Catalytic Iron Research Articles

Batch and Continuous-Flow Iron(II)-Catalyzed Synthesis of Sulfilimines and Sulfoximines using N-Mesyloxycarbamates.

The synthesis of sulfilimines and sulfoximines with N-mesyloxycarbamates in presence of catalytic iron(II) chloride hydrate is described. The use of 1-butylimidazole as a base proved instrumental in enabling a homogeneous reaction mixture compatible with continuous flow processes. The amination is applicable to a wide range of substrates, and affords the desired product in good to excellent yields within 1 to 60 minutes.

Effect of the point mutation H54N on the ferroxidase process of Rana catesbeiana H′ ferritin

Human H and Rana catesbeiana H′ subunits in vertebrate ferritin protein cages catalyze the Fe(II) oxidation by molecular oxygen and promote the ferric oxide biomineral synthesis. By depositing iron biomineral, ferritins also prevent potentially toxic reactions products from Fe(II)-based Fenton chemistry. Recent work from our laboratory was aimed to describe the iron pathways within ferritin, from entrance into the cage to the ferroxidase site, and to understand the role played by amino-acid residues in iron trafficking and catalysis. Our approach exploits anomalous X-ray diffraction from ferritin crystals, exposed to a ferrous salt, to track transient iron binding sites along the path towards a well-defined di-iron site where they get oxidized by oxygen. Coupling structure determination with solution kinetic measurements on selected variants, allows validating the role played by key residues on the catalytic iron oxidation. Our previous studies on H′ ferritin indicated the regulatory role played by His54, and by its human counterpart Gln58, on guiding Fe(II) ions to the catalytic site. Here, we have investigated the effects induced by substituting the wild type His54 with Asn54, having different iron coordination properties. We have obtained a series of atomic-resolution crystal structures that provide time-dependent snapshots of iron bound at different locations in the H′ ferritin H54N variant. The comparison with H′ ferritin and H′ ferritin H54Q variant leads to identify a new iron binding site. Our kinetic and structural data support the role of H′ ferritin residue 54 in regulating the access of Fe(II) ions to the catalytic site.

Hypobaric hypoxia regulates iron metabolism in rats.

Intermittent hypobaric hypoxia can produce a protective effect on both the nervous system and non-nervous system tissues. Intermittent hypobaric hypoxia preconditioning has been shown to protect rats from cardiac ischemia-reperfusion injury by decreasing cardiac iron levels and reactive oxygen species (ROS) production, thereby decreasing oxidative stress to achieve protection. However, the specific mechanism underlying the protective effect of hypobaric hypoxia is unclear. To shed light on this phenomenon, we subjected Sprague-Dawley rats to hypobaric hypoxic preconditioning (8 hours/day). The treatment was continued for 4 weeks. We then measured the iron content in the heart, liver, spleen, and kidney. The iron levels in all of the assessed tissues decreased significantly after hypobaric hypoxia treatment, corroborating previous results that hypobaric hypoxia may produce its protective effect by decreasing ROS production by limiting the levels of catalytic iron in the tissue. We next assessed the expression levels of several proteins involved in iron metabolism (transferrin receptor, L-ferritin, and ferroportin1 [FPN1]). The increased transferrin receptor and decreased L-ferritin levels after hypobaric hypoxia were indicative of a low-iron phenotype, while FPN1 levels remained unchanged. We also examined hepcidin, transmembrane serine proteases 6 (TMPRSS6), erythroferrone (ERFE), and erythropoietin (EPO) levels, all of which play a role in the regulation of systemic iron metabolism. The expression of hepcidin decreased significantly after hypobaric hypoxia treatment, whereas the expression of TMPRSS6 and ERFE and EPO increased sharply. Finally, we measured serum iron and total iron binding capacity in the serum, as well as red blood cell count, mean corpuscular volume, hematocrit, red blood cell distribution width SD, and red blood cell distribution width CV. As expected, all of these values increased after the hypobaric hypoxia treatment. Taken together, our results show that hypobaric hypoxia can stimulate erythropoiesis, which systemically draws iron away from nonhematopoietic tissue through decreased hepcidin levels.

