Redox State Of Iron Research Articles

New insights into the mechanism of iron transport through the bacterial Ftr system present in pathogens.

Iron is an essential nutrient in bacteria. Its ferrous form, mostly present in low oxygen and acidic pH environments, can be imported using the specific Ftr-type transport system, which encompasses the conserved FtrABCD system found in pathogenic bacteria such as Bordetella, Brucella and Burkholderia. The nonpathogenicity and versatile metabolism of Rubrivivax gelatinosus make it an ideal model to study the FtrABCD system. Here, we report a new aspect of its regulation and the role of the periplasmic proteins FtrA and FtrB using in vivo and in vitro approaches. We investigated the metal binding mode and redox state of copper and iron to FtrA by crystallography and biophysical methods. An 'as isolated' FtrA protein from the bacterial periplasm contained a copper ion (Cu+ ) identified by electron paramagnetic resonance (EPR). Copper is coordinated by four conserved side chains (His and Met) in the primary metal site. Structural analysis of R. gelatinosus FtrA and FtrA homologues revealed that copper binding induces a rearrangement of the His95 imidazole ring, releasing thereafter space, as well as both Asp45 and Asp92 side chains, for iron binding in the secondary metal site. EPR highlighted that FtrA can oxidize the bound ferrous ion into the ferric form by reducing the bound Cu2+ into Cu+ , both metal sites being separated by 7 Å. Finally, we showed that FtrB binds iron and not copper. These results provide new insights into the mechanism of ferrous iron utilization by the conserved FtrABCD iron transporter for which we propose a new functional model.

Iron and sulfur reduction caused by different growth seasons inhibits cadmium transfer in the soil-rice system

Effects of iron and sulfur redox states in the soil caused by different growth seasons on Cd uptake by rice remain unclear. In this study, three early rice cultivars and three late rice cultivars were cultivated in a double-cropping system in Cd-contaminated paddy fields. The total Cd accumulation of early rice cultivars was 20.5–51.1 µg plant−1, and the Cd concentration in grains was 0.19–0.73 mg kg−1, significantly lower than those of late rice cultivars by 8–15 times and 3–9 times, respectively. The filling and mature stages were identified as the most crucial stages of Cd uptake by both early and late rice cultivars. The growth season of early rice cultivars was characterized by more abundant rainfall and lower soil Eh than that for late rice cultivars. Therefore, the abundances of Fe-reducing bacteria (FeRB, 36.9–39%) and S-reducing bacteria (SRB, 1.77–2.79%) were higher during the filling and mature stages of early rice. They primarily belonged to the Clostridium, Geobacter, and Desulfuromonadales genera. Stimulation of FeRB and SRB activity promoted Fe(III) and S reduction and increased the content of Fe2+ and S2- in rhizosphere soil. This promoted the binding of Cd to amorphous Fe oxides and sulfides or Fe sulfides, thereby decreasing the available Cd content. Moreover, the Cd in the iron plaque (IP) and Cd transfer from IP to roots were lower in early rice. These findings suggest that maintaining high moisture content in the soil during the filling and mature stages, especially for late rice cultivars, could efficiently reduce Cd uptake by rice planted in contaminated soil.

Structural, redox and isotopic behaviors of iron in geological silicate glasses: A NRIXS study of Lamb-Mössbauer factors and force constants

We report a combined conventional Mössbauer and synchrotron Nuclear Resonant Inelastic X-ray Scattering (NRIXS) study of a series of basalt, andesite, dacite, and rhyolite glasses at temperatures ranging from 5 to 1223 K. These glasses were synthesized under controlled oxygen fugacities and span a wide range of Fe3+/Fetot ratios. As expected from theory, we find that in these glasses, the inverse of the Lamb-Mössbauer factor (the recoil-free fraction) correlates linearly with the fraction of the conventional Mössbauer spectrum that can be ascribed to Fe3+. Extrapolating the linear relationships to pure Fe3+ and Fe2+ endmembers yields a ratio for the Lamb-Mössbauer factors C of Fe3+ and Fe2+ of 1.203 ± 0.017 (1σ), with no clear dependence on the chemical composition of the glass. We show that the materials studied follow the harmonic approximation up to 1223 K for olivine and 773 K for basaltic glass, allowing us to extrapolate C in temperature. The temperature-dependence of C is well approximated by the formula C=1.203±0.033T/300 over the temperature range 50–750 K. This calibration is used to correct previous redox ratio determinations. We investigate how chemical composition and iron redox state influence the mean force constant of iron in glasses, a key driver of Fe isotopic fractionation during magma generation and differentiation. As previously documented by Dauphas et al. (2014), we find clear correlation between bond strength and iron redox ratio that is secondarily modulated by coordination effects in more alkali-rich felsic magmas. New data on simplified glass compositions reveal that network modifiers, notably K and Na, seem to exert an important control on Fe2+ bond strengths. The refined ratio of Mössbauer factors for Fe2+ and Fe3+ is used to improve on calibrations of iron redox state in geological glasses using XANES spectroscopy. We reevaluate the Fe3+/Fetot ratio of MORBs based on previously published XANES and Mössbauer data. 0.087±0.005

