Do shale oil and gas production activities impact ambient air quality? A comprehensive study of 12 years of chemical concentrations and well production data from the Barnett Shale region of Texas.

Starting around 2008, there was rapid expansion of oil and natural gas (ONG) production into more heavily populated areas within the Dallas-Fort Worth metroplex in the Barnett Shale region of Texas. This colocation raised concerns regarding the effect of ONG activities on chemical levels in the air. In the current study, we examined the potential impacts of ONG activity on the types and concentrations of chemicals in ambient air in the Barnett Shale. Volatile organic compound (VOC) concentrations from 6-12 years (2008-2019) of hourly ambient air monitoring data from 15 monitors (4 monitors had ≥ 10 years of data) were compared to several metrics of ONG activity (number of active wells, natural gas production, condensate production) within a 2-mile radius of each monitor. Monitoring sites were also classified into urban, suburban, and rural areas as a surrogate for nearby vehicular emission sources. Analyses of this huge dataset showed that both peak and mean chemical concentrations of lighter alkane hydrocarbons (e.g., ethane) were most impacted by the number of gas wells. Levels of heavier alkanes (e.g., pentane) were increased by condensate production and at monitors located in areas with greater urbanicity, and therefore higher vehicular emissions. The levels of unsaturated alkynes (e.g., ethylene) were entirely driven by urbanicity and were unaffected by nearby ONG activity. The same pattern was seen with the ratio of iso:n-pentane, which is contrary to the findings of others and suggests an area for future research. Aromatic hydrocarbons were impacted by multiple emissions sources and did not show the same patterns as non-aromatic VOCs. No VOC concentrations were at levels of concern for human health or odor based on comparison to Texas air monitoring comparison values. Overall, ONG activities impact air quality, but this must be evaluated in the context of other emission sources such as automobiles.

Soil-Weathered CuO Nanoparticles Compromise Foliar Health and Pigment Production in Spinach (Spinacia oleracea).

In this study, spinach plants exposed to fresh/unweathered (UW) or weathered (W) copper compounds in soil were analyzed for growth and nutritional composition. Plants were exposed for 45 days to freshly prepared or soil-aged (35 days) nanoparticulate CuO (nCuO), bulk-scale CuO (bCuO), or CuSO4 at 0 (control), 400, 400, and 40 mg/kg of soil, respectively. Foliar health, gas exchange, pigment content (chlorophyll and carotenoid), catalase and ascorbate peroxidase enzymes, gene expression, and Cu bioaccumulation were evaluated along with SEM imagery for select samples. Foliar biomass was higher in UW control (84%) and in UW ionic treatment (87%), compared to the corresponding W treatments (p ≤ 0.1). Root catalase activity was increased by 110% in UW bCuO treatment as compared to the W counterpart; the value for the W ionic treatment was increased by 2167% compared to the UW counterpart (p ≤ 0.05). At 20 days post-transplantation, W nCuO-exposed plants had ∼56% lower carotenoid content compared to both W control and the UW counterpart (p ≤ 0.05). The findings indicate that over the full life cycle of spinach plant the weathering process significantly deteriorates leaf pigment production under CuO exposure in particular and foliar health in general.

Improvement of nutrient elements and allicin content in green onion (Allium fistulosum) plants exposed to CuO nanoparticles.

With the exponential growth of nanomaterial production in the last years, nano copper (Cu)-based compounds are gaining more consideration in agriculture since they can work as pesticides or fertilizers. Chinese scallions (Allium fistulosum), which are characterized by their high content of the antioxidant allicin, were the chosen plants for this study. Spectroscopic and microscopic techniques were used to evaluate the nutrient element, allicin content, and enzyme antioxidant properties of scallion plants. Plants were harvested after growing for 80 days at greenhouse conditions in soil amended with CuO particles [nano (nCuO) and bulk (bCuO)] and CuSO4 at 75-600 mg/kg]. Two-photon microscopy images demonstrated the particulate Cu uptake in nCuO and bCuO treated roots. In plants exposed to 150 mg/kg of the Cu-based compounds, root Cu content was higher in plants treated with nCuO compared with bCuO, CuSO4, and control (p ≤ 0.05). At 150 mg/kg, nCuO increased root Ca (86%), root Fe (71%), bulb Ca (74%), and bulb Mg (108%) content, compared with control (p ≤ 0.05). At the same concentration, bCuO reduced root Ca (67%) and root Mg (33%), compared with control (p ≤ 0.05). At all concentrations, nCuO and CuSO4 increased leaf allicin (56-187% and 42-90%, respectively), compared with control (p ≤ 0.05). The antioxidant enzymes were differentially affected by the Cu-based treatments. Overall, the data showed that nCuO enhances nutrient and allicin contents in scallion, which suggests they might be used as a nanofertilizer for onion production.