Protocol and baseline data for a prospective open-label explorative randomized single-center comparative study to determine the effects of various intravenous iron preparations on markers of oxidative stress and kidney injury in chronic kidney disease (IRON-CKD)

BackgroundIntravenous (IV) iron is frequently used to treat iron deficiency/anemia in patients who are unable to tolerate oral iron or the oral iron is not sufficient toreplete iron requirements. However, safety concerns regarding the potential increase in oxidative stress and other adverse effects persist and it remains unclear whether all iron preparations are equivalent. Indeed, the comparative risk of adverse events with IV iron preparations has not been extensively assessed.We hypothesize that IV iron leads to changes in oxidative stress, endothelial function, and potential renal damage depending on the iron formulation (related to the generation of “free” or catalytic labile iron) and this may result in more tubular and glomerular injury manifested as increased proteinuria and raised neutrophil gelatinase–associated lipocalin (NGAL) levels in patients with chronic kidney disease (CKD).MethodsIRON-CKD is a prospective, open-label, explorative, randomized, single-center study designed to compare the safety and efficacy of three parenteral iron preparations: low-molecular-weight iron dextran–Cosmofer, iron sucrose–Venofer, and iron isomaltoside–Monofer. The study includes 40 adults who have established CKD stages 3–5 and serum ferritin (SF) of less than 200 μg/L or transferrin saturation (TS) of less than 20% (or both); they were randomly assigned in a 1:1:1:1 ratio to 200 mg iron dextran, 200 mg iron sucrose, 200 mg iron isomaltoside, or 1000 mg iron isomaltoside. After randomization, participants undergo baseline assessments and then an iron infusion. Each participant is followed up at 2 h, day 1, week 1, and months 1 and 3. At each follow-up visit, patients undergo clinical review, measurement of pulse wave velocity (PWV), blood tests for renal function, and collection of serum/plasma samples for oxidative stress and inflammatory markers.The primary outcomes are measures of oxidative stress, inflammatory markers, and markers of acute renal injury in comparison with baseline measures of each iron preparation and between each of the iron preparations. Secondary objectives include effects on hematinic profiles and hemoglobin concentrations, changes in arterial stiffness, incidence of significant side effects, and change in patients’ quality of life.ResultsBetween October 2015 and April 2018, 521 individuals were identified as potential participants; 216 were contacted, 56 expressed an interest, 49 attended a screening visit, and 40 were confirmed to meet the eligibility criteria and were randomly assigned. The mean age was 58.3 (standard error of the mean 4.4) years, and 23 (58%) were male. All patients were white and English-speaking. The mean SF was 66.6 μg/L, TS was 21.2%, and hemoglobin was 121.6 g/L at randomization for the whole group. The mean estimated glomerular filtration rate was 27.8 mL/min, the urinary protein/creatinine ratio was 104.3 mg/mmol, and CRP was 6.65 mg/L.DiscussionIRON-CKD will provide important information on the short-term effects of three preparations of IV iron in CKD patients with biochemical functional or absolute iron deficiency on measures of oxidative stress, inflammation, endothelial function, and renal injury.Trial registrationEuropean Clinical Trials Database (EudraCT) number 2010-020452-64.

Isoprene Formation from Isoamyl Alcohol in Microchannels of a Converter Modified with Nanoscale Catalytic Iron–Chromium-Containing Systems

A two-stage process for producing isoprene from isoamyl alcohol has been developed through the successive implementation of the steps of dehydration of isoamyl alcohol and dehydrogenation of resulting isoamylenes using porous ceramic converters obtained by self-propagating high-temperature synthesis, with the converter pores bearing Fe–Cr systems synthesized by the sol–gel technique and containing subnanosized iron and chromium particles. It has been shown that the bimetallic catalytic system obtained by separate deposition of Fe and Cr has the highest activity toward the isoamylenes studied in the dehydrogenation reaction; on this catalyst system using 24-fold dilution with water and a temperature of 600°C, the isoprene yield is up to ~10%, selectivity is ~23%, and productivity is ~0.13 Lisoprene/(h gact.comp). The conversion of the isoamylene fraction is 46%.

