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Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O2) concentrations. Here, we have reprogrammed cellular hypoxia (low O2) signaling via gas tunnel engineering of prolyl hydroxylase 2 (PHD2), a non‐heme iron dependent O2 sensor. Using computational modeling and protein engineering techniques, we identify a gas tunnel and critical residues therein that limit the flow of O2 to PHD2's catalytic core. We show that systematic modification of these residues can open the constriction topology of PHD2's gas tunnel. Using kinetic stopped‐flow measurements with NO as a surrogate diatomic gas, we demonstrate up to 3.5‐fold enhancement in its association rate to the iron center of tunnel‐engineered mutants. Our most effectively designed mutant displays 9‐fold enhanced catalytic efficiency (kcat/KM=830±40 M−1 s−1) in hydroxylating a peptide mimic of hypoxia inducible transcription factor HIF‐1α, as compared to WT PHD2 (kcat/KM=90±9 M−1 s−1). Furthermore, transfection of plasmids that express designed PHD2 mutants in HEK‐293T mammalian cells reveal significant reduction of HIF‐1α and downstream hypoxia response transcripts under hypoxic conditions of 1 % O2. Overall, these studies highlight activation of PHD2 as a new pathway to reprogram hypoxia responses and HIF signaling in cells. [ABSTRACT FROM AUTHOR]

Non-heme iron-dependent sulfoxide/selenoxide synthases (NHISS) constitute a unique metalloenzyme class capable of installing a C–S/Se bond onto histidine to generate thio/selenoimidazole antioxidants, such as ergothioneine and ovothiol. These natural products are increasingly recognized for their health benefits. Among associated ergothioneine-biosynthetic enzymes, type IV EgtBs stand out, as they exhibit low sequence similarity with other EgtB subfamilies due to their recent divergence from the ovothiol-biosynthetic enzyme OvoA. Herein, we present crystal structures of two representative EgtB-IV enzymes, offering insights into the basis for this evolutionary convergence and enhancing our understanding of NHISS active site organization more broadly. The ability to interpret how key residues modulate substrate specificity and regioselectivity has implications for downstream identification of divergent reactivity within the NHISS family. To this end, we identify a previously unclassified clade of OvoA-like enzymes with a seemingly hybrid set of characteristics, suggesting they may represent an evolutionary intermediate between OvoA and EgtB-IV. [Display omitted] • Hercynine-bound structures offer insights into the convergent evolution of EgtB-IV • EgtB-IV cannot be converted into OvoA through simple active site mutations • An active site lid-loop dictates cysteine specificity in OvoA-like enzymes • A potential evolutionary intermediate between OvoA and EgtB-IV is presented Ireland et al. report X-ray crystal structures of type IV EgtB enzymes involved in ergothioneine biosynthesis. The results shed light on the convergent evolution of sulfoxide synthase EgtB-IV, which features closest homology to ovothiol-biosynthetic OvoA. Additionally, they clarify the structural basis for EgtB-IV's specificity for cysteine as the S-donor. [ABSTRACT FROM AUTHOR]

The work presented here investigates whether the TpMesFe+ moiety, which had been previously found to mediate the dioxygenation of cysteine and cysteamine, can do the same with simple thiols, which are not functionalized by an amine group. Accordingly, the complexes TpMesFeSR (R=Ph, iPr) were synthesized and after full characterization exposed to O2. They were found to react unselectively to yield sulfinates, disulfides, and thiosulfones as products. The sulfinate complex TpMesFeO2S‐p‐Tol was prepared independently and found to be air‐stable, suggesting that the additional products are not formed via overoxidation or subsequent reactions of coordinated sulfinate product. To investigate whether the larger reaction space, opened up due to the missing amine donor is responsible, a Tp‐ligand functionalized by a pyridyl donor was employed. A corresponding Tp‐iron(II) chlorido complex was prepared and converted through salt metathesis reactions into the respective thiolate analogues, which were fully characterized and structurally authenticated. However, subsequent O2 reactivity studies did not show an improved selectivity. The reason may lie in a more open reaction pocket in combination with an electronic situation which is less ideal than in case of TpMesFe(cysteamine). [ABSTRACT FROM AUTHOR]

As one of the most common iron-chelating agents, deferoxamine (DFO) rapidly chelates iron in the body. Moreover, it does not compete for the iron characteristic of hemoglobin in the blood cells, which is common in the clinical treatment of iron poisoning. Iron is a trace element necessary to maintain organism normal life activities. Iron deficiency can lead to anemia, whereas iron overload can cause elevated levels of cellular oxidative stress and cell damage. As a consequence, detection of the iron content in tissues and blood is of great significance. The traditional techniques for detecting the iron content include inductively coupled plasma-mass spectrometry and atomic absorption spectrometry, which cannot be used for imaging purposes. Laser ablation-ICP-MS and synchrotron radiation micro-X-ray fluorescence can map the concentration and distribution of iron in tissues. However, these methods can only be used to measure the total iron levels in blood or tissues. In recent years, due to the deepening understanding of iron metabolism, diseases related to iron overload have attracted increasing attention. Therefore, we took advantage of the properties of DFO in terms of chelating iron and investigated different sampling times following DFO injection in the tail vein of mice. We used mass spectrometry imaging (MSI) technology to detect the DFO and ferrioxamine content in the blood and different tissues to indirectly characterize the non-heme iron content. [ABSTRACT FROM AUTHOR]

Hemoglobin hydrolysate is derived from the enzymatic degradation of hemoglobin. This work aimed to evaluate whether hemoglobin hydrolysate promotes the absorption of non-heme iron and the safety of absorbed iron in mice by analyzing the iron binding content, iron circulation, and liver homeostasis. We found that hemoglobin hydrolysate promoted the absorption of non-heme iron with high efficiency in duodenum by spontaneously binding non-heme iron during digestion, and increased hepatic iron content by up-regulating divalent metal transporter 1, zinc transporter 14, but hepatic iron content only increased at 3 weeks. Duodenal iron entered the blood through ferroportin without restriction at 3 weeks, and excessive iron entered the liver and then affected the hepatocyte membranes permeability and lipid synthesis through oxidative stress. With the prolongation of dietary intervention, the up-regulated hepcidin acted on the ferroportin to restrict excess iron from entering the blood, and then the hepatic homeostasis recovered. In addition, hemoglobin hydrolysate enhanced the hepatic antioxidant capacity. Taken together, hemoglobin hydrolysate has a strong ability to promote the absorption of non-heme iron in vivo, and the absorbed iron is relatively safe due to the regulation of hepcidin.