Synthesis of ferrian and ferro-saponites: Implications for the structure of (Fe,Mg)-smectites formed under reduced conditions

Abstract Clay minerals are widely distributed on the surface of Earth, Mars, and Ceres in the solar systems. Among numerous clay minerals, smectites can record the history of the environment through the exchange of interlayer cations with those in water or through redox reactions with the atmosphere. Therefore, characterization of chemical compositions and crystal structures of smectites is crucial for revealing the paleoenvironment. For instance, the crystal structure within octahedral sheets of iron-bearing smectites changes to trioctahedral sheets under reduced or dioctahedral sheets under oxidizing conditions. Orbital infrared and X-ray diffraction (XRD) analyses by Mars orbiters/rovers revealed the presence of (Fe,Mg)-smectites on the surface of Mars; however, it has been difficult to characterize the properties of these (Fe,Mg)-smectites, which are rare on the surface of Earth. In this study, we synthesized ferrian (ferric ion-rich) and ferrous (ferrous ion-rich) (Fe,Mg)-saponite and revealed the effect of valence states and iron contents on the crystal structures. These saponites were synthesized using a hydrothermal method under reduced conditions. The crystal structures and valence states of iron were analyzed by XRD, Fourier-transform infrared spectroscopy, transmission electron microscopy, Mössbauer spectroscopy, and X-ray absorption near edge measurements. The synthesized clays were trioctahedral swelling clays and were identified as saponites. The valence state of iron in these synthesized saponites is altered by oxygen and a reducing agent in water; however, the trioctahedral structures are maintained under both oxidizing and reduced conditions, following a reversible reaction. This mechanism can be interpreted by the desorption and adsorption of hydrogen in the hydroxyls of the octahedral sheets of the smectite layers. The maximum basal spacing of the (02l) lattice plane in the octahedral sheets was defined by compiling various smectite data. When the basal spacing of (02l) is larger than the maximum in dioctahedral smectites, smectite can be identified as trioctahedral smectite. The redox state of iron in the octahedral sheet cannot be determined from the basal spacing of (02l). We revealed that the iron content in the trioctahedral sheet has a linear relationship with lattice parameter b. This provides a method to estimate the iron content in saponite from XRD data. The XRD profiles of smectites found at the Yellowknife Bay on Mars can be explained only by trioctahedral smectites, and the iron content in the octahedral sheet is roughly estimated to be 0.5–1.7 in a half-unit cell. These results indicate that the presence of (Fe,Mg)-saponite implies a reduced environment during the formation and that this iron-bearing saponite has both oxidation and reduction capabilities depending on the environment.

