Ferrous Oxidation State Research Articles

Impact of Quaternary Structure upon Bacterial Cytochrome c Peroxidases: Does Homodimerization Matter?

All known active forms of diheme bacterial cytochrome c peroxidase (bCcP) enzymes are described by a homodimeric state. Further, the majority of bCcPs reported display activity only when the high-potential electron transfer heme of the protein (Fe(H)) is reduced to the ferrous oxidation state. Reduction of Fe(H) results in a set of conformational changes allowing for the low-potential peroxidatic heme (Fe(L)) to adopt a high-spin, five-coordinate state that is capable of binding substrate. Here we examine the impact of dimerization upon the activity of the Shewanella oneidensis (So) bCcP by the preparation of single charge-reversal mutants at the dimer interface and use the resulting constructs to illustrate why dimerization is likely a requirement for activity in bCcPs. The E258K mutant is found to form a monomeric state in solution as characterized by size exclusion chromatography and analytical ultracentrifugation analyses. The resulting E258K monomer has an unfolding stability comparable to that of wild-type So bCcP and an activity that is only slightly diminished (k(cat)/K(m) = 23 × 10(6) M(-1) s(-1)). Spectroscopic and potentiometric analyses reveal that while the thermodynamic stability of the activated form of the enzyme is unchanged (characterized by the E(m) value of the Fe(H)(II)/Fe(H)(III) couple), the kinetic stability of the activated form of the enzyme has been greatly diminished upon generation of the monomer. Together, these data suggest a model in which dimerization of bCcP enzymes is required to stabilize the lifetime of the activated form of the enzyme against reoxidation of Fe(H) and deactivation of Fe(L).

Molecular Mechanism of Hepcidin-Mediated Ferroportin Internalization Requires Ferroportin Lysines, Not Tyrosines or JAK-STAT

Ferroportin is the primary means of cellular iron efflux and a key component of iron metabolism. Hepcidin regulates Fpn activity by inducing its internalization and degradation. The mechanism of internalization is reported to require JAK2 activation, phosphorylation of Fpn tyrosine residues 302 and 303, and initiation of transcription through STAT3 phosphorylation. These findings suggest Fpn may be a target for therapeutic intervention through JAK2 modulation. To evaluate the proposed mechanism, Fpn internalization was assessed using several techniques combined with reagents that specifically recognized cell-surface Fpn. Invitro results demonstrated that Hepc-induced Fpn internalization did not require JAK2 or phosphorylation of Fpn residues 302 and 303, nor did it induce JAK-STAT signaling. Invivo, inhibition of JAK2 had no effect on Hepc-induced hypoferremia. However, internalization was delayed by mutation of two Fpn lysine residues that may be targets of ubiquitination.

Role of K+ binding residues in stabilization of heme spin state of Leishmania major peroxidase

The endogenous cation in peroxidases may contribute to the type of heme coordination. Here a series of ferric and ferrous derivatives of wild-type Leishmania major peroxidase (LmP) and of engineered K+ site mutants of LmP, lacking potassium cation binding site, has been examined by electronic absorption spectroscopy at 25°C. Using UV–visible spectrophotometry, we show that the removal of K+ binding site causes substantial changes in spin states of both the ferric and ferrous forms. The spectral changes are interpreted to be, most likely, due to the formation of a bis-histidine coordination structure in both the ferric and ferrous oxidation states at neutral pH 7.0. Stopped flow spectrophotometric techniques revealed that characteristics of Compound I were not observed in the K+ site double mutants in the presence of H2O2. Similarly electron donor oxidation rate was two orders less for the K+ site double mutants compared to the wild type. These data show that K+ functions in preserving the protein structure in the heme surroundings as well as the spin state of the heme iron, in favor of the enzymatically active form of LmP.

Are the biogeochemical cycles of carbon, nitrogen, sulfur, and phosphorus driven by the “FeIII–FeII redox wheel” in dynamic redox environments?

