Browsing by Subject "Iron"
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Item Biophysical and Bioanalytical Analysis of the Iron-ome in Mitochondria Isolated from Saccharomyces cerevisiae(2011-08-08) Garber Morales, Jessica H.An integrative biophysical and bioanalytical approach to studying the Fe distribution in isolated mitochondria was developed. This procedure involved large-scale growths, the inclusion of a chelator in isolation buffers and an anaerobic isolation protocol. Electron microscopy confirmed that mitochondrial membranes were intact and that samples were largely devoid of contaminants. The Fe-ome-the sum of all Fe species in mitochondria--was studied using a combination of EPR, Mossbauer Spectroscopy, Electron Absorption, ICP-MS and Protein analysis. Isolated mitochondria were packed prior to analysis to improve the S/N ratio. The residual buffer content of sample pellets was determined by use of a radio-labeled buffer. There was essentially no difference in the packing efficiency of mitochondria isolated from respiring and fermenting cells. The determined packing factor, 0.80, was used to calculate concentrations of individual species in neat mitochondria. The Fe-omes of mitochondria isolated from cells grown on respiring, respirofermenting and fermenting media were determined. Neat mitochondria contained ~ 750 mM Fe, regardless of whether the cells had been grown on respiring or fermenting media. The Fe distribution of respirofermenting samples (which can undergo respiration and fermentation simultaneously) was nearly identical to that of respiring mitochondria. Fermenting samples had a very different Fe-distribution. Nearly 40 % of the iron in respiring mitochondria was present in respiratory complexes including cytochrome c, cytochrome bc1, succinate dehydrogenase, and cytochrome c oxidase. Fermenting mitochondria contain an Fe-ome dominated by non-protein centers. Approximately 80 % of the Fe was present as a combination of nonheme HS Fe2+, nonheme Fe3+ and Fe3+ nanoparticles. These centers were present in roughly equal amounts. The remaining 20 % of the Fe was present as respiratory complexes which have concentrations ~ 1/2 to 1/3 that of respiring mitochondria. A model is presented in which the nonheme HS Fe2+ species serves as a feedstock for Fe/S and heme biosynthesis. When the cell is growing on respiring media, this metabolic reservoir diminishes as respiratory complexes are constantly synthesized. Under fermentative growth, the metabolic pool increases due to the reduced demand for respiration-related prosthetic groups.Item Chemical enhanced oil recovery utilizing alternative alkalis(2013-05) Unomah, Michael Ogechukwuka; Pope, G. A.This study explores alternative alkaline agents other than sodium carbonate for ASP process on reactive and non-reactive crude oil recovery at 55oC and 100oC. The alkalis studied were sodium metaborate, pH of 10-10.5, and a sodium silicate/borax mixture, pH of 11. Sodium metaborate showed very optimistic results similar to sodium carbonate studies. Sodium metaborate ASP floods recovered 97-99% of residual oil after waterflood in Berea sandstone at 55oC. The oil saturation in the core after the chemical flood was between 0.5-2%. Sodium metaborate ASP floods recovered 96% of the tertiary oil with a residual oil saturation of 2.6% in Bentheimer sandstone at 100oC. More importantly, the retention of surfactant was very low with the use of metaborate in Berea, Bentheimer and high clay content reservoir cores. 0.18 mg/g rock (68%) and 0.07 mg/g rock (30%) of surfactant was retained in Berea and Bentheimer respectively with the use of sodium metaborate. Sodium metaborate ASP floods recovered 96% and 98% of residual oil with a final oil saturation of 4.8% and 0.56% at 100oC and 55oC respectively in reservoir rock. The retention in reservoir core was 0.13 mg/g (48%) and 0.29 mg/g (80%) at 100oC and 55oC respectively. Sodium borax/metasilicate had a lower tertiary oil recovery due to higher surfactant retention in Berea sandstone. The ASP flood recovered 81% and 86% of tertiary oil at 100oC and 55oC respectively. The retention was 0.326 mg/g (97%) and 0.267 mg/g (98%). The last section involves treatment and reduction of reservoir cores containing clays and iron minerals. Reservoirs exist as anaerobic and reduced environments and these conditions must be emulated in laboratory experiments. Exposure of reservoir cores to aerobic conditions causes an oxidizing environment in the core leading to higher surfactant retention in the laboratory than the field. Dithionite was used to reduce reservoir cores and produce lower surfactant retention closer to field tests. Proper reduced conditions also improved oil recovery. Dithionite must be buffered with sodium