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 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.

Evidence that a respiratory shield in Escherichia coli protects a low-molecular-mass FeII pool from O2-dependent oxidation.

Iron is critical for virtually all organisms, yet major questions remain regarding the systems-level understanding of iron in whole cells. Here, we obtained Mössbauer and EPR spectra of Escherichia coli cells prepared under different nutrient iron concentrations, carbon sources, growth phases, and O2 concentrations to better understand their global iron content. We investigated WT cells and those lacking Fur, FtnA, Bfr, and Dps proteins. The coarse-grain iron content of exponentially growing cells consisted of iron-sulfur clusters, variable amounts of nonheme high-spin FeII species, and an unassigned residual quadrupole doublet. The iron in stationary-phase cells was dominated by magnetically ordered FeIII ions due to oxyhydroxide nanoparticles. Analysis of cytosolic extracts by size-exclusion chromatography detected by an online inductively coupled plasma mass spectrometer revealed a low-molecular-mass (LMM) FeII pool consisting of two iron complexes with masses of ∼500 (major) and ∼1300 (minor) Da. They appeared to be high-spin FeII species with mostly oxygen donor ligands, perhaps a few nitrogen donors, and probably no sulfur donors. Surprisingly, the iron content of E. coli and its reactivity with O2 were remarkably similar to those of mitochondria. In both cases, a "respiratory shield" composed of membrane-bound iron-rich respiratory complexes may protect the LMM FeII pool from reacting with O2 When exponentially growing cells transition to stationary phase, the shield deactivates as metabolic activity declines. Given the universality of oxidative phosphorylation in aerobic biology, the iron content and respiratory shield in other aerobic prokaryotes might be similar to those of E. coli and mitochondria.

Recovery of mrs3Δmrs4Δ Saccharomyces cerevisiae Cells under Iron-Sufficient Conditions and the Role of Fe580.

Mrs3 and Mrs4 are mitochondrial inner membrane proteins that deliver an unidentified cytosolic iron species into the matrix for use in iron-sulfur cluster (ISC) and heme biosynthesis. The Mrs3/4 double-deletion strain (ΔΔ) grew slowly in iron-deficient glycerol/ethanol medium but recovered to wild-type (WT) rates in iron-sufficient medium. ΔΔ cells grown under both iron-deficient and iron-sufficient respiring conditions acquired large amounts of iron relative to WT cells, indicating iron homeostatic dysregulation regardless of nutrient iron status. Biophysical spectroscopy (including Mössbauer, electron paramagnetic resonance, and electronic absorption) and bioanalytical methods (liquid chromatography with online inductively coupled plasma mass spectrometry detection) were used to characterize these phenotypes. Anaerobically isolated mitochondria contained a labile iron pool composed of a nonheme high-spin FeII complex with primarily O and N donor ligands, called Fe580. Fe580 likely serves as feedstock for ISC and heme biosynthesis. Mitochondria from respiring ΔΔ cells grown under iron-deficient conditions were devoid of Fe580, ISCs, and hemes; most iron was present as FeIII nanoparticles. O2 likely penetrates the matrix of slow-growing poorly respiring iron-deficient ΔΔ cells and reacts with Fe580 to form nanoparticles, thereby inhibiting ISC and heme biosynthesis. Mitochondria from iron-sufficient ΔΔ cells contained ISCs, hemes, and Fe580 at concentrations comparable to those of WT mitochondria. The matrix of these mutant cells was probably sufficiently anaerobic to protect Fe580 from degradation by O2. An ∼1100 Da manganese complex, an ∼1200 Da zinc complex, and an ∼5000 Da copper species were also present in ΔΔ and WT mitochondrial flow-through solutions. No lower-mass copper complex was evident.

Mössbauer Spectra of Mouse Hearts Reveal Age-dependent Changes in Mitochondrial and Ferritin Iron Levels.

