Ferric Ligands Research Articles

Construction of Fe3Se4@NiSe2 heterogeneous structures with low overpotentials for oxygen evolution reaction

Hydrogen generation from electrochemical water splitting (EWS) is considered as one of the best ways to store renewable energy. To synthesize the highly effective electrocatalyst with low cost is vital to promote the larger-scale development of EWS technology. To improve the activity of NiSe2, Fe3Se4@NiSe2 heterostructures is designed and synthesized by solvothermal method and selenization treatment. Ni(OH)2 nanosheets inducing the growth of nickel iron metal-organic framework (NiFe-MOF) made up of Ni node and organic ferric ligand is designed as the precursor to gain the hierarchical morphology. The morphology, electron state and composition of Fe3Se4@NiSe2 are engineered through adjusting the condition of selenidation treatment. Benefiting from the tuned electron state of Ni, Se elements, hierarchical morphology and lower Rct value, the optimized Fe3Se4@NiSe2 electrode exhibits ultralow overpotentials (110 mV) for oxygen evolution reaction (OER) to drive 10 mA cm−2, small Tafel slope (29.32 mV dec−1) and high Faraday efficiency (FE = 99.50 %) in 1 M KOH. The construction of Fe3Se4@NiSe2 provide novel electrocatalyst as an outstanding non-precious electrocatalysts for OER.

Ligand-dependent inequivalence of the α and β subunits of ferric human hemoglobin bound to haptoglobin

Haptoglobin (Hp) prevents extra-erythrocytic hemoglobin- (Hb-)mediated damage. Hp binds αβ dimers of Hb, displaying heme-based reactivity. Here, kinetics and thermodynamics of cyanide, thiocyanate and imidazole binding to ferric human Hb (Hb(III)), Hb(III) dimers complexed with the human Hp phenotypes 1-1 and 2-2 (Hp1-1:Hb(III) and Hp2-2:Hb(III), respectively), and α(III) and β(III) chains are reported and analyzed in parallel with fluoride and azide binding properties (at pH 7.0 and 20.0 °C). Cyanide and fluoride bind to Hb(III), Hp1-1:Hb(III), Hp2-2:Hb(III), α(III), and β(III) with a simple behavior. In contrast, azide, thiocyanate and imidazole binding to Hb(III), Hp1-1:Hb(III) and Hp2-2:Hb(III) follows a two-step process, whereas ligand binding to α(III) and β(III) chains follows a simple behavior. However, azide, thiocyanate and imidazole binding to Hb(III), Hp1-1:Hb(III) and Hp2-2:Hb(III) is characterized by a simple equilibrium, reflecting the compensation of kinetic parameters. The fast and the slow step of azide, thiocyanate and imidazole binding to Hb(III), Hp1-1:Hb(III) and Hp2-2:Hb(III) mirror the ligand binding properties of the β(III) and α(III) chains, respectively. Values of kinetic and thermodynamic parameters for binding of ferric ligands to Hp1-1:Hb(III) and Hp2-2:Hb(III) match very well with those obtained for ligation of Hb(III) and α(III) and β(III) chains, confirming the ligand-dependent kinetic inequivalence of α(III) and β(III) subunits. However, a variation between tetrameric Hb(III) on one side and Hp1-1:Hb(III), Hp2-2:Hb(III), α(III), and β(III) on the other side for the rate-limiting step (likely referable to the dissociation of heme-coordinated H2O from the heme-Fe(III) atom) suggests a structural change(s) upon dimers to tetramer assembly in Hb(III).

Fluoride and azide binding to ferric human hemoglobin:haptoglobin complexes highlights the ligand-dependent inequivalence of the α and β hemoglobin chains.