Catalytic Allylic Oxidation to Generate Vinylogous Acyl Sulfonates from Vinyl Sulfonates.

Regioselective formation of vinylogous acyl sulfonates was accomplished via the allylic oxidation of the corresponding vinyl sulfonates. The reaction progressed through the agency of catalytic iron(III) chloride catalysis with tert-Butyl hydroperoxide (TBHP) as the stoichiometric oxidant. Tolerance of other functional groups, including some other allylic and benzylic sites, was observed.

Role of Combined Lipoic Acid and Vitamin D3 on Astrocytes as a Way to Prevent Brain Ageing by Induced Oxidative Stress and Iron Accumulation.

Brain ageing is a complex multifactorial process characterized by gradual and continuous loss of neuronal functions. It is hypothesized that at the basis of brain ageing as well as age-related diseases, there is an impairment of the antioxidant defense system leading to an increase of oxidative stress. In this study, two different biological aspects involved in brain ageing and neurodegeneration have been investigated: oxidative stress and iron accumulation damage. In primary mouse astrocytes, the stimulation with 50 μM lipoic acid (LA) and 100 nM vitamin D (vitD) was first investigated in a time-course study to determine the dosages to be used in combination and then in a permeability test using an in vitro blood-brain barrier. In a second set of experiments, the role of oxidative stress was investigated pretreating astrocytes with 200 μM H2O2 for 30 min. The ability of vitD and LA alone and combined together to prevent or repair the damage caused by oxidative stress was investigated after 24 h of stimulation by the MTT test, mitochondrial membrane potential measurement, and Western blot analysis. To induce neurodegeneration, cells were pretreated with 300 μM catalytic iron for 6 days and then treated with vitD and LA alone and combined for additional 6 days to investigate the protection exerted by combination, analyzing viability, ROS production, iron concentration, and activation of intracellular pathways. In our study, the combination of LA and vitD showed beneficial effects on viability of astrocytes, since the substances are able to cross the brain barrier. In addition, combined LA and vitD attenuated the H2O2-induced apoptosis through the mitochondrial-mediated pathway. The combination was also able to counteract the adverse conditions caused by iron, preventing its accumulation. All these data support the hypothesis of the synergistic and cooperative activity exerted by LA and vitD in astrocytes indicating a possible new strategy to slow down ageing.

Chemical Generation of Hydroxyl Radical for Oxidative 'Footprinting'.

For almost four decades, hydroxyl radical chemically generated by Fenton chemistry has been a mainstay for the oxidative 'footprinting' of macromolecules. In this article, we start by reviewing the application of chemical generation of hydroxyl radical to the development of oxidative footprinting of DNA and RNA and the subsequent application of the method to oxidative footprinting of proteins. We next discuss a novel strategy for generating hydroxyl radicals by Fenton chemistry that immobilizes catalytic iron on a solid surface (Pyrite Shrink Wrap laminate) for the application of nucleic acid and protein footprinting. Pyrite Shrink-Wrap Laminate is fabricated by depositing pyrite (Fe-S2, aka 'fool's gold') nanocrystals onto thermolabile plastic (Shrinky Dink). The laminate can be thermoformed into a microtiter plate format into which samples are deposited for oxidation. We demonstrate the utility of the Pyrite Shrink-Wrap Laminate for the chemical generation of hydroxyl radicals by mapping the surface of the T-cell co-stimulatory protein Programmed Death - 1 (PD-1) and the interface of the complex with its ligand PD-L1. We have developed and validated an affordable and reliable benchtop method of hydroxyl radical generation that will broaden the application of protein oxidative footprinting. Due to the minimal equipment required to implement this method, it should be easily adaptable by many laboratories with access to mass spectrometry.

Iron, Hepcidin, and Death in Human AKI.

