Ferrous Binding Research Articles

Proton Transfer via Arginine with Suppressed pKa Mediates Catalysis by Gentisate and Salicylate Dioxygenase.

Gentisate and salicylate 1,2-dioxygenases (GDO and SDO) facilitate aerobic degradation of aromatic rings by inserting both atoms of dioxygen into their substrates, thereby participating in global carbon cycling. The role of acid-base catalysts in the reaction cycles of these enzymes is debatable. We present evidence of the participation of a proton shuffler during catalysis by GDO and SDO. The pH dependence of Michaelis-Menten parameters demonstrates that a single proton transfer is mandatory for the catalysis. Measurements at variable temperatures and pHs were used to determine the standard enthalpy of ionization (ΔHion°) of 51 kJ/mol for the proton transfer event. Although the observed apparent pKa in the range of 6.0-7.0 for substrates of both enzymes is highly suggestive of a histidine residue, ΔHion° establishes an arginine residue as the likely proton source, providing phylogenetic relevance for this strictly conserved residue in the GDO family. We propose that the atypical 3-histidine ferrous binding scaffold of GDOs contributes to the suppression of arginine pKa and provides support for this argument by employing a 2-histidine-1-carboxylate variant of the enzyme that exhibits elevated pKa. A reaction mechanism considering the role of the proton source in stabilizing key reaction intermediates is proposed.

Preparation, characterization, and bioavailability evaluation of antioxidant phosvitin peptide-ferrous complex.

Iron deficiency anemia (IDA) is one of the commonest global nutritional deficiency diseases, and the low bioavailability of iron is a key contributing factor. The peptide-iron complex could be used as a novel iron supplement to improve iron bioavailability. In this study, antioxidant low molecular weight (<3 kDa) phosvitin peptide (named PP-4) was separated to prepare a phosvitin peptide-ferrous complex (named PP-4-Fe); then the structural conformation of PP-4-Fe was characterized and its bioavailability by in vitro digestion was evaluated. The results showed that PP-4 had good ferrous-binding activity with 96.14 ± 2.86 μg Fe2+ mg-1 , and had a strong antioxidant effect with 995.61 ± 79.75 μmol TE mg-1 in 2,2'-azinobis'3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 62.3 ± 3.95 μmol FeSO4 mg-1 in ferric ion reducing antioxidant power (FRAP). After ferrous binding, the FRAP activity of PP-4-Fe, enhanced by 1.8 times, formed a more ordered structure with an increase in α-helix and decrease in γ-random coil. The ferrous binding sites of PP-4 involved were the amino, carboxyl, imidazole, and phosphate groups. The PP-4-Fe complex displayed excellent gastrointestinal stability and antioxidant effects during digestion. The iron dialysis percentage of PP-4-Fe was 74.59% ± 0.68%, and increased to 81.10% ± 0.89% with the addition of 0.25 times vitamin C (VC). This indicated that PP-4-Fe displayed excellent bioavailability and VC in sufficient quantities had a synergistic effect on improving bioavailability. This study demonstrated that antioxidant phosvitin peptide was an efficient delivery system to protect ferrous ions and suggested that the phosvitin peptide-ferrous complex has strong potential as a ferrous supplement. © 2023 Society of Chemical Industry.

Effects of Fe2+/Fe3+ Binding to Human Frataxin and Its D122Y Variant, as Revealed by Site-Directed Spin Labeling (SDSL) EPR Complemented by Fluorescence and Circular Dichroism Spectroscopies.

Frataxin is a highly conserved protein whose deficiency results in the neurodegenerative disease Friederich’s ataxia. Frataxin’s actual physiological function has been debated for a long time without reaching a general agreement; however, it is commonly accepted that the protein is involved in the biosynthetic iron-sulphur cluster (ISC) machinery, and several authors have pointed out that it also participates in iron homeostasis. In this work, we use site-directed spin labeling coupled to electron paramagnetic resonance (SDSL EPR) to add new information on the effects of ferric and ferrous iron binding on the properties of human frataxin in vitro. Using SDSL EPR and relating the results to fluorescence experiments commonly performed to study iron binding to FXN, we produced evidence that ferric iron causes reversible aggregation without preferred interfaces in a concentration-dependent fashion, starting at relatively low concentrations (micromolar range), whereas ferrous iron binds without inducing aggregation. Moreover, our experiments show that the ferrous binding does not lead to changes of protein conformation. The data reported in this study reveal that the currently reported binding stoichiometries should be taken with caution. The use of a spin label resistant to reduction, as well as the comparison of the binding effect of Fe2+ in wild type and in the pathological D122Y variant of frataxin, allowed us to characterize the Fe2+ binding properties of different protein sites and highlight the effect of the D122Y substitution on the surrounding residues. We suggest that both Fe2+ and Fe3+ might play a relevant role in the context of the proposed FXN physiological functions.

Identification of potential indoleamine 2, 3-dioxygenase 1 (IDO1) inhibitors by an FBG-based 3D QSAR pharmacophore model

As a rate-limiting enzyme in the major pathway of l-tryptophan catabolism, indoleamine 2, 3-dioxygenase 1 (IDO1) has been regarded as a potential target for the treatment of a variety of diseases. To identify the key molecular features and extend the structural variety of IDO1 inhibitors, a ferrous binding group (FBG)-based 3D-QSAR pharmacophore model was constructed, validated and used for virtual screening herein. The Specs database was initially filtered based on drug-like rules, and was subsequently screened by the pharmacophore model. Molecular docking simulations and similarity analysis were then followed to prioritize twenty compounds for biological tests. Among them, compound VS-13 exhibited moderate inhibitory activity against IDO1, and could be subjected to further structure-activity relationship studies and structural optimization.