Haptoglobin (Hp) binds human hemoglobin (Hb), contributing to prevent extra-erythrocytic Hb-induced damage. Hp forms preferentially complexes with αβ dimers, displaying heme-based reactivity. Here, kinetics and thermodynamics of fluoride and azide binding to ferric human Hb (Hb(III)) complexed with the human Hp phenotypes 1-1 and 2-2 (Hp1-1:Hb(III) and Hp2-2:Hb(III), respectively) are reported (pH 7.0 and 20.0°C). Fluoride binds to Hp1-1:Hb(III) and Hp2-2:Hb(III) with a one-step kinetic and equilibrium behavior. In contrast, kinetics of azide binding to and dissociation from Hp1-1:Hb(III)(-N3-) and Hp2-2:Hb(III)(-N3-) follow a two-step process. However, azide binding to Hp1-1:Hb(III) and Hp2-2:Hb(III) is characterized by a simple equilibrium, reflecting the compensation of kinetic parameters. The fast and the slow step of azide binding to Hp1-1:Hb(III) and Hp2-2:Hb(III) should reflect azide binding to the ferric β and α chains, respectively, as also proposed for the similar behavior observed in Hb(III). Present results highlight the ligand-dependent kinetic inequivalence of Hb subunits in the ferric form, reflecting structural differences between the two subunits in the interaction with some ferric ligands.

The importance of reductive mechanisms for intestinal uptake of iron from ferric maltol and ferric nitrilotriacetic acid (NTA)

Intestinal iron absorption is thought to proceed with iron mainly in the ferrous form, yet the novel iron complex, ferric maltol is an effective oral preparation. Although possessing a high oil: water partition coefficient, ferric maltol does not diffuse across the intestine but donates its iron to the endogenous uptake system. Reduction of the ferric iron in the gut lumen appears to precede iron uptake both from ferric maltol and from ferric nitrilotriacetic acid (NTA) which is a non-penetrating iron ligand. Uptake of radiolabelled iron (59Fe) into isolated fragments of rat small intestine was inhibited by the ferrous chelator, bathophenanthroline sulphonate (BPS) and enhanced at low concentrations by the reducing agent ascorbic acid. Spectrophotometric evidence was obtained that ferrous ions are generated from these ferric complexes in the presence of ascorbic acid and other reducing agents. The rate of ferrous ion formation was independent of ferric maltol concentration at low ascorbic acid levels and decreased with increasing ferric maltol concentration at higher levels of ascorbate. Maltol has a high affinity for ferric ions and may delay reduction at higher concentrations. By contrast, a higher rate of ferrous ion generation was seen with ferric NTA and this increased with iron ligand concentration. Washings from the intestinal lumen also brought about ferrous ion formation from these ferric ligands. Gel filtration revealed these reducing factors to be of low molecular weight. The washings, however, interfered with 59Fe uptake into the isolated fragments, but when reducing fractions only from the filtered washings were used, enhanced iron uptake was seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonreductive Iron Uptake Mechanism in the Marine Alveolate Chromera velia

Chromera velia is a newly cultured photosynthetic marine alveolate. This microalga has a high iron requirement for respiration and photosynthesis, although its natural environment contains less than 1 nm of this metal. We found that this organism uses a novel mechanism of iron uptake, differing from the classic reductive and siderophore-mediated iron uptake systems characterized in the model yeast Saccharomyces cerevisiae and present in most yeasts and terrestrial plants. C. velia has no trans-plasma membrane electron transfer system, and thus cannot reduce extracellular ferric chelates. It is also unable to use hydroxamate siderophores as iron sources. Iron uptake from ferric citrate by C. velia is not inhibited by a ferrous chelator, but the rate of uptake is strongly decreased by increasing the ferric ligand (citrate) concentration. The cell wall contains a large number of iron binding sites, allowing the cells to concentrate iron in the vicinity of the transport sites. We describe a model of iron uptake in which aqueous ferric ions are first concentrated in the cell wall before being taken up by the cells without prior reduction. We discuss our results in relation to the strategies used by the phytoplankton to take up iron in the oceans.

Heme Ligand Binding Properties and Intradimer Interactions in the Full-length Sensor Protein Dos from Escherichia coli and Its Isolated Heme Domain