Therapeutic Targeting of TET-Dioxygenase Deficiency in Myeloid Malignancies

Background: TET-dioxygenases (TET1, TET2, and TET3) are key epigenetic regulators, which require molecular oxygen, αKG, and Fe 2+ to progressively oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine, leading to the demethylation of mCpGs at promoter and enhancer, a key step for mounting an efficient transcription profile and thereby differentiation. Loss of function TET2 mutation (TET2 MT) is frequently observed in myeloid neoplasms (MN). Despite extensive studies of the biochemical mechanisms underlying distorted myeloid differentiation and neoplastic evolution of TET2 MT HSPCs, targeted therapies are lagging. Here, we report the therapeutic utility of preferential targeting of TET2 mutant and TET-dioxygenase deficient cells either by restoring the lost activity to induce differentiation and death or by inhibiting the residual activity to induce synthetic lethality.Methods: Genetic as well as pharmacologic in vitro and in vivo models of AML were used to validate specific targeting of TET2 mutant cells. Knockout and over-expression in the isogenic background of different cells were used to understand the pro-survival and proliferative mechanism of TET2 loss in myeloid cells. The efficacy of small molecule pharmacophore targeting TET-dioxygenase was evaluated in vitro in cell free and cell culture as well as in vivo in a mouse competitive transplant model to demonstrate the proof of therapeutic concept.Result: Ascorbate, used as an activator of αKG/Fe 2+ dependent dioxygenase including TETs, functions via maintaining the redox state of iron (II) in the dioxygenase catalytic site with a half-maximal effective concentration of 13.8 ± 1.3 µM for enhancing TET2activity as measured by an ELISA for 5hmC. However, the reported intracellular cellular concentrations of ascorbate in human remains in the millimolar range, well above the saturating concentration required for TET-dioxygenases activation. Interestingly, ascorbate failed to activate TET in primary mononuclear cells isolated from myeloid neoplasia patients with TET2 mutations. Consistent with the in-vitro observation, we found that ascorbate treatment in physiologically relevant doses has no significant effect in syngeneic murine models (p=0.1). TET-inactivation is a complex phenomenon that is controlled by context-dependent post-translational modifications including lysine acetylation by acetyltransferases and class I and II deacetylases. Therefore, the anti-leukemic effect of the supraphysiological doses of ascorbate in humans and mice leukemia models may not be dependent on TET activation by ascorbate.On the other hand, a comprehensive analysis of the configurations of TET2 MT in myeloid neoplasia (n= 2617) demonstrated a remarkable exclusivity with 2-hydroxyglutarate (2-HG) producing neomorphic IDH1/2 MT. Ectopic inducible expression of IDH1/2 MT induced synthetic lethality of malignant TET2 MT cells. Therefore, themutual exclusivity of TET2 MT and neomorphic IDH1/2 MT is due to synthetic lethality caused by 2-HG production. In addition, we demonstrate that sequential deletion of TET1 and TET3 leads to growth arrest in cellular models, which has never been observed in any myeloid malignancies. Altogether, these observations suggest that malignant TET2 mutant myeloid cells may be vulnerable to inhibition of residual TET-activity coming from TET1 and TET3.Therefore, we synthesized TET-specific inhibitor on 2HG scaffold using iterative computer-aided design, characterized in cell-free as well as cell culture model systems, and selected TETi76 with no cytotoxic effect in normal bone marrow-derived CD34 + cells. TETi76 selectively inhibits TET-dioxygenase activity with IC50 of 1.5, 9.4, and 8.8 μmol/L for TET1/2/3, and it restricts clonal outgrowth of TET2 MT both in vitro and in vivo in malignant and nonmalignant clonal hematopoiesis of indeterminate potential (CHIP).Conclusion: Taken together, here we demonstrated that anti-cancer activity of ascorbate may not come from its role as a TET activator and that a minimum level of TET-dioxygenase activity is required for cell survival, rendering TET2 -mutant malignant cells selectively vulnerable to inhibitors of TET-function. DisclosuresMaciejewski: Regeneron: Consultancy; Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Alexion: Consultancy.

Decontamination of radioactive cesium and the redox state of Iron in the soil

Decontamination of radioactive cesium and the redox state of Iron in the soil

Iron Oxidation by a Fused Cytochrome-Porin Common to Diverse Iron-Oxidizing Bacteria

ABSTRACTIron (Fe) oxidation is one of Earth’s major biogeochemical processes, key to weathering, soil formation, water quality, and corrosion. However, our understanding of microbial contribution is limited by incomplete knowledge of microbial iron oxidation mechanisms, particularly in neutrophilic iron oxidizers. The genomes of many diverse iron oxidizers encode a homolog to an outer membrane cytochrome (Cyc2) shown to oxidize iron in two acidophiles. Phylogenetic analyses show Cyc2 sequences from neutrophiles cluster together, suggesting a common function, though this function has not been verified in these organisms. Therefore, we investigated the iron oxidase function of heterologously expressed Cyc2 from a neutrophilic iron oxidizer Mariprofundus ferrooxydans PV-1. Cyc2PV-1 is capable of oxidizing iron, and its redox potential is 208 ± 20 mV, consistent with the ability to accept electrons from Fe2+ at neutral pH. These results support the hypothesis that Cyc2 functions as an iron oxidase in neutrophilic iron-oxidizing organisms. The results of sequence analysis and modeling reveal that the entire Cyc2 family shares a unique fused cytochrome-porin structure, with a defining consensus motif in the cytochrome region. On the basis of results from structural analyses, we predict that the monoheme cytochrome Cyc2 specifically oxidizes dissolved Fe2+, in contrast to multiheme iron oxidases, which may oxidize solid Fe(II). With our results, there is now functional validation for diverse representatives of Cyc2 sequences. We present a comprehensive Cyc2 phylogenetic tree and offer a roadmap for identifying cyc2/Cyc2 homologs and interpreting their function. The occurrence of cyc2 in many genomes beyond known iron oxidizers presents the possibility that microbial iron oxidation may be a widespread metabolism.

