Ferric State Research Articles

Electron self-exchange and self-amplified posttranslational modification in the hemoglobins from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002

Many heme proteins undergo covalent attachment of the heme group to a protein side chain. Such posttranslational modifications alter the thermodynamic and chemical properties of the holoprotein. Their importance in biological processes makes them attractive targets for mechanistic studies. We have proposed a reductively driven mechanism for the covalent heme attachment in the monomeric hemoglobins produced by the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 (GlbN) (Nothnagel et al. in J Biol Inorg Chem 16:539-552, 2011). These GlbNs coordinate the heme iron with two axial histidines, a feature that distinguishes them from most hemoglobins and conditions their redox properties. Here, we uncovered evidence for an electron exchange chain reaction leading to complete heme modification upon substoichiometric reduction of GlbN prepared in the ferric state. The GlbN electron self-exchange rate constants measured by NMR spectroscopy were on the order of 10(2)-10(3) M(-1) s(-1) and were consistent with the proposed autocatalytic process. NMR data on ferrous and ferric Synechococcus GlbN in solution indicated little dependence of the structure on the redox state of the iron or cross-link status of the heme group. This allowed the determination of lower bounds to the cross-exchange rate constants according to Marcus theory. The observations illustrate the ability of bishistidine hemoglobins to undergo facile interprotein electron transfer and the chemical relevance of such transfer for covalent heme attachment.

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.

Structural complex of sterol 14α-demethylase (CYP51) with 14α-methylenecyclopropyl-Δ7-24, 25-dihydrolanosterol

Sterol 14α-demethylase (CYP51) that catalyzes the removal of the 14α-methyl group from the sterol nucleus is an essential enzyme in sterol biosynthesis, a primary target for clinical and agricultural antifungal azoles and an emerging target for antitrypanosomal chemotherapy. Here, we present the crystal structure of Trypanosoma (T) brucei CYP51 in complex with the substrate analog 14α-methylenecyclopropyl-Δ7-24,25-dihydrolanosterol (MCP). This sterol binds tightly to all protozoan CYP51s and acts as a competitive inhibitor of F105-containing (plant-like) T. brucei and Leishmania (L) infantum orthologs, but it has a much stronger, mechanism-based inhibitory effect on I105-containing (animal/fungi-like) T. cruzi CYP51. Depicting substrate orientation in the conserved CYP51 binding cavity, the complex specifies the roles of the contact amino acid residues and sheds new light on CYP51 substrate specificity. It also provides an explanation for the effect of MCP on T. cruzi CYP51. Comparison with the ligand-free and azole-bound structures supports the notion of structural rigidity as the characteristic feature of the CYP51 substrate binding cavity, confirming the enzyme as an excellent candidate for structure-directed design of new drugs, including mechanism-based substrate analog inhibitors.

Role of the tertiary and quaternary structure in the formation of bis-histidyl adducts in cold-adapted hemoglobins

All tetrameric hemoglobins from Antarctic fish, including that from Trematomus bernacchii, HbTb form in the ferric state, promptly and distinctively from all the other tetrameric hemoglobins, a mixture of aquo-met at the α subunits and bis-histidyl adduct (hemichrome) at the β subunits. The role of the tertiary and quaternary structure in the hemichrome formation is unknown. Here we report the cloning, expression, purification, spectroscopic and computational characterization of the β-chain of HbTb (β-HbTb). Similarly to the human β-chains, β-HbTb self-assembles to form the homotetramer β4-HbTb; however, the latter quantitatively forms reversible ferric and ferrous bis-histidyl adducts, which are only partially present in the human tetramer (β4-HbA). A molecular dynamics study of the isolated β subunit of the two Hbs indicates that the ability to form hemichrome is an intrinsic feature of the chain; moreover, the greater propensity of β-HbTb to form the bis-histidyl adduct is probably linked to the higher flexibility of the CD loop region. On the bases of these experimental and computational results on the isolated chain, the influence of the quaternary structure on the stability of the endogenous ferrous and ferric hexa-coordination is also discussed.