Dos from Escherichia coli is a bacterial gas sensor protein comprising a heme-containing gas sensor domain and a phosphodiesterase catalytic domain. Using a combination of static light scattering and gel filtration experiments, we established that, as are many other sensor proteins, the full-length protein is dimeric. The full-length dimer (association constant <10 nm) is more stable than the dimeric heme domain (association constant approximately 1 mum), and the dimer interface presumably includes both sensor and catalytic domains. Ultrafast spectroscopic studies showed little influence of the catalytic domain on kinetic processes in the direct vicinity of the heme. By contrast, the properties of ligand (CO and O(2)) binding to the heme in the sensor domain, occurring on a microsecond to second time scale, were found to be influenced by (i) the presence of the catalytic domain, (ii) the dimerization state, and in dimers, (iii) the ligation state of the other subunit. These results imply allosteric interactions within dimers. Steady-state titrations demonstrated marked cooperativity in oxygen binding to both the full-length protein and the isolated heme domain, a feature not reported to date for any dimeric sensor protein. Analysis of a variety of time-resolved experiments showed that Met-95 plays a major role in the intradimer interactions. The intrinsic binding and dissociation rates of Met-95 to the heme were modulated approximately 10-fold by intradimer and sensor-catalytic domain interactions. Dimerization effects were also observed for cyanide binding to the ferric heme domains, suggesting a similar role for Met-95 in ferric proteins.

Secretion of iron-complexing ligands from Closterium aciculare (Charophyceae, Chlorophyta) under iron-deficient conditions

Siderophores, ferric iron-specific ligands, are produced by many species of prokaryotes, fungi and higher plants under iron-limited conditions. We report the possibility of siderophore production by a freshwater eukaryotic microalga Closterium aciculare under iron-deficient conditions. Closterium aciculare secreted a substance that is reactive in the chrome azurol S (CAS) assay in an iron-deficient, chemically defined medium. The final concentrations of the CAS-reactive substance were 0.17 and 0.35 μM for some 0.1 g 1−1 of C. aciculare in the medium with noniron and 10 nM iron addition, respectively. The amount of the CAS-reactive substance increased along with the increase in cell density of C. aciculare, whereas the addition of iron with nonlimiting concentration (100 nM) to the medium completely repressed the production of the CAS-reactive substance. Because C. aciculare did not produce the CAS-reactive substance in the iron-replete medium, the CAS-reactive substance was considered to be a siderophore.

Fluorescent effector as a probe of the allosteric equilibrium in methemoglobin

A fluorescent analogue of diphosphoglycerate (DPG), hydroxy-pyrenetrisulfonate (HPT), was used as a probe of the allosteric equilibrium of methemoglobin. Like DPG, HPT binds, one per tetramer, with a higher affinity to deoxyHb than to oxyHb. Once bound, the HPT fluorescence is quenched by energy transfer to the hemes. HPT can thus serve as a probe of the conformational state of the hemoglobin tetramer: a higher quenching indicates a stronger binding and therefore, more of the deoxy conformation. Since HPT binds to the same site as DPG, it can be displaced by DPG in order to determine the fluorescence intensity of the free HPT under the same conditions, to correct for the inner filter effect. The high spin ferric ligands, such as water and F, showed less fluorescence (more of the deoxy state) than low spin cyano-metHb. The aquo-metHb samples showed a reversion to the oxyHb conformation above pH 7, as expected due to the acid–alkaline transition forming hydroxy-metHb. Effectors such as bezafibrate, which do not bind to the same site as DPG, show an increase in the deoxy-like characteristics.

Quaternary structure dynamics and carbon monoxide binding kinetics of hemoglobin valency hybrids

The kinetics of CO binding and changes in quaternary structure for symmetric valency hybrids of human hemoglobin have been extensively studied by laser photolysis techniques. Both alpha+beta and alpha beta+ hybrids were studied with five different ferric ligands, over a broad range of CO concentrations and photolysis levels. After full CO photolysis, the hybrid tetramers switch extensively and rapidly (< 200 microseconds) to the T quaternary structure. Both R --> T and T --> R transition rates for valency hybrid tetramers with 0 and 1 bound CO have been obtained, as well as the CO association rates for alpha and beta subunits in the R and T states. The results reveal submillisecond R reversible T interconversion, and, for the first time, the changes in quaternary rates and equilibria due to binding a single CO per tetramer have been resolved. The data also show significant alpha-beta differences in quaternary dynamics and equilibria. The allosteric constants do not vary with the spin states of the ferric subunits as predicted by the Perutz stereochemical model. For the alpha beta+CN hybrid the kinetics are heterogeneous and imply partial conversion to a T-like state with very low (seconds) R reversible T interconversion.

