Ferric State Research Articles

Non-oxidative effects of the flavonoid-rich fraction of Lasianthera africana leaves on human haemoglobin

ABSTRACT Several plant phytochemicals have been reported to possess therapeutic properties. However, most of them are capable of causing oxidation of the human haemoglobin into the ferric state making them hazardous to cells and tissues. This study was aimed at investigating the non-oxidative effect of the flavonoid-rich fraction of Lasianthera africana leaves (LAFRF) on human haemoglobin. Isolation and purification of LAFRF was carried out by solvent extraction, partitioning and ion-exchange chromatography. Six test groups were setup as follows: A reaction tube containing 800 µl of oxyhaemoglobin (OxyHb) served as the normal control, while the test groups were made up of different concentrations of LAFRF (5, 10, 20, 40 and 80 mg/ml) incubated with 100 µl of OxyHb. The non-oxidative effects of the LAFRF on the human haemoglobin was assessed by UV-visible spectroscopy. The results indicated that LAFRF possess non-oxidative effects on human haemoglobin, as it did not cause any disappearance of the absorbance maxima at 576 nm and 541 nm at all concentrations investigated. Furthermore, a concentration-dependent increase in the absorbance of the treated OxyHb was recorded when compared to the untreated OxyHb, thus signifying its potential as a safe pharmacological agent for in vivo investigations.

A heme-binding protein produced by Haemophilus haemolyticus inhibits non-typeable Haemophilus influenzae.

Commensal bacteria serve as an important line of defense against colonisation by opportunisitic pathogens, but the underlying molecular mechanisms remain poorly explored. Here, we show that strains of a commensal bacterium, Haemophilus haemolyticus, make hemophilin, a heme-binding protein that inhibits growth of the opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) in culture. We purified the NTHi-inhibitory protein from H. haemolyticus and identified the hemophilin gene using proteomics and a gene knockout. An x-ray crystal structure of recombinant hemophilin shows that the protein does not belong to any of the known heme-binding protein folds, suggesting that it evolved independently. Biochemical characterisation shows that heme can be captured in the ferrous or ferric state, and with a variety of small heme-ligands bound, suggesting that hemophilin could function under a range of physiological conditions. Hemophilin knockout bacteria show a limited capacity to utilise free heme for growth. Our data suggest that hemophilin is a hemophore and that inhibition of NTHi occurs by heme starvation, raising the possibility that competition from hemophilin-producing H. haemolyticus could antagonise NTHi colonisation in the respiratory tract.

Characterization of the Cofactors Involved in Non-Enzymatic Metmyoglobin Reduction In Vitro

ObjectivesConsumers’ meat purchasing decisions are strongly influenced by color. Myoglobin is the primary meat pigment that contributes to meat color. Myoglobin consists of an iron-containing heme ring and amino acids in the form of globin chains. Both the state of the heme iron and the type of ligand affects meat color. The consumer-preferred bright cherry-red color oxymyoglobin is formed when the iron is in the ferrous state and oxygen bind to the heme. The oxidation of oxymyoglobin or deoxymyoglobin results in the formation of the brown color, ferric metmyoglobin. Predominant metmyoglobin accumulation negatively impacts consumer purchasing choices. Although muscle type and pre- and post-harvest factors can influence meat discoloration, meat has an inherent ability to reduce metmyoglobin through enzymatic pathways, mitochondria-mediated pathways, and non-enzymatic mechanisms. In the enzymatic pathway, an electron from NADH is transferred to metmyoglobin by an enzyme and an electron carrier; while in mitochondria-mediated pathway, an electron from the electron-transport chain is transferred via cytochromes. Previous research speculated the role of non-enzymatic pathway in meat color; however, limited studies have characterized the cofactors present in a meat system. The objectives of this study were to characterize cofactors in non-enzymatic metmyoglobin reduction and determine the effect of storage temperature and postmortem muscle pH in-vitro.Materials and MethodsPurified equine metmyoglobin was reduced in the presence of combinations of electron carriers and donors. Methylene blue and cytochrome c were evaluated as the electron carriers, and NADH and ascorbate were considered as the electron donors. The cofactors were held at 4 and 25°C to determine temperature effects on the reduction of metmyoglobin, and the same cofactor combinations were evaluated at pH of 5.2, 5.6, 6.0, and 6.4 to reflect postmortem muscle pH. Spectrophotometry was utilized to monitor the rates of metmyoglobin reduction. The experiments were replicated five times, and the data were analyzed using the Mixed Procedure of SAS.ResultsThe results indicated that methylene blue was a significantly more effective electron carrier than cytochrome c with both electron donors, ascorbate and NADH. EDTA had no impact on the non-enzymatic metmyoglobin reducing the ability of methylene blue (P = 0.91). Temperature and pH had cofactor specific effects on the non-enzymatic reduction of metmyoglobin. Lower temperature resulted in an increased non-enzymatic metmyoglobin reduction for methylene blue regardless of electron donor (ascorbate, P = 0.03, NADH, P = 0.04). As pH increased, the non-enzymatic metmyoglobin reducing activity reduced significantly in the presence of NADH and methylene blue.ConclusionIn conclusion, the characteristics of the cofactors at specific temperatures and pH impacted the non-enzymatic reduction of metmyoglobin. Further, current in vitro research indicated that non-enzymatic metmyoglobin reduction is possible at lower temperature and meat pH.