Purpose Iron’s fluctuation between the II (ferrous) and III (ferric) oxidation states has been coined as the “FeIII–FeII redox wheel.” Numerous studies have coupled the “iron redox wheel” with the biogeochemical cycle of carbon (C), nitrogen (N), sulfur (S), or phosphorus (P) individually in soils or sediments, but evidence suggests that the FeIII–FeII redox wheel drives the biogeochemical cycles interactively in a fluctuating redox microenvironment. The interactions of the FeIII–FeII redox wheel with the biogeochemical cycles of C, N, S, and P in the fluctuating redox environments were reviewed in this paper.

Kinetics of α-Globin Binding to α-Hemoglobin Stabilizing Protein (AHSP) Indicate Preferential Stabilization of Hemichrome Folding Intermediate

Human α-hemoglobin stabilizing protein (AHSP) is a conserved mammalian erythroid protein that facilitates the production of Hemoglobin A by stabilizing free α-globin. AHSP rapidly binds to ferrous α with association (k'(AHSP)) and dissociation (k(AHSP)) rate constants of ≈10 μm(-1) s(-1) and 0.2 s(-1), respectively, at pH 7.4 at 22 °C. A small slow phase was observed when AHSP binds to excess ferrous αCO. This slow phase appears to be due to cis to trans prolyl isomerization of the Asp(29)-Pro(30) peptide bond in wild-type AHSP because it was absent when αCO was mixed with P30A and P30W AHSP, which are fixed in the trans conformation. This slow phase was also absent when met(Fe(3+))-α reacted with wild-type AHSP, suggesting that met-α is capable of rapidly binding to either Pro(30) conformer. Both wild-type and Pro(30)-substituted AHSPs drive the formation of a met-α hemichrome conformation following binding to either met- or oxy(Fe(2+))-α. The dissociation rate of the met-α·AHSP complex (k(AHSP) ≈ 0.002 s(-1)) is ∼100-fold slower than that for ferrous α·AHSP complexes, resulting in a much higher affinity of AHSP for met-α. Thus, in vivo, AHSP acts as a molecular chaperone by rapidly binding and stabilizing met-α hemichrome folding intermediates. The low rate of met-α dissociation also allows AHSP to have a quality control function by kinetically trapping ferric α and preventing its incorporation into less stable mixed valence Hemoglobin A tetramers. Reduction of AHSP-bound met-α allows more rapid release to β subunits to form stable fully, reduced hemoglobin dimers and tetramers.

Detection of Nano-Oxidation Sites on the Surface of Hemoglobin Crystals Using Tip-Enhanced Raman Scattering

Hemoglobin nanocrystals were analyzed with tip-enhanced Raman scattering (TERS), surface-enhanced resonance Raman scattering (SERRS) and conventional resonance Raman scattering (RRS) using 532 nm excitation. The extremely high spatial resolution of TERS enables selective enhancement of heme, protein, and amino acid bands from the crystal surface not observed in the SERRS or RRS spectra. Two bands appearing at 1378 and 1355 cm(-1) assigned to the ferric and ferrous oxidation state marker bands, respectively, were observed in both TERS and SERRS spectra but not in the RRS spectrum of the bulk sample. The results indicate that nanoscale oxidation changes are occurring at the hemoglobin crystal surface. These changes could be explained by oxygen exchange at the crystal surface and demonstrate the potential of the TERS technique to obtain structural information not possible with conventional Raman microscopy.

Characterization of Heme Ligation Properties of Rv0203, a Secreted Heme Binding Protein Involved in Mycobacterium tuberculosis Heme Uptake

