Heme Molecule Research Articles

Artemisinin-resistant Plasmodium falciparum Kelch13 mutant proteins display reduced heme-binding affinity and decreased artemisinin activation.

The potency of frontline antimalarial drug artemisinin (ART) derivatives is triggered by heme-induced cleavage of the endoperoxide bond to form reactive heme-ART alkoxy radicals and covalent heme-ART adducts, which are highly toxic to the parasite. ART-resistant (ART-R) parasites with mutations in the Plasmodium falciparum Kelch-containing protein Kelch13 (PfKekch13) exhibit impaired hemoglobin uptake, reduced yield of hemoglobin-derived heme, and thus decreased ART activation. However, any direct involvement of PfKelch13 in heme-mediated ART activation has not been reported. Here, we show that the purified recombinant PfKelch13 wild-type (WT) protein displays measurable binding affinity for iron and heme, the main effectors for ART activation. The heme-binding property is also exhibited by the native PfKelch13 protein from parasite culture. The two ART-R recombinant PfKelch13 mutants (C580Y and R539T) display weaker heme binding affinities compared to the ART-sensitive WT and A578S mutant proteins, which further translates into reduced yield of heme-ART derivatives when ART is incubated with the heme molecules bound to the mutant PfKelch13 proteins. In conclusion, this study provides the first evidence for ART activation via the heme-binding propensity of PfKelch13. This mechanism may contribute to the modulation of ART-R levels in malaria parasites through a novel function of PfKelch13.

Modulatory potentials of nitric oxide in obesity-induced endothelial dysfunction

Obesity is caused by a significant increase in adipose tissue due to excessive caloric intake that exceeds energy expenditure. Obesity has emerged as a significant public health issue in both poor resource and economic developed nations, owing to its increased incidence and links to chronic diseases, such osteoarthritis, hypertension, non-alcoholic fatty liver disease (NAFLD), and cardiovascular diseases (CVDs). It is believed that uncoupled eNOS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, xanthine oxidase, lipoxygenase, cyclooxygenase, microsomal P-450 enzymes, and pro-oxidant heme molecules are responsible for the drastic decrease in NO production and the relative increase in reactive oxygen species (ROS) secretion, which are considered as the molecular mechanisms associated with obesity-induced endothelial dysfunction. Vascular endothelium defines a monolayer of cells, which lies between the vascular smooth muscle cells (VSMCs) and the vessel lumen. It performs a number of protective actions facilitated by the release of nitric oxide (NO), a soluble gas that is generated in endothelial cells by the amino acid L-arginine through the action of endothelial nitric oxide synthase (eNOS). The maintenance of vascular homeostasis is facilitated by various biological actions of NO, such as modulation of vascular dilator tone, control of local cell growth, and protection of the endothelium from the harmful effects of cellular components in blood circulation, while maintaining normal endothelial functions. Decreased peripheral arterial endothelium-dependent dilation (EDD) in response to mechanical (intravascular shear) or chemical (acetylcholine) stimuli suggests that decreased NO bioavailability has been recognised as major molecular mechanisms associated with the progress and development of obesity-induced endothelial dysfunction. In this review, the modulatory effects of NO in obesity-induced endothelial dysfunction will be discussed.