Iron is a key mediator of AKI in animal models, but data on circulating iron parameters in human AKI are limited. We examined results from the ARF Trial Network study to assess the association of plasma catalytic iron, total iron, transferrin, ferritin, free hemoglobin, and hepcidin with 60-day mortality. Participants included critically ill patients with AKI requiring RRT who were enrolled in the study. Of the 807 study participants, 409 (51%) died by day 60. In both unadjusted and multivariable adjusted models, higher plasma concentrations of catalytic iron were associated with a significantly greater risk of death, as were lower concentrations of hepcidin. After adjusting for other factors, patients with catalytic iron levels in the highest quintile versus the lowest quintile had a 4.06-fold increased risk of death, and patients with hepcidin levels in the lowest quintile versus the highest quintile of hepcidin had a 3.87-fold increased risk of death. These findings were consistent across multiple subgroups. Other iron markers were also associated with death, but the magnitude of the association was greatest for catalytic iron and hepcidin. Higher plasma concentrations of catalytic iron and lower concentrations of hepcidin are each independently associated with mortality in critically ill patients with AKI requiring RRT. These findings suggest that plasma concentrations of catalytic iron and hepcidin may be useful prognostic markers in patients with AKI. Studies are needed to determine whether strategies to reduce catalytic iron or increase hepcidin might be beneficial in this patient population.

Practical preparation of diphenylmethyl ethers from 2-diphenylmethoxypyridine using catalytic iron(iii) chloride.

A novel facile synthetic method for producing diphenylmethyl (DPM) ethers from 2-diphenylmethoxypyridine was developed. A variety of DPM ethers was successfully achieved with high yield via treatment of alcohols with 2-diphenylmethoxypyridine in the presence of catalytic FeCl3. The procedure is a practical and efficient synthetic procedure to protect various alcohols, and it can be applied to prepare bioactive compounds.

Changes in Regioselectivity of H Atom Abstraction during the Hydroxylation and Cyclization Reactions Catalyzed by Hyoscyamine 6β-Hydroxylase.

Hyoscyamine 6β-hydroxylase (H6H) is an αKG-dependent nonheme iron oxidase that catalyzes the oxidation of hyoscyamine to scopolamine via two separate reactions: hydroxylation followed by oxidative cyclization. Both of these reactions are expected to involve H atom abstraction from each of two adjacent carbon centers (C6 vs C7) in the substrate. During hydroxylation, there is a roughly 85:1 preference for H atom abstraction from C6 versus C7; however, this inverts to a 1:16 preference during cyclization. Furthermore, 18O incorporation experiments in the presence of deuterated substrate are consistent with the catalytic iron(IV)-oxo complex being able to support the coordination of an additional ligand during hydroxylation. These observations suggest that subtle differences in the substrate binding configuration can have significant consequences for the catalytic cycle of H6H.

Two clusters of residues contribute to the activity and substrate specificity of Fm1, a bifunctional oleate and linoleate desaturase of fungal origin

Polyunsaturated fatty acids (PUFAs) have important industrial, physiological, and nutritional properties. Plants use the sequential activities of FAD2 and FAD3 desaturases to convert 18:1Δ9 to the important PUFA 18:3Δ9,12,15, whereas the fungus Fusarium verticillioides 7600 uses the bifunctional desaturase Fm1 for both reactions. Here, we used a combination of sequence comparisons, structural modeling, and mutagenesis experiments to investigate Fm1's regioselectivity and identified two functionally relevant clusters of residues that contribute to Fm1 activity. We found that cluster I (Leu153, Phe157, and His194), located near the catalytic iron ions, predominantly affects activity, whereas cluster II (Tyr280, His284, and Leu287), located in a helix forming the entrance to the substrate-binding pocket, mainly specifies 15-desaturation. Individual or combined substitutions of cluster II residues substantially reduced 15-desaturation. The combination of F157W from cluster I with Y280L, H284V, and L287T from cluster II created an increased-activity variant that almost completely lost the ability to desaturate at C15 and acted almost exclusively as a 12-desaturase. No variants were identified in which 15-desaturation occurred in the absence of 12-desaturation. Fm1 displayed only traces of activity with C16 substrate, but several cluster I variants exhibited increased activity with both 18:1 and 16:1 substrates, converting 16:1Δ9 to 16:3Δ9,12,15, consistent with Fm1 performing sequential v + 3 desaturation reactions at C12 and then C15. We propose that cluster II residues interact with the substrate headgroup when the acyl chain contains both Δ9 and Δ12 double bonds, in which case C15 becomes positioned adjacent to the di-iron site enabling a second v + 3 desaturation.