The secreted Mycobacterium tuberculosis (Mtb) heme binding protein Rv0203 has been shown to play a role in Mtb heme uptake. In this work, we use spectroscopic (absorption, electron paramagnetic resonance, and magnetic circular dichrosim) methods to further characterize the heme coordination environments of His-tagged and native protein forms, Rv0203-His and Rv0203-notag, respectively. Rv0203-His binds the heme molecule through bis-His coordination and is low-spin in both ferric and ferrous oxidation states. Rv0203-notag is high-spin in both oxidation states and shares spectroscopic similarity with pentacoordinate oxygen-ligated heme proteins. Mutagenesis experiments determined that residues Tyr59, His63, and His89 are required for Rv0203-notag to efficiently bind heme, reinforcing the hypothesis based on our previous structural and mutagenesis studies of Rv0203-His. While Tyr59, His63, and His89 are required for the binding of heme to Rv0203-notag, comparison of the absorption spectra of the Rv0203-notag mutants suggests the heme ligand may be the hydroxyl group of Tyr59, although an exogenous hydroxide cannot be ruled out. Additionally, we measured the heme affinities of Rv0203-His and Rv0203-notag using stopped flow techniques. The rates for binding of heme to Rv0203-His and Rv0203-notag are similar, 115 and 133 μM(-1) s(-1), respectively. However, the heme off rates differ quite dramatically, whereby Rv0203-His gives biphasic dissociation kinetics with fast and slow rates of 0.0019 and 0.0002 s(-1), respectively, and Rv0203-notag has a single off rate of 0.082 s(-1). The spectral and heme binding affinity differences between Rv0203-His and Rv0203-notag suggest that the His tag interferes with heme binding. Furthermore, these results imply that the His tag has the ability to stabilize heme binding as well as alter heme ligand coordination of Rv0203 by providing an unnatural histidine ligand. Moreover, the heme affinity of Rv0203-notag is comparable to that of other heme transport proteins, implying that Rv0203 may act as an extracellular heme transporter.

CO2-water–basalt interaction. Low temperature experiments and implications for CO2 sequestration into basalts

CO2-water–basaltic glass batch experiments were performed in order to study the feasibility of low temperature CO2 sequestration into basalts including the key reactions and chemical mass transfer associated to progressive water–rock interaction. The experiments were carried out at 40°C for up to 260days with initial dissolved CO2 concentrations ranging from 24 to 305mmol/kg. Alteration minerals were identified on the basaltic glass grain surfaces and in the matrix, consisting of poorly crystalline Ca–Mg–Fe carbonates, Fe-hydroxides and/or oxy-hydroxides and Ca–Mg–Fe clays. Other cryptocrystalline phases were identified by variable amounts of Al, Si and Fe reflected by the secondary mineral compositions. The water chemistry was monitored during the experiments and was characterized by an increase in Si, Ca, Mg and Na with time, whereas Al, Fe and CO2 decreased. The dissolution of basaltic glass in CO2-rich waters was observed to be incongruent with the overall water composition and secondary mineralogy depending on reaction progress and pH. The pH was determined primarily by the initial CO2 concentration and its ionization constants, the amount of dissolved basaltic glass and by the mass and composition of secondary minerals formed. Initially, the pH increased rapidly from <4.5 to ∼4.5–6. Under these conditions, Mg and Ca were observed to be mobile and dissolved stoichiometrically relative to Na, whereas Si, Al and Fe were immobile. Upon quantitative CO2 mineralization, the pH increased to >6.5 and the mobility of most elements consequently decreased including Mg and Ca. The experimental results indicate that increased aqueous CO2 concentrations modify considerably the natural water–basalt reaction path. The mineralization of CO2 into carbonates was rapid and controlled by the initial CO2 concentration and rock to water ratio, with the composition of the carbonates depending on the availability of Ca, Mg and Fe and the oxidation state of Fe. At pH <5.5, Fe was in the ferrous oxidation state resulting in the formation of Fe-rich carbonates with the incorporation of Ca and Mg as well as the formation of Ca–(Mg)–Fe smectites. At pH >5.5, the mobility of Fe decreased due to oxidation of Fe(II) to Fe(III) and subsequent formation of ferrihydrite. At pH >6.5, the experimental solutions became progressively supersaturated with calcite, zeolites and Mg-rich clays limiting the mobility of Ca and Mg. The results indicate that reactions between clays (Ca–(Mg)–Fe smectites) and carbonates at pH <6.5, and zeolites, clays (Ca–Mg–Fe smectites) and carbonates at pH >6.5, control together the availability of Ca, Mg and Fe, playing a key role for low temperature CO2 mineralization and sequestration into mafic rocks.