bicarbonate to maintain the reducing power of dithionite. Dithionite oxidation by ferric iron and water causes hydroxyl ion consumption and pH decrease. The EH and iron concentration of the effluents must be monitored to determine the success of the core reduction. Effluent EH matching injected values and iron concentration close to the mineral solubility in brine should be used as benchmark for the success of core reductionItem Development of an alkaline redox flow battery : from fundamentals to benchtop prototype(2015-05) Arroyo Currás, Netzahualcóyotl; Bard, Allen J.; Crooks, Richard M; Mullins, Charles B; Rose, Michael; Yu, GuihuaThis work presents the first alkaline redox flow battery (a-RFB) based on the coordination chemistry of cobalt(III/II) and iron(III/II) with amino-alcohol ligands in concentrated NaOH([subscript aq]). The a-RFB was developed by carrying out systematic structural and electrochemical characterizations of various redox-active coordination compounds to find the most suitable candidates for electrochemical energy storage. In the characterization studies, particular attention was given to the redox couple Fe(III/II)- TEA, where TEA = triethanolamine, because of its importance in the fields of supramolecular chemistry, magnetic memory films, and electrochemical energy storage. The structures of Fe(III)-TEA in the solid state and in alkaline solution are reported for the first time. Moreover, experimental evidence is presented for the existence of an EC reaction in the heterogeneous reduction of Fe(III/II)-TEA in concentrated base. Furthermore, experiments were carried out to study the reactivity of Fe(II)-TEA with O2. This is important because O2 reacts spontaneously with Fe(II)-TEA to produce hydrogen peroxide, decreasing the charging-discharging capacity of the a-RFB. The reduction of oxygen by Fe(II)-TEA in concentrated base was studied by UV-Vis spectroscopy and coulometric titrations. Additionally, a new method for the quick identification of redox couples with slow EC reactions, k[subscript f] < 0.1 s-1, is presented. The new method is based on scanning electrochemical microscopy (SECM) and consists of creating a thin-layer cell between the tip and substrate electrode. During analysis of a redox couple, the tip reports a current transient proportional to the decaying concentration of the product of the E reaction, from which an apparent forward rate constant for the C reaction can be determined. This method was designed for the field of RFB research, where the identification of redox couples with no EC reactions is necessary to ensure that a battery can run for thousands of cycles. Lastly, surface oxidation of polycrystalline Ir ultramicroelectrodes was studied by the surface interrogation mode of SECM (SI-SECM), using Fe(II)-TEA as the titrant. This was done to demonstrate the existence of hydrous oxides of Ir(IV) and Ir(V) prior to the onset of oxygen evolution in concentrated base. Numerical simulations were carried out using commercial software and were used to validate the experimental results reported in this work.Item FBXL5 Is Required for the Maintenance of Cellular Andsystemic Iron Homeostasis(2013-01-16) Ruiz, Julio Cesar Francisco; Bruick, Richard Keith, Ph.D.Iron is an essential element for most living organisms. Due to its chemical properties, iron plays an important role in many vital biochemical processes. Both iron excess and deficiency have detrimental effects in human health. Therefore, iron metabolism must be tight regulated. Maintenance of cellular iron homeostasis requires coordinate posttranscriptional regulation of iron metabolism genes by Iron Regulatory Proteins 1 and 2 (IRP1 and IRP2). IRP2 is targeted for proteasomal degradation in iron replete cells by the E3 ubiquitin ligase complex containing F-box and Leucine-rich Repeat Protein 5 (FBXL5). Depletion of FBXL5 leads to aberrant accumulation of IRP2 and misregulation of IRP2 under high iron conditions, underscoring FBXL5 importance in regulation of iron metabolism. Interestingly, FBXL5 is regulated in an inverse fashion to IRP2 as it is stabilized under iron-replete conditions and preferentially degraded when iron or oxygen becomes limiting. However, FBXL5Õs iron- and oxygen-dependent regulation and its role in the maintenance of systemic iron homeostasis are poorly understood. Biochemical and molecular biology assays revealed that FBXL5 features a hemerythrin-like domain that serves as a direct sensor of cellular iron as well as oxygen availability and subsequently governs FBXL5Õs own stability. Importantly, in vivo deletion of the ubiquitously-expressed murine Fbxl5 gene results in a failure to sense increased cellular iron availability, accompanied by constitutive IRP2 accumulation and misexpression of