Iron in the Promotion and Initiation of Cancer How Free Iron Accelerates Predisposing Insulin Resistance

Iron is physiologically essential to life, but biochemically it is harmful because of its evident -but unappreciated- oxidative and inflammatory tissue power when it accumulates, is dosed in excess, or is free; and that because, after entering the body, unlike any other metal, its elimination is almost non-existent in man; thus, metal is a powerful promoter of chronic degenerative diseases, from diabetes, neurodegeneration to cancer, through extensive coronary and cardio-cerebrovascular disease; modifying its clinical expressivity and accelerating its severity. Iron is a powerful oxidizing and inflammatory agent, and its accumulation causes and promotes the proliferation of cancer cells in particular, both in animals and in humans. Free and accumulated iron triggers a powerful uncontrolled Cell Proliferation, permanently feeding the survival of the neoplastic cell. After more than 50 years of experimental and preclinical studies, it is clearly demonstrating the carcinogenicity of iron; and this is also proven in humans, from breast cancer and endometrium, in women, to cancer of the colon-rectum, prostate, and pancreas in men. In Western men and women, the reductions in iron deposits have an important anti-tumor and preventive effect for the development of cancer or diabetes, two entities biologically interrelated by the states of Resistance to Insulin, an inflammatory state that favors the development of malignant neoplasms, and can accelerate its aggressiveness. It is the chronic excess of insulin or its Tissue Resistance, the biological event and the clinical syndrome that increases the cancerous power of excess iron, both silent epidemics in modern man. Moderate increases in body iron levels increase the risk of acquiring cancer, and raise the level of their mortality. And its deficiency or chelation in vivo decreases the Tumor growth (Wang F, Elliott RL, Head JF: Inhibitory effect of deferoxamine mesylate and low iron diet on the 13762NF rat mammary adenocarcinoma Anticancer Res. 1999 Jan-Feb; 19 (1A): 445-50). If excess iron mediates and increases the risk of cancer associated with Insulin resistance, any subject with this syndrome can minimize any associated health risks (and their increased risk of cancer), avoiding iron-rich diets and donating blood with regularity; Iron is the metal that causes “exponential” and punctual mutations and fusion of genes through chromosomal translocations, constituting the greatest risk factor for human carcinogenesis. Iron is physiologically essential for life but biochemically dangerous. Chronic accumulation of iron causes pantropic organ damage and excess body iron play an important role in carcinogenesis, coronary artery disease, neurodegenerative disease, stroke and inflammatory disorders. Iron is very slowly excreted from humans once it is absorbed into the body. The significance of iron excess has been markedly underestimated, despite the fact that iron overloading disorders are as common place in the US white population. Iron-overload and catalytic iron promotes activation of oxidative responsive transcription factors and pro-inflammatory cytokines that increase cancer extension and aggravate them. There is accumulative evidence for iron as a carcinogenic metal in epidemiological, clinical, animal, and cell culture studies. The role of iron in various cancers, such as colorectal and liver cancer was demonstrated. Recent advancements on the molecular mechanisms of iron carcinogenesis evolved the Insulin-resistance generation and promotion, fisiopatologic condition that is not only permissive, but may be generated cancer and promoting it. Unlike other nutritional metals, iron is highly conserved: toxicity due to excess iron can occur either acutely after a single dose or chronically due to excessive accumulation in the body from diet. In vivo studies have demonstrated that an iron deficiency induced by either feeding a low iron diet injecting the iron chelator deferoxamine mesylate decreases tumor growth (Wang F, Elliott RL, Head JF: Inhibitory effect of deferoxamine mesylate and low iron diet on the 13762NF rat mammary adenocarcinoma Anticancer Res. 1999 Jan-Feb;19(1A):445-50). Iron supplementation has at times proven ineffective and even detrimental to health. Thus, iron excess may mediate the increased cancer risk associated with insulin resistance and heme-rich diets, and subjects who are insulin resistant can minimize any health risk associated with iron overload by avoiding heme-rich flesh foods and donating blood regularly. The energy that sustains cancer cells derived preferentially from glycolysis depends on the gene p53 deficiency-iron induced. This nutrient is postulated to contribute to the initiation of cancer in vivo, but iron overload initiates and sustain cancer development if chronic infection or insulin resistance conditions are present. Cancer cells require considerably more iron than normal cells. Since iron catalytic can induce driver point mutation and create fusion genes through chromosomal translocations, iron overload is one of the most important risk factors in human carcinogenesis. Because free iron may play a catalytic role in “spontaneous” mutagenesis, moderately elevated iron stores increased overall risk for cáncer.