Iron Deficiency Anemia Caused By Malabsorption from Proton Pump Inhibition

Introduction: Iron deficiency anemia (IDA) not responding to oral iron replacement usually requires a hematologic evaluation. 48 patients taking a proton pump inhibitor (PPI) and not responding to oral iron replacement were found to have an elevated serum gastrin (SG). No patient had gastrointestinal bleeding, gastric resection, bariatric surgery, or menorrhagia. Other causes for iron malabsorption such as celiac disease or helicobacter infection were not present. 94 percent responded to intravenous iron (IV iron). Methods: All patients previously had undergone diagnostic gastrointestinal evaluations. Testing for celiac disease and helicobacter infection was negative. Gastric biopsies did not demonstrate atrophy. Most referrals were from gastroenterologists. Results: 94% responded to IV iron with a rise in their hemoglobin of >/= 2 grams per cent. 83 percent (40/48) were women. Iron dextran (ID) at a fixed dose of 825 mgm was given to 85% of the patients. Twelve of these 41 patients were given a second infusion of ID as the first dose did not produce a satisfactory response. Ferric carboxymaltose and ferumoxytol were each given once at the fixed recommended dose, and second infusions was not necessary. Four patients received iron sucrose at a weight based dose, and a second series of infusions were not necessary. One patient responded to ferumoxytol after a suboptimal response to iron dextran. An elevated SG was defined as >100 pg/mL. The average SG was 370.25 pg/mL (114 to 2101 pg/mL). Hemoglobin rose an average of 3.35 gram% (9.56 to 12.91 gm%). The change in hemoglobin was minus 0.4 to plus 7.0 gm% with a baseline hemoglobin ranging between 6.6 to 14.3 gm% and rising between 9.3 to 16.2 gram%. Ferritin rose an average of 14.8 to 158 ng/mL with baseline ferritin ranging between 3 to 73 ng/mL and rising between 22 to 659 ng/mL The average MCV rose from 75.89 to 84.93 fL with baseline MCV ranging between 61 to 93 fL rising between 69 to 96 fL The average iron saturation rose from 7.49 to 22.89% with baseline saturation ranging between 2 to 34% and rising between 10 to 39%. Discussion: Dietary iron consists of both heme and non heme iron. Heme iron is derived from the hemoglobin and myoglobin in animal food sources such as meat, seafood, and poultry. Heme iron is in the ferrous (II) oxidation state, is easily absorbable, and contributes 10% or somewhat more of total absorbed iron. Non heme iron is in the ferric (III) form and is derived from plants and iron fortified food. Normally 1-2 mgm of iron is absorbed daily. Heme iron is well absorbed after its release by pancreatic enzymes. Non heme iron is less well absorbed and requires acid secretion from gastric parietal cells for the denaturing of ingested proteins and subsequent proteolysis. PPI causes decreased hydrogen ion (H+) production by inhibiting the hydrogen/potassium pump within gastric parietal cells. The elevated SG derives from G cell hyperplasia as a response to the lowered H+ activity caused by PPI. The decreased H+ activity inhibits the release of ferric iron from non animal sources. Iron absorption occurs in the proximal duodenum through the action of a brush border ferrireductase such as duodenal cytochrome B which reduces ferric iron to ferrous iron. With less ferric iron available for reduction less ferrous iron is absorbed, and iron deficiency results. Intravenous iron fully corrected the IDA in 94% of treated patients. Two of the 3 non responders were obese and only received one infusion of ID. Perhaps a second infusion might have been beneficial. However no relation between weight, response, and ID dosing could be detected. Both patients had normal hemoglobins before the iron infusion but were very symptomatic from their iron deficiency. Both patients experienced a rise in their hemoglobin (1.9 gram% and 1.4 gm%). The third non responder actually had a fall in the hemoglobin from 10.5 to 10.1 gm%. No clear explanation was apparent. No clear explanation for the female predominance was apparent. Conclusion: In 2009 119 million prescriptions for PPI were written in the USA. The gastrointestinal literature suggests that anemia from PPI is uncommon. Very likely IDA due to IM from PPI is much more common than recognized and should be considered for any iron deficient patients without evidence for other causes for IDA. Intravenous iron is highly effective. Disclosures No relevant conflicts of interest to declare.