Cardiac function requires continuous high levels of energy, and so iron, a critical player in mitochondrial respiration, is an important component of the heart. Hearts from 57Fe-enriched mice were evaluated by Mössbauer spectroscopy. Spectra consisted of a sextet and two quadrupole doublets. One doublet was due to residual blood, whereas the other was due to [Fe4S4]2+ clusters and low-spin FeII hemes, most of which were associated with mitochondrial respiration. The sextet was due to ferritin; there was no evidence of hemosiderin, a ferritin decomposition product. Iron from ferritin was nearly absent in young hearts, but increased steadily with age. EPR spectra exhibited signals similar to those of brain, liver, and human cells. No age-dependent EPR trends were apparent. Hearts from HFE-/- mice with hemochromatosis contained slightly more iron overall than controls, including more ferritin and less mitochondrial iron; these differences typify slightly older hearts, perhaps reflecting the burden due to this disease. HFE-/- livers were overloaded with ferritin but had low mitochondrial iron levels. IRP2-/- hearts contained less ferritin than controls but normal levels of mitochondrial iron. Hearts of young mice born to an iron-deficient mother contained normal levels of mitochondrial iron and no ferritin; the heart from the mother contained low ferritin and normal levels of mitochondrial iron. High-spin FeII ions were nearly undetectable in heart samples; these were evident in brains, livers, and human cells. Previous Mössbauer spectra of unenriched diseased human hearts lacked mitochondrial and blood doublets and included hemosiderin features. This suggests degradation of iron-containing species during sample preparation.

Structure and Magnetization Dynamics of Dy-Fe and Dy-Ru Bonded Complexes.

We present an investigation of isostructural complexes that feature unsupported direct bonds between a formally trivalent lanthanide ion (Dy3+ ) and either a first-row (Fe) or a second-row (Ru) transition metal (TM) ion. The sterically rigid, yet not too bulky ligand PyCp22- (PyCp22- =[2,6-(CH2 C5 H3 )2 C5 H3 N]2- ) facilitates the isolation and characterization of PyCp2 Dy-FeCp(CO)2 (1; d(Dy-Fe)=2.884(2) Å) and PyCp2 Dy-RuCp(CO)2 (2; d(Dy-Ru)=2.9508(5) Å). Computational and spectroscopic studies suggest strong TM→Dy bonding interactions. Both complexes exhibit field-induced slow magnetic relaxation with effectively identical energy barriers to magnetization reversal. However, in going from Dy-Fe to Dy-Ru bonding, we observed faster magnetic relaxation at a given temperature and larger direct and Raman coefficients, which could be due to differences in the bonding and/or spin-phonon coupling contributions to magnetic relaxation.

Structures, Interconversions, and Spectroscopy of Iron Carbonyl Clusters with an Interstitial Carbide: Localized Metal Center Reduction by Overall Cluster Oxidation.

The syntheses, interconversions, and spectroscopic properties of a set of iron carbonyl clusters containing an interstitial carbide are reported. This includes the low temperature X-ray structures of the six-iron clusters (Y)2[Fe6(µ6-C)(µ2-CO)4(CO)12] (1a-c; where Y = NMe4, NEt4, PPh4); the five-iron cluster [Fe5(µ5-C)(CO)15] (3); and the novel formulation of the five-iron cluster (NMe4)2[Fe5(µ5-C)(µ2-CO)(CO)13] (4). Also included in this set is the novel charge-neutral cluster, [Fe6(µ6-C)(CO)18] (2), for which we were unable to obtain a crystallographic structure. As synthetic proof for the identity of 2, we performed a closed loop of interconversions within a family of crystallographically defined species (1, 3, and 4): [Fe6]2- → [Fe6]0 → [Fe5]0 → [Fe5]2- → [Fe6]2-. The structural, spectroscopic, and electronic properties of this "missing link" cluster 2 were investigated by IR, Raman, XPS, and Mössbauer spectroscopies-as well as by DFT calculations. A single νCO feature (1965 cm-1) in the IR spectrum of 2, as well as a prominent Raman feature (νsymm = 1550 cm-1), are consistent with the presence of terminal carbonyls and a {(µ6-C)Fe6} arrangement of iron centers around the central carbide. The XPS of 2 exhibits a higher energy Fe 2p3/2 feature (707.4 eV) as compared to that of 1 (705.5 eV), consistent with the two-electron oxidation induced by treatment of 1 with two equivalents of [Fc](PF6) under CO atmosphere (for the two added CO ligands). DFT calculations indicate two axial and four equatorial Fe sites in 1, all of which have the same or similar oxidation states, for example, two Fe(0) and four Fe(+0.5). These assignments are supported by Mössbauer spectra for 1, which exhibit two closely spaced quadrupole doublets with δ = 0.076 and 0.064 mm s-1. The high-field Mössbauer spectrum of 2 (4.2 K) exhibits three prominent quadrupole doublets with δ = -0.18, -0.11, and +0.41 mm s-1. This indicates three pairs of chemically equivalent Fe sites. The first two pairs arise from irons of a similar oxidation state, while the last pair arises from irons in a different oxidation state, indicating a mixed-valent cluster. Variable field Mössbauer spectra for 2 were simulated assuming these two groups and a diamagnetic ground state. Taken together, the Mössbauer results and DFT calculations for 2 indicate two axial Fe(II) sites and four equatorial sites of lower valence, probably Fe(0). In the DFT optimized pentagonal bipyramidal structure for 2, the Fe(II)-Ccarbide distances are compressed (∼1.84 Å), while the Fe(0)-Ccarbide distances are elongated (∼2.05 Å). Analysis of the formulations for 1 (closo-square bipyramid) and 2 (nido-pentagonal bipyramid) is considered in the context of the textbook electron-counting rules of 14n+2 and 14n+4 for closo and nido clusters, respectively. This redox-dependent intracluster disproportionation of Fe oxidation states is concluded to arise from changes in bonding to the central carbide. A similar phenomenon may be promoted by the central carbide of the FeMoco cluster of nitrogenase, which may in turn stimulate N2 reduction.