Metalloenzymes catalyze a wide range of chemical transformations, with the active site residues playing a key role in modulating chemical reactivity and selectivity. Unlike smaller synthetic catalysts, a metalloenzyme active site is embedded in a larger protein, which makes interrogation of electronic properties and geometric features with quantum mechanical calculations challenging. Here we implement the ability to fetch crystallographic structures from the Protein Data Bank and analyze the metal binding sites in the program molSimplify. We show the usefulness of the newly created protein3D class to extract the local environment around non‐heme iron enzymes containing a two histidine motif and prepare 372 structures for quantum mechanical calculations. Our implementation of protein3D serves to expand the range of systems molSimplify can be used to analyze and will enable high‐throughput study of metal‐containing active sites in proteins. [ABSTRACT FROM AUTHOR]

AbstractContext Metformin (Met) has a protective effect against cardiac ischemia and reperfusion (I/R) injury.Objective This study uncovered the Met effect on ferroptosis in cardiac I/R.Materials and methods Sprague-Dawley rats underwent cardiac I/R treatment (ischaemia 30 min; reperfusion 24 h) (I/R group), and administered intravenously with Met (200 mg/kg) (I/R + Met group). Haematoxylin–eosin staining, Prussian blue staining, immunohistochemistry and transmission electron microscope were conducted on cardiac tissues. H9c2 cells underwent oxygen-glucose deprivation/reoxygenation (OGD/R group) and treated by Met (0.1 mM) (OGD/R + Met group). Adenosine monophosphate-activated protein kinase α (AMPKα) siRNA was transfected into OGD/R-induced H9c2 cells. Cell counting kit-8 (CCK-8) assay, dichloro-dihydro-fluorescein diacetate (DCFH-DA) and JC-1 staining were conducted on H9c2 cells. Ferroptosis-related indicators and gene expression were detected by enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot.Results In cardiac I/R rat, Met decreased heart and serum MDA, cardiac and serum non-heme iron, and serum CK-MB and LDH (inhibition rate: 50.0%, 48.8%, 47.6%, 29.5%, 30.6% and 34.7%, respectively), relieved cardiac tissue ferroptosis and mitochondria damage, increased fraction shortening and ejection fraction (157.5% and 146.2% on day 28, respectively), up-regulated AMPKα and down-regulated NOX4 in cardiac tissues. In OGD/R-induced H9c2 cells, Met (0.1 mM) increased cell viability (promotion rate: 170.0%), decreased non-heme iron and MDA (inhibition rate: 30.1% and 47.9%, respectively), relieved ferroptosis, up-regulated AMPKα and down-regulated NOX4. AMPKα silencing abrogated these effects of Met on the OGD/R-induced H9c2 cells.Discussion and conclusions Met shows effectiveness in relieving ferroptosis in cardiac I/R. In the future, Met may be an effective drug for relieving ferroptosis in cardiac I/R patients clinically.

Carotenoid cleavage dioxygenases (CCDs) constitute a superfamily of enzymes that are found in all domains of life where they play key roles in the metabolism of carotenoids and apocarotenoids as well as certain phenylpropanoids such as resveratrol. Interest in these enzymes stems not only from their biological importance but also from their remarkable catalytic properties including their regioselectivity, their ability to accommodate diverse substrates, and the additional activities (e.g., isomerase) that some of these enzyme possess. X-ray crystallography is a key experimental approach that has allowed detailed investigation into the structural basis behind the interesting biochemical features of these enzymes. Here, we describe approaches used by our lab that have proven successful in generating single crystals of these enzymes in resting or ligand-bound states for high-resolution X-ray diffraction analysis.

A bulky, tridentate phenolate ligand (ImPh2NNOtBu) was used to synthesise the first example of a mononuclear, facial, N,N,O‐bound iron(II) benzoylformate complex, [Fe(ImPh2NNOtBu)(BF)] (2). The X‐ray crystal structure of 2 reveals that the iron centre is pentacoordinate (τ=0.5), with a vacant site located cis to the bidentate BF ligand. The Mössbauer parameters of 2 are consistent with high‐spin iron(II), and are very close to those reported for α‐ketoglutarate‐bound non‐heme iron enzyme active sites. According to NMR and UV‐vis spectroscopies, the structural integrity of 2 is retained in both coordinating and non‐coordinating solvents. Cyclic voltammetry studies show that the iron centre has a very low oxidation potential and is more prone to electrochemical oxidation than the redox‐active phenolate ligand. Complex 2 reacts with NO to form a S=3/2 {FeNO}7 adduct in which NO binds directly to the iron centre, according to EPR, UV‐vis, IR spectroscopies and DFT analysis. Upon O2 exposure, 2 undergoes oxidative decarboxylation to form a diiron(III) benzoate complex, [Fe2(ImPh2NNOtBu)2(μ2‐OBz)(μ2‐OH)2]+ (3). A small amount of hydroxylated ligand was also observed by ESI‐MS, hinting at the formation of a high‐valent iron(IV)‐oxo intermediate. Initial reactivity studies show that 2 is capable of oxygen atom transfer reactivity with O2, converting methyl(p‐tolyl)sulfide to sulfoxide. [ABSTRACT FROM AUTHOR]

Metformin (Met) has a protective effect against cardiac ischemia and reperfusion (I/R) injury. This study uncovered the Met effect on ferroptosis in cardiac I/R. Sprague-Dawley rats underwent cardiac I/R treatment (ischaemia 30 min; reperfusion 24 h) (I/R group), and administered intravenously with Met (200 mg/kg) (I/R + Met group). Haematoxylin–eosin staining, Prussian blue staining, immunohistochemistry and transmission electron microscope were conducted on cardiac tissues. H9c2 cells underwent oxygen-glucose deprivation/reoxygenation (OGD/R group) and treated by Met (0.1 mM) (OGD/R + Met group). Adenosine monophosphate-activated protein kinase α (AMPKα) siRNA was transfected into OGD/R-induced H9c2 cells. Cell counting kit-8 (CCK-8) assay, dichloro-dihydro-fluorescein diacetate (DCFH-DA) and JC-1 staining were conducted on H9c2 cells. Ferroptosis-related indicators and gene expression were detected by enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. In cardiac I/R rat, Met decreased heart and serum MDA, cardiac and serum non-heme iron, and serum CK-MB and LDH (inhibition rate: 50.0%, 48.8%, 47.6%, 29.5%, 30.6% and 34.7%, respectively), relieved cardiac tissue ferroptosis and mitochondria damage, increased fraction shortening and ejection fraction (157.5% and 146.2% on day 28, respectively), up-regulated AMPKα and down-regulated NOX4 in cardiac tissues. In OGD/R-induced H9c2 cells, Met (0.1 mM) increased cell viability (promotion rate: 170.0%), decreased non-heme iron and MDA (inhibition rate: 30.1% and 47.9%, respectively), relieved ferroptosis, up-regulated AMPKα and down-regulated NOX4. AMPKα silencing abrogated these effects of Met on the OGD/R-induced H9c2 cells. Met shows effectiveness in relieving ferroptosis in cardiac I/R. In the future, Met may be an effective drug for relieving ferroptosis in cardiac I/R patients clinically. [ABSTRACT FROM AUTHOR]