Nutritional Status of Tomato (Solanum lycopersicum) Fruit Grown in Fusarium-Infested Soil: Impact of Cerium Oxide Nanoparticles.

In this study, the impact of cerium oxide nanoparticles on the nutritional value of tomato (Solanum lycopersicum) fruit grown in soil infested with Fusarium oxysporum f. sp. lycopersici was investigated in a greenhouse pot study. Three-week old seedlings of Bonny Best tomato plants were exposed by foliar and soil routes to nanoparticle CeO2 (NP CeO2) and cerium acetate (CeAc) at 0, 50, and 250 mg/L and transplanted into pots containing a soil mixture infested with the Fusarium wilt pathogen. Fruit biomass, water content, diameter, and nutritional content (lycopene, reducing and total sugar) along with elemental composition, including Ce, were evaluated. Fruit Ce concentration was below the detection limit in all treatments. Foliar exposure to NP CeO2 at 250 increased the fruit dry weight (67%) and lycopene content (9%) in infested plants, compared with the infested untreated control. Foliar exposure to CeAc at 50 mg/L reduced fruit fresh weight (46%) and water content (46%) and increased the fruit lycopene content by 11% via root exposure as compared with the untreated infested control. At 250 mg/L, CeAc increased fruit dry weight (94%), compared with the infested untreated control. Total sugar content decreased in fruits of infested plants exposed via roots to NP CeO2 at 50 mg/kg (63%) and 250 mg/kg (54%), CeAc at 50 mg/kg (46%), and foliarly at 50 mg/L (50%) and 250 mg/L (50%), all compared with the infested untreated control. Plants grown in Fusarium-infested soil had decreased fruit dry weight (42%) and lycopene content (17%) and increased total sugar (60%) and Ca content (140%), when compared with the noninfested untreated control (p ≤ 0.05). Overall, the data suggested minimal negative effects of NP CeO2 on the nutritional value of tomato fruit while simultaneously suppressing Fusarium wilt disease.

Backbone resonance assignments and secondary structure of the apo-Drosophila melanogaster frataxin homolog (Dfh).

Friedreich's ataxia, the most prevalent hereditary ataxia, is caused by a patient's inability to produce a viable form of the protein frataxin. Frataxin plays an essential role in cellular iron regulation and has been shown to participate in the assembly of iron-sulfur (Fe-S) clusters under a variety of roles, including modulating persulfide production and directing Fe(II) delivery to the assembly scaffold protein. While the activity and structure of multiple eukaryotic frataxin orthologs have been characterized, the fly ortholog has numerous advantages over other orthologs with regards to protein stability, its activity towards Fe-S cluster assembly and its stability for forming stable proteins partner assemblies. Given the obvious advantages for studying the Drosophila melanogaster frataxin homolog (Dfh) over its orthologs, we have undertaken a structural characterization of apo-Dfh as the first step towards solving the solution structure of the protein alone and in complex with protein partners within the Fe-S cluster assembly pathway.

ZnO nanoparticles increase photosynthetic pigments and decrease lipid peroxidation in soil grown cilantro (Coriandrum sativum).