Mitochondrial Iron-Sulfur Cluster Activity and Cytosolic Iron Regulate Iron Traffic in Saccharomyces cerevisiae.

An ordinary differential equation-based mathematical model was developed to describe trafficking and regulation of iron in growing fermenting budding yeast. Accordingly, environmental iron enters the cytosol and moves into mitochondria and vacuoles. Dilution caused by increasing cell volume is included. Four sites are regulated, including those in which iron is imported into the cytosol, mitochondria, and vacuoles, and the site at which vacuolar Fe(II) is oxidized to Fe(III). The objective of this study was to determine whether cytosolic iron (Fecyt) and/or a putative sulfur-based product of iron-sulfur cluster (ISC) activity was/were being sensed in regulation. The model assumes that the matrix of healthy mitochondria is anaerobic, and that in ISC mutants, O2 diffuses into the matrix where it reacts with nonheme high spin Fe(II) ions, oxidizing them to nanoparticles and generating reactive oxygen species. This reactivity causes a further decline in ISC/heme biosynthesis, which ultimately gives rise to the diseased state. The ordinary differential equations that define this model were numerically integrated, and concentrations of each component were plotted versus the concentration of iron in the growth medium and versus the rate of ISC/heme biosynthesis. Model parameters were optimized by fitting simulations to literature data. The model variant that assumed that both Fecyt and ISC biosynthesis activity were sensed in regulation mimicked observed behavior best. Such "dual sensing" probably arises in real cells because regulation involves assembly of an ISC on a cytosolic protein using Fecyt and a sulfur species generated in mitochondria during ISC biosynthesis and exported into the cytosol.

A mathematical model of iron import and trafficking in wild-type and Mrs3/4ΔΔ yeast cells.

BACKGROUND: Iron plays crucial roles in the metabolism of eukaryotic cells. Much iron is trafficked into mitochondria where it is used for iron-sulfur cluster assembly and heme biosynthesis. A yeast strain in which Mrs3/4, the high-affinity iron importers on the mitochondrial inner membrane, are deleted exhibits a slow-growth phenotype when grown under iron-deficient conditions. However, these cells grow at WT rates under iron-sufficient conditions. The object of this study was to develop a mathematical model that could explain this recovery on the molecular level. RESULTS: A multi-tiered strategy was used to solve an ordinary-differential-equations-based mathematical model of iron import, trafficking, and regulation in growing Saccharomyces cerevisiae cells. At the simplest level of modeling, all iron in the cell was presumed to be a single species and the cell was considered to be a single homogeneous volume. Optimized parameters associated with the rate of iron import and the rate of dilution due to cell growth were determined. At the next level of complexity, the cell was divided into three regions, including cytosol, mitochondria, and vacuoles, each of which was presumed to contain a single form of iron. Optimized parameters associated with import into these regions were determined. At the final level of complexity, nine components were assumed within the same three cellular regions. Parameters obtained at simpler levels of complexity were used to help solve the more complex versions of the model; this was advantageous because the data used for solving the simpler model variants were more reliable and complete relative to those required for the more complex variants. The optimized full-complexity model simulated the observed phenotype of WT and Mrs3/4ΔΔ cells with acceptable fidelity, and the model exhibited some predictive power. CONCLUSIONS: The developed model highlights the importance of an FeII mitochondrial pool and the necessary exclusion of O2 in the mitochondrial matrix for eukaryotic iron-sulfur cluster metabolism. Similar multi-tiered strategies could be used for any micronutrient in which concentrations and metabolic forms have been determined in different organelles within a growing eukaryotic cell.

