Mössbauer and EPR detection of iron trafficking kinetics and possibly labile iron pools in whole Saccharomyces cerevisiae cells.

The kinetics of iron trafficking in whole respiring Saccharomyces cerevisiae cells were investigated using Mössbauer and EPR spectroscopies. The Mössbauer-active isotope 57Fe was added to cells growing under iron-limited conditions; cells were analyzed at different times post iron addition. Spectroscopic changes suggested that the added 57Fe initially entered the labile iron pool, and then distributed to vacuoles and mitochondria. The first spectroscopic feature observed, ∼ 3 min after adding 57Fe plus a 5 to 15 min processing dead time, was a quadrupole doublet typical of nonheme high-spin FeII. This feature likely arose from labile FeII pools in the cell. At later times (15-150 min), magnetic features due to S = 5/2 FeIII developed; these likely arose from FeIII in vacuoles. Corresponding EPR spectra were dominated by a g = 4.3 signal from the S = 5/2 FeIII ions that increased in intensity over time. Developing at a similar rate was a quadrupole doublet typical of S = 0 [Fe4S4]2+ clusters and low-spin FeII hemes; such centers are mainly in mitochondria, cytosol, and nuclei. Development of these features was simulated using a published mathematical model, and simulations compared qualitatively well with observations. In the five sets of experiments presented, all spectroscopic features developed within the doubling time of the cells, implying that the detected iron trafficking species are physiologically relevant. These spectroscopy-based experiments allow the endogenous labile iron pool within growing cells to be detected without damaging or altering the pool, as definitely occurs using chelator-probe detection and possibly occurs using chromatographic separations.

Yeast Mitochondria Import Aqueous FeII and, When Activated for Iron-Sulfur Cluster Assembly, Export or Release Low-Molecular-Mass Iron and Also Export Iron That Incorporates into Cytosolic Proteins.

Iron-sulfur cluster (ISC) assembly occurs in both mitochondria and cytosol. Mitochondria are thought to export a low-molecular-mass (LMM) iron and/or sulfur species which is used as a substrate for cytosolic ISC assembly. This species, called X-S or (Fe-S)int, has not been directly detected. Here, an assay was developed in which mitochondria were isolated from 57Fe-enriched cells and incubated in various buffers. Thereafter, mitochondria were separated from the supernatant, and both fractions were investigated by ICP-MS-detected size exclusion liquid chromatography. Aqueous 54FeII in the buffer declined upon exposure to intact 57Fe-enriched mitochondria. Some 54Fe was probably surface-absorbed but some was incorporated into mitochondrial iron-containing proteins when mitochondria were activated for ISC biosynthesis. When activated, mitochondria exported/released two LMM nonproteinaceous iron complexes. One species, which comigrated with an Fe-ATP complex, developed faster than the other Fe species, which also comigrated with phosphorus. Both were enriched in 54Fe and 57Fe, suggesting that the added 54Fe entered a pre-existing pool of 57Fe, which was also the source of the exported species. When 54Fe-loaded 57Fe-enriched mitochondria were mixed with isolated cytosol and activated, multiple cytosolic proteins became enriched with Fe. No incorporation was observed when 54Fe was added directly to the cytosol in the absence of mitochondria. This suggests that a different Fe source in mitochondria, the one enriched mainly with 57Fe, was used to export a species that was ultimately incorporated into cytosolic proteins. Iron from buffer was imported into mitochondria fastest, followed by mitochondrial ISC assembly, LMM iron export, and cytosolic ISC assembly.

Labile Iron Pool of Isolated Escherichia coli Cytosol Likely Includes Fe-ATP and Fe-Citrate but not Fe-Glutathione or Aqueous Fe.