The growth of the nanotechnology industry has raised concerns about its environmental impacts. In particular, the effect on terrestrial plants, which are the primary producers of the global food chain, is widely debated. In this study, cilantro plants (Coriandrum sativum) were cultivated for 35 days in soil amended with ZnO nanoparticles (N ZnO), bulk ZnO (B ZnO) and ZnCl2 (ionic/I Zn) at 0-400 mg/kg. Photosynthetic pigments, lipid peroxidation, 1NMR-based metabolic, and ICP-based metallomic profiles were evaluated. All Zn compounds increased the chlorophyll content by at least 50%, compared to control. Only N ZnO at 400 mg/kg decreased lipid peroxidation by 70%. 1NMR data showed that all compounds significantly changed the carbinolic-based compounds, compared with control. Highest root and shoot uptake of Zn was observed at B 400 and I 100, respectively. Results of this study corroborates that N ZnO at a concentration <400 mg/kg improved photosynthesis pigments and the defense response in cilantro plants cultivated in organic soil.

Finding the conditions for the beneficial use of ZnO nanoparticles towards plants-A review.

Zinc oxide nanoparticles (ZnO NPs) have a wide range of applications in cosmetics, electrical, and optical industries. The wide range of applications of ZnO NPs, especially in personal care products, suggest they can reach major environmental matrices causing unforeseen effects. Recent literature has shown conflicting findings regarding the beneficial or detrimental effects of ZnO NPs towards terrestrial biota. In this review we carried out a comprehensive survey about beneficial, as well as detrimental aspects, of the ZnO NPs exposure toward various terrestrial plants. A careful scrutiny of the literature indicates that at low concentrations (about 50 mg/kg), ZnO NPs have beneficial effects on plants. Conversely, at concentrations above 500 mg/kg they may have detrimental effects, unless there is a deficiency of Zn in the growing medium. This review also remarks the critical role of the biotic and abiotic factors that may elevate or ameliorate the impact of ZnO NPs in terrestrial plants.

Plant uptake and translocation of contaminants of emerging concern in soil.

The advent of industrialization has led to the discovery of a wide range of chemicals designed for multiple uses including plant protection. However, after use, most of the chemicals and their derivatives end up in soil and water, interacting with living organisms. Plants, which are primary producers, are intentionally or unintentionally exposed to several chemicals, serving as a vehicle for the transfer of products into the food chain. Although the exposure of pesticides towards plants has been witnessed over a long time in agricultural production, other chemicals have attracted attention very recently. In this review, we carried out a comprehensive overview of the plant uptake capacity of various contaminants of emerging concern (CEC) in soil, such as pesticides, polycyclic aromatic hydrocarbons, perfluorinated compounds, pharmaceutical and personal care products, and engineered nanomaterials. The uptake pathways and overall impacts of these chemicals are highlighted. According to the literature, bioaccumulation of CEC in the root part is higher than in aerial parts. Furthermore, various factors such as plant species, pollutant type, and microbial interactions influence the overall uptake. Lastly, environmental factors such as soil erosion and temperature can also affect the CEC bioavailability towards plants.

Role of Cerium Compounds in Fusarium Wilt Suppression and Growth Enhancement in Tomato ( Solanum lycopersicum).

The use of nanoparticles in plant protection may reduce pesticide usage and contamination and increase food security. In this study, three-week-old Solanum lycopersicum seedlings were exposed, by root or foliar pathways, to CeO2 nanoparticles and cerium acetate at 50 and 250 mg/L prior to transplant into sterilized soil. One week later, the soil was inoculated with the fungal pathogen Fusarium oxysporum f. sp. lycopersici (1 g/kg), and the plants were cultivated to maturity in a greenhouse. Disease severity, biomass/yield, and biochemical and physiological parameters were analyzed in harvested plants. Disease severity was significantly reduced by 250 mg/L of nano-CeO2 and CeAc applied to the soil (53% and 35%, respectively) or foliage (57% and 41%, respectively), compared with non-treated infested controls. Overall, the findings show that nano-CeO2 has potential to suppress Fusarium wilt and improve the chlorophyll content in tomato plants.

In vitro characterization of a novel Isu homologue from Drosophila melanogaster for de novo FeS-cluster formation.