Towards understanding of lanthanide-transition metal bonding: investigations of the first Ce-Fe bonded complex.

The syntheses, structural, and magnetic characterization of three new organometallic Ce complexes stabilized by PyCp22- (PyCp22- = [2,6-(CH2C5H3)2C5H3N]2-) are reported. Complex 1 provides the first example of a crystallographically characterized unsupported Ce-Fe bond in a molecular compound. Results from IR spectroscopy and computational analyses suggest weaker Fe → Ce electron-donation than in a previously reported Dy-Fe bonded species.

Transition metal redox switches for reversible "on/off" and "slow/fast" single-molecule magnet behaviour in dysprosium and erbium bis-diamidoferrocene complexes.

Single-molecule magnets (SMMs) are considered viable candidates for next-generation data storage and quantum computing. Systems featuring switchability of their magnetization dynamics are particularly interesting with respect to accessing more complex logic gates and device architectures. Here we show that transition metal based redox events can be exploited to enable reversible switchability of slow magnetic relaxation of magnetically anisotropic lanthanide ions. Specifically, we report anionic homoleptic bis-diamidoferrocene complexes of Dy3+ (oblate) and Er3+ (prolate) which can be reversibly oxidized by one electron to yield their respective charge neutral redox partners (Dy: [1]- , 1; Er: [2]- , 2). Importantly, compounds 1 and 2 are thermally stable which allowed for detailed studies of their magnetization dynamics. We show that the Dy3+[1]- /1 system can function as an "on"/"off" or a "slow"/"fast" redox switchable SMM system in the absence or presence of applied dc fields, respectively. The Er3+ based [2]- /2 system features "on"/"off" switchability of SMM properties in the presence of applied fields. Results from electrochemical investigations, UV-vis-NIR spectroscopy, and 57Fe Mössbauer spectroscopy indicate the presence of significant electronic communication between the mixed-valent Fe ions in 1 and 2 in both solution and solid state. This comparative evaluation of redox-switchable magnetization dynamics in low coordinate lanthanide complexes may be used as a potential blueprint toward the development of future switchable magnetic materials.

Ferric ions accumulate in the walls of metabolically inactivating Saccharomyces cerevisiae cells and are reductively mobilized during reactivation.

Mössbauer and EPR spectra of fermenting yeast cells before and after cell wall (CW) digestion revealed that CWs accumulated iron as cells transitioned from exponential to post-exponential growth. Most CW iron was mononuclear nonheme high-spin (NHHS) Fe(III), some was diamagnetic and some was superparamagnetic. A significant portion of CW Fe was removable by EDTA. Simulations using an ordinary-differential-equations-based model suggested that cells accumulate Fe as they become metabolically inactive. When dormant Fe-loaded cells were metabolically reactivated in Fe-deficient bathophenanthroline disulfonate (BPS)-treated medium, they grew using Fe that had been mobilized from their CWs AND using trace amounts of Fe in the Fe-deficient medium. When grown in Fe-deficient medium, Fe-starved cells contained the lowest cellular Fe concentrations reported for a eukaryotic cell. During metabolic reactivation of Fe-loaded dormant cells, Fe(III) ions in the CWs of these cells were mobilized by reduction to Fe(II), followed by release from the CW and reimport into the cell. BPS short-circuited this process by chelating mobilized and released Fe(II) ions before reimport; the resulting Fe(II)(BPS)3 complex adsorbed on the cell surface. NHHS Fe(II) ions appeared transiently during mobilization, suggesting that these ions were intermediates in this process. In the presence of chelators and at high pH, metabolically inactive cells leached CW Fe; this phenomenon probably differs from metabolic mobilization. The iron regulon, as reported by Fet3p levels, was not expressed during post-exponential conditions; Fet3p was maximally expressed in exponentially growing cells. Decreased expression of the iron regulon and metabolic decline combine to promote CW Fe accumulation.