The existence of labile iron pools (LFePs) in biological systems has been recognized for decades, but their chemical composition remains uncertain. Here, the LFeP in cytosol from Escherichia coli was investigated. Mössbauer spectra of whole vs lysed cells indicated significant degradation of iron-sulfur clusters (ISCs), even using an unusually gentle lysis procedure; this demonstrated the fragility of ISCs. Moreover, the released iron contributed to the non-heme high-spin Fe(II) species in the cell, which likely included the LFeP. Cytosol batches isolated from cells grown with different levels of iron supplementation were passed through a 3 kDa cutoff membrane, and resulting flow-through-solutions (FTSs) were subjected to SEC-ICP-MS. Mössbauer spectroscopy was used to evaluate the oxidation states of standards. FTSs exhibited iron-detected peaks likely due to different forms of Fe-citrate and Fe-nucleotide triphosphate complexes. Fe-Glutathione (GSH) complexes were not detected using physiological concentrations of GSH mixed with either Fe(II) or Fe(III); Fe(II)-GSH was concluded not to be a significant component of the LFeP in E. coli under physiological conditions. Aqueous iron was also not present in significant concentrations in isolated cytosol and is unlikely a major component of the pool. Fe appeared to bind ATP more tightly than citrate, but ATP also hydrolyzed on the timescale of tens of hours. Isolated cytosol contained excess ligands that coordinated the added Fe(II) and Fe(III). The LFeP in healthy metabolically active cells is undoubtedly dominated by the Fe(II) state, but the LFeP is redox-active such that a fraction might be present as stable and soluble Fe(III) complexes especially under oxidatively stressed cellular conditions.

Low-temperature Mössbauer spectroscopy of organs from 57Fe-enriched HFE(-/-) hemochromatosis mice: an iron-dependent threshold for generating hemosiderin.

Hereditary hemochromatosis is an iron-overload disease most often arising from a mutation in the Homeostatic Fe regulator (HFE) gene. HFE organs become overloaded with iron which causes damage. Iron-overload is commonly detected by NMR imaging, but the spectroscopic technique is insensitive to diamagnetic iron. Here, we used Mössbauer spectroscopy to examine the iron content of liver, spleen, kidney, heart, and brain of 57Fe-enriched HFE(-/-) mice of ages 3-52 wk. Overall, the iron contents of all investigated HFE organs were similar to the same healthy organ but from an older mouse. Livers and spleens were majorly overloaded, followed by kidneys. Excess iron was generally present as ferritin. Iron-sulfur clusters and low-spin FeII hemes (combined into the central quadrupole doublet) and nonheme high-spin FeII species were also observed. Spectra of young and middle-aged HFE kidneys were dominated by the central quadrupole doublet and were largely devoid of ferritin. Collecting and comparing spectra at 5 and 60 K allowed the presence of hemosiderin, a decomposition product of ferritin, to be quantified, and it also allowed the diamagnetic central doublet to be distinguished from ferritin. Hemosiderin was observed in spleens and livers from HFE mice, and in spleens from controls, but only when iron concentrations exceeded 2-3 mM. Even in those cases, hemosiderin represented only 10-20% of the iron in the sample. NMR imaging can identify iron-overload under non-invasive room-temperature conditions, but Mössbauer spectroscopy of 57Fe-enriched mice can detect all forms of iron and perhaps allow the process of iron-overloading to be probed in greater detail.

Might nontransferrin-bound iron in blood plasma and sera be a nonproteinaceous high-molecular-mass FeIII aggregate?

The HFE (Homeostatic Fe regulator) gene is commonly mutated in hereditary hemochromatosis. Blood of (HFE)(-/-) mice and of humans with hemochromatosis contains toxic nontransferrin-bound iron (NTBI) which accumulates in organs. However, the chemical composition of NTBI is uncertain. To investigate, HFE(-/-) mice were fed iron-deficient diets supplemented with increasing amounts of iron, with the expectation that NTBI levels would increase. Blood plasma was filtered to obtain retentate and flow-through solution fractions. Liquid chromatography detected by inductively coupled plasma mass spectrometry of flow-through solutions exhibited low-molecular-mass iron peaks that did not increase intensity with increasing dietary iron. Retentates yielded peaks due to transferrin (TFN) and ferritin, but much iron in these samples adsorbed onto the column. Retentates treated with the chelator deferoxamine (DFO) yielded a peak that comigrated with the Fe-DFO complex and originated from iron that adhered to the column in the absence of DFO. Additionally, plasma from younger and older 57Fe-enriched HFE mice were separately pooled and concentrated by ultrafiltration. After removing contributions from contaminating blood and TFN, Mössbauer spectra were dominated by features due to magnetically interacting FeIII aggregates, with greater intensity in the spectrum from the older mice. Similar features were generated by adding 57FeIII to "pseudo plasma". Aggregation was unaffected by albumin or citrate at physiological concentrations, but DFO or high citrate concentrations converted aggregated FeIII into high-spin FeIII complexes. FeIII aggregates were retained by the cutoff membrane and adhered to the column, similar to the behavior of NTBI. A model is proposed in which FeII entering blood is oxidized, and if apo-TFN is unavailable, the resulting FeIII ions coalesce into FeIII aggregates, a.k.a. NTBI.