Heme ligation and redox chemistry in two bacterial thiosulfate dehydrogenase (TsdA) enzymes

Thiosulfate dehydrogenases (TsdAs) are bidirectional bacterial di-heme enzymes that catalyze the interconversion of tetrathionate and thiosulfate at measurable rates in both directions. In contrast to our knowledge of TsdA activities, information on the redox properties in the absence of substrates is rather scant. To address this deficit, we combined magnetic CD (MCD) spectroscopy and protein film electrochemistry (PFE) in a study to resolve heme ligation and redox chemistry in two representative TsdAs. We examined the TsdAs from Campylobacter jejuni, a microaerobic human pathogen, and from the purple sulfur bacterium Allochromatium vinosum. In these organisms, the enzyme functions as a tetrathionate reductase and a thiosulfate oxidase, respectively. The active site Heme 1 in both enzymes has His/Cys ligation in the ferric and ferrous states and the midpoint potentials (Em) of the corresponding redox transformations are similar, −185 mV versus standard hydrogen electrode (SHE). However, fundamental differences are observed in the properties of the second, electron transferring, Heme 2. In C. jejuni, TsdA Heme 2 has His/Met ligation and an Em of +172 mV. In A. vinosum TsdA, Heme 2 reduction triggers a switch from His/Lys ligation (Em, −129 mV) to His/Met (Em, +266 mV), but the rates of interconversion are such that His/Lys ligation would be retained during turnover. In summary, our findings have unambiguously assigned Em values to defined axial ligand sets in TsdAs, specified the rates of Heme 2 ligand exchange in the A. vinosum enzyme, and provided information relevant to describing their catalytic mechanism(s).

A sustainable process for recovery of potash fertilizer from glauconite through simultaneous production of pigment grade red oxide

A sustainable process is proposed and developed for the recovery of potash fertilizer from glauconitic clay with simultaneous production of pigment grade iron oxide. Potassium is a part of stable structure (dioctahedral T-O-T) in glauconite and locked in alumino-silicate matrix. In order to break this matrix and maximize potassium dissolution, the glauconite sample containing 5.6% K2O and 31.9% Fe2O3 was subjected to reduction with coke followed by oxidation cooling (redox roasting). The process simultaneously releases potash and convert iron in the matrix to ferric state. The redox roasted sample was leached in hydrochloric acid and the leach liquor containing 6.1 g/L of potassium and 30.2 g/L of iron was subjected to solvent extraction using mixed solvent system (20% Aliquat 336 and 15% TBP) to separate iron prior to recovery of potash. Based on McCabe–Thiele plot, two stage counter current extraction at O:A ratio of 1:0.75 and three stage stripping at O:A ratio of 0.4:1 are sufficient to remove >99% of iron from the leach solution. The strip solution was hydrothermally treated to produce red iron oxide suitable for pigment application and iron free raffinate was treated to produce fertilizer grade potassium chloride.