Early intraoperative iron-binding proteins are associated with acute kidney injury after cardiac surgery

ObjectivesIron regulation is an important modifier of renal ischemia–reperfusion injury, but the role of iron-binding proteins during cardiopulmonary bypass remains unclear. The goal was to characterize iron-binding proteins throughout ischemia–reperfusion injury to determine their association with acute kidney injury development. MethodsA prospective observational cohort of adult patients who underwent cardiac surgery (n = 301) was obtained, and acute kidney injury was defined by Kidney Disease Improving Global Outcomes. Serum ferritin, transferrin saturation, and urine hepcidin-25 were measured. ResultsIntraoperative serum ferritin was lower at the start of cardiopulmonary bypass (P = .005) and 1-hour cardiopulmonary bypass (P = .001) in patients with acute kidney injury versus patients without acute kidney injury. Lower serum ferritin and higher transferrin saturation at 1-hour cardiopulmonary bypass were independent predictors of acute kidney injury (serum ferritin odds ratio, 0.66; 95% confidence interval [CI], 0.48-0.91; transferrin saturation odds ratio, 1.26; 95% CI, 1.02-1.55) and improved model discrimination (area under the curve [AUC], 0.76; 95% CI, 0.67-0.85) compared with clinical prediction alone (AUC, 0.72; 95% CI, 0.62-0.81; ΔAUC and net reclassification index, P = .01). Lower ferritin, higher transferrin saturation at 1-hour cardiopulmonary bypass, and lower urine hepcidin-25 at postoperative day 1 were also independent predictors for acute kidney injury development, and this model demonstrated an AUC of 0.80 (0.72-0.87), which was superior to clinical prediction (ΔAUC P = .002, integrated discrimination improvement and net reclassification index P = .003). ConclusionsOur findings suggest that lower levels of intraoperative iron-binding proteins may reflect an impaired capacity to rapidly handle catalytic iron released during cardiopulmonary bypass, leading to kidney injury. These data highlight the importance of iron homeostasis in human ischemia–reperfusion injury and suggest it is a potentially modifiable risk during cardiac surgery. Intraoperative detection of incipient acute kidney injury may be feasible and could be used as an enrichment strategy for clinical trials.

Adapting to oxygen: 3-Hydroxyanthrinilate 3,4-dioxygenase employs loop dynamics to accommodate two substrates with disparate polarities