Mössbauer-based molecular-level decomposition of the Saccharomyces cerevisiae ironome, and preliminary characterization of isolated nuclei.

One hundred proteins in Saccharomyces cerevisiae are known to contain iron. These proteins are found mainly in mitochondria, cytosol, nuclei, endoplasmic reticula, and vacuoles. Cells also contain non-proteinaceous low-molecular-mass labile iron pools (LFePs). How each molecular iron species interacts on the cellular or systems' level is underdeveloped as doing so would require considering the entire iron content of the cell-the ironome. In this paper, Mössbauer (MB) spectroscopy was used to probe the ironome of yeast. MB spectra of whole cells and isolated organelles were predicted by summing the spectral contribution of each iron-containing species in the cell. Simulations required input from published proteomics and microscopy data, as well as from previous spectroscopic and redox characterization of individual iron-containing proteins. Composite simulations were compared to experimentally determined spectra. Simulated MB spectra of non-proteinaceous iron pools in the cell were assumed to account for major differences between simulated and experimental spectra of whole cells and isolated mitochondria and vacuoles. Nuclei were predicted to contain ∼30 µM iron, mostly in the form of [Fe4S4] clusters. This was experimentally confirmed by isolating nuclei from 57Fe-enriched cells and obtaining the first MB spectra of the organelle. This study provides the first semi-quantitative estimate of all concentrations of iron-containing proteins and non-proteinaceous species in yeast, as well as a novel approach to spectroscopically characterizing LFePs.

Cooperative redox and spin activity from three redox congeners of sulfur-bridged iron nitrosyl and nickel dithiolene complexes.

The synthesis of sulfur-bridged Fe-Ni heterobimetallics was inspired by Nature's strategies to "trick" abundant first row transition metals into enabling 2-electron processes: redox-active ligands (including pendant iron-sulfur clusters) and proximal metals. Our design to have redox-active ligands on each metal, NO on iron and dithiolene on nickel, resulted in the observation of unexpectedly intricate physical properties. The metallodithiolate, (NO)Fe(N2S2), reacts with a labile ligand derivative of [NiII(S2C2Ph2)]0, NiDT, yielding the expected S-bridged neutral adduct, FeNi, containing a doublet {Fe(NO)}7. Good reversibility of two redox events of FeNi led to isolation of reduced and oxidized congeners. Characterization by various spectroscopies and single-crystal X-ray diffraction concluded that reduction of the FeNi parent yielded [FeNi]-, a rare example of a high-spin {Fe(NO)}8, described as linear FeII(NO-). Mössbauer data is diagnostic for the redox change at the {Fe(NO)}7/8 site. Oxidation of FeNi generated the 2[FeNi]+⇌[Fe2Ni2]2+ equilibrium in solution; crystallization yields only the [Fe2Ni2]2+ dimer, isolated as PF6- and BArF- salts. The monomer is a spin-coupled diradical between {Fe(NO)}7 and NiDT+, while dimerization couples the two NiDT+ via a Ni2S2 rhomb. Magnetic susceptibility studies on the dimer found a singlet ground state with a thermally accessible triplet excited state responsible for the magnetism at 300 K (χMT = 0.67 emu·K·mol-1, µeff = 2.31 µB), and detectable by parallel-mode EPR spectroscopy at 20 to 50 K. A theoretical model built on an H4 chain explains this unexpected low energy triplet state arising from a combination of anti- and ferromagnetic coupling of a four-radical molecular conglomerate.

Yeast cells depleted of the frataxin homolog Yfh1 redistribute cellular iron: Studies using Mössbauer spectroscopy and mathematical modeling.

