 to 131.00 ± 3 mg kg−1. The characterization of the protein SPOTS associated with mercury was performed by electrospray ionisation tandem mass spectrometry (ESI–MS/MS), and 10 proteins were identified. Bioinformatics analyses showed that most of the proteins found linked to mercury were involved in cellular component processes and biological processes. For the most part, protein sequences have cellular functions comprising catalytic, binding, sense of localization, and metabolic processes. Image 10701 • Metalloproteomic study in samples of hepatic tissue from Arapaima gigas. • Mercury-associated metal binding protein in Amazonian fish. • Elucidation of mercury toxicity mechanisms in ichthyofauna at the molecular and metallomic. • Mercury detected in 18 protein stains at different concentrations. [ABSTRACT FROM AUTHOR]