IRP2 target genes. FBXL5-null mice die during embryogenesis, though viability is restored by simultaneous deletion of the IRP2, but not IRP1, gene. Fbxl5 heterozygous mice behave like their wild type littermates when fed an iron-sufficient diet. However, unlike wild type mice that manifest decreased hematocrit and hemoglobin levels when fed a low-iron diet, Fbxl5 heterozygotes maintain normal hematologic values due to increased iron absorption. IRP2Õs responsiveness to low iron is specifically enhanced in the duodena of the heterozygotes and is accompanied by increased expression of the Divalent Metal Transporter-1. These results confirm FBXL5Õs role in the in vivo maintenance of cellular and systemic iron homeostasis and reveal a privileged role for the intestine in their regulation by virtue of its unique FBXL5 iron sensitivity.Item FBXL5: Sensor and Regulator of Mammalian Iron Homeostasis(2011-08-26T17:34:52Z) Salahudeen, Ameen Abdulla; Bruick, Richard K.While iron is an important cofactor for many proteins, the chemical properties of iron that favor its biological roles can lead to toxic side reactions that damage macromolecules. Cellular iron homeostasis is maintained by the coordinate posttranscriptional regulation of gene products responsible for iron uptake, release, utilization, and storage. This process is mediated by Iron Regulatory Proteins (IRPs) that bind to Iron Responsive Elements (IREs) in the mRNAs of these genes. When iron bioavailability is low IRPs bind IREs within these mRNAs, affecting their subsequent translation or stability. When cellular free iron availability is high, IRPs are preferentially degraded by the proteasome. An SCF E3 ubiquitin ligase complex containing the FBXL5 protein regulates this process as a function of cellular iron and oxygen concentrations. This process occurs through the stability of FBXL5, which accumulates under iron and oxygen replete conditions and is targeted for degradation upon iron depletion. FBXL5 contains an iron- and oxygen -sensing hemerythrin domain that acts as a ligand-binding regulatory switch mediating its stability. As a result, FBXL5 directly senses iron and oxygen levels to serve as a regulator of cellular iron homeostasis.Item Hierarchical three-dimensional Fe-Ni hydroxide nanosheet arrays on carbon fiber electrodes for oxygen evolution reaction(2014-05) O'Donovan-Zavada, Robert Anthony; Manthiram, ArumugamAs demands for alternative sources of energy increase over the coming decades, water electrolysis will play a larger role in meeting our needs. The oxygen evolution reaction (OER) component of water electrolysis suffers from slow kinetics. An efficient, inexpensive, alternative electrocatalyst is needed. We present here high-activity, low onset potential, stable catalyst materials for OER based on a hierarchical network architecture consisting of Fe and Ni coated on carbon fiber paper (CFP). Several compositions of Fe-Ni electrodes were grown on CFP using a hydrothermal method, which produced an interconnected nanosheet network morphology. The materials were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). Electrochemical performance of the catalyst was examined by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). The best electrodes showed favorable activity (23 mA/cm², 60 mA/mg), onset potential (1.42 V vs. RHE), and cyclability.Item Investigating the Roles of Vacuoles in Iron Trafficking in Saccharomyces cerevisiae(2013-11-27) Cockrell, Allison LeighTransition metals play essential roles in biological systems, but Fe can also be toxic to cells. In order to maintain this balance between necessity and toxicity mechanisms are employed for regulating and storing intracellular Fe. In Saccharomyces cerevisiae, vacuoles are responsible for sequestering, storing, and supplying Fe to the cytosol. Many of the proteins and regulatory pathways involved in Fe trafficking and storage in S. cerevisiae have been identified, but the forms of Fe which are involved in these processes have not been fully characterized. In these studies, biophysical and bioanalytical techniques were used to study intracellular Fe distributions in S. cerevisiae cells and organelles. Ultimately, Fe-containing species were biophysically characterized and absolute Fe concentrations in cells and organelles were quantified. The motivation for these studies stemmed from previous studies which revealed that the majority of the whole-cell Fe is a non-heme, high-spin (NHHS) form of Fe^(3+). This Fe is not localized to the mitochondria. The purpose of these studies was to determine if the vacuoles contained this NHHS Fe^(3+). A large-scale isolation procedure was developed to obtain purified vacuoles from S. cerevisiae and to investigate the Fe in these organelles. M?ssbauer