The neurodegenerative disease Friedreich's ataxia arises from a deficiency of frataxin, a protein that promotes iron-sulfur cluster (ISC) assembly in mitochondria. Here, primarily using Mössbauer spectroscopy, we investigated the iron content of a yeast strain in which expression of yeast frataxin homolog 1 (Yfh1), oxygenation conditions, iron concentrations, and metabolic modes were varied. We found that aerobic fermenting Yfh1-depleted cells grew slowly and accumulated FeIII nanoparticles, unlike WT cells. Under hypoxic conditions, the same mutant cells grew at rates similar to WT cells, had similar iron content, and were dominated by FeII rather than FeIII nanoparticles. Furthermore, mitochondria from mutant hypoxic cells contained approximately the same levels of ISCs as WT cells, confirming that Yfh1 is not required for ISC assembly. These cells also did not accumulate excessive iron, indicating that iron accumulation into yfh1-deficient mitochondria is stimulated by O2. In addition, in aerobic WT cells, we found that vacuoles stored FeIII, whereas under hypoxic fermenting conditions, vacuolar iron was reduced to FeII. Under respiring conditions, vacuoles of Yfh1-deficient cells contained FeIII, and nanoparticles accumulated only under aerobic conditions. Taken together, these results informed a mathematical model of iron trafficking and regulation in cells that could semiquantitatively simulate the Yfh1-deficiency phenotype. Simulations suggested partially independent regulation in which cellular iron import is regulated by ISC activity in mitochondria, mitochondrial iron import is regulated by a mitochondrial FeII pool, and vacuolar iron import is regulated by cytosolic FeII and mitochondrial ISC activity.

Influence of Metal Identity on Light-Induced Switchable Adsorption in Azobenzene-Based Metal-Organic Frameworks.

Energy-efficient capture and release of small gas molecules, particularly carbon dioxide (CO2) and methane (CH4), are of significant interest in academia and industry. Porous materials such as metal-organic frameworks (MOFs) have been extensively studied, as their ultrahigh porosities and tunability enable significant amounts of gas to be adsorbed while also allowing specific applications to be targeted. However, because of the microporous nature of MOFs, the gas adsorption performance is dominated by high uptake capacity at low pressures, limiting their application. Hence, methods involving stimuli-responsive materials, particularly light-induced switchable adsorption (LISA), offer a unique alternative to thermal methods. Here, we report the mechanism of a well-known LISA system, the azobenzene-based material PCN-250, for CO2 and CH4 adsorption. There is a noticeable difference in the LISA effect dependent on the metal cluster involved, with the most significant being PCN-250-Al, where the adsorption can change by 83.1% CH4 and 56.1% CO2 at 298 K and 1 bar and inducing volumetric storage changes of 36.2 and 33.9 cm3/cm3 at 298 K between 5 and 85 bar (CH4) and 2 and 9 bar (CO2), respectively. Using UV light in both single-crystal X-ray diffraction and gas adsorption testing, we show that upon photoirradiation, the framework undergoes a "localized heating" phenomenon comparable to an increase of 130 K for PCN-250-Fe and improves the working capacity. This process functions because of the constrained nature of the ligand, preventing the typical trans-to-cis isomerization observed in free azobenzene. In addition, we observed that the degree of localized heating is highly dependent on the metal cluster involved, with the series of isostructural PCN-250 systems showing variable performance based upon the degree of interaction between the ligand and the metal center.

Cis-Divacant Octahedral Fe(II) in a Dimensionally Reduced Family of 2-(Pyridin-2-yl)pyrrolide Complexes.