Glassy magnetic cronstedtite signatures in Mukundpura CM2 chondrite based on magnetic and Mössbauer studies

AbstractWe have studied the Mukundpura CM2 meteorite for magnetic properties as a function of temperature and magnetic field, as well as its Mössbauer spectrum, at room and low temperatures (up to 5 K). We find that the high temperature paramagnetic phase is followed by two magnetic transitions: a weak transition near 125 K and a strong transition at 8 K. The weak (125 K) magnetic phase can be attributed to complex Fe2+–Fe3+ constituents present in the meteorite. The absence of the characteristic sextet corresponding to magnetite in Mossbauer spectrum indicates that this magnetic phase is not magnetite, which, if present, must be in insignificant amount. The 8 K magnetic ordering is superimposed with weak ferromagnetic ordering, showing spin‐glass transition. The Mössbauer spectrum taken at 5 K substantiates the observed spin‐glassy nature, as very large hyperfine field ~32 T is recorded, causing localized subordering leading to spin‐glass behavior. The Mössbauer spectra also confirm that iron is mainly present in serpentine‐group minerals, both in ferrous and ferric states. The complete serpentinization of basic silicates indicates aggressive hydrous alteration. These results show that the observed spin‐glass signature is a characteristic feature of the cronstedtite phase in CM meteorites. This feature is unique to carbonaceous CM chondrites and could be used for nondestructive, quick, and independent classification of this rare class of meteorites. Furthermore, the absence of olivine and the presence of cronstedtite in Mossbauer spectra show that the degree of aqueous alteration observed is the most severe in Mukundpura CM2 meteorite, as compared to many other CM2 meteorites. The degree of aqueous alteration in CM2 carbonaceous chondrites increases in the sequence: Paris, Murchison, Murray, Mighei, Nogoya, Cold Bokkeveld, and Mukundpura.

Redox‐State‐Mediated Regulation of Cytochrome c Release in Apoptosis Revealed by Surface‐Enhanced Raman Scattering on Nickel Substrates

AbstractThe interaction of cytochrome c (Cyt c) with cardiolipin (CL) is believed to play an important role in the initial events of apoptosis. Herein, we investigate the structural changes of CL‐bound Fe2+Cyt c and the correlation with Cyt c release through surface‐enhanced Raman spectroscopy (SERS) on nickel substrates. The SERS results together with molecular dynamics simulation reveal that Fe2+Cyt c undergoes autoxidation and a relatively larger conformational alteration after binding with CL, inducing higher peroxidase activity of Cyt c and higher permeability of the CL membrane compared with those induced by the Fe3+Cyt c. The proapoptotic activity and SERS effect of the Ni nanostructures allow the in situ study of the redox‐state‐dependent Cyt c release from isolated mitochondria, which reveals for the first time that the ferrous state of Cyt c most likely plays a more important role in triggering apoptosis.

Redox-State-Mediated Regulation of Cytochrome c Release in Apoptosis Revealed by Surface-Enhanced Raman Scattering on Nickel Substrates.

The interaction of cytochrome c (Cyt c) with cardiolipin (CL) is believed to play an important role in the initial events of apoptosis. Herein, we investigate the structural changes of CL-bound Fe2+ Cyt c and the correlation with Cyt c release through surface-enhanced Raman spectroscopy (SERS) on nickel substrates. The SERS results together with molecular dynamics simulation reveal that Fe2+ Cyt c undergoes autoxidation and a relatively larger conformational alteration after binding with CL, inducing higher peroxidase activity of Cyt c and higher permeability of the CL membrane compared with those induced by the Fe3+ Cyt c. The proapoptotic activity and SERS effect of the Ni nanostructures allow the in situ study of the redox-state-dependent Cyt c release from isolated mitochondria, which reveals for the first time that the ferrous state of Cyt c most likely plays a more important role in triggering apoptosis.