Association of Catalytic Iron With Cardiovascular Disease

The ability of iron to cycle reversibly between its ferrous and ferric oxidation states is essential for the biological functions of iron but may contribute to vascular injury through the generation of powerful oxidant species. We examined the association between chemical forms of iron that can participate in redox cycling, often referred to as "catalytic" or "labile" iron, and cardiovascular disease (CVD). In our cross-sectional study of 496 participants, 85 had CVD. Serum catalytic iron was measured using the bleomycin-detectable iron assay that detects biologically active iron. The odds of existing CVD for subjects in the upper third of catalytic iron were 10 times that of subjects with lower catalytic iron in unadjusted analyses. The association was decreased by 1/2 by age adjustment, but little additional attenuation occurred after adjusting for age, Framingham Risk Score, estimated glomerular filtration rate, hypertension status, high-density lipoprotein cholesterol, and systolic blood pressure, with the association remaining strong and significant (odds ratio 3.8, 95% confidence interval 1.4 to 10.1). In conclusion, we provide preliminary evidence for a strong detrimental association between high serum catalytic iron and CVD even after adjusting for several co-morbid conditions; however, broader prospective studies are needed to confirm these findings, which would support therapeutic trials to assess the beneficial effects of iron chelators on CVD.

Enzyme Reactivation by Hydrogen Peroxide in Heme-based Tryptophan Dioxygenase

An intriguing mystery about tryptophan 2,3-dioxygenase is its hydrogen peroxide-triggered enzyme reactivation from the resting ferric oxidation state to the catalytically active ferrous form. In this study, we found that such an odd Fe(III) reduction by an oxidant depends on the presence of L-Trp, which ultimately serves as the reductant for the enzyme. In the peroxide reaction with tryptophan 2,3-dioxygenase, a previously unknown catalase-like activity was detected. A ferryl species (δ = 0.055 mm/s and ΔE(Q) = 1.755 mm/s) and a protein-based free radical (g = 2.0028 and 1.72 millitesla linewidth) were characterized by Mössbauer and EPR spectroscopy, respectively. This is the first compound ES-type of ferryl intermediate from a heme-based dioxygenase characterized by EPR and Mössbauer spectroscopy. Density functional theory calculations revealed the contribution of secondary ligand sphere to the spectroscopic properties of the ferryl species. In the presence of L-Trp, the reactivation was demonstrated by enzyme assays and by various spectroscopic techniques. A Trp-Trp dimer and a monooxygenated L-Trp were both observed as the enzyme reactivation by-products by mass spectrometry. Together, these results lead to the unraveling of an over 60-year old mystery of peroxide reactivation mechanism. These results may shed light on how a metalloenzyme maintains its catalytic activity in an oxidizing environment.

Reactivity of Deoxy- and Oxyferrous Dehaloperoxidase B from Amphitrite ornata: Identification of Compound II and Its Ferrous–Hydroperoxide Precursor

Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. The bifunctional nature of DHP as a globin peroxidase appears to be at odds with the traditional starting oxidation state for each individual activity. Namely, reversible oxygen binding is only mediated via a ferrous heme in globins, and peroxidase activity is initiated from ferric centers and to the exclusion of the oxyferrous oxidation state from the peroxidase cycle. Thus, to address what appears to be a paradox, herein we report the details of our investigations into the DHP catalytic cycle when initiated from the deoxy- and oxyferrous states using biochemical assays, stopped-flow UV-visible, and rapid-freeze-quench electron paramagnetic resonance spectroscopies, and anaerobic methods. We demonstrate the formation of Compound II directly from deoxyferrous DHP B upon its reaction with hydrogen peroxide and show that this occurs both in the presence and in the absence of trihalophenol. Prior to the formation of Compound II, we have identified a new species that we have preliminarily attributed to a ferrous-hydroperoxide precursor that undergoes heterolysis to generate the aforementioned ferryl intermediate. Taken together, the results demonstrate that the oxyferrous state in DHP is a peroxidase competent starting species, and an updated catalytic cycle for DHP is proposed in which the ferric oxidation state is not an obligatory starting point for the peroxidase catalytic cycle of dehaloperoxidase. The data presented herein provide a link between the peroxidase and oxygen transport activities, which furthers our understanding of how this bifunctional enzyme is able to unite its two inherent functions in one system.