3-Hydroxyanthranilate 3,4-dioxygenase (HAO) is an iron-dependent protein that activates O2 and inserts both oxygen atoms into 3-hydroxyanthranilate (3-HAA). An intriguing question is how HAO can rapidly bind O2, even though local O2 concentrations and diffusion rates are relatively low. Here, a close inspection of the HAO structures revealed that substrate- and inhibitor-bound structures exhibit a closed conformation with three hydrophobic loop regions moving toward the catalytic iron center, whereas the ligand-free structure is open. We hypothesized that these loop movements enhance O2 binding to the binary complex of HAO and 3-HAA. We found that the carboxyl end of 3-HAA triggers changes in two loop regions and that the third loop movement appears to be driven by an H-bond interaction between Asn27 and Ile142 Mutational analyses revealed that N27A, I142A, and I142P variants cannot form a closed conformation, and steady-state kinetic assays indicated that these variants have a substantially higher Km for O2 than WT HAO. This observation suggested enhanced hydrophobicity at the iron center resulting from the concerted loop movements after the binding of the primary substrate, which is hydrophilic. Given that O2 is nonpolar, the increased hydrophobicity at the iron center of the binary complex appears to be essential for rapid O2 binding and activation, explaining the reason for the 3-HAA-induced loop movements. Because substrate binding-induced open-to-closed conformational changes are common, the results reported here may help further our understanding of how oxygen is enriched in nonheme iron-dependent dioxygenases.

Catalytic iron-carbene intermediate revealed in a cytochrome c carbene transferase

Recently, heme proteins have been discovered and engineered by directed evolution to catalyze chemical transformations that are biochemically unprecedented. Many of these nonnatural enzyme-catalyzed reactions are assumed to proceed through a catalytic iron porphyrin carbene (IPC) intermediate, although this intermediate has never been observed in a protein. Using crystallographic, spectroscopic, and computational methods, we have captured and studied a catalytic IPC intermediate in the active site of an enzyme derived from thermostable Rhodothermus marinus (Rma) cytochrome c High-resolution crystal structures and computational methods reveal how directed evolution created an active site for carbene transfer in an electron transfer protein and how the laboratory-evolved enzyme achieves perfect carbene transfer stereoselectivity by holding the catalytic IPC in a single orientation. We also discovered that the IPC in Rma cytochrome c has a singlet ground electronic state and that the protein environment uses geometrical constraints and noncovalent interactions to influence different IPC electronic states. This information helps us to understand the impressive reactivity and selectivity of carbene transfer enzymes and offers insights that will guide and inspire future engineering efforts.

Physiological functions of ferroportin in the regulation of renal iron recycling and ischemic acute kidney injury.

Renal iron recycling preserves filtered iron from urinary excretion. However, it remains debated whether ferroportin (FPN), the only known iron exporter, is functionally involved in renal iron recycling and whether renal iron recycling is required for systemic iron homeostasis. We deleted FPN in whole nephrons by use of a Nestin-Cre and in the distal nephrons and collecting ducts, using a Ksp-Cre, and investigated its impacts on renal iron recycling and systemic iron homeostasis. FPN deletion by Nestin-Cre, but not by Ksp-Cre, caused excess iron retention and increased ferritin heavy chain (FTH1) specifically in the proximal tubules and resulted in the reduction of serum and hepatic iron. The systemic iron redistribution was aggravated, resulting in anemia and the marked downregulation of hepatic hepcidin in elderly FPN knockout (KO)/Nestin-Cre mice. Similarly, in iron-deficient FPN KO/Nestin-Cre mice, the renal iron retention worsened anemia with the activation of the erythropoietin-erythroferrone-hepcidin pathway and the downregulation of hepatic hepcidin. Hence, FPN likely located at the basolateral membrane of the proximal tubules to export iron into the circulation and was required for renal iron recycling and systemic iron homeostasis particularly in elderly and iron-deficient mice. Moreover, FPN deletion in the proximal tubules alleviated ischemic acute kidney injury, possibly by upregulating FTH1 to limit catalytic iron and by priming antioxidant mechanisms, indicating that FPN could be deleterious in the pathophysiology of ischemic acute kidney injury (AKI) and thus may be a potential target for the prevention and mitigation of ischemic AKI.