New insights into the mechanism of electron transfer within flavohemoglobins: tunnelling pathways, packing density, thermodynamic and kinetic analyses

Flavohemoglobins (FlavoHb) are metalloenzymes catalyzing the reaction of nitric oxide dioxygenation. The iron cation of the heme group needs to be preliminarily reduced to the ferrous state to be catalytically competent. This reduction is triggered by a flavin adenine dinucleotide (FAD) prosthetic group which is localized in a distinct domain of the protein. In this paper we obtain new insights into the internal long range electron transfer (over ca. 12 Å) using a combination of experimental and computational approaches. Employing a time-resolved pulse radiolysis technique we report the first direct measurement of the FADH˙→ HemeFe(III) electron transfer rate. A rate constant of (6.8 ± 0.5) × 10(3) s(-1) is found. A large panel of computational approaches are used to provide the first estimation of the thermodynamic characteristics of the internal electron transfer step within flavoHb: both the driving force and the reorganization energy are estimated as a function of the protonated state of the flavin semi-quinone. We also report an analysis of the electron pathways involved in the tunnelling of the electron through the aqueous interface between the globin and the flavin domains.

Studies of iron(ii) and iron(iii) complexes with fac-N2O, cis-N2O2 and N2O3 donor ligands: models for the 2-His 1-carboxylate motif of non-heme iron monooxygenases

Enzymes in the oxygen-activating class of mononuclear non-heme iron oxygenases (MNOs) contain a highly conserved iron center facially ligated by two histidine nitrogen atoms and one carboxylate oxygen atom that leave one face of the metal center (three binding sites) open for coordination to cofactor, substrate, and/or dioxygen. A comparative family of [Fe(II/III)(N(2)O(n))(L)(4-n))](±x), n = 1-3, L = solvent or Cl(-), model complexes, based on a ligand series that supports a facially ligated N,N,O core that is then modified to contain either one or two additional carboxylate chelate arms, has been structurally and spectroscopically characterized. EPR studies demonstrate that the high-spin d(5) Fe(III)g = 4.3 signal becomes more symmetrical as the number of carboxylate ligands decreases across the series Fe(N(2)O(3)), Fe(N(2)O(2)), and Fe(N(2)O(1)), reflecting an increase in the E/D strain of these complexes as the number of exchangeable/solvent coordination sites increases, paralleling the enhanced distribution of electronic structures that contribute to the spectral line shape. The observed systematic variations in the Fe(II)-Fe(III) oxidation-reduction potentials illustrate the fundamental influence of differential carboxylate ligation. The trend towards lower reduction potential for the iron center across the [Fe(III)(N(2)O(1))Cl(3)](-), [Fe(III)(N(2)O(2))Cl(2)](-) and [Fe(III)(N(2)O(3))Cl](-) series is consistent with replacement of the chloride anions with the more strongly donating anionic O-donor carboxylate ligands that are expected to stabilize the oxidized ferric state. This electrochemical trend parallels the observed dioxygen sensitivity of the three ferrous complexes (Fe(II)(N(2)O(1)) < Fe(II)(N(2)O(2)) < Fe(II)(N(2)O(3))), which form μ-oxo bridged ferric species upon exposure to air or oxygen atom donor (OAD) molecules. The observed oxygen sensitivity is particularly interesting and discussed in the context of α-ketoglutarate-dependent MNO enzyme mechanisms.

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.

Probing a Complex of Cytochromecand Cardiolipin by Magnetic Circular Dichroism Spectroscopy: Implications for the Initial Events in Apoptosis

Oxidation of cardiolipin (CL) by its complex with cytochrome c (cyt c) plays a crucial role in triggering apoptosis. Through a combination of magnetic circular dichroism spectroscopy and potentiometric titrations, we show that both the ferric and ferrous forms of the heme group of a CL:cyt c complex exist as multiple conformers at a physiologically relevant pH of 7.4. For the ferric state, these conformers are His/Lys- and His/OH(-)-ligated. The ferrous state is predominantly high-spin and, most likely, His/-. Interconversion of the ferric and ferrous conformers is described by a single midpoint potential of -80 ± 9 mV vs SHE. These results suggest that CL oxidation in mitochondria could occur by the reaction of molecular oxygen with the ferrous CL:cyt c complex in addition to the well-described reaction of peroxides with the ferric form.

Chemical proprieties of the iron-quinone complex in mutated reaction centers of Rb. sphaeroides

We investigated type II bacterial photosynthetic reaction centers, which contain a quinone - iron complex (QA-Fe-QB) on their acceptor side. Under physiological conditions it was observed mainly in a reduced high spin state but its low spin ferrous states were also observed. Therefore, it was suggested that it might regulate the dynamical properties of the iron–quinone complex and the protonation and deprotonation events in its neighbourhood. In order to get insight into the molecular mechanism of the NHFe low spin state formation, we preformed Mössbauer studies of a wild type of Rb. sphaeroides and its two mutated forms. Our Mössbauer measurements show that the hydrophobicity of the QA binding site can be crucial for stabilization of the high spin ferrous state of NHFe.