دراسة دور الهيم اوكسجينيز-1 في المرضى الذين يعانون من السمنة

الهيم اوكسجينيز-1 هو عبارة إنزيم محفز يعمل على تكسر جزيئات الهيم إلى بيليفيردين، وحديدالحر، وبيليروبين، وأول أكسيد الكربون. تهدف هذه الدراسة إلى معرفة مستويات ودور (HO-1) في مرضى السمنة ضد الإجهاد التأكسدي وعلاقته بالسمنة. تضمنت الدراسة 139 عينة تضم (84بدناء و55 اشخاص غير بدناء). تم تقسيم كلا مجاميع الدراسة قسمت إلى أربع مجاميع بالاعتماد على مؤشر كتلة الجسم. تم جمع عينات الدم اشخاص بدناء ومجاميع صحية (رجال ونساء) في مستشفى اليرموك والمركز الصحي لأبحاث السكري في الفترة بين ديسمبر2022/ الى يونيو/2023. تم قياس بعض المتغيرات الكيميائية الحيوية لجميع مجموعات الدراسة، والتي تشمل: - تحديد مستويات HO-1 في المصل باستخدام تقنية ELISAو ملف الدهون والسكر الصائم FSG بالطريقة الانزيمية. حيث وجد أن مستويات مؤشر كتلة الجسم تزداد بشكل ملحوظ في مجموعة السمنة من النوع الثاني ومجموعة السمنة من النوع الأول ومجموعة الوزن الزائد مقارنة بمجاميع وزن الطبيعي. كما أظهرت النتائج زيادة معنوية (p≤ 0.05) في محيط الخصر الى الورك (WHR) في مجاميع السمنة من النوع الثاني والسمنة من النوع الأول ومجموعة الوزن الزائد مقارنة بمجوعة الوزن الطبيعي. كذلك أظهرت النتائج أن مستويات HO-1 عالية في مرضى السمنة من النوع الاول ومجموعة السمنة من النوع الثاني مقارنةً مع مجاميع الوزن الطبيعي الوزن الزائد. اظهر التحليل الاحصائي أن مستوى HO-1 ترتبط سلبيا مع مؤشر كتلة الجسم في مجموعة الوزن الطبيعي G1))، بينما إيجابية مع مجاميع السمنة من النوع الثاني والسمنة من النوع الأول والوزن الزائد. نستنتج من هذه الدراسة أن خط دفاع الجسم الأول ضد هجوم الإجهاد التأكسدي هو HO-1. هذا الإنزيم الواقي للخلايا يقلل من الإجهاد التأكسدي وهو حيوي للتحكم في تكون الدهون، وهو أمر حاسم لتطور أمراض التمثيل الغذائي ومضاعفاتها.

In-depth structure-function profiling of the complex formation between clotting factor VIII and heme

Background and aimsBlood disorders, such as sickle cell disease, and other clinical conditions are often accompanied by intravascular hemolytic events along with the development of severe coagulopathies. Hemolysis, in turn, leads to the accumulation of Fe(II/III)-protoporphyrin IX (heme) in the intravascular compartment, which can trigger a variety of proinflammatory and prothrombotic reactions. As such, heme binding to the blood coagulation proteins factor VIII (FVIII), fibrinogen, and activated protein C with functional consequences has been demonstrated earlier. MethodsWe herein present an in-depth characterization of the FVIII-heme interaction at the molecular level and its (patho-)physiological relevance through the application of biochemical, biophysical, structural biology, bioinformatic, and diagnostic tools. ResultsFVIII has a great heme-binding capacity with seven heme molecules associating with the protein. The respective binding sites were identified by investigating heme binding to FVIII-derived peptides in combination with molecular docking and dynamic simulation studies of the complex as well as cryo-electron microscopy, revealing three high-affinity and four moderate heme-binding motifs (HBMs). Furthermore, the relevance of the FVIII-heme complex formation was characterized in physiologically relevant assay systems, revealing a ~ 50 % inhibition of the FVIII cofactor activity even in the protein-rich environment of blood plasma. ConclusionOur study provides not only novel molecular insights into the FVIII-heme interaction and its physiological relevance, but also strongly suggests the reduction of the intrinsic pathway and the accentuation of the final clotting step (by, for example, fibrinogen crosslinking) in hemolytic conditions as well as a future perspective in the context of FVIII substitution therapy of hemorrhagic events in hemophilia A patients.

Fabrication of Electronically Conductive Protein-Heme Nanowires for Power Harvesting.

Electronically conductive protein-based materials can enable the creation of bioelectronic components and devices from sustainable and nontoxic materials, while also being well-suited to interface with biological systems, such as living cells, for biosensor applications. However, as proteins are generally electrical insulators, the ability to render protein assemblies electroactive in a tailorable manner can usher in a plethora of useful materials. Here, an approach to fabricate electronically conductive protein nanowires is presented by aligning heme molecules in proximity along protein filaments, with these nanowires also possessing charge transfer abilities that enable energy harvesting from ambient humidity. The heme-incorporated protein nanowires demonstrate electron transfer over micrometer distances, with conductive atomic force microscopy showing individual nanowires having comparable conductance to other previously characterized heme-based bacterial nanowires. Exposure of multilayer nanowire films to humidity produces an electrical current, presumably through water molecules ionizing carboxyl groups in the filament and creating an unbalanced total charge distribution that is enhanced by the heme. Incorporation of heme and potentially other metal-center porphyrin molecules into protein nanostructures could pave the way for structurally- and electrically-defined protein-based bioelectronic devices.

Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit

Filamentous fungi are critical in the transition to a more sustainable food system. While genetic modification of these organisms has promise for enhancing the nutritional value, sensory appeal, and scalability of fungal foods, genetic tools and demonstrated use cases for bioengineered food production by edible strains are lacking. Here, we develop a modular synthetic biology toolkit for Aspergillus oryzae, an edible fungus used in fermented foods, protein production, and meat alternatives. Our toolkit includes a CRISPR-Cas9 method for gene integration, neutral loci, and tunable promoters. We use these tools to elevate intracellular levels of the nutraceutical ergothioneine and the flavor-and color molecule heme in the edible biomass. The strain overproducing heme is red in color and is readily formulated into imitation meat patties with minimal processing. These findings highlight the promise of synthetic biology to enhance fungal foods and provide useful genetic tools for applications in food production and beyond.

Hematin anhydride (β-hematin): An analogue to malaria pigment hemozoin possesses nonlinearity

Hematin anhydride (β-hematin), the synthetic analogue of the malaria pigment, “hemozoin”, is a heme dimer produced by reciprocal covalent bonds among carboxylic acid groups on the protoporphyrin-IX ring and the iron atom present in the two adjacent heme molecules. Hemozoin is a disposal product formed from the digestion of hemoglobin present in the red blood cells infected with hematophagous malaria parasites. Besides, as the parasites invade red blood cells, hemozoin crystals are eventually released into the bloodstream, where they accumulate over time in tissues. Severe malaria infection leads to significant dysfunction in vital organs such as the liver, spleen, and brain in part due to the autoimmune response to the excessive accumulation of hemozoin in these tissues. Also, the amount of these crystals in the vasculature correlates with disease progression. Thus, hemozoin is a unique indicator of infection used as a malaria biomarker and hence, used as a target for the development of antimalarial drugs. Hence, exploring various properties of hemozoin is extremely useful in the direction of diagnosis and cure. The present study focuses on finding one of the unknown properties of β-hematin in physiological conditions by using the Z-scan technique, which is simple, sensitive, and economical. It is observed that hemozoin possesses one of the unique material properties, i.e., nonlinearity with a detection limit of ∼ 15 µM. The self-defocusing action causes β-hematin to exhibit negative refractive nonlinearity. The observed data is analyzed with a thermal lensing model. We strongly believe that our simple and reliable approach to probing the nonlinearity of β-hematin will provide fresh opportunities for malaria diagnostics & cure in the near future.

Pathway crosstalk between the central metabolic and heme biosynthetic pathways in Phanerochaete chrysosporium

A comprehensive analysis to survey heme-binding proteins produced by the white-rot fungus Phanerochaete chrysosporium was achieved using a biotinylated heme–streptavidin beads system. Mitochondrial citrate synthase (PcCS), glyceraldehyde 3-phosphate dehydrogenase (PcGAPDH), and 2-Cys thioredoxin peroxidase (mammalian HBP23 homolog) were identified as putative heme-binding proteins. Among these, PcCS and PcGAPDH were further characterized using heterologously expressed recombinant proteins. Difference spectra of PcCS titrated with hemin exhibited an increase in the Soret absorbance at 414 nm, suggesting that the axial ligand of the heme is a His residue. The activity of PcCS was strongly inhibited by hemin with Ki oxaloacetate of 8.7 μM and Ki acetyl-CoA of 5.8 μM. Since the final step of heme biosynthesis occurred at the mitochondrial inner membrane, the inhibition of PcCS by heme is thought to be a physiological event. The inhibitory mode of the heme was similar to that of CoA analogues, suggesting that heme binds to PcCS at His347 at the AcCoA–CoA binding site, which was supported by the homology model of PcCS. PcGAPDH was also inhibited by heme, with a lower concentration than that for PcCS. This might be caused by the different location of these enzymes. From the integration of these phenomena, it was concluded that metabolic regulations by heme in the central metabolic and heme synthetic pathways occurred in the mitochondria and cytosol. This novel pathway crosstalk between the central metabolic and heme biosynthetic pathways, via a heme molecule, is important in regulating the metabolic balance (heme synthesis, ATP synthesis, flux balance of the tricarboxylic acid (TCA) cycle and cellular redox balance (NADPH production) during fungal aromatic degradation.Key points• A comprehensive survey of heme-binding proteins in P. chrysosporium was achieved.• Several heme-binding proteins including CS and GAPDH were identified.• A novel metabolic regulation by heme in the central metabolic pathways was found.