The final stage of Fe deficiency is Fe deficiency anaemia, with repercussions for human health, especially in children under 5 years of age. Studies conducted in Brazilian public daycare centres show high prevalence of anaemia. The present study aims to evaluate the availability of Fe in the meals of the Municipal Centers of Early Childhood Education in Maceió. The experimental design comprises selection of algorithms, menu evaluation, calculation of the estimates, comparison between the estimates obtained and the recommendations, and analysis of correlation between meal constituents, and of the concordance between the absorbable Fe estimates. Four algorithms were selected and a monthly menu consisting of 22 d. The correlation analysis showed a moderate positive correlation to animal tissue (AT) v. non-heme iron (r = 0·42; P = 0·04), and negative to AT v. Ca (r = −0·54; P = 0·09) and Ca v. phytates (r = −0·46, P = 0·03). Estimates of absorbable Fe ranged from 0·23 to 0·44 mg/d. The amount of Fe available, unlike the total amount of Fe offered, does not meet the nutritional recommendations on most school days. The Bland–Altman analysis indicated that the Monsen and Balinfty and Rickard et al. showed greater agreement. The results confirm the need to adopt strategies to increase the availability of Fe in school meals. [ABSTRACT FROM AUTHOR]

Carotenoid cleavage dioxygenases (CCDs) comprise a superfamily of mononuclear non-heme iron proteins that catalyze the oxygenolytic fission of alkene bonds in carotenoids to generate apocarotenoid products. Some of these enzymes exhibit additional activities such as carbon skeleton rearrangement and trans-cis isomerization. The group also includes a subfamily of enzymes that split the interphenyl alkene bond in molecules such as resveratrol and lignostilbene. CCDs are involved in numerous biological processes ranging from production of light-sensing chromophores to degradation of lignin derivatives in pulping waste sludge. These enzymes exhibit unique features that distinguish them from other families of non-heme iron enzymes. The distinctive properties and biological importance of CCDs have stimulated interest in their modes of catalysis. Recent structural, spectroscopic, and computational studies have helped clarify mechanistic aspects of CCD catalysis. Here, we review these findings emphasizing common and unique properties of CCDs that enable their variable substrate specificity and regioselectivity. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.

Abstract Background Heme and non-heme irons are two forms of iron in the diet. Few studies have evaluated the association between heme iron intake and the risk of gastric cancer (GC). We aimed to investigate the association between heme, non-heme and total iron intake and risk of GC in Iran. Methods In a hospital-based case–control study, nutritionists interviewed 178 pathologically confirmed GC patients and 276 controls using a valid Diet History Questionnaire. Multiple logistic regression model was used to estimate Odds Ratios (OR) and 95% Confidence Intervals (CIs) for iron intake and risk of GC. Results Subjects in the highest tertile of total iron intake were 46% less likely to get GC than those in the lowest (OR = 0.54, 95% CI: 0.32–0.92), however, the associations were not significant for intake of heme and non-heme iron. The risk of GC in the highest tertile of total meat intake was 2.51 times higher than the lowest. We found significant associations between GC and chicken (OR = 2.95; 95% CI: 1.66–5.22) and fish intake (OR = 1.89; 95% CI: 1.09–3.27), However, we found no associations between the risk of GC and intake of red meat, salted fish, and liver. Conclusion Total iron intake was associated with a lower risk of GC which could be partly due to the high prevalence of anemia in Iran. Although, we could not find any significant association between the risk of GC and the intake of heme and non-hem iron among the Iranian population.

The author will outline the research history of the main issues addressed in this paper. The author has worked on this research himself. XDH, which is responsible for purine degradation, is present in various organisms. However, conversion to XO only occurs in mammals. The molecular mechanism of this conversion was elucidated in this study. The physiological and pathological significance of this conversion is presented. Finally, enzyme inhibitors were successfully developed, two of which are used as therapeutic agents for gout. Their wide application potential is also discussed. [ABSTRACT FROM AUTHOR]

Carotenoid cleavage dioxygenases (CCDs) use a nonheme Fe(II) cofactor to split alkene bonds of carotenoid and stilbenoid substrates. The iron centers of CCDs are typically five-coordinate in their resting states, with solvent occupying an exchangeable site. The involvement of this iron-bound solvent in CCD catalysis has not been experimentally addressed, but computational studies suggest two possible roles. 1) Solvent dissociation provides a coordination site for O2, or 2) solvent remains bound to iron but changes its equilibrium position to allow O2 binding and potentially acts as a proton source. To test these predictions, we investigated isotope effects (H2O versus D2O) on two stilbenoid-cleaving CCDs, Novosphingobium aromaticivorans oxygenase 2 (NOV2) and Neurospora crassa carotenoid oxygenase 1 (CAO1), using piceatannol as a substrate. NOV2 exhibited an inverse isotope effect (k H/k D ∼ 0.6) in an air-saturated buffer, suggesting that solvent dissociates from iron during the catalytic cycle. By contrast, CAO1 displayed a normal isotope effect (k H/k D ∼ 1.7), suggesting proton transfer in the rate-limiting step. X-ray absorption spectroscopy on NOV2 and CAO1 indicated that the protonation states of the iron ligands are unchanged within pH 6.5-8.5 and that the Fe(II)-aquo bond is minimally altered by substrate binding. We pinpointed the origin of the differential kinetic behaviors of NOV2 and CAO1 to a single amino acid difference near the solvent-binding site of iron, and X-ray crystallography revealed that the substitution alters binding of diffusible ligands to the iron center. We conclude that solvent-iron dissociation and proton transfer are both associated with the CCD catalytic mechanism.

Thiol dioxygenases are a subset of non-heme mononuclear iron oxygenases that catalyze the O2-dependent oxidation of thiol-bearing substrates to yield sulfinic acid products. Cysteine dioxygenase (CDO) and 3-mercaptopropionic acid (3MPA) dioxygenase (MDO) are the most extensively characterized members of this enzyme family. As with many non-heme mononuclear iron oxidase/oxygenases, CDO and MDO exhibit an obligate-ordered addition of organic substrate before dioxygen. As this substrate-gated O2-reactivity extends to the oxygen-surrogate, nitric oxide (NO), EPR spectroscopy has long been used to interrogate the [substrate:NO:enzyme] ternary complex. In principle, these studies can be extrapolated to provide information about transient iron-oxo intermediates produced during catalytic turnover with dioxygen. In this work, we demonstrate that cyanide mimics the native thiol-substrate in ordered-addition experiments with MDO cloned from Azotobacter vinelandii (AvMDO). Following treatment of the catalytically active Fe(II)-AvMDO with excess cyanide, addition of NO yields a low-spin (S = 1/2) (CN/NO)-Fe-complex. Continuous wave and pulsed X-band EPR characterization of this complex produced in wild-type and H157N variant AvMDO reveal multiple nuclear hyperfine features diagnostic of interactions within the first- and outer-coordination sphere of the enzymatic Fe-site. Spectroscopically validated computational models indicate simultaneous coordination of two cyanide ligands replaces the bidentate (thiol and carboxylate) coordination of 3MPA allowing for NO-binding at the catalytically relevant O2-binding site. This promiscuous substrate-gated reactivity of AvMDO with NO provides an instructive counterpoint to the high substrate-specificity exhibited by mammalian CDO for l-cysteine. [ABSTRACT FROM AUTHOR]