Indirect Electrochemical Oxidation of Chlorophenols in Dilute Aqueous Solutions

The indirect electrochemical oxidation of chlorophenols was studied. The process involved the simultaneous reduction of water to produce protons that were used for the subsequent electrochemical reduction of oxygen to generate hydrogen peroxide. In the presence of ferrous ions, a Fenton’s reagent was produced. In the process, upon reaction with hydrogen peroxide, ferrous ions were converted to the ferric state and were then regenerated at the cathode to recover ferrous ion—an important reactant in Fenton’s reagent. A series of chlorophenols, including 4-mono-, 6-dichloro-, and 4-trichlorophenol, were studied. The degradation of monochlorophenols proceeded in decreasing order: 3-chlorophenol>2-chlorophenol∼4-chlorophenol; for dichloerophenols, the order was: 3, 5-dichlorophenol>3, 4-dichlorophenol>2, 3-dichlorophenol>2, 5-dichlorophenol>2, 4-dichlorophenol>2, 6-dichlorophenol;and for trichloro-phenols, the order followed: 2, 3, 5-trichlorophenol>2, 3, 6-trichlorophenol>2, 4, 5-trichlorophenol>2, 4, 6-trichlorophenol. The results indicated that the number and the relative locations (i.e., ortho, meta, or para) of chlorine substitution on the benzene ring played an important role in determining the rate and extent of chlorophenol oxidation. Regardless of the type of chlorine-substituted phenol, two major reaction pathways were responsible for the intermediate degradation of chlorophenols: hydroxyl radical attack (hydroxylation) and dechlorination. The detailed kinetics of the degradation of chlorophenols was studied in detail using 2-chlorophenol. Formic acid and acetic acid were the major end products that, in combination, contributed to 70% of the residual total organic carbon (TOC). Three other minor intermediates, 2-chloro-hydroquinone (2-Cl-HQ), 2-chloro-catechol (2-Cl-CA), and 4-hydroxyl,2,2’-dichlorodiphenyl ether (HDCDH), were observed; each contributed to a small percentage of the residual TOC.

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.

The role of a disulfide bridge in the stability and folding kinetics of Arabidopsis thaliana cytochrome c6A

Cytochrome c6A is a eukaryotic member of the Class I cytochrome c family possessing a high structural homology with photosynthetic cytochrome c6 from cyanobacteria, but structurally and functionally distinct through the presence of a disulfide bond and a heme mid-point redox potential of +71mV (vs normal hydrogen electrode). The disulfide bond is part of a loop insertion peptide that forms a cap-like structure on top of the core α-helical fold. We have investigated the contribution of the disulfide bond to thermodynamic stability and (un)folding kinetics in cytochrome c6A from Arabidopsis thaliana by making comparison with a photosynthetic cytochrome c6 from Phormidium laminosum and through a mutant in which the Cys residues have been replaced with Ser residues (C67/73S). We find that the disulfide bond makes a significant contribution to overall stability in both the ferric and ferrous heme states. Both cytochromes c6A and c6 fold rapidly at neutral pH through an on-pathway intermediate. The unfolding rate for the C67/73S variant is significantly increased indicating that the formation of this region occurs late in the folding pathway. We conclude that the disulfide bridge in cytochrome c6A acts as a conformational restraint in both the folding intermediate and native state of the protein and that it likely serves a structural rather than a previously proposed catalytic role.

Femtosecond UV Studies of the Electronic Relaxation Processes in Cytochrome c

We report on an experimental study with UV and visible ultrafast time-gated emission and transient absorption of the early photodynamics of horse heart Cytochrome c in both ferric and ferrous redox states. A clear separation in time and energy of tryptophan and haem emission is observed. Excitation of the haem via resonant energy transfer from the tryptophan residue is observed in the subsequent haem electronic relaxation. Different Trp-haem energy transfer time constants of the ferrous and ferric forms are obtained. An almost instantaneous relaxation to the lowest singlet excited state (corresponding to the so-called Q band) characterizes the earliest electronic dynamics of the haem, independent of excitation energy, while dark intermediate states govern the ground-state recovery. The information gathered in these two experiments and in the literature allows us to propose a simple scheme for the electronic relaxation leading to ligand dissociation.