Iron acquisition byCryptococcus neoformans

Iron is an essential element for the growth and metabolism of microbial cells. Most pathogenic microbes elaborate powerful iron chelating agents (siderophores) to mobilize iron from ferric ligands. The pathogenic yeast, Cryptococcus neoformans has not been found to produce siderophores and its mechanism of iron acquisition is unknown. This investigation explored an alternative pathway for iron acquisition by examining the interactions of iron with the cell surface. Iron uptake experiments were conducted utilizing radiolabelled ferrous iron and ferric iron chelates, with evidence for the presence of iron(II) receptors and the generation of ferrous iron by surface reduction. Hyperbolic kinetics were found when 59FeII was presented to the organism and uptake was blocked with bathophenanthroline sulphonate, an Fe2+ chelator. The yeast also acquired iron as [59Fe3+]-citrate and [59Fe3+]-pyrophosphate while bathophenanthroline sulphonate reduced the acquisition of these ferric ligands by 48% and 52% respectively. Pre-incubation with either ferric ligand also reduced iron acquisition by 50%. KCN inhibited uptake of iron(II) by 90% and uptake of [59Fe3+]-pyrophosphate and [59Fe3+]-citrate by 46% and 56% respectively; dinitrophenol had no effect on these processes. The data suggest that C. neoformans can (i) generate ferrous iron at the cell surface via a reduction of ferric chelates, with the subsequent acquisition of the ferrous iron, and (ii) acquire iron through the interaction of ferric chelates with a surface component.

Isolation and Stability of Partially Oxidized Intermediates of Carp Hemoglobin: Kinetics of CO Binding to the Mono- and Triferric Species

The monoliganded and triliganded forms of the asymmetric valency hybrids of carp hemoglobin were isolated using high-performance liquid chromatography. These partially oxidized hybrids were shown to be sufficiently stable to permit the measurement of the kinetics of CO binding. The effects of protons and inositol hexaphosphate on the rates of these reactions were examined. The kinetics of CO recombination with these partially oxidized derivatives were compared to the kinetics of CO binding to the fully ferrous molecule. To a first approximation, the kinetic behavior of the monoferric derivative was consistent with a small shift in the T<==>R equilibrium in favor of the R state. The presence of three ferric ligands resulted in a still greater shift in the conformational equilibrium in favor of the R state. The kinetic behavior of the triferric molecule was similar, but not identical, to that of a fully ferrous molecule which is triliganded with CO. The properties of both asymmetric valency hybrids were responsive to the nature of the ligand; i.e., the rate of CO binding was increased more by the presence of cyanide on the ferric hemes than by water. Not all of the data could be accommodated within the two-state model. For example, there was evidence of an altered T state in the case of the tricyanomet derivative at low pH in the presence of inositol hexaphosphate.

6] Allosteric equilibrium measurements with hemoglobin valency hybrids

Publisher Summary This chapter discusses the allosteric equilibrium measurements with hemoglobin (Hb) valency hybrids samples.There is a need to correct for the influence of a slightly oxidized sample. Although the oxidation levels can usually be maintained below 5%, this level is worse than it seems when dealing with a tetrameric system. This problem may be aggravated in Hb mutants, natural or artificial, in which the rate of autoxidation is increased. Thus, an understanding of how ferric subunits affect their neighbors within an Hb tetramer is important in order to compare the ligand-binding parameters of different samples. The use of highly oxidized solutions provides another method to study the triply liganded form. With different ferric ligands and the use of external effectors– whether the two-state model is still operational at the triply liganded level can be determined. By comparing different ferric ligands with varying amounts of high- and low-spin ferric hemes, the coupling of the spin state and the allosteric equilibrium can be studied.