Background: Heme and non-heme irons are two forms of iron in the diet. Few studies have evaluated the association between heme iron intake and the risk of gastric cancer (GC). We aimed to investigate the association between heme, non-heme and total iron intake and risk of GC in Iran. Methods: In a hospital-based case–control study, nutritionists interviewed 178 pathologically confirmed GC patients and 276 controls using a valid Diet History Questionnaire. Multiple logistic regression model was used to estimate Odds Ratios (OR) and 95% Confidence Intervals (CIs) for iron intake and risk of GC. Results: Subjects in the highest tertile of total iron intake were 46% less likely to get GC than those in the lowest (OR = 0.54, 95% CI: 0.32–0.92), however, the associations were not significant for intake of heme and non-heme iron. The risk of GC in the highest tertile of total meat intake was 2.51 times higher than the lowest. We found significant associations between GC and chicken (OR = 2.95; 95% CI: 1.66–5.22) and fish intake (OR = 1.89; 95% CI: 1.09–3.27), However, we found no associations between the risk of GC and intake of red meat, salted fish, and liver. Conclusion: Total iron intake was associated with a lower risk of GC which could be partly due to the high prevalence of anemia in Iran. Although, we could not find any significant association between the risk of GC and the intake of heme and non-hem iron among the Iranian population. [ABSTRACT FROM AUTHOR]

Highly selective C-H functionalization remains an ongoing challenge in organic synthetic methodologies. Biocatalysts are robust tools for achieving these difficult chemical transformations. Biocatalyst engineering has often required directed evolution or structure-based rational design campaigns to improve their activities. In recent years, machine learning has been integrated into these workflows to improve the discovery of beneficial enzyme variants. In this work, we combine a structure-based self-supervised machine learning framework, MutComputeX, with classical molecular dynamics simulations to down select mutations for rational design of a non-heme iron-dependent lysine dioxygenase, LDO. This approach consistently resulted in functional LDO mutants and circumvents the need for extensive study of mutational activity before-hand. Our rationally designed single mutants purified with up to 2-fold higher expression yields than WT and displayed higher total turnover numbers (TTN). Combining five such single mutations into a pentamutant variant, LPNYI LDO, leads to a 40 % improvement in the TTN (218±3) as compared to WT LDO (TTN=160±2). Overall, this work offers a low-barrier approach for those seeking to synergize machine learning algorithms with pre-existing protein engineering strategies., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Soluble methane monooxygenase (sMMO) facilitates the conversion of methane to methanol at a non-heme FeIV2 intermediate MMOHQ, which is formed in the active site of the sMMO hydroxylase component (MMOH) during the catalytic cycle. Other biological systems also employ high-valent FeIV sites in catalysis; however, MMOHQ is unique as Nature's only identified FeIV2 intermediate. Previous 57Fe Mössbauer spectroscopic studies have shown that MMOHQ employs antiferromagnetic coupling of the two FeIV sites to yield a diamagnetic cluster. Unfortunately, this lack of net spin prevents the determination of the local spin state (Sloc) of each of the irons by most spectroscopic techniques. Here, we use Fe Kβ X-ray emission spectroscopy (XES) to characterize the local spin states of the key intermediates of the sMMO catalytic cycle, including MMOHQ trapped by rapid-freeze-quench techniques. A pure XES spectrum of MMOHQ is obtained by subtraction of the contributions from other reaction cycle intermediates with the aid of Mössbauer quantification. Comparisons of the MMOHQ spectrum with those of known Sloc = 1 and Sloc = 2 FeIV sites in chemical and biological models reveal that MMOHQ possesses Sloc = 2 iron sites. This experimental determination of the local spin state will help guide future computational and mechanistic studies of sMMO catalysis. [ABSTRACT FROM AUTHOR]

Purpose: We aimed to assess the long-term association of total, heme, non-heme, and supplemental iron intake and risk of type 2 diabetes (T2D). Methods: PubMed, Scopus, and Web of Science were searched to October 2021. Two researchers extracted data in duplicate and rated the certainty in the estimates using the GRADE approach. Random-effects models were applied to estimate the relative risks (RRs) and 95% CIs. Dose–response associations were modeled by a one-stage weighted mixed-effects meta-analysis. Results: Eleven prospective cohort studies 323,788 participants and 28,837 incident cases of T2D were included. High versus low category meta-analysis indicated that higher heme iron intake was associated with a 20% higher risk of T2D (95% CI 1.07, 1.35; I2 = 77%, n = 11; GRADE = moderate). Dose–response analysis indicated a positive monotonic association, wherein each 1 mg/day increment in heme iron intake was related to a 16% higher risk (95% CI 1.03, 1.30). No significant relationship was detected between dietary intakes of total, non-heme, and supplemental iron and risk of T2D (GRADE = very low). Conclusions: In summary, higher heme iron intake was associated with a higher risk of T2D. Our results are in line with existing evidence indicating that adopting a Western-style dietary pattern, rich in dietary sources of heme iron, was associated with a higher risk of T2D. Registry and registry number: The protocol of this systematic review was registered at PROSPERO (registration number: CRD42021226835). [ABSTRACT FROM AUTHOR]

Carotenoid cleavage dioxygenases (CCDs) are non-heme iron-containing enzymes found in all domains of life that generate biologically important apocarotenoids. Prior studies have revealed a critical role for a conserved 4-His motif in forming the CCD iron center. By contrast, the roles of other active site residues in catalytic function, including maintenance of the stringent regio- and stereo-selective cleavage activity, typically exhibited by these enzymes have not been thoroughly investigated. Here, we examined the functional and structural importance of active site residues in an apocarotenoid-cleaving oxygenase (ACO) from Synechocystis Most active site substitutions variably lowered maximal catalytic activity without markedly affecting the Km value for the all-trans-8-apocarotenol substrate. Native C15-C15 cleavage activity was retained in all ACO variants examined suggesting that multiple active site residues contribute to the enzymes regioselectivity. Crystallographic analysis of a nearly inactive W149A-substituted ACO revealed marked disruption of the active site structure, including loss of iron coordination by His-238 apparently from an altered conformation of the conserved second sphere Glu-150 residue. Gln- and Asp-150-substituted versions of ACO further confirmed the structural/functional requirement for a Glu side chain at this position, which is homologous to Glu-148 in RPE65, a site in which substitution to Asp has been associated with loss of enzymatic function in Leber congenital amaurosis. The novel links shown here between ACO active site structure and catalytic activity could be broadly applicable to other CCD members and provide insights into the molecular pathogenesis of vision loss associated with an RPE65 point mutation.