The extracellular proteome of two Bifidobacterium species reveals different adaptation strategies to low iron conditions

BackgroundBifidobacteria are among the first anaerobic bacteria colonizing the gut. Bifidobacteria require iron for growth and their iron-sequestration mechanisms are important for their fitness and possibly inhibit enteropathogens. Here we used combined genomic and proteomic analyses to characterize adaptations to low iron conditions of B. kashiwanohense PV20-2 and B. pseudolongum PV8-2, 2 strains isolated from the feces of iron-deficient African infants and selected for their high iron-sequestering ability.ResultsAnalyses of the genome contents revealed evolutionary adaptation to low iron conditions. A ferric and a ferrous iron operon encoding binding proteins and transporters were found in both strains. Remarkably, the ferric iron operon of B. pseudolongum PV8-2 is not found in other B. pseudolongum strains and likely acquired via horizontal gene transfer. The genome B. kashiwanohense PV20-2 harbors a unique region encoding genes putatively involved in siderophore production.Additionally, the secretomes of the two strains grown under low-iron conditions were analyzed using a combined genomic-proteomic approach. A ferric iron transporter was found in the secretome of B. pseudolongum PV8-2, while ferrous binding proteins were detected in the secretome of B. kashiwanohense PV20-2, suggesting different strategies to take up iron in the strains. In addition, proteins such as elongation factors, a glyceraldehyde-3-phosphate dehydrogenase, and the stress proteins GroEL and DnaK were identified in both secretomes. These proteins have been previously associated with adhesion of lactobacilli to epithelial cells.ConclusionAnalyses of the genome and secretome of B. kashiwanohense PV20-2 and B. pseudolongum PV8-2 revealed different adaptations to low iron conditions and identified extracellular proteins for iron transport. The identified extracellular proteins might be involved in competition for iron in the gastrointestinal tract.

Optimization of Enzymatic Hydrolysis for Producing Ferrous Binding Peptides from Horse Mackerel (Trachurus japonicus) Processing Byproducts

Optimization of Enzymatic Hydrolysis for Producing Ferrous Binding Peptides from Horse Mackerel (Trachurus japonicus) Processing Byproducts

Lipoprotein MtsA of MtsABC in Streptococcus pyogenes primarily binds ferrous ion with bicarbonate as a synergistic anion

Lipoprotein MtsA is a critical component of MtsABC responsible for iron binding and transport in the Gram-positive bacterium Streptococcus pyogenes. The present collective experimental data establish that Fe 2+ is the primary binding ion for MtsA under optimal physiologically relevant conditions. The binding affinities of MtsA to metal ions are Fe 2+ > Fe 3+ >Cu 2+ > Mn 2+ > Zn 2+. We report for the first time that bicarbonate is required as a synergistic anion for stable ferrous binding to MtsA, similar to the iron binding in human transferrin. This work provides valuable information, which helps to understand iron metabolism in bacteria, and creates a basis for developing strategies to suppress bacterial infection.

Diel variations in iron speciation in northern Australian shelf waters

Iron in northern Australian shelf waters was found predominantly in fine (< 0.2 μm) 8-hydroxyquinoline-reactive form or in larger (1.0 μm) particulate form. Partitioning into the larger size fraction dominated during a period of high turbidity following a cyclonic resuspension event. Only a small component of the iron pool reacts rapidly with the strong ferrous binding agent ferrozine. The concentration of the ferrozine-reactive component is strongly dependent on light intensity with maximum concentration observed at peak light intensity. The close correlation between ferrozine-reactive iron concentration and light intensity suggests a fine balance between Fe(III) reduction and Fe(II) oxidation with the steady state concentration observed being strongly influenced by light induced changes in redox kinetics. An observed lack of association between particulate iron concentrations and the concentration of ferrozine-reactive iron suggests that the soluble rather than the particulate iron pool is most influenced by light. The chromophore may be an Fe(III)-organic complex with the strong iron binding ligand that is now recognised to be present in seawater.

Uptake of iron byGeotrichum candidum, a non-siderophore producer

Geotrichum candidum (isolate 1–9) pathogenic on citrus fruits, appears to lack siderophore production. Iron uptake byG. candidum is mediated by two distinct iron-regulated, energy-and temperature-dependent transport systems that require sulfhydryl groups. One system exhibits specificity for either ferric or ferrous iron, whereas the other exhibits specificity for ferrioxamine-B-mediated iron uptake and presumably other hydroxamate siderophores. Radioactive iron uptake from59FeCl3 showed an optimum at pH 6 and 35° C, and Michaelis-Menten kinetics (apparentKm = 3 μm,Vmax = 0.054 nmol · mg−1 · min−1). The maximal rate of Fe2+ uptake was higher than Fe3+ (Vmax = 0.25 nmol · mg−1 · min−1) but theKm was identical. Reduction of ferric to ferrous iron prior to transport could not be detected. The ferrioxamine B system exhibits an optimum at pH 6 and 40° C and saturation kinetics (Km = 2 μM,Vmax = 0.22 nmol · mg−1 · min−1). The two systems were distinguished as two separate entities by negative reciprocal competition, and on the basis of differential response to temperature and phenazine methosulfate. Mossbauer studies revealed that cells fed with either57FeCl3 or57FeCl2 accumulated unknown ferric and ferrous binding metabolites.