CO 2–water–basalt interaction. Numerical simulation of low temperature CO 2 sequestration into basalts

The interaction between CO 2-rich waters and basaltic glass was studied using reaction path modeling in order to get insight into the water–rock reaction process including secondary mineral composition, water chemistry and mass transfer as a function of CO 2 concentration and reaction progress ( ξ). The calculations were carried out at 25–90 °C and pCO 2 to 30 bars and the results were compared to recent experimental observations and natural systems. A thermodynamic dataset was compiled from 25 to 300 °C in order to simulate mineral saturations relevant to basalt alteration in CO 2-rich environment including revised key aqueous species for mineral dissolution reactions and apparent Gibbs energies for clay and carbonate solid solutions observed to form in nature. The dissolution of basaltic glass in CO 2-rich waters was found to be incongruent with the overall water composition and secondary mineral formation depending on reaction progress and pH. Under mildly acid conditions in CO 2 enriched waters (pH <6.5), SiO 2 and simple Al–Si minerals, Ca–Mg–Fe smectites and Ca–Mg–Fe carbonates predominated. Iron, Al and Si were immobile whereas the Mg and Ca mobility depended on the mass of carbonate formed and water pH. Upon quantitative CO 2 mineralization, the pH increased to >8 resulting in Ca–Mg–Fe smectite, zeolites and calcite formation, reducing the mobility of most dissolved elements. The dominant factor determining the reaction path of basalt alteration and the associated element mobility was the pH of the water. In turn, the pH value was determined by the concentration of CO 2 and extent of reaction. The composition of the carbonates depended on the mobility of Ca, Mg and Fe. At pH <6.5, Fe was in the ferrous oxidation state resulting in the formation of Fe-rich carbonates with the incorporation of Ca and Mg. At pH >8, the mobility of Fe and Mg was limited due to the formation of clays whereas Ca was incorporated into calcite, zeolites and clays. Competing reactions between clays (Ca–Fe smectites) and carbonates at low pH, and zeolites and clays (Mg–Fe smectites) and carbonates at high pH, controlled the availability of Ca, Mg and Fe, playing a key role for low temperature CO 2 mineralization and sequestration into basalts. Several problems of the present model point to the need of improvement in future work. The determinant factors linking time to low temperature reaction path modeling may not only be controlled by the primary dissolving phase, which presents challenges concerning non-stoichiometric dissolution, the leached layer model and reactive surface area, but may include secondary mineral precipitation kinetics as rate limiting step for specific reactions such as retrieved from the present reaction path study.

Crystal Structures of Parasponia and Trema Hemoglobins: Differential Heme Coordination Is Linked to Quaternary Structure

Hemoglobins from the plants Parasponia andersonii (ParaHb) and Trema tomentosa (TremaHb) are 93% identical in primary structure but differ in oxygen binding constants in accordance with their distinct physiological functions. Additionally, these proteins are dimeric, and ParaHb exhibits the unusual property of having different heme redox potentials for each subunit. To investigate how these hemoglobins could differ in function despite their shared sequence identity and to determine the cause of subunit heterogeneity in ParaHb, we have measured their crystal structures in the ferric oxidation state. Furthermore, we have made a monomeric ParaHb mutant protein (I43N) and measured its ferrous/ferric heme redox potential to test the hypothesized link between quaternary structure and heme heterogeneity in wild-type ParaHb. Our results demonstrate that TremaHb is a symmetric dimeric hemoglobin similar to other class 1 nonsymbiotic plant hemoglobins but that ParaHb has structurally distinct heme coordination in each of its two subunits that is absent in the monomeric I43N mutant protein. A mechanism for achieving structural heterogeneity in ParaHb in which the Ile(101(F4)) side chain contacts the proximal His(105(F8)) in one subunit but not the other is proposed. These results are discussed in the context of the evolution of plant oxygen transport hemoglobins, and other potential functions of plant hemoglobins.