Four-coordinate transition-metal complexes can adopt a diverse array of coordination geometries, with square planar and tetrahedral coordination being the most prevalent. Previously, we reported the synthesis of a trinuclear Fe(II) complex, Fe3TPM2, supported by a 3-fold-symmetric 2-pyridylpyrrolide ligand [i.e., tris(5-(pyridin-2-yl)-1H-pyrrol-2-yl)methane] that featured a rare cis-divacant octahedral (CDO) geometry at each Fe(II) center. Here, a series of truncated 2-pyridylpyrrolide ligands are described that support mono- and binuclear Fe(II) complexes that also exhibit CDO geometries. Metalation of the tetradentate ligand bis[5-(pyridin-2-yl)-1H-pyrrol-2-yl]methane (H2BPM) in tetrahydrofuran (THF) results in the binuclear complex Fe2(BPM)2(THF)2 in which both Fe(II) ions are octahedrally coordinated. The coordinated THF solvent ligands are labile: THF dissociation leads to Fe2(BPM)2, which features five-coordinate Fe(II) ions. The Fe-Fe distance in these binuclear complexes can be elongated by ligand methylation. Metalation of bis[5-(6-methylpyridin-2-yl)-1H-pyrrol-2-yl]methane (H2BPMMe) in THF leads to the formation of four-coordinate, CDO Fe(II) centers in Fe(BPMMe)2. Further ligand truncation affords bidentate ligands 2-(1H-pyrrol-2-yl)pyridine (PyrPyrrH) and 2-methyl-6-(1H-pyrrol-2-yl)pyridine (PyrMePyrrH). Metalation of these ligands in THF affords six-coordinate complexes Fe(PyrPyrr)2(THF)2 and Fe(PyrMePyrr)2(THF)2. Dissociation of labile solvent ligands provides access to four-coordinate Fe(II) complexes. Ligand disproportionation at Fe(PyrPyrr)2 results in the formation of Fe(PyrPyrr)3 and Fe(0). Ligand methylation suppresses this disproportionation and enables isolation of Fe(PyrMePyrr)2, which is rigorously CDO. Complete ligand truncation, by separating the 2-pyridylpyrrolide ligands into the constituent monodentate pyridyl and pyrrolide donors, affords Fe(Pyr)2(Pyrr)2 in which Fe(II) is tetrahedrally coordinated. Computational analysis indicates that the potential energy surface that dictates the coordination geometry in this family of four-coordinate complexes is fairly flat in the vicinity of CDO coordination. These synthetic studies provide the structural basis to explore the implications of CDO geometry on Fe-catalyzed reactions.

Thermal decarboxylation for the generation of hierarchical porosity in isostructural metal-organic frameworks containing open metal sites.

The effect of metal-cluster redox identity on the thermal decarboxylation of a series of isostructural metal-organic frameworks (MOFs) with tetracarboxylate-based ligands and trinuclear µ3-oxo clusters was investigated. The PCN-250 series of MOFs can consist of various metal combinations (Fe3, Fe/Ni, Fe/Mn, Fe/Co, Fe/Zn, Al3, In3, and Sc3). The Fe-based system can undergo a thermally induced reductive decarboxylation, producing a mixed valence cluster with decarboxylated ligand fragments subsequently eliminated to form uniform mesopores. We have extended the analysis to alternative monometallic and bimetallic PCN-250 systems to observe the cluster's effect on the decarboxylation process. Our results suggest that the propensity to undergo decarboxylation is directly related to the cluster redox accessibility, with poorly reducible metals, such as Al, In, and Sc, unable to thermally reduce at the readily accessible temperatures of the Fe-containing system. In contrast, the mixed-metal variants are all reducible. We report improvements in gas adsorption behavior, significantly the uniform increase in the heat of adsorption going from the microporous to hierarchically induced decarboxylated samples. This, along with Fe oxidation state changes from 57Fe Mössbauer spectroscopy, suggests that reduction occurs at the clusters and is essential for mesopore formation. These results provide insight into the thermal behavior of redox-active MOFs and suggest a potential future avenue for generating mesoporosity using controlled cluster redox chemistry.

The Pyrococcus furiosus ironome is dominated by [Fe4S4]2+ clusters or thioferrate-like iron depending on the availability of elemental sulfur.