Disproportionation of H2O2 Mediated by Diiron-Peroxo Complexes as Catalase Mimics.

Heme iron and nonheme dimanganese catalases protect biological systems against oxidative damage caused by hydrogen peroxide. Rubrerythrins are ferritine-like nonheme diiron proteins, which are structurally and mechanistically distinct from the heme-type catalase but similar to a dimanganese KatB enzyme. In order to gain more insight into the mechanism of this curious enzyme reaction, non-heme structural and functional models were carried out by the use of mononuclear [FeII(L1–4)(solvent)3](ClO4)2 (1–4) (L1 = 1,3-bis(2-pyridyl-imino)isoindoline, L2 = 1,3-bis(4′-methyl-2-pyridyl-imino)isoindoline, L3 = 1,3-bis(4′-Chloro-2-pyridyl-imino)isoindoline, L4 = 1,3-bis(5′-chloro-2-pyridyl-imino)isoindoline) complexes as catalysts, where the possible reactive intermediates, diiron-perroxo [FeIII2(μ-O)(μ-1,2-O2)(L1-L4)2(Solv)2]2+ (5–8) complexes are known and well-characterized. All the complexes displayed catalase-like activity, which provided clear evidence for the formation of diiron-peroxo species during the catalytic cycle. We also found that the fine-tuning of iron redox states is a critical issue, both the formation rate and the reactivity of the diiron-peroxo species showed linear correlation with the FeIII/FeII redox potentials. Their stability and reactivity towards H2O2 was also investigated and based on kinetic and mechanistic studies a plausible mechanism, including a rate-determining hydrogen atom transfer between the H2O2 and diiron-peroxo species, was proposed. The present results provide one of the first examples of a nonheme diiron-peroxo complex, which shows a catalase-like reaction.

Magnetic resonance imaging (MRI) of pharmacological ascorbate-induced iron redox state as a biomarker in subjects undergoing radio-chemotherapy

Pharmacological ascorbate (P-AscH-) combined with standard of care (SOC) radiation and temozolomide is being evaluated in a phase 2 clinical trial (NCT02344355) in the treatment of glioblastoma (GBM). Previously published data demonstrated that paramagnetic iron (Fe3+) catalyzes ascorbate's oxidation to form diamagnetic iron (Fe2+). Because paramagnetic Fe3+ may influence relaxation times observed in MR imaging, quantitative MR imaging of P-AscH--induced changes in redox-active Fe was assessed as a biomarker for therapy response.Gel phantoms containing either Fe3+ or Fe2+ were imaged with T2* and quantitative susceptibility mapping (QSM). Fifteen subjects receiving P-AscH- plus SOC underwent T2* and QSM imaging four weeks into treatment. Subjects were scanned: pre-P-AscH- infusion, post-P-AscH- infusion, and post-radiation (3–4 h between scans). Changes in T2* and QSM relaxation times in tumor and normal tissue were calculated and compared to changes in Fe3+ and Fe2+ gel phantoms. A GBM mouse model was used to study the relationship between the imaging findings and the labile iron pool.Phantoms containing Fe3+ demonstrated detectable changes in T2* and QSM relaxation times relative to Fe2+ phantoms. Compared to pre-P-AscH-, GBM T2* and QSM imaging were significantly changed post-P-AscH- infusion consistent with conversion of Fe3+ to Fe2+. No significant changes in T2* or QSM were observed in normal brain tissue. There was moderate concordance between T2* and QSM changes in both progression free survival and overall survival. The GBM mouse model showed similar results with P-AscH- inducing greater changes in tumor labile iron pools compared to the normal tissue. ConclusionsT2* and QSM MR-imaging responses are consistent with P-AscH- reducing Fe3+ to Fe2+, selectively in GBM tumor volumes and represent a potential biomarker of response. This study is the first application using MR imaging in humans to measure P-AscH--induced changes in redox-active iron.