Potent Activation of Indoleamine 2,3-Dioxygenase by Polysulfides.

Indoleamine 2,3-dioxygenase (IDO1) is a heme enzyme that catalyzes the oxygenation of the indole ring of tryptophan to afford N-formylkynurenine. This activity significantly suppresses the immune response, mediating inflammation and autoimmune reactions. These consequential effects are regulated through redox changes in the heme cofactor of IDO1, which autoxidizes to the inactive ferric state during turnover. This change in redox status increases the lability of the heme cofactor leading to further suppression of activity. The cell can thus regulate IDO1 activity through the supply of heme and reducing agents. We show here that polysulfides bind to inactive ferric IDO1 and reduce it to the oxygen-binding ferrous state, thus activating IDO1 to maximal turnover even at low, physiologically significant concentrations. The on-rate for hydrogen disulfide binding to ferric IDO1 was found to be >106 M-1 s-1 at pH 7 using stopped-flow spectrometry. Fe K-edge XANES and EPR spectroscopy indicated initial formation of a low-spin ferric sulfur-bound species followed by reduction to the ferrous state. The μM affinity of polysulfides for IDO1 implicates these polysulfides as important signaling factors in immune regulation through the kynurenine pathway. Tryptophan significantly enhanced the relatively lower-affinity binding of hydrogen sulfide to IDO1, inspiring the use of the small molecule 3-mercaptoindole (3MI), which selectively binds to and activates ferric IDO1. 3MI sustains turnover by catalytically transferring reducing equivalents from glutathione to IDO1, representing a novel strategy of upregulating innate immunosuppression for treatment of autoimmune disorders. Reactive sulfur species are thus likely unrecognized immune-mediators with potential as therapeutic agents through these interactions with IDO1.

Strongly Coupled Redox-Linked Conformational Switching at the Active Site of the Non-Heme Iron-Dependent Dioxygenase, TauD.

2-Oxoglutarate (2OG)-dependent dioxygenases catalyze C-H activation while performing a wide range of chemical transformations. In contrast to their heme analogues, non-heme iron centers afford greater structural flexibility with important implications for their diverse catalytic mechanisms. We characterize an in situ structural model of the putative transient ferric intermediate of 2OG:taurine dioxygenase (TauD) by using a combination of spectroelectrochemical and semiempirical computational methods, demonstrating that the Fe(III/II) transition involves a substantial, fully reversible, redox-linked conformational change at the active site. This rearrangement alters the apparent redox potential of the active site between -127 mV for reduction of the ferric state and +171 mV for oxidation of the ferrous state of the 2OG-Fe-TauD complex. Structural perturbations exhibit limited sensitivity to mediator concentrations and potential pulse duration. Similar changes were observed in the Fe-TauD and taurine-2OG-Fe-TauD complexes, thus attributing the reorganization to the protein moiety rather than the cosubstrates. Redox-difference infrared spectra indicate a reorganization of the protein backbone in addition to the involvement of carboxylate and histidine ligands. Quantitative modeling of the transient redox response using two alternative reaction schemes across a variety of experimental conditions strongly supports the proposal for intrinsic protein reorganization as the origin of the experimental observations.

A unique ferrous iron binding mode is associated with large conformational changes for the transport protein FpvC of Pseudomonas aeruginosa.