Photosystem II (PSII) performs oxidation of water and reduction of plastoquinone through light-induced electron transfer. Electron transfer reactions at individual redox cofactors are controlled by their redox potentials, and the forward and backward electron flows in PSII are regulated by tuning them. It is, thus, crucial to accurately estimate the redox potentials of the cofactors and their shifts by environmental changes to understand the regulatory mechanisms in PSII. Fourier-transform infrared (FTIR) spectroelectrochemistry combined with a light-induced difference technique is a powerful method to investigate the mechanisms of the redox reactions in PSII. In this review, we introduce the methodology and the application of this method in the studies of the iron-quinone complex, which consists of two plastoquinone molecules, QA and QB, and the non-heme iron, on the electron-acceptor side of PSII. It is shown that FTIR spectroelectrochemistry is a useful method not only for estimating the redox potentials but also for detecting the reactions of nearby amino-acid residues coupled with the redox reactions. [ABSTRACT FROM AUTHOR]

Iron-containing metalloenzymes that contain the 2-His-1-Carboxylate facial triad at their active site are well known for their ability to activate molecular oxygen and catalyse a broad range of oxidative transformations. Many of these reactions are synthetically challenging, and developing small molecular iron-based catalysts that can achieve similar reactivity and selectivity remains a long-standing goal in homogeneous catalysis. This review focuses on the development of bioinspired facial N,N,O ligands that model the 2-His-1-Carboxylate facial triad to a greater degree of structural accuracy than many of the polydentate N-donor ligands commonly used in this field. By developing robust, well-defined N,N,O facial ligands, an increased understanding could be gained of the factors governing enzymatic reactivity and selectivity.

Iron deficiency is considered as a common problem facing the general world population. In the current research, experiments were conducted to evaluate the effects of aqueous extracts of Acacia senegal (Gum Arabic, GA), Spinacia oleracea (spinach), Zea mays (corn), and Capsicum annuum (red chili pepper) on iron and ferritin levels in rats. Vitamin C and fructose levels were first estimated in these plants by using High-Performance Liquid Chromatography (HPLC). The results showed that the GA extract contained the highest level of fructose (853 mg.L-1 ), followed by red chili pepper (635 mg.L-1 ), corn (521 mg.L-1 ), and spinach (271 mg.L-1 ) extracts. Also, the results of vitamin C estimation showed levels of 3.376, 0.645, 0.579, and 0.347 mg/ml in the extracts of spinach, red chili pepper, corn, and GA, respectively. Next, thirty male albino rats (age 8-12 weeks, weight 120-200 g) were divided randomly into six groups, each kept in a separate polypropylene rat cage. Treatments of rats with various plant extracts were conducted following a CRD complete random design, with five replicates for each treatment. The results of lipid profile test revealed that GA treatment caused the highest reduction in the serum levels of TC, TG, LDL, VLDL, and HDL in the treated rats (76.25, 55.25,23, 15.25, and 38 mg/dl, respectively), as compared to the other plant extracts. Evidence from this study suggests that the higher serum level of fructose in the group of rats treated with all plant extracts enhances the process of ferritin formation, possibly via increasing the level of bioavailable iron [ABSTRACT FROM AUTHOR]

Objective: The current study evaluated the associations between different forms and sources of Fe and breast cancer risk in Southern Chinese women.Design: Case-control study. We collected data on the consumption of Fe from different forms and food sources by using a validated FFQ. Multivariable logistic regression and restricted cubic spline (RCS) analysis was used to reveal potential associations between Fe intake and breast cancer risk.Setting: A case-control study of women at three major hospitals in Guangzhou, China.Participants: From June 2007 to March 2019, 1591 breast cancer cases and 1622 age-matched controls were recruited.Results: In quartile analyses, Fe from plants and Fe from white meat intake were inversely associated with breast cancer risk, with OR of 0·65 (95 % CI 0·47, 0·89, Ptrend = 0·006) and 0·76 (95 % CI 0·61, 0·96, Ptrend = 0·014), respectively, comparing the highest with the lowest quartile. No associations were observed between total dietary Fe, heme or non-heme Fe, Fe from meat or red meat and breast cancer risk. RCS analysis demonstrated J-shaped associations between total dietary Fe, non-heme Fe and breast cancer, and reverse L-shaped associations between heme Fe, Fe from meat and Fe from red meat and breast cancer.Conclusion: Fe from plants and white meat were inversely associated with breast cancer risk. Significant non-linear J-shaped associations were found between total dietary Fe, non-heme Fe and breast cancer risk, and reverse L-shaped associations were found between heme Fe, Fe from meat or red meat and breast cancer risk. [ABSTRACT FROM AUTHOR]

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Iron deficiency is the most common micronutrient deficiency worldwide. While iron deficiency is known to suppress embryonic organogenesis, its effect on the adult organ in the context of clinically relevant damage has not been considered. Here we report that iron deficiency is a risk factor for nephrotoxic intrinsic acute kidney injury of the nephron (iAKI). Iron deficiency exacerbated cisplatin-induced iAKI by markedly increasing non-heme catalytic iron and Nox4 protein which together catalyze production of hydroxyl radicals followed by protein and DNA oxidation, apoptosis and ferroptosis. Crosstalk between non-heme catalytic iron/Nox4 and downstream oxidative damage generated a mutual amplification cycle that facilitated rapid progression of cisplatin-induced iAKI. Iron deficiency also exacerbated a second model of iAKI, rhabdomyolysis, via increasing catalytic heme-iron. Heme-iron induced lipid peroxidation and DNA oxidation by interacting with Nox4-independent mechanisms, promoting p53/p21 activity and cellular senescence. Our data suggests that correcting iron deficiency and/or targeting specific catalytic iron species are strategies to mitigate iAKI in a wide range of patients with diverse forms of kidney injury. [Display omitted] • Iron deficiency increases renal non-heme catalytic iron and Nox4 in cisplatin mice. • Nox4 interacts with non-heme catalytic iron to drive hydroxyl radical renal injury. • Non-heme catalytic iron/Nox4 crosstalks with downstream oxidative damage. • This crosstalk amplifies to facilitate rapid progression of cisplatin-iAKI. • Iron deficiency increases heme catalytic iron and cellular senescence in Rh kidneys. [ABSTRACT FROM AUTHOR]