α-Glucosidase isoform G contributes to heme detoxification in Rhodnius prolixus and its knockdown affects Trypanosoma cruzi metacyclogenesis

The triatomine bug Rhodnius prolixus is a hematophagous hemipteran and a primary vector of Trypanosoma cruzi, the causative agent of Chagas' disease (CD), in Central America and Northern South America. Blood-feeding poses significant challenges for hematophagous organisms, particularly due to the release of high doses of pro-oxidant free heme during hemoglobin digestion. In this arthropod, most of the free heme in the gut is aggregated into hemozoin (Hz), an inert and non-oxidative biocrystal. Two major components present in the perimicrovillar membranes (PMM) of triatomine insects have been previously implicated in heme crystallization: lipids and the biochemical marker of the PMM, the enzyme α-glucosidase. In this study, we investigated the role of R. prolixus α-glucosidase isoform G (Rp-αGluG) in heme detoxification and the effects of its knockdown on the insect physiology. The effect of α-glucosidase isoform G (αGluG) knockdown on T. cruzi proliferation and metacyclogenesis was also investigated. Initially, a 3D structure of Rp-αGluG was predicted by comparative modeling and then subjected to molecular docking with the heme molecule, providing in silico support for understanding the process of Hz biocrystallization. Next, adult females of R. prolixus were challenged with RNAi against Rp-αGluG (dsαGluG) to assess physiological and phenotypic changes caused by its knockdown. Our data show that the group challenged with dsαGluG produced less Hz, resulting in more intact hemoglobin available in the digestive tract. These animals also laid fewer eggs, which had a lower hatching rate. In addition, T. cruzi metacyclogenesis was significantly lower in the dsαGluG group. The present work demonstrates the importance of Rp-αGluG in heme detoxification, the digestive and reproductive physiology of R. prolixus, as well as its influence on the life cycle of T. cruzi. Since heme neutralization is a vital process for hematophagous bugs, our study provides useful information for the development of new strategies targeting the Hz formation and potentially affecting the vectorial transmission of Chagas disease.

N-unsubstituted Imidazoles: Design, Synthesis, and Antimicrobial Evaluation.

All the current antifungal azoles have one substituted nitrogen atom in their imidazole or triazole rings. In this study, eleven imine and amine derivatives of imidazole, in which both nitrogen atoms of the imidazole ring are unsubstituted, were designed and synthesized. Imine derivatives were prepared by condensation of imidazole-4-carboxaldehyde with appropriate amines, and then in the next step, using sodium borohydride, the imines were reduced to amine derivatives. Docking studies reveal unsubstituted nitrogen atom of the imidazole ring coordinated well with the heme molecule of the receptor. In vitro, antimicrobial evaluation was tested on Candida albicans, E. coli, and Staphylococcus aureus. Based on the results of the antimicrobial study, compound 10, which contains 4-chlorobenzyl moiety, proved to be the most potent compound against Candida albicans, and it was more active than the reference drug fluconazole and showed comparable activity to amphotericin B. Compounds 10 and 11 and compounds 8, 10 and 11 showed significant responses against E. coli and Staphylococcus aureus respectively. It is concluded that compound 10 can be acted as a new lead compound to find new azoles antifungal.

Wolbachia Ferrochelatase as a potential drug target against filarial infections

Filarial infections are among the world's most disturbing diseases caused by 3 major parasitic worms; Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi, affecting more than 500 million people worldwide. Currently used drugs for mass drug administration (MDA) have been met with several challenges including the development of complications in individuals with filaria co-infections and parasitic drug resistance. The filarial endosymbiont, Wolbachia, has emerged as an attractive therapeutic target for filariasis elimination, due to the dependence of the filaria on this endosymbiont for survival. Here, we target an important enzyme in the Wolbachia heme biosynthetic pathway (ferrochelatase), using high-throughput virtual screening and molecular dynamics with MM-PBSA calculations. We identified four drug candidates; Nilotinib, Ledipasvir, 3-benzhydryloxy-8-methyl-8-azabicyclo[3.2.1]octane, and 2-(4-Amino-piperidin-1-yl)-ethanol as potential small molecules inhibitors as they could compete with the enzyme's natural substrate (Protoporphyrin IX) for active pocket binding. This prevents the worm from receiving the heme molecule from Wolbachia for their growth and survival, resulting in their death. This study which involved targeting enzymes in biosynthetic pathways of the parasitic worms' endosymbiont (Wolbachia), has proven to be an alternative therapeutic option leading to the discovery of new drugs, which will help facilitate the elimination of parasitic infections.