Vibrational Coherence Spectroscopy of the Heme Domain in the CO-Sensing Transcriptional Activator CooA

Femtosecond vibrational coherence spectroscopy was used to investigate the low-frequency vibrational dynamics of the heme in the carbon monoxide oxidation activator protein (CooA) from the thermophilic anaerobic bacterium Carboxydothermus hydrogenoformans (Ch-CooA). Low frequency vibrational modes are important because they are excited by the ambient thermal bath (k(B)T = 200 cm(-1)) and participate in thermally activated barrier crossing events. However, such modes are nearly impossible to detect in the aqueous phase using traditional spectroscopic methods. Here, we present the low frequency coherence spectra of the ferric, ferrous, and CO-bound forms of Ch-CooA in order to compare the protein-induced heme distortions in its active and inactive states. Distortions take place predominantly along the coordinates of low-frequency modes because of their weak force constants, and such distortions are reflected in the intensity of the vibrational coherence signals. A strong mode near ~90 cm(-1) in the ferrous form of Ch-CooA is suggested to contain a large component of heme ruffling, consistent with the imidazole-bound ferrous heme crystal structure, which shows a significant protein-induced heme distortion along this coordinate. A mode observed at ~228 cm(-1) in the six-coordinate ferrous state is proposed to be the ν(Fe-His) stretching vibration. The observation of the Fe-His mode indicates that photolysis of the N-terminal α-amino axial ligand takes place. This is followed by a rapid (~8.5 ps) transient absorption recovery, analogous to methionine rebinding in photolyzed ferrous cytochrome c. We have also studied CO photolysis in CooA, which revealed very strong photoproduct state coherent oscillations. The observation of heme-CO photoproduct oscillations is unusual because most other heme systems have CO rebinding kinetics that are too slow to make the measurement possible. The low frequency coherence spectrum of the CO-bound form of Ch-CooA shows a strong vibration at ~230 cm(-1) that is broadened and up-shifted compared to the ν(Fe-His) of Rr-CooA (216 cm(-1)). We propose that the stronger Fe-His bond is related to the enhanced thermal stability of Ch-CooA and that there is a smaller (time dependent) tilt of the histidine ring with respect to the heme plane in Ch-CooA. The appearance of strong modes at ~48 cm(-1) in both the ferrous and CO-bound forms of Ch-CooA is consistent with coupling of the heme doming distortion to the photolysis reaction in both samples. Upon CO binding and protein activation, a heme mode near 112 ± 5 cm(-1) disappears, probably indicating a decreased heme saddling distortion. This reflects changes in the heme environment and geometry that must be associated with the conformational transition activating the DNA-binding domain. Protein-specific DNA binding to the CO-bound form of Ch-CooA was also investigated, and although the CO rebinding kinetics are significantly perturbed, there are negligible changes in the low-frequency vibrational spectrum of the heme.

Spectroscopic, catalytic and binding properties of Bacillus subtilis NO synthase-like protein: Comparison with other bacterial and mammalian NO synthases

Genome sequencing has shown the presence of genes coding for NO-synthase (NOS)-like proteins in bacteria. The roles and properties of these proteins remain unclear. UV–visible spectroscopy was used to characterize the recombinant NOS-like protein from Bacillus subtilis (bsNOS) in its ferric and ferrous states in the presence of various Fe III- and Fe II-heme-ligands and of a series of l-arginine ( l-arg) analogs. BsNOS exhibited several spectroscopic and binding properties in common with Bacillus anthracis NOS (baNOS) that were clearly different from those of tetrahydrobiopterin (H4B)-free mammalian NOS oxygenase domains (mNOS oxys) and of Staphylococcus aureus NOS (saNOS). Interestingly, bsNOS and baNOS that do not contain H4B exhibited properties much closer to those of H4B-containing mNOS oxys. Moreover, bsNOS was found to efficiently catalyze the oxidation of l-arginine into l-citrulline by H 2O 2, whereas H4B-free mNOS oxys exhibited low activities for this reaction.