We present the synthesis and coordination chemistry of a bulky, tripodal N,N,O ligand, ImPh2NNOtBu (L), designed to model the 2‐His‐1‐carboxylate facial triad (2H1C) by means of two imidazole groups and an anionic 2,4‐di‐tert‐butyl‐subtituted phenolate. Reacting K‐L with MCl2 (M = Fe, Zn) affords the isostructural, tetrahedral non‐heme complexes [Fe(L)(Cl)] (1) and [Zn(L)(Cl)] (2) in high yield. The tridentate N,N,O ligand coordination observed in their X‐ray crystal structures remains intact and well‐defined in MeCN and CH2Cl2 solution. Reacting 2 with NaSPh affords a tetrahedral zinc thiolate complex, [Zn(L)(SPh)] (4), that is relevant to isopenicillin N synthase (IPNS) biomimicry. Cyclic voltammetry studies demonstrate the ligand's redox non‐innocence, where phenolate oxidation is the first electrochemical response observed in K‐L, 2 and 4. However, the first electrochemical oxidation in 1 is iron‐centred, the assignment of which is supported by DFT calculations. Overall, ImPh2NNOtBu provides access to well‐defined mononuclear, monoligated, N,N,O‐bound metal complexes, enabling more accurate structural modelling of the 2H1C to be achieved. [ABSTRACT FROM AUTHOR]

Background: Gestational diabetes (GDM) is a pregnancy-related glucose intolerance with significant implications for maternal and fetal health. Calcium is essential for insulin secretion and metabolism, while iron intake may also impact GDM. This case-control study was conducted to investigate the relationship between calcium and iron intake with the risk of GDM. Methods: GDM was defined as Fasting Blood Sugar>92mg/dL or 75g Oral-Glucose-Tolerance-Test 120-minutes>153mg/dL. A 168-Item food-frequency-questionnaire was used to collect dietary calcium and iron intake from 24-40 weeks of gestation. The impact of total iron, red, processed/unprocessed meat consumption, calcium, and dairy intake on GDM were investigated. Results: A total of 229 GDM and 205 non-GDM women (18-45 years) participated. GDM group had higher pre-pregnancy weight, weight gain, and pre-pregnancy BMI. Across all models, GDM risk significantly increased in the third and fourth quartiles of iron intake. The fourth quartile had an Odds Ratio (OR) of 2.68 (CI 95%, 4.89-1.56; P<0.001) compared to the reference. Heme-iron consumption in the fourth quartiles increased GDM risk. In the second calcium intake model, ORs for the second, third, and fourth quartiles were 0.51 (CI 95%, 0.91-0.25), 0.43 (CI 95%, 0.77-0.24), and 0.35 (CI 95%, 0.63-0.19), respectively (P<0.001 all), reducing GDM risk by 50-65% compared to the first quartile. Dairy consumption in all quartiles of the first and second models was associated with lower GDM risk. Conclusions: Consumption of heme-iron through red and processed meat associated with an increased chance of developing GDM. Dairy intake reduces the chances of developing GDM in pregnant women.

Cooking utensils may leach toxic metals or trace elements into food. Iron is an essential nutrient and both iron deficiency and iron excess can affect optimal health. The aim of the study was to determine non-heme iron (NHI) and some antioxidant parameters of rats fed banga soup (BS) prepared using different utensils. Twentyfive Wistar albino rats were used for the study. They were allowed to acclimatize for one week before commencement of the experiment. Group 1 served as control. Rats in Group 2, 3, 4 and 5 were administered BS cooked using cast iron pot (CIP), aluminium pot (AP), blended mixture (BM) of BS and aqueous tween 80 respectively. Rats in Group 1 to 5 received tap water daily and standard laboratory diet (feed) throughout the experiment period of 28 days. There were no significant difference in aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) activities, albumin (Alb) and total protein (TP) in the serum and liver of the entire experimental Groups. Significant decrease were observed in total phenol content (TPC), total antioxidant capacity (TAC), ferric reducing antioxidant power (FRAP) and ascorbic oxidase (AO) activity in the serum and NHI content in the serum, liver and kidney of Group 2, 3 and 4 when compared with Group 1 and 5. Keywords: Banga soup; non-heme iron; antioxidant; aluminium pot; cast iron pot

The potential of the redox couple FeIV=O / FeIII–O is of interest for the reactivity of the high‐valent nonheme iron oxidants in enzymes and bioinspired small molecule systems but, unfortunately, experimentally it so far is very poorly described. Discussed are three computational methods that are used in combination with available experimental data derived from titrations of FeIV=O species with ferrocene derivatives in dry acetonitrile, and from spectroelectrochemical titrations of FeIII–OH complexes in wet acetonitrile, i.e. describing the FeIV=O / FeIII–OH couple – both data sets are known to have some ambiguities. First, a DFT‐based method is used to compute the E° values of 14 FeIV=O / FeIII–O couples with an error margin of around 110 mV. A subset of four species of the original data set is used to evaluate a DLPNO‐CCSD(T) based approach, and another subset of complexes, where the spectroelectrochemically determined FeIV=O / FeIII–OH potentials are also known, are used for a Bordwell‐Polanyi analysis, which also yield pKa values. It is shown that the three approaches lead to a consistent picture but due to possible ambiguities with the experimental data, it currently is not possible to fully evaluate the accuracy of the used approaches. [ABSTRACT FROM AUTHOR]

Thebaine 6- O -demethylase (T6ODM) is an Fe(II)/2-oxoglutarate-dependent dioxygenase catalysing two oxidative O -demethylation reactions in morphine biosynthesis. Its crystal structure revealed a large active site pocket which is at least two times larger than necessary to accommodate a substrate (thebaine or oripavine) molecule. Since so far no crystal structures have been obtained for enzyme-substrate complex, which is necessary to explain the enzyme regiospecificity towards the C6-bound methoxy group, in this work we used computational methods and multi-parametric surface plasmon resonance measurements to elucidate the most likely structure of this complex and the reaction mechanism starting therefrom. Results of simulations and experiments unanimously indicate that the enzyme-substrate complex of T6ODM has a 1:2 stoichiometry. The key residues responsible for substrate binding are: Val-128, Glu-133, Met-150 and Agr-219 for the substrate in the distal position, and Asp-144, Leu-235 and Leu-353 for the proximal substrate molecule. QM/MM and DFT calculations show that the oxo ligand is bound trans to His-295 and the enzyme catalyzes hydroxylation of the C6-bound methoxy group according to the established rebound mechanism. The final stage of the demethylation reaction, which includes deformylation and enol-keton tautomerization steps, is most likely catalysed by water molecules and takes place in the solvent. [ABSTRACT FROM AUTHOR]