Effects of Geochemical and Environmental Parameters on Abiotic Organic Chemistry Driven by Iron Hydroxide Minerals

AbstractGeological conditions play a significant role in prebiotic/abiotic organic chemistry, especially when reactive minerals are present. Previous studies of the prebiotic synthesis of amino acids and other products in mineral‐containing systems have shown that a diverse array of compounds can be produced, depending on the experimental conditions. However, these previous experiments have not simulated the effects of varying geochemical conditions, in which factors such as pH, iron redox state, or chemical concentrations may vary over time and space in a natural environment. In geochemical systems that contain overlapping gradients, many permutations of individual conditions could exist and affect the outcome of an organic reaction network. We investigated reactions of pyruvate and glyoxylate, two compounds that are central to the emergence of metabolism, in simulated geological gradients of redox, pH, and ammonia concentration. Our results show that the positioning of pyruvate/glyoxylate reactions in this environmental parameter space determines the organic product distribution that results. Therefore, the distribution pattern of amino acids and alpha‐hydroxy acids produced prebiotically in a system reflects the specific reaction conditions, and would be distinct at various locations in an environment depending on local geochemistry. This is significant for origin of life chemistry in which the composition and function of oligomers could be affected by the environmentally driven distribution of monomers available. Also, for astrobiology and planetary science where organic distribution patterns are sometimes considered as a possible biosignature, it is important to consider environmentally driven abiotic organic reactions that might produce similar effects.

Hydroxylamine Complexes of Cytochrome c': Influence of Heme Iron Redox State on Kinetic and Spectroscopic Properties.

Hydroxylamine (NH2OH or HA) is a redox-active nitrogen oxide that occurs as a toxic intermediate in the oxidation of ammonium by nitrifying and methanotrophic bacteria. Within ammonium containing environments, HA is generated by ammonia monooxygenase (nitrifiers) or methane monooxygenase (methanotrophs). Subsequent oxidation of HA is catalyzed by heme proteins, including cytochromes P460 and multiheme hydroxylamine oxidoreductases, the former contributing to emissions of N2O, an ozone-depleting greenhouse gas. A heme-HA complex is also a proposed intermediate in the reduction of nitrite to ammonia by cytochrome c nitrite reductase. Despite the importance of heme-HA complexes within the biogeochemical nitrogen cycle, fundamental aspects of their coordination chemistry remain unknown, including the effect of the Fe redox state on heme-HA affinity, kinetics, and spectroscopy. Using stopped-flow UV-vis and resonance Raman spectroscopy, we investigated HA complexes of the L16G distal pocket variant of Alcaligenes xylosoxidans cytochrome c'-α (L16G AxCP-α), a pentacoordinate c-type cytochrome that we show binds HA in its Fe(III) (Kd ∼ 2.5 mM) and Fe(II) (Kd = 0.0345 mM) states. The ∼70-fold higher HA affinity of the Fe(II) state is due mostly to its lower koff value (0.0994 s-1 vs 11 s-1), whereas kon values for Fe(II) (2880 M-1 s-1) and Fe(III) (4300 M-1 s-1) redox states are relatively similar. A comparison of the HA and imidazole affinities of L16G AxCP-α was also used to predict the influence of Fe redox state on HA binding to other proteins. Although HA complexes of L16G AxCP-α decompose via redox reactions, the lifetime of the Fe(II)HA complex was prolonged in the presence of excess reductant. Spectroscopic parameters determined for the Fe(II)HA complex include the N-O stretching vibration of the NH2OH ligand, ν(N-O) = 906 cm-1. Overall, the kinetic trends and spectroscopic benchmarks from this study provide a foundation for future investigations of heme-HA reaction mechanisms.

Structure of NaFeSiO4, NaFeSi2O6, and NaFeSi3O8 glasses and glass-ceramics

AbstractThe crystallization of iron-containing sodium silicate phases holds particular importance, both in the management of high-level nuclear wastes and in geosciences. Here, we study three as-quenched glasses and their heat-treated chemical analogs, NaFeSiO4, NaFeSi2O6, and NaFeSi3O8 (with nominal stoichiometries from feldspathoid, pyroxene, and feldspar mineral groups, i.e., Si/Fe = 1, 2, and 3, respectively) using various techniques. Phase analyses revealed that as-quenched NaFeSiO4 could not accommodate all Fe in the glass phase (some Fe crystallizes as Fe3O4), whereas as-quenched NaFeSi2O6 and NaFeSi3O8 form amorphous glasses. NaFeSi2O6 glass is the only composition that crystallizes into its respective isochemical crystalline polymorph, i.e., aegirine, upon isothermal heat-treatment. As revealed by Mössbauer spectroscopy, iron is predominantly present as fourfold-coordinated Fe3+ in all glasses, though it is present as sixfold-coordinated Fe3+ in the aegirine crystals (NaFeSi2O6), as expected from crystallography. Thus, Na-Fe silicate can form a crystalline phase in which it is octahedrally coordinated, even though it is mostly tetrahedrally coordinated in the parent glasses. Thermal behavior, magnetic properties, iron redox state (including Fe K-edge X-ray absorption), and vibrational properties (Raman spectra) of the above compositions are discussed.