Occurrence and formation of endogenous histidine hexa‐coordination in cold‐adapted hemoglobins

Spectroscopic and crystallographic evidence of endogenous (His) ligation at the sixth coordination site of the heme iron has been reported for monomeric, dimeric, and tetrameric hemoglobins (Hbs) in both ferrous (hemochrome) and ferric (hemichrome) oxidation states. In particular, the ferric bis- histidyl adduct represents a common accessible ordered state for the β chains of all tetrameric Hbs isolated from Antarctic and sub-Antarctic fish. Indeed, the crystal structures of known tetrameric Hbs in the bis-His state are characterized by a different binding state of the α and β chains. An overall analysis of the bis-histidyl adduct of globin structures deposited in the Protein Data Bank reveals a marked difference between hemichromes in tetrameric Hbs compared to monomeric/dimeric Hbs. Herein, we review the structural, spectroscopic and stability features of hemichromes in tetrameric Antarctic fish Hbs. The role of bis-histidyl adducts is also addressed in a more evolutionary context alongside the concept of its potential physiological role.

Lipid binding to cytoglobin leads to a change in haem co-ordination: a role for cytoglobin in lipid signalling of oxidative stress

Cytoglobin is a recently discovered hexa-co-ordinate haemoglobin that does not appear to function as a classical oxygen-binding protein. Its function is unknown and studies on the effects of changes in its expression have not decisively determined its role within the cell. In the present paper, we report that the protein is transformed from hexa-co-ordinate to penta-co-ordinate on binding a lipid molecule. This transformation occurs with the ferric oxidation state of the protein, but not the ferrous state, indicating that this process only occurs under an oxidative environment and may thus be related to redox-linked cell signalling mechanisms. Oleate binds to the protein in a 1:1 stoichiometry and with high affinity (K(d)=0.7 μM); however, stopped-flow kinetic measurements yield a K(d) value of 110 μM. The discrepancy between these K(d) values may be rationalized by recognizing that cytoglobin is a disulfide-linked dimer and invoking co-operativity in oleate binding. The lipid-induced transformation of cytoglobin from hexa-co-ordinate to penta-co-ordinate does not occur with similar hexa-co-ordinate haemoglobins such as neuroglobin, and therefore appears to be a unique property of cytoglobin among the haemoglobin superfamily. The lipid-derived transformation may explain why cytoglobin has enhanced peroxidatic activity, converting lipids into various oxidized products, a property virtually absent from neuroglobin and much decreased in myoglobin. We propose that the binding of ferric cytoglobin to lipids and their subsequent transformation may be integral to the physiological function of cytoglobin, generating cell signalling lipid molecules under an oxidative environment.

Spectroscopic and Mechanistic Investigations of Dehaloperoxidase B fromAmphitrite ornata

Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. Of the two DHP isoenzymes identified to date, much of the recent focus has been on DHP A, whereas very little is known pertaining to the activity, substrate specificity, mechanism of function, or spectroscopic properties of DHP B. Herein, we report the recombinant expression and purification of DHP B, as well as the details of our investigations into its catalytic cycle using biochemical assays, stopped-flow UV-visible, resonance Raman, and rapid freeze-quench electron paramagnetic resonance spectroscopies, and spectroelectrochemistry. Our experimental design reveals mechanistic insights and kinetic descriptions of the dehaloperoxidase mechanism which have not been previously reported for isoenzyme A. Namely, we demonstrate a novel reaction pathway in which the products of the oxidative dehalogenation of trihalophenols (dihaloquinones) are themselves capable of inducing formation of oxyferrous DHP B, and an updated catalytic cycle for DHP is proposed. We further demonstrate that, unlike the traditional monofunctional peroxidases, the oxyferrous state in DHP is a peroxidase-competent starting species, which suggests that the ferric oxidation state may not be an obligatory starting point for the enzyme. The data presented herein provide a link between the peroxidase and oxygen transport activities which furthers our understanding of how this bifunctional enzyme is able to unite its two inherent functions in one system.