The aim of the work was to study the intranuclear distribution of nucleophosmin (B23), coilin (p80), AgNOR proteins, and nonheme iron in the neurons of the human substantia nigra. Fragments of the human midbrain (n = 10) were shown to have no signs of neurodegeneration. The material was fixed in zinc–ethanol–formaldehyde, a special fixative that provides high preservation of antigenic determinants and tissue structures. This work revaled a new data on molecular and structural organization of the nucleoli of neurons in human substantia nigra. It was found that, in the nuclei of substantia nigra neurons, there is only one large nucleolus, and its size varies between individuals (from 4.2 ± 0.4 to 6.2 ± 0.6 μm in diameter). Nucleophosmin is concentrated solely in the nucleolus and is unevenly distributed in it. Cajal bodies found in the nucleus of most substantia nigra neurons are coiline-containing structures of round or oval shape and about 1 μm in size. Coilin accumulation has also been noted in numerous intranuclear microstructures, which significantly differ in their localization and morphological characteristics from typical Cajal bodies. AgNOR proteins are present in the substantia nigra neurons and are very variably distributed. Nonheme iron was detected in the nucleoli ofof some substantia nigra neurons. In some cases iron was concentrated in the giant fibrillar center (GFC). The data obtained will contribute to a clearer understanding of the role of nucleolar proteins in the functioning of dopaminergic neurons in the human brain and the determination of molecular sensors of nucleolar stress observed during neurodegeneration. [ABSTRACT FROM AUTHOR]

Cooking utensils may leach toxic metals or trace elements into food. Iron is an essential nutrient and both iron deficiency and iron excess can affect optimal health. The aim of the study was to determine non-heme iron (NHI) and some antioxidant parameters of rats fed banga soup (BS) prepared using different utensils. Twentyfive Wistar albino rats were used for the study. They were allowed to acclimatize for one week before commencement of the experiment. Group 1 served as control. Rats in Group 2, 3, 4 and 5 were administered BS cooked using cast iron pot (CIP), aluminium pot (AP), blended mixture (BM) of BS and aqueous tween 80 respectively. Rats in Group 1 to 5 received tap water daily and standard laboratory diet (feed) throughout the experiment period of 28 days. There were no significant difference in aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) activities, albumin (Alb) and total protein (TP) in the serum and liver of the entire experimental Groups. Significant decrease were observed in total phenol content (TPC), total antioxidant capacity (TAC), ferric reducing antioxidant power (FRAP) and ascorbic oxidase (AO) activity in the serum and NHI content in the serum, liver and kidney of Group 2, 3 and 4 when compared with Group 1 and 5. [ABSTRACT FROM AUTHOR]

Novel functions emerge from novel structures. To develop efficient catalytic systems for challenging chemical transformations, chemists often seek inspirations from enzymatic catalysis. A large number of iron complexes supported by nitrogen‐rich multidentate ligands have thus been developed to mimic oxo‐transfer reactivity of dioxygen‐activating metalloenzymes. Such efforts have significantly advanced our understanding of the reaction mechanisms by trapping key intermediates and elucidating their geometric and electronic properties. Critical to the success of this biomimetic approach is the design and synthesis of elaborate ligand systems to balance the thermodynamic stability, structural adaptability, and chemical reactivity. In this Concept article, representative design strategies for biomimetic atom‐transfer chemistry are discussed from the perspectives of "ligand builders". Emphasis is placed on how the primary coordination sphere is constructed, and how it can be elaborated further by rational design for desired functions. [ABSTRACT FROM AUTHOR]

Oxygen affinity is an important property of metalloproteins that helps elucidate their reactivity profile and mechanism. Heretofore, oxygen affinity values were determined either using flash photolysis and polarography techniques that require expensive instrumentation, or using oxygen titration methods which are erroneous at low nanomolar and at high millimolar oxygen concentrations. Here, we describe an inexpensive, easy-to-setup, and a one-pot method for oxygen affinity measurements that uses the enzyme chlorite dismutase (Cld) as a precise in situ oxygen source. Using this method, we measure thermodynamic and kinetic oxygen affinities (Kd and KM) of different classes of heme and non-heme metalloproteins involved in oxygen transport, sensing, and catalysis. The method enables oxygen affinity measurements over a wide concentration range from 10 nM to 5 mM which is unattainable by simply diluting oxygen-saturated buffers. In turn, we were able to precisely measure oxygen affinities of a model set of eight different metalloproteins with affinities ranging from 48 ± 3 nM to 1.18 ± 0.03 mM. Overall, the Cld method is easy and inexpensive to set up, requires significantly lower quantities of protein, enables precise oxygen affinity measurements, and is applicable for proteins exhibiting nanomolar-to-millimolar affinity values. [ABSTRACT FROM AUTHOR]

[Display omitted] • Myoglobin (Mb) oxidation could promote oxidation of myofibrillar protein (MP). • Mb oxidation affected the water retention of MP. • MP water retention was closely linked to hemin iron and non-hemin iron. The impact of oxidized myoglobin (Mb) on myofibrillar protein (MP) oxidation and water retention was investigated. Results showed that the oxidation of Mb increased with increasing concentration of oxidized linoleic acid (OLA). In the presence of 100 mmol/L OLA, hemin iron decreased by 62.07 % compared to the control group. Further investigation showed that mild oxidation of Mb (≤10 mmol/L OLA) increased the water retention and the absolute value of the zeta potential of MP, whereas excessive oxidation (>10 mmol/L OLA) decreased these properties. With the increase of Mb oxidation, the carbonyl content in MP increased, and α-helices changed to random helix. And the tertiary structure changed. Pearson correlation analysis suggested that oxidized Mb affected the water retention of MP, which was closely related to hemin iron and non-hemin iron. In conclusion, OLA induced Mb oxidation, further promoted MP oxidation and affected its water retention. [ABSTRACT FROM AUTHOR]

Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O 2 ) concentrations. Here, we have reprogrammed cellular hypoxia (low O 2 ) signaling via gas tunnel engineering of prolyl hydroxylase 2 (PHD2), a non-heme iron dependent O 2 sensor. Using computational modeling and protein engineering techniques, we identify a gas tunnel and critical residues therein that limit the flow of O 2 to PHD2's catalytic core. We show that systematic modification of these residues can open the constriction topology of PHD2's gas tunnel. Using kinetic stopped-flow measurements with NO as a surrogate diatomic gas, we demonstrate up to 3.5-fold enhancement in its association rate to the iron center of tunnel-engineered mutants. Our most effectively designed mutant displays 9-fold enhanced catalytic efficiency ( k cat / K M = 830 ± 40 M -1 s -1 ) in hydroxylating a peptide mimic of hypoxia inducible transcription factor HIF-1α, as compared to WT PHD2 ( k cat / K M = 90 ± 9 M -1 s -1 ). Furthermore, transfection of plasmids that express designed PHD2 mutants in HEK-293T mammalian cells reveal significant reduction of HIF-1α and downstream hypoxia response transcripts under hypoxic conditions of 1% O 2 . Overall, these studies highlight activation of PHD2 as a new pathway to reprogram hypoxia responses and HIF signaling in cells.