Anthropogenic Anoxic History of the Tuvalu Atoll Recorded as Annual Black Bands in Coral

Atoll islands are small, low-lying and highly vulnerable to sea level rise (SLR). Because these islands are fully composed of the skeletons from coral reef creatures, the healthy coral ecosystem plays a pivotal role in island resilience against SLR. The environmental deterioration of reefs caused by increases in the human population has been recently reported, but the timing and process are unknown. We investigated the annual black bands in a coral boring core from Fongafale Island, the capital of Tuvalu, which is a symbolic atoll country that is being submerged due to SLR. The iron redox state and microbial gene segments in the coral skeleton might be new environmental indicators that reveal the linkage between anthropogenic activity and coral reef ecosystems. Our findings provide the first demonstration that iron sulfide has formed concentrated black layers since 1991 under the seasonal anoxic conditions inside coral annual bands. Since the 1990s, increasing human activity and domestic waste-induced eutrophication has promoted sludge and/or turf algae proliferation with the subsequent seasonal destruction, resulting in sulfate reduction by anaerobic bacteria. With the recent climate variability, these anthropogenic effects have induced the mass mortality of branching corals, deteriorated the coral reef ecosystem and deprived the resilience of the island against SLR.

Multicopper oxidase of Acinetobacter baumannii: Assessing its role in metal homeostasis, stress management and virulence

A putative multicopper oxidase, encoded as CopA in the proteome of Acinetobacter baumannii 19606, and designated as AbMCO, was expressed heterologously in E. coli (pET-28a) and purified by Ni-NTA affinity chromatography. The purified AbMCO exhibited in vitro oxidase activities upon exogenous addition of ≥1 μM copper ions. Kinetic studies revealed its phenol oxidase activity as it could catalyze the oxidation of substrates viz. 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), guaiacol, pyrogallol and catechol. Additionally, AbMCO displayed siderophore oxidase activity which depicted its role in metal homeostasis and protection from the toxic redox states of copper and iron. Importantly, expression of abMCO increased manifold upon challenge with high concentrations of copper sulphate (CuSO4, 1.5 mM) and sodium chloride (NaCl, 700 mM) which suggested its protective role in stress adaptation and management. Intra-macrophage assay of abMCO-expressing and abMCO-non expressing cells depicted no significant change in the survival rate of A. baumannii inside the macrophages. These findings indicate that A. baumannii encodes a multicopper oxidase, conferring copper tolerance and survival under stress conditions but had no role in virulence of this pathogen.

Interest of a New Method for Free Plasma Heme Related Species Dosages in Sickle Cell Disease and Beta Thalassemia