Pseudomonas aeruginosa secretes pyoverdine, a major siderophore to get access to iron, an essential nutrient. Pyoverdine scavenges ferric iron in the bacterial environment with the resulting complex internalized by bacteria. Releasing of iron from pyoverdine in the periplasm involves an iron reduction by an inner membrane reductase and two solute-binding proteins (SBPs) FpvC and FpvF in association with their ABC transporter. FpvC and FpvF belong to two different subgroups of SBPs within the structural cluster A: FpvC and FpvF were proposed to be a metal-binding protein and a ferrisiderophore-binding protein respectively. Here, we report the redox state and the binding mode of iron to FpvC. We first solved the crystal structure of FpvC bound to a fortuitous Ni2+ by single anomalous dispersion method. Using a different protein purification strategy, we determined the structure of FpvC with manganese and iron, which binds to FpvC in a ferrous state as demonstrated by electron paramagnetic resonance. FpvC is the first example of a hexahistidine metal site among SBPs in which the Fe2+ redox state is stabilized under aerobic conditions. Using biophysics methods, we showed that FpvC reversibly bind to a broad range of divalent ions. The structure of a mutant mimicking the apo FpvC reveals a protein in an open state with large conformational changes when compared with the metal-bound FpvC. These results highlight that the canonical metal site in FpvC is distinct from those yet described in SBPs and they provide new insights into the mechanism of PVD-Fe dissociation in P.aeruginosa.

Identification of Intermediates in Peroxidase Catalytic Cycle of a DNAzyme Possessing Heme

Abstract Heme in the ferric state (heme(Fe3+)) binds to G-quadruplex DNAs to form stable complexes that exhibit enhanced peroxidase activities. The complexes are considered DNAzymes possessing heme as a prosthetic group (heme-DNAzymes), and have been extensively investigated as promising catalysts for a variety of applications. On ESR and stopped-flow measurements, an iron(IV)oxo porphyrin π-cation radical known as Compound I was detected in reaction mixtures of heme-DNAzymes and hydrogen peroxide. This finding not only resolved the long-standing issue of the mechanism underlying the enhancement of the peroxidase activity of heme(Fe3+) in the scaffold of a G-quadruplex DNA, but also provided new insights as to the design of novel heme-DNAzymes.

A spectroelectrochemical investigation of the heme‐based sensor DevS from Mycobacterium tuberculosis: a redox versus oxygen sensor

Tuberculosis is one of the oldest known infectious diseases, responsible for millions of deaths annually around the world. The ability of Mycobacteriumtuberculosis (Mtb) to enter into a dormant state has been considered integral to the success of this bacterium as a human pathogen. One of the key systems involved in regulating the entrance into dormancy is the differentially expressed in virulent strain sensor protein (DevS) [(dormancy survival sensor protein (DosS)]. However, the physiological signal for DevS has remained unclear since it was first shown to be a heme-based sensor with conflicting reports on whether it is a redox or an oxygen sensor. To address this question and provide a better understanding of the electronic properties of this protein, we present here, for the first time, a series of spectroelectrochemistry measurements of the full-length holo DevS in anaerobic conditions as well as bound to CO, NO, imidazole (Imz), cyanide, and O2 . An interesting feature of this protein is its ability to bind Imz even in the ferrous state, implying small-molecule analogues could be designed as potential regulators. Nonetheless, a midpoint potential (Em ) value of +10mV [vs normal hydrogen electrode (NHE)] for DevS as measured under anaerobic conditions is much higher than the expected cytosolic potential for Mtb or even within stimulated macrophages (~-270 mV vs NHE), indicating this sensor works in a reduced ferrous state. These data, along with the high oxygen affinity and very slow auto-oxidation rate of DevS, provides evidence that it is not a redox sensor. Overall, this study validates the biological function of DevS as an oxygen sensor directly involved in the dormancy/latency of Mtb.