Coordination modes of tyrosinate-ligated catalase-type heme enzymes: Magnetic circular dichroism studies of Plexaura homomalla allene oxide synthase, Mycobacterium avium ssp . paratuberculosis protein-2744c, and bovine liver catalase in their ferric and ferrous states

Bovine liver catalase (BLC), catalase-related allene oxide synthase (cAOS) from Plexaura homomalla, and a recently isolated protein from the cattle pathogen Mycobacterium avium ssp . paratuberculosis (MAP-2744c (MAP)) are all tyrosinate-ligated heme enzymes whose crystal structures have been reported. cAOS and MAP have low (< 20%) sequence similarity to, and significantly different catalytic functions from, BLC. cAOS transforms 8 R-hydroperoxy-eicosatetraenoic acid to an allene epoxide, whereas the MAP protein is a putative organic peroxide-dependent peroxidase. To elucidate factors influencing the functions of these and related heme proteins, we have investigated the heme iron coordination properties of these tyrosinate-ligated heme enzymes in their ferric and ferrous states using magnetic circular dichroism and UV–visible absorption spectroscopy. The MAP protein shows remarkable spectral similarities to cAOS and BLC in its native Fe(III) state, but clear differences from ferric proximal heme ligand His93Tyr Mb (myoglobin) mutant, which may be attributed to the presence of an Arg +- N ω-H···¯O-Tyr (proximal heme axial ligand) hydrogen bond in the first three heme proteins. Furthermore, the spectra of Fe(III)-CN¯, Fe(III)-NO, Fe(II)-NO (except for five-coordinate MAP), Fe(II)-CO, and Fe(II)-O 2 states of cAOS and MAP, but not H93Y Mb, are also similar to the corresponding six-coordinate complexes of BLC, suggesting that a tyrosinate (Tyr-O¯) is the heme axial ligand trans to the bound ligands in these complexes. The Arg +- N ω-H to ¯O-Tyr hydrogen bond would be expected to modulate the donor properties of the proximal tyrosinate oxyanion and, combined with the subtle differences in the catalytic site structures, affect the activities of cAOS, MAP and BLC.

Fe isotope fractionation caused by translocation of iron during growth of bean and oat as models of strategy I and II plants

The determination of the plant-induced Fe-isotopic fractionation is a promising tool to better quantify their role in the geochemical Fe cycle and possibly to identify the physiological mechanisms of Fe uptake and translocation in plants. Here we explore the isotope fractionation caused by translocation of Fe during growth of bean and oat as representatives of strategy I and II plants. Plants were grown on a nutrient solution supplemented with Fe(III)-EDTA and harvested at three different ages. We used the technique of multi-collector ICP-MS to resolve the small differences in the stable iron isotope compositions of plants. Total bean plants, regardless of their age, were found to be enriched in the light iron isotopes by −1.2‰ relative to the growth solution throughout. During growth plants internally redistributed isotopes where young leaves increasingly accumulated the lighter isotopes whereas older leaves and the total roots were simultaneously depleted in light iron isotopes. Oat plants were also enriched in the light iron isotopes but during growth the initial isotope ratio maintained in all organs at all growth stages. We conclude that isotope fractionation in bean as a representative of strategy I plants is a result of translocation or re-translocation processes. Furthermore we assume that both uptake and translocation of Fe in oat maintains the irons’ ferric state, or that Fe is always bound to high-mass ligands, so that isotope fractionation is virtually absent in these plants. However, in contrast to our previous study in which strategy II plants were grown on soil substrate, oat plants grown on Fe(III)-EDTA contain iron that enriches 54Fe by 0.5 permil over 56Fe. A possible explanation for the enrichment is the prevalence of a constitutive reductive uptake mechanism of iron in the nutrient solution used which is non-deficient in iron.

Amphitrite ornata Dehaloperoxidase (DHP): Investigations of Structural Factors That Influence the Mechanism of Halophenol Dehalogenation Using “Peroxidase-like” Myoglobin Mutants and “Myoglobin-like” DHP Mutants