FeS-clusters are utilized by numerous proteins within several biological pathways that are essential for life. In eukaryotes, the primary FeS-cluster production pathway is the mitochondrial iron-sulfur cluster (ISC) pathway. In Saccharomyces cerevisiae, de novo FeS-cluster formation is accomplished through coordinated assembly with the substrates iron and sulfur by the scaffold assembly protein "Isu1". Sulfur for cluster assembly is provided by cysteine desulfurase "Nfs1", a protein that works in union with its accessory protein "Isd11". Frataxin "Yfh1" helps direct cluster assembly by serving as a modulator of Nfs1 activity, by assisting in the delivery of sulfur and Fe(ii) to Isu1, or more likely through a combination of these and other possible roles. In vitro studies on the yeast ISC machinery have been limited, however, due to the inherent instability of recombinant Isu1. Isu1 is a molecule prone to degradation and aggregation. To circumvent Isu1 instability, we have replaced yeast Isu1 with the fly ortholog to stabilize our in vitro ISC assembly system and assist us in elucidating molecular details of the yeast ISC pathway. Our laboratory previously observed that recombinant frataxin from Drosophila melanogaster has remarkable stability compared to the yeast ortholog. Here we provide the first characterization of D. melanogaster Isu1 (fIscU) and demonstrate its ability to function within the yeast ISC machinery both in vivo and in vitro. Recombinant fIscU has physical properties similar to that of yeast Isu1. It functions as a stable dimer with similar Fe(ii) affinity and ability to form two 2Fe-2S clusters as the yeast dimer. The fIscU and yeast ISC proteins are compatible in vitro; addition of Yfh1 to Nfs1-Isd11 increases the rate of FeS-cluster formation on fIscU to a similar extent observed with Isu1. Finally, fIscU expressed in mitochondria of a yeast strain lacking Isu1 (and its paralog Isu2) is able to completely reverse the deletion phenotypes. These results demonstrate fIscU can functionally replace yeast Isu1 and it can serve as a powerful tool for exploring molecular details within the yeast ISC pathway.

Inhibition of the Human ABC Efflux Transporters P-gp and BCRP by the BDE-47 Hydroxylated Metabolite 6-OH-BDE-47: Considerations for Human Exposure.

High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [(3H)-paclitaxel and (3H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity. These findings suggest that some effects previously attributed to BDE-47 in biological systems may actually be due to 6-OH-BDE-47. Considerations for human exposure are discussed.

Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8.

The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeast Saccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)] in vitro and in vivo and that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

SecA drives transmembrane insertion of RodZ, an unusual single-span membrane protein.

The transmembrane (TM) helices of most type II single-span membrane proteins (S-SMPs) of Escherichia coli occur near the N-terminus, where the cell's targeting mechanisms can readily identify it as it emerges from the ribosome. However, the TM helices of a few S-SMPs, such as RodZ, occur a hundred or more residues downstream from the N-terminus, which raises fundamental questions about targeting and assembly. Because of RodZ's novelty and potential usefulness for understanding TM helix insertion in vivo, we examined its membrane targeting and assembly. We used RodZ constructs containing immunotags before the TM domain to assess membrane insertion using proteinase K digestion. We confirmed the N(in)-C(out) (type II) topology of RodZ and established the absence of a targeting signal other than the TM domain. RodZ was not inserted into the membrane under SecA depletion conditions or in the presence of sodium azide, which is known to inhibit SecA. Insertion failed when the TM proton gradient was abolished with Carbonyl cyanide m-chlorophenyl hydrazone. Insertion also failed when RodZ was expressed in SecE-depleted E. coli, indicating that the SecYEG translocon is required for RodZ assembly. Protease accessibility assays of RodZ in other E. coli depletion strains revealed that insertion is independent of SecB, YidC, and SecD/F. Insertion was found to be only weakly dependent on the signal recognition particle pathway: insertion was weakly dependent on the Ffh but independent of FtsY. We conclude that membrane insertion of RodZ requires only the SecYEG translocon, the SecA ATPase motor, and the TM proton motive force.

Crystal structure and characterization of particulate methane monooxygenase from Methylocystis species strain M.

Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Previous biochemical and structural studies of pMMO have focused on preparations from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. A pMMO from a third organism, Methylocystis species strain M, has been isolated and characterized. Both membrane-bound and solubilized Methylocystis sp. strain M pMMO contain ~2 copper ions per 100 kDa protomer and exhibit copper-dependent propylene epoxidation activity. Spectroscopic data indicate that Methylocystis sp. strain M pMMO contains a mixture of Cu(I) and Cu(II), of which the latter exhibits two distinct type 2 Cu(II) electron paramagnetic resonance (EPR) signals. Extended X-ray absorption fine structure (EXAFS) data are best fit with a mixture of Cu-O/N and Cu-Cu ligand environments with a Cu-Cu interaction at 2.52-2.64 Å. The crystal structure of Methylocystis sp. strain M pMMO was determined to 2.68 Å resolution and is the best quality pMMO structure obtained to date. It provides a revised model for the pmoA and pmoC subunits and has led to an improved model of M. capsulatus (Bath) pMMO. In these new structures, the intramembrane zinc/copper binding site has a different coordination environment from that in previous models.

Key players and their role during mitochondrial iron-sulfur cluster biosynthesis.

Iron-sulfur clusters are multifaceted iron-containing cofactors coordinated and utilized by numerous proteins in nearly all biological systems. Fe-S-cluster-containing proteins help direct pathways essential for cell viability and participate in biological applications ranging from nucleotide biosynthesis and stability, protein translation, enzyme catalysis, and mitochondrial metabolism. Fe-S-containing proteins function by utilizing the unique electronic and chemical properties inherent in the Fe containing cofactor. Fe-S clusters are constructed of inorganic iron and sulfide arranged in a distinct caged structural makeup ranging from [Fe(2) -S(2) ], [Fe(3) -S(4) ], [Fe(4) -S(4) ], up to [Fe(8) -S(8) ] clusters. In eukaryotes, cluster activity is controlled in part at the assembly level and the major pathway for cluster production exists within the mitochondria. Recent insight into the pathway of mitochondrial cluster assembly has come from new in vivo and in vitro reports that provided direct insight into how all protein partners within the assembly pathway interact. However, we are only just beginning to understand the role of each protein within this complex pageant that is mitochondrial Fe-S cluster assembly. In this report we present results, using the yeast model for mitochondrial assembly, to describe the molecular details of how important proteins in the pathway coordinate for cluster assembly.

Emerging fungal infections among children: A review on its clinical manifestations, diagnosis, and prevention.

The incidence of fungal infections is increasing at an alarming rate, presenting an enormous challenge to healthcare professionals. This increase is directly related to the growing population of immunocompromised individuals especially children resulting from changes in medical practice such as the use of intensive chemotherapy and immunosuppressive drugs. Although healthy children have strong natural immunity against fungal infections, then also fungal infection among children are increasing very fast. Virtually not all fungi are pathogenic and their infection is opportunistic. Fungi can occur in the form of yeast, mould, and dimorph. In children fungi can cause superficial infection, i.e., on skin, nails, and hair like oral thrush, candida diaper rash, tinea infections, etc., are various types of superficial fungal infections, subcutaneous fungal infection in tissues under the skin and lastly it causes systemic infection in deeper tissues. Most superficial and subcutaneous fungal infections are easily diagnosed and readily amenable to treatment. Opportunistic fungal infections are those that cause diseases exclusively in immunocompromised individuals, e.g., aspergillosis, zygomycosis, etc. Systemic infections can be life-threatening and are associated with high morbidity and mortality. Because diagnosis is difficult and the causative agent is often confirmed only at autopsy, the exact incidence of systemic infections is difficult to determine. The most frequently encountered pathogens are Candida albicans and Aspergillus spp. But other fungi such as non-albicans Candida spp. are increasingly important.

Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly.

Frataxin, a conserved nuclear-encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two, Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone in the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural module to improve our understanding of the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry. Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into an Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly.

Oxidation of methane by a biological dicopper centre.