Effects of an Amendment on Cadmium Transportation in the Rhizosphere Soil-Rice System

The remediation of cadmium (Cd) contaminated paddy soils has become an important issue in the field of remediation of agricultural soils contaminated by heavy metals. The iron (Fe) redox cycle (referring to the fluctuation of iron between the ferrous (Ⅱ) and ferric (Ⅲ) oxidation states) exhibits a unique role in the transportation of Cd in the soil-rice system. The exploration of practical remediation strategies for Cd from the perspective of the Fe redox cycle is expected to obtain some state-of-the-art technologies and products to reduce Cd accumulation in rice grains. In this study, an amendment was selected and a field experiment was carried out to investigate the effects of this amendment on Cd transportation from the rhizosphere soil to the Fe plaque, and further to different rice tissues at four different growth stages, and to highlight some possible mechanisms by which the Fe redox cycle controls Cd availability in rice paddy fields. The results showed that the amendment induced the formation of Fe sulfides, which co-precipitated with Cd, reducing the NH4Ac-extractable Cd content in rhizosphere soils at the tillering, jointing, and filling stages; the oxidation of Fe sulfides increased the NH4Ac-extractable Cd content in the rhizosphere soil at the maturing stage; the formation of Fe sulfides in rhizosphere soils impeded the migration of Fe(Ⅱ) from the rhizosphere soil to the root surface, decreasing the content of DCB-extractable Fe and Cd in Fe plaques at the tillering and filling stages; the amendment inhibited Cd transportation from the roots to other tissues, increasing the proportion of Cd in the roots at the jointing, filling, and maturing stages, but decreasing the proportion in the straws at the jointing, filling, and maturing stages, and in the rice grain at the maturing stage. These findings provide a theoretical basis for the exploration and application of the amendment, and have significance in the field of remediation of Cd-contaminated paddy soils.

Hereditary methemoglobinemia caused by Hb M-Hyde Park (Hb M-Akita) (HBB:c.277C > T; p.His93Tyr).

Healthy human blood contains only atrace amount of methemoglobin (HbM), less than 1%. In HbM iron is present in the oxidized ferric state (Fe3+) not in the reduced ferrous form (Fe2+) and this reduces the ability of hemoglobin to bind oxygen. The described rare hemoglobin variant HbM‑Hyde Park (also known as HbM-Akita) results from the substitution of amino acid tyrosine by histidine at position 93of the beta-globin chain of hemoglobin. The rare Hb variant Hb M‑Hyde Park (Hb M‑Akita) is mainly inherited autosomal dominant and causes methemoglobinemia. Due to the low frequency of inherited HbMvariants, the diagnosis is challenging. Here, we here report on afamily with Hb M‑Hyde Park (Hb M‑Akita) whose members demonstrated Hb M > 10%, but were, asymptomatic except for chronic cyanosis. Due to human mobility and migration other hemogobin variants, such as beta-thalassemia minor have spread to Austria . Agenetic combination of two different hemoglobin variants may result in severe anemia. Genetic counseling for patients with hemoglobin variants, including Hb M‑Hyde Park (Hb M‑Akita) and beta-thalassemia minor, is essential.

Experimental and theoretical studies of the porphyrin ligand effect on the electronic structure and reactivity of oxoiron(iv) porphyrin π-cation-radical complexes.

Oxoiron(IV) porphyrin π-cation-radical complexes (Cpd I) have been studied as models for reactive intermediates called compound I in cytochromes P450, peroxidases, and catalases. It has been well known that the electronic structure and reactivity of Cpd I are modulated by the substitutedposition and the electron-withdrawing ability of the substituent. However, there still remain two major questions: (1) how many electronegative halogen atoms should be introduced in the meso-phenyl group to switch the porphyrin π-cation-radical state of Cpd I? (2) How does the electron-withdrawing effect of the substituent modulate the reactivity of Cpd I? To answer these two questions, we here performed experimental and theoretical studies on the electron-withdrawing effect of the meso-substituent. We gradually increased the electron-withdrawing effect by increasing the number of fluorine atoms in the meso-phenyl group. Spectroscopic analyses of these Cpd I models reveal that the porphyrin radical state shifts from having a2u radical character to having a1u radical character with an increase in the number of the fluorine atoms inthe phenyl group, and the ground state of Cpd I switches from the a2u state to the a1u state when four fluorine atoms are introduced in the meso-phenyl group. The switch of the radical state is predicted well by LC-BLYP, but not by the commonly used B3LYP. The theoretical calculations indicate that the electron-withdrawing substituent makes Cpd I more reactive by stabilizing the ferric porphyrin state (product state) more than the Cpd I state (reactant state), generating a larger free energy change in the oxygenation reaction (ΔG) of Cpd I.