Dehaloperoxidase (DHP), discovered in the marine terebellid polychaete Amphitrite ornata, is the first heme-containing globin with a peroxidase activity. The sequence and crystal structure of DHP argue that it evolved from an ancient O(2) transport and storage globin. Thus, DHP retains an oxygen carrier function but also has the ability to degrade halophenol toxicants in its living environment. Sperm whale myoglobin (Mb) in the ferric state has a peroxidase activity ∼10 times lower than that of DHP. The catalytic activity enhancement observed in DHP appears to have been generated mainly by subtle changes in the positions of the proximal and distal histidine residues that appeared during DHP evolution. Herein, we report investigations into the mechanism of action of DHP derived from examination of "peroxidase-like" Mb mutants and "Mb-like" DHP mutants. The dehalogenation ability of wild-type Mb is augmented in the peroxidase-like Mb mutants (F43H/H64L, G65T, and G65I Mb) but attenuated in the Mb-like T56G DHP variant. X-ray crystallographic data show that the distal His residues in G65T Mb and G65I are positioned ∼0.3 and ∼0.8 Å, respectively, farther from the heme iron compared to that in the wild-type protein. The H93K/T95H double mutant Mb with the proximal His shifted to the "DHP-like" position has an increased peroxidase activity. In addition, a better dehaloperoxidase (M86E DHP) was generated by introducing a negative charge near His89 to enhance the imidazolate character of the proximal His. Finally, only minimal differences in dehalogenation activities are seen among the exogenous ligand-free DHP, the acetate-bound DHP, and the distal site blocker L100F DHP mutant. Thus, we conclude that binding of halophenols in the internal binding site (i.e., distal cavity) is not essential for catalysis. This work provides a foundation for a new structure-function paradigm for peroxidases and for the molecular evolution of the dual-function enzyme DHP.

Hepcidin and iron‐mediated resistance to malaria

The survival of higher organisms requires constant vigilance against encroachment by potentially pathogenic microorganisms. Every portal of entry, and every potential niche, must be defended simultaneously against a wide spectrum of invaders. Each of these invaders – whether symbiotic or potentially pathogenic – has adopted their own cunning strategy to avoid detection and elimination, and to hijack the host's nutritional resources and metabolic apparatus to multiply and spread. In response to these threats humans have evolved a bewildering array of innate and adaptive defenses of which our knowledge is still only in its infancy. Among the innate defenses are mechanisms designed to withhold nutritional resources from the invaders and, of all the nutrients, iron is by far the most critical. It lies at the epicenter of most host‐pathogen battles for resource control; it is the equivalent of crude oil in global geopolitics. But 32% of the earth's crust is composed of iron (and 88% of its core) making it the most abundant element on the planet, so why should it be the subject of such blatant avarice? And what is the evidence that it is the focus of a fierce arms race between host and pathogen? The avarice is generally explained by the very low solubility of iron compounds in their usual oxidized ferric state and by the usefulness of iron's redox potentials, which make it a suitable co‐factor for numerous enzymes and pathways. The evidence for its centrality in host‐pathogen competition comes, inter alia , from the following sources: (i) most bacteria have made a disproportionate genetic investment in processes for iron acquisition; (ii) many of these genes are concentrated in islands of high pathogenicity, are important in defining niche specificity, and …

Influence of the Covalent Heme–Protein Bonds on the Redox Thermodynamics of Human Myeloperoxidase

Myeloperoxidase (MPO) is the most abundant neutrophil enzyme and catalyzes predominantly the two-electron oxidation of ubiquitous chloride to generate the potent bleaching hypochlorous acid, thus contributing to pathogen killing as well as inflammatory diseases. Its catalytic properties are closely related with unique posttranslational modifications of its prosthetic group. In MPO, modified heme b is covalently bound to the protein via two ester linkages and one sulfonium ion linkage with a strong impact on its (electronic) structure and biophysical and chemical properties. Here, the thermodynamics of the one-electron reduction of the ferric heme in wild-type recombinant MPO and variants with disrupted heme-protein bonds (M243V, E242Q, and D94V) have been investigated by thin-layer spectroelectrochemistry. It turns out that neither the oligomeric structure nor the N-terminal extension in recombinant MPO modifies the peculiar positive reduction potential (E°' = 0.001 V at 25 °C and pH 7.0) or the enthalpy or entropy of the Fe(III) to Fe(II) reduction. By contrast, upon disruption of the MPO-typical sulfonium ion linkage, the reduction potential is significantly lower (-0.182 V). The M243V mutant has an enthalpically stabilized ferric state, whereas its ferrous form is entropically favored because of the loss of rigidity of the distal H-bonding network. Exchange of an adjacent ester bond (E242Q) induced similar but less pronounced effects (E°' = -0.094 V), whereas in the D94V variant (E°' = -0.060 V), formation of the ferrous state is entropically disfavored. These findings are discussed with respect to the chlorination and bromination activity of the wild-type protein and the mutants.