Characterization of conformational changes and noncovalent complexes of myoglobin by electrospray ionization mass spectrometry, circular dichroism and fluorescence spectroscopy

Electrospray ionization mass spectrometry (ESI-MS) was employed to monitor the heme release and the conformational changes of myoglobin (Mb) under different solvent conditions, and to observe ligand bindings of Mb. ESI-MS, complemented by circular dichroism and fluorescence spectroscopy, was used to study the mechanism of acid- and organic solvent-induced denaturation by probing the changes in the secondary and the tertiary structure of Mb. The results obtained show that complete disruption of the heme-protein interactions occurs when Mb is subjected to one of the following solution conditions: pH 3.2-3.6, or solution containing 20-30% acetonitrile or 40-50% methanol. Outside these ranges, Mb is present entirely in its native state (binding with a heme group) or as apomyoglobin (i.e. without the heme). Spectroscopic data demonstrate that the denaturation mechanism of Mb induced by acid may be significantly different from that by the organic solvent. Low pH reduces helices in Mb, whereas certain organic content level in solution results in the loss of the tertiary structure. ESI-MS conditions were established to observe the H(2)O- and CO-bound Mb complexes, respectively. H(2)O binding to metmyoglobin (17,585 Da), where the heme iron is in the ferric oxidation state, is observed in ESI-MS. CO binding to Mb (17,595 Da), on the other hand, can be only observed after the heme iron is reduced to the ferrous form. Therefore, ESI-MS combined with spectroscopic techniques provides a useful means for probing the formation of ligand-binding complexes and characterizing protein conformational changes.

Trema and Parasponia Hemoglobins Reveal Convergent Evolution of Oxygen Transport in Plants

All plants contain hemoglobins that fall into distinct phylogenetic classes. The subset of plants that carry out symbiotic nitrogen fixation expresses hemoglobins that scavenge and transport oxygen to bacterial symbiotes within root nodules. These "symbiotic" oxygen transport hemoglobins are distinct in structure and function from the nonoxygen transport ("nonsymbiotic") Hbs found in all plants. Hemoglobins found in two closely related plants present a paradox concerning hemoglobin structure and function. Parasponia andersonii is a nitrogen-fixing plant that expresses a symbiotic hemoglobin (ParaHb) characteristic of oxygen transport hemoglobins in having a pentacoordinate ferrous heme iron, moderate oxygen affinity, and a relatively rapid oxygen dissociation rate constant. A close relative that does not fix nitrogen, Trema tomentosa, expresses hemoglobin (TremaHb) sharing 93% amino acid identity to ParaHb, but its phylogeny predicts a typical nonsymbiotic hemoglobin with a hexacoordinate heme iron, high oxygen affinity, and slow oxygen dissociation rate constant. Here we characterize heme coordination and oxygen binding in TremaHb and ParaHb to investigate whether or not two hemoglobins with such high sequence similarity are actually so different in functional behavior. Our results indicate that the two proteins resemble nonsymbiotic hemoglobins in the ferric oxidation state and symbiotic hemoglobins in the ferrous oxidation state. They differ from each other only in oxygen affinity and oxygen dissociation rate constants, two factors key to their different functions. These results demonstrate distinct mechanisms for convergent evolution of oxygen transport in different phylogenetic classes of plant hemoglobins.

Trafficking of heme and porphyrins in metazoa.

Trafficking of heme and porphyrins in metazoa.

Mössbauer characterisation of Fe–polygalacturonate as a medicine for human anaemia: the effect of iron concentration

57Fe Mossbauer spectroscopy was used to study the effect of iron concentration on the oxidation state and microenvironments of iron in Fe–polygalacturonate compounds prepared by a novel method from pectin. The iron concentration of the coordination compounds was determined by inductively coupled plasma optical emission spectrometry analysis. The Mossbauer spectra of the studied compounds could be decomposed into three markedly different quadrupole doublets referring to three microenvironments. Two of these have ferrous and one has ferric oxidation state. In the applied concentration range the relative occurrence of the ferric component was found to increase considerably with iron concentration. At the same time, with increasing iron concentration the relative occurrence characteristic of the three components showed saturation behaviour up to the iron concentration at which for each pair of galacturonic acid units there is on average one iron atom in the system, which iron concentration value is interpreted as to be related to the complete fill up of certain iron complexation sites of the polygalacturonate chains.