From Ferrous HRP to Compound I and Back: The Role of Protein and the Mediators in Reversible Redox Transitions

Highly oxidized iron species play key role in the catalysis of many heme and non-heme enzymes due to their ability to activate substrates via electron or hydrogen atom transfer. In Nature, such highly reactivate and unstable states are typically generated by activation and reduction of molecular oxygen by ferrous porphyrin with the ensuing transfer of redox equivalents onto the cofactor and/or protein itself. Oxygen activation is also associated with a variety of side-reactions, including generation of ROS and self-inactivation. This study investigates alternative approaches to sustaining catalytic activity of highly-oxidized heme species via direct oxidation of cofactor-bound solvent ligand under externally applied electric potential. We developed experimental framework that allows to quickly and reversibly traverse a range of oxidation states of heme cofactor between ferrous iron and Compound I while monitoring redox changes spectroscopically in the UV-Vis or IR domains. We investigate the role of redox mediators, their structures and redox potentials on the ability to catalyze electron transfer to and from the active site of horseradish peroxidase. We show that self-catalyzed modification of the protein moiety enhances the rate the electron transfer, eventually enabling sustained redox transitions in the absence of a mediator. Our results also demonstrate the role on intra-protein solvent molecule as an intrinsic reductant, rationalizing spontaneous conversion of Compounds I and II to the ferric state.

Magnetic and Dielectric Properties of LiFePO₄

A LiFePO₄ was prepared using the ball milling. The X-ray diffraction patterns of the sample were measured, and the results confirmed that the sample’s structure was orthorhombic with space group Pnma. The particle size and morphology of the sample prepared by the ball mill method confirmed by FE-SEM. The magnetization curves of the sample were measured using a vibrating sample magnetometer at temperatures of 4.2 to 295 K at 1000 Oe. The Néel temperature (TN) and spin-reorientation temperature (TS) were found to be 51.5 and 25 K, respectively. We investigated the magnetic hyperfine interaction using Mössbauer spectrometry at various temperatures between 4.2 and 295 K. At temperatures below TN, the Mössbauer spectra of the sample were exhibited eight absorption lines resulting from the magnetic dipole and electric quadruple interaction. The Fe ions state of sample at all temperatures were found to be ferrous state (Fe2+) ions. Debye temperature (θD) of 433 ± 5 K was obtained for the sample. The permeability and permittivity were obtained using a Network analyzer.

X-ray driven reduction of Cpd I of Catalase-3 from N. crassa reveals differential sensitivity of active sites and formation of ferrous state

Catalases are biotechnologically relevant enzymes because of their applications in food technology, bioremediation, and biomedicine. The dismutation of hydrogen peroxide occurs in two steps; in the first one, the enzyme forms an oxidized compound I (Cpd I) and in the second one, the enzyme is reduced to the ferric state. In this research work, we analyzed the reduction of Cpd I by X-ray radiation damage during diffraction experiments in crystals of CAT-3, a Large-Size Subunit Catalase (LSC) from Neurospora crassa. A Multi-Crystal Data collection Strategy was applied in order to obtain the Cpd I structure at a resolution of 2.2 Å; this intermediate was highly sensitive to X-ray and was easily reduced at very low deposited radiation dose, causing breakage of the Fe=O bond. The comparison of the structures showed reduced intermediates and also evidenced the differential sensitivity per monomer. The resting ferric state was reduced to the ferrous state, an intermediate without a previous report in LSC. The chemically obtained Cpd I and the X-ray reduced intermediates were identified by UV-visible microspectrometry coupled to data collection. The differential sensitivity and the formation of a ferrous state are discussed, emphasizing the importance of the correct interpretation in the oxidation state of the iron heme.