Vast world reserves of methane gas are underutilized as a feedstock for the production of liquid fuels and chemicals owing to the lack of economical and sustainable strategies for the selective oxidation of methane to methanol. Current processes to activate the strong C-H bond (104 kcal mol(-1)) in methane require high temperatures, are costly and inefficient, and produce waste. In nature, methanotrophic bacteria perform this reaction under ambient conditions using metalloenzymes called methane monooxygenases (MMOs). MMOs thus provide the optimal model for an efficient, environmentally sound catalyst. There are two types of MMO. Soluble MMO (sMMO) is expressed by several strains of methanotroph under copper-limited conditions and oxidizes methane with a well-characterized catalytic di-iron centre. Particulate MMO (pMMO) is an integral membrane metalloenzyme produced by all methanotrophs and is composed of three subunits, pmoA, pmoB and pmoC, arranged in a trimeric alpha(3)beta(3)gamma(3) complex. Despite 20 years of research and the availability of two crystal structures, the metal composition and location of the pMMO metal active site are not known. Here we show that pMMO activity is dependent on copper, not iron, and that the copper active site is located in the soluble domains of the pmoB subunit rather than within the membrane. Recombinant soluble fragments of pmoB (spmoB) bind copper and have propylene and methane oxidation activities. Disruption of each copper centre in spmoB by mutagenesis indicates that the active site is a dicopper centre. These findings help resolve the pMMO controversy and provide a promising new approach to developing environmentally friendly C-H oxidation catalysts.

Arsenic binding and transfer by the ArsD As(III) metallochaperone.

ArsD is a metallochaperone that delivers trivalent metalloids [As(III) or Sb(III)] to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. Interaction with ArsD increases the affinity of ArsA for As(III), conferring resistance to environmental concentrations of arsenic. Previous genetic analysis suggested that ArsD residues Cys12, Cys13, and Cys18 are involved in the transfer of As(III) to ArsA. Here X-ray absorption spectroscopy was used to show that As(III) is coordinated with three sulfur atoms, consistent with the three cysteine residues forming the As(III) binding site. Two single-tryptophan derivatives of ArsD exhibited quenching of intrinsic protein fluorescence upon binding of As(III) or Sb(III), which allowed estimation of the rates of binding and affinities for metalloids. Substitution of Cys12, Cys13, or Cys18 decreased the affinity for As(III) more than 10-fold. Reduced glutathione greatly increased the rate of binding of As(III) to ArsD but did not affect binding of As(III) to ArsA. This suggests that in vivo cytosolic As(III) might be initially bound to GSH and transferred to ArsD and then to ArsAB, which pumps the metalloid out of the cell. The As(III) chelator dimercaptosuccinic acid did not block the transfer from ArsD to ArsA, consistent with channeling of the metalloid from one protein to the other, as opposed to release and rebinding of the metalloid. Finally, transfer of As(III) from ArsD to ArsA occurred in the presence of MgATP at 23 degrees C but not at 4 degrees C. Neither MgADP nor MgATP-gamma-S could replace MgATP. These results suggest that transfer occurs with a conformation of ArsA that transiently forms during the catalytic cycle.

Structure of the two transmembrane Cu+ transport sites of the Cu+ -ATPases.

Cu(+)-ATPases drive metal efflux from the cell cytoplasm. Paramount to this function is the binding of Cu(+) within the transmembrane region and its coupled translocation across the permeability barrier. Here, we describe the two transmembrane Cu(+) transport sites present in Archaeoglobus fulgidus CopA. Both sites can be independently loaded with Cu(+). However, their simultaneous occupation is associated with enzyme turnover. Site I is constituted by two Cys in transmembrane segment (TM) 6 and a Tyr in TM7. An Asn in TM7 and Met and Ser in TM8 form Site II. Single site x-ray spectroscopic analysis indicates a trigonal coordination in both sites. This architecture is distinct from that observed in Cu(+)-trafficking chaperones and classical cuproproteins. The high affinity of these sites for Cu(+) (Site I K(a)=1.3 fM(-1), Site II K(a)=1.1 fM(-1)), in conjunction with reversible direct Cu(+) transfer from chaperones, points to a transport mechanism where backward release of free Cu(+) to the cytoplasm is largely prevented.