and EPR analysis revealed that the primary form of Fe in vacuoles is a mononuclear, NHHS Fe^(3+) species. A second form of Fe was also observed as superparamagnetic ferric phosphate nanoparticles (NP). By investigating model compounds of Fe and polyphosphate we determined that a shift in vacuolar pH induces the conversion between NHHS Fe^(3+) and NP. These results showed that there are at least two forms of Fe in vacuoles, and that the ratio of these two forms is dependent upon the pH of these organelles. Biophysical analyses of whole cells also revealed the presence of low concentrations of a non-heme, high-spin Fe^(2+) species. The goal of these next projects was to determine if this NHHS Fe^(2+) species was localized to the cytosol. Genetic strains lacking or over-expressing the vacuolar Fe import protein Ccc1p were studied by M?ssbauer spectroscopy (?CCC1 and CCC1-up, respectively). ?CCC1 cells showed low vacuolar Fe (NHHS Fe3+ and NP), and increased NHHS Fe^(2+). We hypothesize that this NHHS Fe^(2+) is cytosolic Fe. We also propose that this NHHS Fe^(2+) is involved in the regulating intracellular Fe levels. CCC1-up cells accumulated more Fe than wild-type (WT) cells, and showed elevated levels of vacuolar Fe (NHHS Fe^(3+) and NP). These cells also accumulated high levels of NHHS Fe^(2+). The CCC1-up cells exhibited an adenine deficient phenotype, where the cells developed a red color during growth. With excess adenine the levels of NHHS Fe^(2+) declined, which indicated that this Fe accumulation was related to adenine deficiency. We conclude that adenine deficiency leads to the accumulation of a sequestered (possibly vacuolar) form of NHHS Fe^(2+). Overall, we have identified two separate pools of NHHS Fe^(2+) in ?CCC1 and CCC1-up cells. In ?CCC1 cells the NHHS Fe^(2+) pool is localized to the cytosol and is sensed by the cell. In CCC1-up cells the NHHS Fe^(2+) is sequestered from the Fe regulatory mechanism- possibly in the vacuoles. These data have helped us better understand the roles of vacuoles in Fe trafficking and the dynamics of vacuolar Fe trafficking.Item An Iron Sensing E3 Ubiquitin Ligase Regulates Iron Homeostasis(2011-08-26T17:35:16Z) Thompson, Joel William; Bruick, Richard K.Human iron homeostasis must be tightly regulated to provide sufficient iron for vital cellular processes while preventing the toxic accumulation of free iron. IRP2 plays a critical role in cellular iron homeostasis by coordinating the posttranscriptional regulation of a variety of genes involved in iron metabolism. Posttranslational regulation of IRP2 is essential for its ability to maintain cellular iron homeostasis. The protein is stabilized under iron deficient conditions but polyubiquitinated and degraded by the proteasome when iron is plentiful. However, the E3 ubiquitin ligase that targets IRP2 for degradation is unknown. Moreover, the mechanisms cells use to sense iron levels and correlate changes of this metabolite to differences in IRP2 stability remain poorly understood. To identify the E3 ubiquitin ligase responsible for IRP2 degradation, a high throughput RNAi screen was conducted. The top hit from the screen, FBXL5, interacts with and polyubiquitinates IRP2. Interestingly, FBXL5 is inversely regulated to IRP2. The protein is stabilized under conditions of excess iron and destabilized when iron is limiting. Deletion experiments identified the N terminus of FBXL5 as the region of the protein required for its iron dependent regulation. Bioinformatics predicted the N terminus encodes an iron binding hemerythrin domain. Consistent with this prediction X-ray crystallography demonstrated that the FBXL5 N-terminal domain adopts a hemerythrin fold with a diiron center. Mutation of iron ligating residues in the hemerythrin domain to abolish iron binding leads to constitutive destabilization of FBXL5. Collectively, these findings indicate that the hemerythrin domain acts as a ligand dependent regulatory switch controlling FBXL5Õs expression. Moreover, these data suggest that iron dependent regulation of FBXL5 exerts reciprocal effects on IRP2 stability. Thus, FBXL5 possesses an iron binding hemerythrin domain enabling cells to gauge bioavailable iron levels and control IRP2 expression accordingly, resulting in a tightly regulated circuit in the maintenance of iron homeostasis.Item The role of iron in the Earth's deep interior(2015-05) Liu, Jin, Ph. D.; Lin, Jung-Fu; Grand, Stephen P; Lassiter, John C; Lavier, Luc L; Li, XiaoqinIron represents almost one third of the total mass of our planet, more than any other element existing in the Earth. Knowledge of the physical and chemical properties of iron-bearing phases at high pressure and temperature (P-T) is crucial for understanding the thermal-chemical state