Introduction: Hemolytic anemia combines 3 components to various extents: extravascular hemolysis, intravascular hemolysis and dyserythropoiesis. Global hemolysis in excess to defense lines induces oxidative and inflammatory syndromes and vascular damages in various organs. Therefore, accurate hemolysis biomarkers are required for a better evaluation of hemolytic disorders, such as sickle cell disease (SCD) or thalassemia syndromes and to evaluate the efficacy of various therapies. Accordingly, we have developed a new spectrophotometric method to measure and calculate several hemolysis biomarkers in plasma or serum including Hemoglobin in various forms (HbO2, HbCO, MetHb), Heme or Hemin bound to albumin or to hemopexin, total bilirubin and total hemopexin. Patients and Method : Blood samples were collected at steady-state for 77 SCD adults (mean age 39.8 ± 10.2 yrs, M/F ratio 0.64) and 23 beta thalassemia patients (mean age 42.7 ± 16 yrs, M/F ratio 0.91); SCD patients (SS or Sb0-Thalassemia) were either treated with Hydroxycarbamide (HU: 27) or not (NT: 50). For comparison, plasma samples from healthy volunteers (HV) were also analyzed. Continuous variables were expressed as means ± SD or medians [interquartile range], depending on their normal or asymmetric distributions. Categorical variables were expressed as numbers (%). Univariate analyses were done using Student's t-test or Mann-Whitney non-parametric test, depending on the distribution. Correlation were analyzed using a spearman test. The dosage methodology is based on the light absorption spectrophotometry of plasma samples using an appropriate mathematical conversion of the signal, reference spectra of the different species and some chemical modifications of the iron redox and ligation states. Results: The levels of plasma Hb were statistically higher in homozygous SCD patients compared to beta-thalassemia patients (p=0.001) and healthy volunteers, with median of 6.3 [3.4-11], 2.6 [1-5.4] and 1.7 [0.5-3] µM respectively (Table 1). Interestingly levels of plasma heme were higher in beta thalassemia patient compared to SCD patients (p=0.0001) and HV, with a mean of 1.05 [0.05-3], 10.5 [3.5-24] and ≤ 0.2 µM level of detection respectively (Table 1). A significant negative correlation was found between heme and hemopexin levels in both diseases (p<0.0001; r=0.85). Among 77 SS or Sb0-Thalassemia patients, 29 were treated by HU without any difference for plasma Hb, plasma Heme and Hemopexin values compared to non-treated patients. Discussion and Conclusions: plasma Hb is a more accurate dosage for intra vascular hemolysis than other biomarkers which are not specific of intra vascular hemolysis and could be biased by other pathological conditions: LDH or ASAT can be increased in hepatic or muscular cytolysis, bilirubin is dependent on the heme oxygenase and glycuronyl transferase activities, and reticulocytes are dependent on erythropoiesis. Our results showed that the intra-vascular hemolysis is more pronounced in SCD compared to beta-thalassemia based on the plasma Hb levels. The elevated plasma heme concentration in beta thalassemia is a new finding that should be investigated in more details. It could reflect the ineffective erythropoiesis or heme export from erythroblasts or macrophages involving hemopexin scavenging. Disclosures Bencheikh: Hemanext: Research Funding. Bartolucci:HEMANEXT: Membership on an entity's Board of Directors or advisory committees; AddMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Kinetic analysis of a globin-coupled diguanylate cyclase, YddV: Effects of heme iron redox state, axial ligands, and heme distal mutations on catalysis

Heme-based oxygen sensors allow bacteria to regulate their activity based on local oxygen levels. YddV, a globin-coupled oxygen sensor with diguanylate cyclase activity from Escherichia coli, regulates cyclic-di-GMP synthesis based on oxygen availability. Stable and active samples of the full-length YddV protein were prepared by attaching it to maltose binding protein (MBP). To better understand the full-length protein's structure, the interactions between its domains were examined by performing a kinetic analysis. The diguanylate cyclase reaction catalyzed by YddV-MBP exhibited Michaelis-Menten kinetics. Its pH optimum was 8.5–9.0, and catalysis required either Mg2+ or Mn2+; other divalent metal ions gave no activity. The most active form of YddV-MBP had a 5-coordinate Fe(III) heme complex; its kinetic parameters were KmGTP 84 ± 21 μM and kcat 1.2 min−1. YddV-MBP with heme Fe(II), heme Fe(II)-O2, and heme Fe(II)-CO complexes had kcat values of 0.3 min−1, 0.95 min−1, and 0.3 min−1, respectively, suggesting that catalysis is regulated by the heme iron's redox state and axial ligand binding. The kcat values for heme Fe(III) complexes of L65G, L65Q, and Y43A YddV-MBP mutants bearing heme distal amino acid replacements were 0.15 min−1, 0.26 min−1 and 0.54 min−1, respectively, implying that heme distal residues play key regulatory roles by mediating signal transduction between the sensing and functional domains. Ultracentrifugation and size exclusion chromatography experiments showed that YddV-MBP is primarily dimeric in solution, with a sedimentation coefficient around 8. The inactive heme-free H93A mutant is primarily octameric, suggesting that catalytically active dimer formation requires heme binding.

Experimental study of amphibole crystallization from the highly magnesian melt of Shiveluch volcano