Pyrococcus furiosus is a hyperthermophilic anaerobic archaeon whose metabolism depends on whether elemental sulfur is (+S0) or is not (-S0) included in growth medium. Under +S0 conditions, expression of respiratory hydrogenase declines while respiratory membrane-bound sulfane reductase and the putative iron-storage protein IssA increase. Our objective was to investigate the iron content of WT and ΔIssA cells under these growth conditions using Mössbauer spectroscopy. WT-S0 cells contained ∼1 mM Fe, with ∼85% present as two spectroscopically distinct forms of S = 0 [Fe4S4]2+ clusters; the remainder was mainly high-spin FeII. WT+S0 cells contained 5 to 9 mM Fe, with 75 to 90% present as magnetically ordered thioferrate-like (TFL) iron nanoparticles. TFL iron was similar to chemically defined thioferrates; both consisted of FeIII ions coordinated by an S4 environment, and both exhibited strong coupling between particles causing high applied fields to have little spectral effect. At high temperatures with magnetic hyperfine interactions abolished, TFL iron exhibited two doublets overlapping those of [Fe4S4]2+ clusters in -S0 cells. This coincidence arose because of similar coordination environments of TFL iron and cluster iron. The TFL structure was more heterogeneous in the presence of IssA. Presented data suggest that IssA may coordinate insoluble iron sulfides as TFL iron, formed as a byproduct of anaerobic sulfur respiration under high iron conditions, which thereby reduces its toxicity to the cell. This was the first Mössbauer characterization of the ironome of an archaeon, and it illustrates differences relative to the iron content of better-studied bacteria such as Escherichia coli.

Mössbauer and LC-ICP-MS investigation of iron trafficking between vacuoles and mitochondria in vma2ΔSaccharomyces cerevisiae.

Vacuoles are acidic organelles that store FeIII polyphosphate, participate in iron homeostasis, and have been proposed to deliver iron to mitochondria for iron-sulfur cluster (ISC) and heme biosynthesis. Vma2Δ cells have dysfunctional V-ATPases, rendering their vacuoles nonacidic. These cells have mitochondria that are iron-dysregulated, suggesting disruption of a putative vacuole-to-mitochondria iron trafficking pathway. To investigate this potential pathway, we examined the iron content of a vma2Δ mutant derived from W303 cells using Mössbauer and EPR spectroscopies and liquid chromatography interfaced with inductively-coupled-plasma mass spectrometry. Relative to WT cells, vma2Δ cells contained WT concentrations of iron but nonheme FeII dominated the iron content of fermenting and respiring vma2Δ cells, indicating that the vacuolar FeIII ions present in WT cells had been reduced. However, vma2Δ cells synthesized WT levels of ISCs/hemes and had normal aconitase activity. The iron content of vma2Δ mitochondria was similar to WT, all suggesting that iron delivery to mitochondria was not disrupted. Chromatograms of cytosolic flow-through solutions exhibited iron species with apparent masses of 600 and 800 Da for WT and vma2∆, respectively. Mutant cells contained high copper concentrations and high concentrations of a species assigned to metallothionein, indicating copper dysregulation. vma2Δ cells from previously studied strain BY4741 exhibited iron-associated properties more consistent with prior studies, suggesting subtle strain differences. Vacuoles with functional V-ATPases appear unnecessary in W303 cells for iron to enter mitochondria and be used in ISC/heme biosynthesis; thus, there appears to be no direct or dedicated vacuole-to-mitochondria iron trafficking pathway. The vma2Δ phenotype may arise from alterations in trafficking of iron directly from cytosol to mitochondria.

The thermally induced decarboxylation mechanism of a mixed-oxidation state carboxylate-based iron metal-organic framework.

Investigations into a thermally generated decarboxylation mechanism for metal site activation and the generation of mesopores in a carboxylate iron-based MOF, PCN-250, have been conducted. PCN-250 exhibits an interesting oxidation state change during thermal treatment under inert atmospheres or vacuum conditions, transitioning from an Fe(iii)3 cluster to a Fe(ii)Fe(iii)2 cluster. To probe this redox event and discern a mechanism of activation, a combination of thermogravimetric analysis, gas sorption, scanning electron microscopy, 57Fe Mössbauer spectroscopy, gas chromatography-mass spectrometry, and X-ray diffraction studies were conducted. The results suggest that the iron-site activation occurs due to ligand decarboxylation above 200 °C. This is also consistent with the generation of a missing cluster mesoporous defect in the framework. The resulting mesoporous PCN-250 maintains high thermal stability, preserving crystallinity after multiple consecutive high-temperature regeneration cycles. Additionally, the thermally reduced PCN-250 shows improvements in the total uptake capacity of methane and CO2.