Hemopexin and albumin inhibit heme-induced macrophage activation while also enabling heme-LPS synergistic promotion of TNF production

Free heme released from hemoglobin contributes to exacerbated inflammation and tissue damage in hemolytic diseases. While a moderate level of free heme does not cause intravascular inflammation by itself, its presence during infection greatly enhances inflammation. Although specific serum proteins have been found to affect heme-induced inflammation, the selective contribution of serum proteins inhibiting macrophage activation by heme or, conversely, amplifying the production of cytokines by macrophages stimulated with heme and microbial molecules, is poorly defined. Here we identified a serum fraction containing proteins with >50 KDa which was capable of inhibiting heme-stimulated TNF production and capable of enabling TNF production under conditions of a heme-LPS synergy. The inhibition of heme-induced TNF production was mimicked by Hemopexin (Hx), human serum albumin (HSA), serum from Hx-knockout mice, and less efficiently by serum from albumin-knockout mice, but not by serum LDL. Hx and HSA inhibited heme-induced ROS generation, MAPK/ Syk phosphorylation and cell death. However, Hx and HSA each also promoted the synergistic relationship between heme and LPS upon TNF production. Serum from Hx-knockout mice was fully capable of enabling this synergy, while serum from albumin-knockout mice was less efficient to promote TNF production under these conditions. Low concentrations of HSA mimicked the ability of serum to enable heme-stimulated IL-1β production after LPS priming, while high concentrations inhibited it. Together, our findings indicate how heme inflammatory effects are restrained in the blood upon sterile hemolysis, yet exacerbate inflammation in the presence of microbes. Moreover, it is interesting to note that opposing effects of serum proteins on heme-induced macrophage activation were selected through evolution, with both effects exerted by Hx and albumin.

Abstract 2845: PpIX synthesis increases PDT resistant cells due to the modulating effect of EGCG on the heme synthesis pathway

Abstract Introduction: Photodynamic therapy (PDT) is a treatment for non melanoma skin cancer. PDT kills cells through reactive oxygen species (ROS), produced by interaction between cellular O2 and an excited photosensitizer (PS) by a specific light. One of the most used PS in dermatology is Protoporphyrin IX (PpIX) and its precursor methyl aminolevulinate (MAL). MAL enters into cells and is converted to PpIX by enzymes of the heme group synthesis pathway. The insertion of ferrous iron into PpIX forms a heme molecule, which has no PS action. A previous study shows that efficacy of PDT, in resistant cells, can be improved by addition of epigallocatechin gallate (EGCG), a green tea polyphenol. Therefore, the aim of this work is to evaluate the rol of EGCG on PPIX synthesis. Material and methods: It was used a A-431 cell line from skin squamous cell carcinoma. The EC50 of EGCG was determined by MTT assay after 24 hours. Clonogenic assay was carried out using EGCG 10, 20 and 40 μM, for 4 and 24 hours of incubation. To evaluate PpIX and ROS, A-431 cells were exposed to EGCG (10, 20, 40 μM) with or without MAL 2 mM. After 4 hours, some plates were irradiated with red light to generate ROS. PpIX and ROS were detected by flow cytometry. Gene expression of enzymes of heme synthesis was analyzed by RT-qPCR. Intracellular iron was measured using a commercial kit.  Results: EC50 determined a concentration of 50 μM of EGCG at 24 hours. EGCG concentrations from 10 μM reduced cell clonogenic capacity, even with only 4 hours of incubation. A-431 cells show PDT resistance with MAL 2 mM and a fluence of 4 J/cm2. The addition of EGCG to MAL-PDT showed a decrement of cell viability with an increase of PpIX and ROS. Besides, incubation with EGCG for 4 hours, triggered an increment of HMBS enzyme and decrease of FECH. In addition, EGCG reduced intracellular iron concentrations. Conclusion: These findings show that EGCG has a PDT enhancing effect, decreasing intracellular iron levels and modulating gene expression of heme synthesis enzymes, which are directly related with high levels of PpIX production and ROS levels, causing the death of resistant cells.Acknowledgements: Postdoctoral FONDECYT Nº 3210618, IDeA I+D FONDEF ID21I10027 Citation Format: Daniela León, Álvaro Gutiérrez, Helga Weber, Ramón Silva, María Elena Reyes, Tamara Viscarra, Kurt Buchegger, Carmen Ili, Priscilla Brebi. PpIX synthesis increases PDT resistant cells due to the modulating effect of EGCG on the heme synthesis pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2845.