Coupling of ferric iron spin and allosteric equilibrium in hemoglobin

The allosteric transition in triply ferric hemoglobin has been studied with different ferric ligands. This valency hybrid permits observation of oxygen or CO binding properties to the single ferrous subunit, whereas the liganded state of the other three ferric subunits can be varied. The ferric hemoglobin (Hb) tetramer in the absence of effectors is generally in the high oxygen affinity (R) state; addition of inositol hexaphosphate induces a transition towards the deoxy (T) conformation. The fraction of T-state formed depends on the ferric ligand and is correlated with the spin state of the ferric iron complexes. High-spin ferric ligands such as water or fluoride show the most T-state, whereas low-spin ligands such as cyanide show the least. The oxygen equilibrium data and kinetics of CO recombination indicate that the allosteric equilibrium can be treated in a fashion analogous to the two-state model. The binding of a low-spin ferric ligand induces a change in the allosteric equilibrium towards the R-state by about a factor of 150 (at pH 6.5), similar to that of the ferrous ligands oxygen or CO; however, each high-spin ferric ligand induces a T to R shift by a factor of 40.

Assessment of the α 1β 2 contact structure of valency hybrid hemoglobins by ultraviolet difference spectra

We have developed a rapid and useful method for purification of valency hybrid hemoglobins (α 2 +β 2 and α 2β 2 +: + denotes ferric heme) from a hemoglobin solution oxidized partially with ferricyanide by preparative high-performance liquid chromatography. This method does not involve the separation of hemoglobin subunits and the reconstitution of ferric and partner ferrous subunits. Using the valency hybrid hemoglobins thus prepared, the effect of the ferric spin state on the α 1β 2 subunit boundary structure was investigated by measuring the ultraviolet difference absorption spectra between the deoxy and the oxy valency hybrids associated with various ferric ligands (fluoride, aquo, azide and cyanide). All derivatives of both α 2 +β 2 and α 2β 2 + showed the difference spectra characteristic of R-T quaternary structural transition. However, the magnitude of the difference spectral peak observed near 288 nm was larger for high-spin derivatives than for low-spin ones. The magnitude of the peak for the valency hybrid hemoglobin was closely correlated with the difference in the free energy of oxygen binding between the R and T states. Since the R state of high-spin hybrids is considered to be identical to that of low-spin hybrids, we concluded from these results that the α 1β 2 subunit boundary structure plays an important role in regulating the oxygen affinity of deoxy T state.

Quaternary structure has little influence on spin states in mixed-spin human methemoglobins.

A key feature of the Perutz stereochemical model for cooperativity in hemoglobin is a strong coupling between quaternary structure and the spin state of the heme iron [Perutz, M. F. (1979) Annu. Rev. Biochem. 48, 327-386]. While this coupling appears to be present for carp azide methemoglobin, it should also be present for all liganded forms of human methemoglobin that exhibit a thermal high-spin in equilibrium low-spin equilibrium. To test this hypothesis, we have measured the changes in spin equilibria upon conversion of six mixed-spin forms of human methemoglobin from the R (high-affinity) to the T (low-affinity) quaternary structure by addition of inositol hexaphosphate. These experiments were done with a sensitive superconducting magnetic susceptibility instrument on solutions at 20 degrees C in 20 mM maleate buffer, pH 6. The data show zero or small increases in high-spin content upon switching from R to T, changes that are equivalent to a relative stabilization of the high-spin form by only 0-300 cal mol-1 heme-1. These changes in energy are far less than the 1200 cal mol-1 heme-1 predicted from the Perutz stereochemical model [Cho, K. C., & Hopfield, J. J. (1979) Biochemistry 18, 5826-5833]. That is, these data do not support a view that the low affinity of the T state is due to restraints acting through the iron-proximal histidine linkage. The mechanistic implications of these results and the differences between species and ferric ligands are discussed.

The active sites of chloroperoxidase and cytochrome P-450-CAM: Comparative spectroscopic and ligand binding properties