The paper reports results of an experimental study of amphibole crystallization from the highly magnesian andesite melt of Shiveluch volcano, Kamchatka. The experiments were carried out in IHPV at 300 MPa and 940–980°С in iron-saturated platinum capsules, using rapid quenching and temperature oscillations (in some experiments). The redox state of iron in the system was measured before and after the experiments using Mössbauer spectroscopy. The maximum size of the experimental amphibole crystals (up to 200 μm) was close to those of natural amphibole phenocrysts in the volcanic rocks of Shiveluch volcano. The experimental data show that the content of octahedrally coordinated Al (Al6) in the amphibole considerably varies with small variations in the intensive parameters (P, T, and fO2) and composition of the melt, and the maximum Al6 concentration can be evaluated only by using a reasonably large dataset of amphibole analyses. A modified 13eCNK method is suggested to calculate the values of Al6 and Fe3+/Fe2+ with regard for the Ti concentration and the probable partial transfer of Mg into site B in high-Mg amphibole. Calculations with this modified technique yield lower Fe3+/Fe2+ and higher Al6 values. Our experimental data show that the temperature of amphibole liquidus crystallization decreases from about 990 to 960°C when the oxygen fugacity drops from NNO + 1.5 to NNO + 0.4. In view of this, the transition from amphibole-bearing to anhydrous mineral assemblage in the magmas of Shiveluch volcano might have been caused by variations of the oxygen fugacity but not water. The application of our geobarometer to amphiboles from Shiveluch volcano (extrusions Krasnaya and Karan) yields the highest pressure estimate of above 1 GPa, corresponding to the P-T conditions of the melting of garnet-bearing amphibolite in the lower crust.

Redox states of hemoglobin determine left ventricle pressure recovery and activity of mitochondrial complex IV in hypoxic rat hearts

Cardiovascular effects were reported to occur in humans and in animal models during transfusion with hemoglobin (Hb)-based oxygen therapeutics. The effects of Hb's iron redox states on cardiac parameters during hypoxia/reoxygenation are however poorly defined. We hypothesize that acute exposures to ferric Hb during hypoxia leads to cardiomyocyte injury and an impaired left ventricular response accompanied by cardiac mitochondrial bioenergetic dysfunction. Recovery of left ventricular functions in an isolated rat heart Langendorff perfusion system was observed following perfusion with ferrous but not with ferric Hb. Ferric Hb induced the development of heart lesions, and impairment of the respiratory chain complex activity. Under normoxia, a sharp decline in cardiac parameters was observed following co-perfusion of low (20 μM) and high (100 μM) ascorbic acid (Asc) with ferrous Hb. This trend continued with ferric Hb co-perfusion, but only at the higher concentration of Asc. These observations suggest that perfusion of the hypoxic heart with ferric Hb increases oxidative stress thereby resulting in cardiac dysfunction. Intervention with Asc to reduce ferric Hb may offer a strategy to control Hb toxicity; however, timing of administration, and dosage of Asc may require individual optimization to target specific redox forms of Hb.

Fungus-initiated catalytic reactions at hyphal-mineral interfaces drive iron redox cycling and biomineralization

The ability of fungi to weather a wide range of minerals influences plant nutrition and enhances global biogeochemical cycles of life-essential elements. The fungus-mineral interface plays a key role in weathering, but the specific mechanisms underlying these processes remain poorly understood. Here, we examined fungal-mineral weathering using hematite and Trichoderma guizhouense. We showed that hematite dissolution increased over cultivation time, with the formation of secondary minerals up to ∼3000 µm −2 at the interfaces after 66 h cultivation. Of the hematite associated with hyphae, approximately 15% was converted to the secondary mineral ferrihydrite. Importantly, superoxide radicals were detected at the hyphal tips and along the whole hyphae. During cultivation, a high concentration (∼1000 nM) of hydroxyl radical was also detected. Synchrotron radiation based spectromicroscopies at fungus-mineral interfaces suggest that fungus hyphae alter the local redox state of iron and thus are redox-active. These findings indicate that fungus-initiated catalytic reactions occur at hyphal-mineral interfaces, in view of the fact that superoxide does not diffuse far from the site of formation. Furthermore, these results also suggest that the catalytic reactions may serve as a new strategy for microbial iron uptake. Together, these findings constitute a significant step forward in understanding the ways that fungi make minerals available to biological systems.

Iron transitions during activation of allosteric heme proteins in cell signaling.

Allosteric heme proteins can fulfill a very large number of different functions thanks to the remarkable chemical versatility of heme through the entire living kingdom. Their efficacy resides in the ability of heme to transmit both iron coordination changes and iron redox state changes to the protein structure. Besides the properties of iron, proteins may impose a particular heme geometry leading to distortion, which allows selection or modulation of the electronic properties of heme. This review focusses on the mechanisms of allosteric protein activation triggered by heme coordination changes following diatomic binding to proteins as diverse as the human NO-receptor, cytochromes, NO-transporters and sensors, and a heme-activated potassium channel. It describes at the molecular level the chemical capabilities of heme to achieve very different tasks and emphasizes how the properties of heme are determined by the protein structure. Particularly, this reviews aims at giving an overview of the exquisite adaptability of heme, from bacteria to mammals.