Pre-conditioning of gingival epithelial cells with sub-apoptotic concentrations of curcumin prevents pro-inflammatory cytokine release.

Plaque-induced gingival inflammation (gingivitis) is ubiquitous in humans. The epithelial barrier reacts to the presence of oral bacteria and induces inflammatory cascades. The objective of this study was to investigate the mechanism by which the small molecule micronutrient curcumin could decrease inflammatory response in vitro to oral bacterium heat-killed Fusobacterium nucleatum as curcumin could be a useful compound for combatting gingivitis already consumed by humans. H400 oral epithelial cell line was pre-conditioned with curcumin and the production of cytokines was measured by enzyme-linked immunosorbent assay (ELISA) and translocation of transcription factors was used to monitor inflammatory responses. Haem oxygenase (HO-1) expression and molecules that HO-1 releases were evaluated for their potential to reduce the quantity of cytokine production. Immunofluorescence microscopy and Western blotting were used to evaluate changes in transcription factor and enzyme location. Pre-conditioning of H400 cells with a sub-apoptotic concentration of curcumin (20 μM) attenuated secretion of Granulocyte-Macrophage - Colony-Stimulating Factor (GM-CSF) and reduced NFkB nuclear translocation. This pre-conditioning caused an increase in nuclear Nrf2; an initial drop (at 8h) followed by an adaptive increase (at 24 h) in glutathione; and an increase in haem oxygenase (HO-1) expression. Inhibition of HO-1 by SnPPIX prevented the curcumin-induced attenuation of GM-CSF production. HO-1 catalyses the breakdown of haem to carbon monoxide, free iron and biliverdin: the HO-1/CO anti-inflammatory pathway. Elevations in carbon monoxide, achieved using carbon monoxide releasing molecule-2 (CORM2) treatment alone abrogated F. nucleatum-induced cytokine production. Biliverdin is converted to bilirubin by biliverdin reductase (BVR). This pleiotropic protein was found to increase in cell membrane expression upon curcumin treatment. Curcumin decreased inflammatory cytokine production induced by Fusobacterium nucleatum in H400 oral epithelial cells. The mechanism of action appears to be driven by the increase of haem oxygenase and the production of carbon monoxide.

QM Calculations Revealed that Outer-Sphere Electron Transfer Boosted O-O Bond Cleavage in the Multiheme-Dependent Cytochrome bd Oxygen Reductase.

The cytochrome bd oxygen reductase catalyzes the four-electron reduction of dioxygen to two water molecules. The structure of this enzyme reveals three heme molecules in the active site, which differs from that of heme-copper cytochrome c oxidase. The quantum chemical cluster approach was used to uncover the reaction mechanism of this intriguing metalloenzyme. The calculations suggested that a proton-coupled electron transfer reduction occurs first to generate a ferrous heme b595. This is followed by the dioxygen binding at the heme d center coupled with an outer-sphere electron transfer from the ferrous heme b595 to the dioxygen moiety, affording a ferric ion superoxide intermediate. A second proton-coupled electron transfer produces a heme d ferric hydroperoxide, which undergoes efficient O-O bond cleavage facilitated by an outer-sphere electron transfer from the ferrous heme b595 to the O-O σ* orbital and an inner-sphere proton transfer from the heme d hydroxyl group to the leaving hydroxide. The synergistic benefits of the two types of hemes rationalize the highly efficient oxygen reduction repertoire for the multi-heme-dependent cytochrome bd oxygen reductase family.

Bio-Electroanalysis Performance of Heme Redox-Center for π-π Interaction Bonding of a Methylene Blue-Graphene Modified Electrode.