Spectral similarities between chloroperoxidase (CPO) and cytochrome P-450 (P-450) have previously been used to suggest endogenous thiolate axial ligation to the CPO heme iron [1–6] as has been established for P-450. Both enzymes exhibit unique hyperporphyrin (‘split Soret’) spectra in their ferrous CO forms [1]. However, no free sulfhydryl group available for ligation to the heme iron have been found in chemical studies of CPO [7]. The t001. Soret Absorption Maxima (nm) and Spin States of the Ligand Complexes of Ferric CPO and P-450-CAM.aLigandCPObP-450-CAMcλ(ϵmM)Spin Statedλ(ϵmM)Spin StatedSH−449(e)ls467(43)flCH3COS−446(62)gls459elsCN−439(92)ls439(78)lsPyridine439(82)l421(111)lsNO437(114)ls430.5(105)lsCH3(CH2)3NC435(105)ls429.5(98)lsN−3432(110)ls427(81)lsSeCN−432(80)ls425(90)lsSCN−429(112)ls425(98)lsImidazole429(100)ls425(98)lsNo−2427.5(101)lseOCN−∼427els418.5(98)lsHCOO−425(121)ls419(106)lsHOCH2CH2SH418(69)ms464(58)fls417 (∼60)ms417(∼60)CH3CH2CH2SH416(74)ms464(∼33)flsCH3SCH3416(93)ms424(91)lsThioxane416(88)ms418(97)lsCH3SSCH3416(93)ms418(98)lsCH3COO−413(97)ms420(98)lsF−409(98)ghsno bindinga0.1 M K+ Phospate, 4 °C.bpH 6.0.cpH 7.0, (Refs.9–11).dls, low spin; ms, high-low mixed spin; hs, high spin.eUnstable.fRed Soret of hyperporphyrin spectrum (Ref. 11).gpH 3.0.t002. Soret Absorption Maxima (nm) of the Ligand Complexes of Ferrous CPO and P-450-CAM.LigandCPOa2 λ(ϵmM)P-450-CAMb2 λ(ϵmM)Me2PhPc2459d2460(98)CN−454(∼140)Not observedCH3(CH2)3NC452(141)452(106)CO444(167)446(120)NO440(114)438(83)a2pH 6.0.b2pH 7.0.c2Dimenthyllphenylphosphine.d2Not determined. MCD [4] and EPR [6] spectroscopic properties of analogous CPO and P-450 derivatives are generally similar, although some differences do exist. To obtain additional information about the active site structure of CPO, we have carried out extensive optical and MCD spectroscopic studies of both ferric and ferrous CPO in the presence of various exogenous ligands. The results obtained with CPO have been compared with those obtained with P-450 [9 - 11, this work] in order to obtain a better understanding of the electronic structure of the heme iron in both proteins.

The effect of ferric ligands on the oxygen affinity of the ferrous subunits in valency hybrid haemoglobins

Oxygen equilibrium curves of various valency hybrid haemoglobins (cyanide, azide, fluoride, aquomet hybrids) have been measured in the presence of inorganic phosphate or inositol hexaphosphate. All the hybrids showed asymmetric oxygen equilibrium curves which is inconsistent with the presence of only two oxygen binding sites. This inconsistency was found to be due to a small amount of oxidation of the ferrous subunits. Except for α 2 + β 2 combined with inositol hexaphosphate, the oxygen affinity was always lower in high compared to low spin hybrids implying that the deoxy quaternary structure is more stable for high spin derivatives. This result is consistent with the stereochemical interpretation of the co-operative effects proposed by Perutz (1970) . However, in the presence of inositol hexaphosphate, α 2 +CN β 2 and α 2 +F β 2 have similar oxygen affinity, showing that here the effect of spin state is no longer dominant. The reason for this is not yet clear. The oxygen binding parameters of the valency hybrids have been analysed according to the two-state allosteric model ( Monod et al. , 1965 ). This model adequately describes the oxygen binding properties in the presence of inositol hexaphosphate but not in phosphate buffer.

Functional properties of partially oxidized trout hemoglobins

This paper reports on a study of the effect of partial oxidation on oxygen and carbon monoxide binding by components I and IV of trout hemoglobin. The O 2 binding equilibria of the various oxidation mixtures show a decrease in the heme-heme interactions as the number of oxidized sites is increased. However the large Bohr effect, characteristic of Hb Trout IV, is maintained unchanged. Similarly the time course of CO combination changes on increasing the fractional oxidation, and the autocatalytic character of the CO binding kinetics is lost; however the pH dependence of the apparent “on” constant in the oxidation mixtures is similar to that characteristic of the native molecule. The results of the O 2 equilibria and of CO binding kinetics may be interpreted in accordance with the two state concerted model suggesting that in the oxidation intermediates there is the fraction of the high affinity (R) conformation. Additional experiments on the effect of azide, and fluoride, ferric ligands which produce a change of spin state of the heme iron, suggest that additional second order conformational changes may also come into play.