Nitrile hydratase (NHase) is a non-heme iron-containing enzyme that has applications in commodity chemical synthesis, pharmaceutical intermediate synthesis, and reclamation of nitrile-(bromoxynil) contaminated land. Mechanistic study of the enzyme has been complicated by the expression of multiple overlapping Fe(III) EPR signals. The individual signals were recently assigned to distinct chemical species with the assistance of DFT calculations. Here, the origins and evolution of the EPR signals from cells overexpressing the enzyme were investigated, with the aims of optimizing the preparation of homogeneous samples of NHase for study and investigating the application of E. coli overexpressing the enzyme for "green" chemistry. It was revealed that nitrile hydratase forms two sets of inactive complexes in vivo over time. One is due to reversible complexation with endogenous carboxylic acids, while the second is due to irreversibly inactivating oxidation of an essential cysteine sulfenic acid. It was shown that the homogeneity of preparations can be improved by employing an anaerobic protocol. The ability of the substrates acrylonitrile and acetonitrile to be taken up by cells and hydrated to the corresponding amides by NHase was demonstrated by EPR identification of the product complexes of NHase in intact cells. The inhibitors butyric acid and butane boronic acid were also taken up by E. coli and formed complexes with NHase in vivo, indicating that care must be taken with environmental variables when attempting microbially assisted synthesis and reclamation. [ABSTRACT FROM AUTHOR]

Non‐heme iron oxygenases contain either monoiron or diiron active sites, and the role of the second iron in the latter enzymes is a topic of particular interest, especially for soluble methane monooxygenase (sMMO). Herein we report the activation of a non‐heme FeIII‐OOH intermediate in a synthetic monoiron system using FeIII(OTf)3 to form a high‐valent oxidant capable of effecting cyclohexane and benzene hydroxylation within seconds at −40 °C. Our results show that the second iron acts as a Lewis acid to activate the iron–hydroperoxo intermediate, leading to the formation of a powerful FeV=O oxidant—a possible role for the second iron in sMMO. [ABSTRACT FROM AUTHOR]

A key step of denitrification, the reduction of toxic nitric oxide to nitrous oxide, is catalysed by cytochrome c‐dependent NO reductase (cNOR). cNOR contains four redox‐active cofactors: three hemes and a nonheme iron (FeB). Heme b3 and FeB constitute the active site, but the specific mechanism of NO‐binding events and reduction is under debate. Here, we used a recently constructed, fully folded and hemylated cNOR variant that lacks FeB to investigate the role of FeB during catalysis. We show that in the FeB‐less cNOR, binding of both NO and O2 to heme b3 still occurs but further reduction is impaired, although to a lesser degree for O2 than for NO. Implications for the catalytic mechanisms of cNOR are discussed. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effects of eating wholegrain rye bread with high or low amounts of phytate on iron status in women under free-living conditions. Methods: In this 12-week, randomized, parallel-design intervention study, 102 females were allocated into two groups, a high-phytate-bread group or a low-phytate-bread group. These two groups were administered: 200 g of blanched wholegrain rye bread/day, or 200 g dephytinized wholegrain rye bread/day. The bread was administered in addition to their habitual daily diet. Iron status biomarkers and plasma alkylresorcinols were analyzed at baseline and post-intervention. Results: Fifty-five females completed the study. In the high-phytate-bread group (n = 31) there was no change in any of the iron status biomarkers after 12 weeks of intervention (p > 0.05). In the low-phytate bread group (n = 24) there were significant decreases in both ferritin (mean = 12%; from 32 ± 7 to 27 ± 6 µg/L, geometric mean ± SEM, p < 0.018) and total body iron (mean = 12%; from 6.9 ± 1.4 to 5.4 ± 1.1 mg/kg, p < 0.035). Plasma alkylresorcinols indicated that most subjects complied with the intervention. Conclusions: In Swedish females of reproductive age, 12 weeks of high-phytate wholegrain bread consumption had no effect on iron status. However, consumption of low-phytate wholegrain bread for 12 weeks resulted in a reduction of markers of iron status. Although single-meal studies clearly show an increase in iron bioavailability from dephytinization of cereals, medium-term consumption of reduced phytate bread under free-living conditions suggests that this strategy does not work to improve iron status in healthy women of reproductive age. [ABSTRACT FROM AUTHOR]

Nitrile hydratases (NHase) catalyze the hydration of nitriles to the corresponding amides. We report on the heterologous expression of various nitrile hydratases. Some of these enzymes have been investigated by others and us before, but sixteen target proteins represent novel sequences. Of 21 target sequences, 4 iron and 16 cobalt containing proteins were functionally expressed from Escherichia coli BL21 (DE3) Gold. Cell free extracts were used for activity profiling and basic characterization of the NHases using the typical NHase substrate methacrylonitrile. Co-type NHases are more tolerant to high pH than Fe-type NHases. A screening for activity on three structurally diverse nitriles was carried out. Two novel Co-dependent NHases from Afipia broomeae and Roseobacter sp. and a new Fe-type NHase from Gordonia hydrophobica were very well expressed and hydrated methacrylonitrile, pyrazine-carbonitrile, and 3-amino-3-(p-toluoyl)propanenitrile. The Co-dependent NHases from Caballeronia jiangsuensis and Microvirga lotononidis, as well as two Fe-dependent NHases from Pseudomonades, were—in addition—able to produce the amide from cinnamonitrile. Summarizing, seven so far uncharacterized NHases are described to be promising biocatalysts.

Hummus, an iron-containing plant-based dish mainly made from chickpea purée, tahini, lemon juice and garlic, could be a valuable source of iron when bioavailable. Since the processing and formulation of food influence iron bioavailability, the present study investigated for the first time, their effects on hummus. Firstly, iron bioaccessibility was assessed on eight samples (prepared according to the screening Hadamard matrix) by in vitro digestion preceding iron dialysis. Then, iron bioavailability of four selected samples was estimated by the in vitro digestion/Caco-2 cell model. Total and dialyzable iron were determined by the atomic absorption spectrometry and ferritin formation was determined using an ELISA kit. Only autoclaving, among other processes, had a significant effect on iron bioaccessibility (+9.5, p < 0.05). Lemon juice had the highest positive effect (+15.9, p < 0.05). Consequently, the effect of its acidic components were investigated based on a full factorial 23 experimental design; no significant difference was detected. Garlic’s effect was not significant, but tahini’s effect was negative (−8.9, p < 0.05). Despite the latter, hummus had a higher iron bioavailability than only cooked chickpeas (30.4 and 7.23 ng ferritin/mg protein, respectively). In conclusion, hummus may be a promising source of iron; further in vivo studies are needed for confirmation.