and evolution of our planet. In this dissertation, I employed the diamond anvil cell (DAC) and synchrotron radiation facilities (e.g., X-ray diffraction and inelastic X-ray scattering spectroscopies) to study the phase stability, sound velocity and/or elasticity of representative iron-bearing phases of the mantle and core, namely ferromagnesite and iron alloys. In the Earth's mantle, iron-bearing magnesite [(Mg,Fe)CO₃] (hereafter called ferromagnesite) has been commonly proposed to be a potential deep-carbon carrier. Studying the spin transition and phase stability of ferromagnesite at high P-T is necessary for our understanding of the deep-carbon storage and the global carbon cycle of the Earth. Based on X-ray diffraction results, the spin crossover in ferromagnesite broadens and shifts toward higher pressures at elevated temperatures up to 1200 K. The rhombohedral ferromagnesite (phase I) is found to transform into a new orthorhombic high-pressure phase (phase II) up to the lower-mantle conditions of approximately 120 GPa and 2400 K. It is conceivable that the high-spin phase I undergoes spin transition into the low-spin phase I approximately at 1400 km, and below 1900 km the high-pressure phase II becomes stable as a major deep-carbon carrier at the deeper parts of the lower mantle. In the Earth's core, the primary constituent is iron that is alloyed with a certain amount of light elements. Studying the velocity-density profiles and elasticity of iron and iron-rich alloys at high P-T is essential for establishing satisfactory geophysical and geochemical models of the core. Based on the measured velocity-density-pressure relationships of bcc-Fe and Fe-Si alloy at high P-T, a strong velocity reduction is found at elevated temperatures. Furthermore, velocity-density profiles of hcp-Fe₀.₈₅Ni₀.₁₀Si₀.₀₅ alloy have been investigated up to 147 GPa using multiple complementary experimental techniques. The derived ρ-V[subscript P] and ρ-V[subscript D] profiles of hcp-Fe₀.₈₅Ni₀.₁₀Si₀.₀₅ exhibit concave curvatures with increasing pressure. The velocity-density profiles and Poisson's ratio of the hcp-Fe alloyed with 5 (±2) wt. % Si and 5% Ni at 6000 K could match seismic observations of the inner core, indicating that silicon can be a potential major light element that satisfies geophysical constraints of the Earth's core.Item Study of anomalous behavior in solution synthesized iron nanoparticles(2012-05) Monson, Todd Charles; Erskine, James L.; Huber, Dale L.; Demkov, Alexander A.; Yao, Zhen; Tsoi, Maxim; Rabenberg, Llewellyn K.The magnetic and physical properties of oxide-free, ligand passivated, iron nanoparticles were studied using superconducting quantum interference device (SQUID) magnetometry and synchrotron based X-ray radiation. Particles used for this study ranged in diameter between 2 and 10 nm, which made it possible to distinguish between bulk and surface effects in the nanoparticles’ properties. Additionally, the effects of two different weakly interacting ligands (2,4-pentanedione and hexaethylene glycol monododecylether) on the nanoparticles’ behavior were studied. The results of this study were compared to theoretical predictions of magnetic transition metal behavior in both thin films and nanoparticles, as well as experimental results from measurements of transition metal clusters formed in an inert carrier gas and measured with a Stern-Gerlach magnet. Magnetometry revealed that the iron nanoparticles have a magnetocrystalline anisotropy an order of magnitude greater than bulk iron. At the same time, these particles exhibit a saturation mass magnetization up to 209 Am2/kg, which is only slightly lower than bulk iron. The structural properties of these particles were characterized using high energy X-ray diffraction analyzed using the atomic pair distribution function method (PDF). The PDF analysis indicates that the Fe particles have a distorted and expanded form of the bcc lattice, which could, at least in part, explain the magnetocrystalline anisotropy of these nanoparticles. X-ray absorption fine structure (XAFS) was used to study the surface properties of the iron nanoparticles and further characterize their structural properties. XAFS showed that oxidized species of iron exist at the nanoparticles’ surface and can be attributed to iron/ligand interactions. The percentage of oxidized species scales with the surface to volume ratio of the nanoparticles, and therefore appears limited to the nanoparticle surface. The layer of Fe(II) species present at the nanoparticles’ surface accounts for the reduction in saturation mass magnetization values (when compared to bulk iron) observed in these particles. XAFS analysis also provided further confirmation of the nanoparticles’ expanded crystalline lattice.