P-228 - Nitric oxide is an epigenetic regulator of gene expression by directly controlling DNA methylation patterns

Previously we demonstrated that nitric oxide (NO) could inhibit the catalytic activity and expression levels of JmjC-domain containing histone demethylases which resulted in significant changes in histone posttranslational modifications. Current studies investigated whether NO could similarly inhibit Ten Eleven Translocation (TET) enzymes which belong to the same family of mononuclear non-heme iron dioxygenases. TET proteins are involved in DNA demethylation by catalyzing the successive oxidation of 5-methylcytosine (5mC) to 5-hydroxy- (5hmC), 5-formly, and 5-carboxy-. Cancer cells treated with physiologic concentrations of NO demonstrated differential changes in TET mRNA/protein expression levels for all three isoforms. We measured changes in 5mC oxidation products in response to NO and found that global levels of 5hmC were markedly decreased in the NO treated cells suggesting inhibition of TET activity. Measurement of TET enzyme activity following cellular NO exposed demonstrated a significant decrease in activity which was by supported by EPR studies showing that NO could directly bind to catalytic non-heme iron. Lastly, the enrichment of 5mC/5hmC at specific NO-regulated genes correlated to changes in their expression indicating a novel mechanisms of gene regulation by NO.

Facile Syntheses and Molecular-Docking of Novel Substituted 3,4-Dimethyl-1H-pyrrole-2-carboxamide/carbohydrazide Analogues with Antimicrobial and Antifungal Properties.

The article describes the use of facile one-pot, high-yielding reactions to synthesize substituted 3,4-dimethyl-1H-pyrrole-2-carboxamides 3a–m and carbohydrazide analogues 5a–l as potential antifungal and antimicrobial agents. The structural identity and purity of the synthesized compounds were assigned based on appropriate spectroscopic techniques. Synthesized compounds were assessed in vitro for antifungal and antibacterial activity. The compounds 5h, 5i and 5j were found to be the most potent against Aspergillus fumigatus, with MIC values of 0.039 mg/mL. The compound 5f bearing a 2, 6-dichloro group on the phenyl ring was found to be the most active broad spectrum antibacterial agent with a MIC value of 0.039 mg/mL. The mode of action of the most promising antifungal compounds (one representative from each series; 3j and 5h) was established by their molecular docking with the active site of sterol 14α-demethylase. Molecular docking studies revealed a highly spontaneous binding ability of the tested compounds in the access channel away from catalytic heme iron of the enzyme, which suggested that the tested compounds inhibit this enzyme and would avoid heme iron-related deleterious side effects observed with many existing antifungal compounds.

N-linoleoylamino acids as chiral probes of substrate binding by soybean lipoxygenase-1

Lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids and their derivatives to produce conjugated diene hydroperoxides. Soybean lipoxygenase-1 (SBLO-1) has been the subject of intensive structural and mechanistic study, but the manner in which this enzyme binds substrates is uncertain. Previous studies suggest that the fatty acyl group of the substrate binds in an internal cavity near the catalytic iron with the polar end at the surface of the protein or perhaps external to the protein. To test this model, we have investigated two pairs of enantiomeric N-linoleoylamino acids as substrates for SBLO-1. If the amino acid moiety binds external to the protein, the kinetics and product distribution should show little or no sensitivity to the stereochemical configuration of the amino acid moiety. Consistent with this expectation, N-linoleoyl-l-valine (LLV) and N-linoleoyl-d-valine (LDV) are both good substrates with kcat/Km values that are equal within error and about 40% higher than kcat/Km for linoleic acid. Experiments with N-linoleoyl-l-tryptophan (LLT) and N-linoleoyl-d-tryptophan (LDT) were complicated by the low critical micelle concentrations (CMC = 6–8 μM) of these substances. Below the CMC, LDT is a better substrate by a factor of 2.7. The rates of oxygenation of LDT and LLT continue to rise above the CMC, with modest stereoselectivity in favor of the d enantiomer. With all of the substrates tested, the major product is the 13(S)-hydroperoxide, and the distribution of minor products is not appreciably affected by the configuration of the amino acid moiety. The absence of stereoselectivity with LLV and LDV, the modest magnitude of the stereoselectivity with LLT and LDT, and the ability micellar forms of LLT and LDT to increase the concentration of available substrate are all consistent with the hypothesis that the amino acid moiety binds largely external to SBLO-1 and interacts with it only weakly.