Hemeprotein detection has motivated extensive research on the direct reaction of a heme molecule and a redox dye. The present study used methylene blue as both donor and acceptor for a redox reaction. First, the solid phases of methylene blue (MB) and graphene (GP) formed a π-π interaction bond at the aromatic rings. The conductivity of GP was better than that of carbon in a carbon electrode (CE). Then, the working CE was modified using strong adsorption of MB/GP on the electrode surface. The surface of the electrode was investigated using a modified and an unmodified electrode. The electrode's properties were studied using voltammograms of redox couple K3[Fe(CN)6]3-/4-. Its reaction was used to find the active area of the modified electrode, which was 1.76 times bigger than that of the unmodified electrode. The surface coverage values of the modified and unmodified electrodes were 8.17 × 10-6 and 1.53 × 10-5 mol/cm2, respectively. This research also studied the application of hemeprotein detection. Hemoglobin (Hb), myoglobin (Mb), and cytochrome c (Cyt. C) were studied by the reaction of Fe (III/II) at the heme-redox center. The electrocatalytic reaction between MB/GP and hemeproteins produced an anodic peak at 0.35 V for Hb, Mb, and Cyt. C. This nanohybrid film enhanced electron transfer between protein molecules and the modified carbon electrode. The amperometric measurements show that the limit of detection was 0.2 µM, 0.3 µM, and 0.1 µM for Hb, Mb, and Cyt. C, respectively. The measurement spanned a linear range of 0.2 µM to 5 µM, 0.3 µM to 5 µM, and 0.1 µM to 0.7 µM for Hb, Mb, and Cyt. C, respectively. Hb showed the lowest sensitivity compared with Mb and Cyt. C due to the role of steric hindrance in the hemeprotein specificity structure. This study offers a simple and efficient fabrication platform for electrochemical sensors for hemeproteins. When compared to other complex immobilization processes, the fabrication method for this sensor has many benefits, including no need for special chemicals and easy preparation and electrode modification-both of which are crucial for the development of electrochemical sensing devices.

The Shr receptor from Streptococcus pyogenes uses a cap and release mechanism to acquire heme–iron from human hemoglobin

Streptococcus pyogenes (group A Streptococcus) is a clinically important microbial pathogen that requires iron in order to proliferate. During infections, S. pyogenes uses the surface displayed Shr receptor to capture human hemoglobin (Hb) and acquires its iron-laden heme molecules. Through a poorly understood mechanism, Shr engages Hb via two structurally unique N-terminal Hb-interacting domains (HID1 and HID2) which facilitate heme transfer to proximal NEAr Transporter (NEAT) domains. Based on the results of X-ray crystallography, small angle X-ray scattering, NMR spectroscopy, native mass spectrometry, and heme transfer experiments, we propose that Shr utilizes a "cap and release" mechanism to gather heme from Hb. In the mechanism, Shr uses the HID1 and HID2 modules to preferentially recognize only heme-loaded forms of Hb by contacting the edges of its protoporphyrin rings. Heme transfer is enabled by significant receptor dynamics within the Shr-Hb complex which function to transiently uncap HID1 from the heme bound to Hb's β subunit, enabling the gated release of its relatively weakly bound heme molecule and subsequent capture by Shr's NEAT domains. These dynamics may maximize the efficiency of heme scavenging by S. pyogenes, enabling it to preferentially recognize and remove heme from only heme-loaded forms of Hb that contain iron.

Depleted uranium causes renal mitochondrial dysfunction through the ETHE1/Nrf2 pathway

The kidney is the main organ affected by acute depleted uranium (DU) toxicity. The mechanism of nephrotoxicity induced by DU is complex and needs to be further explored. This study aimed to elucidate the function of mitochondrial dysfunction in nephrotoxicity generated by DU and confirm the latent mechanism. We verified that DU (2.5–10 mg/kg) caused mitochondrial dysfunction in male rat kidneys and decreased ATP content and the mitochondrial membrane potential. In addition, melatonin (20 mg/kg), as an antioxidant, alleviated DU-induced oxidative stress and mitochondrial dysfunction in male rats, further reducing kidney damage caused by DU. These results indicate that mitochondrial dysfunction plays a vital role in DU nephrotoxicity. When ethylmalonic encephalopathy 1 (ETHE1) was knocked down, DU-induced oxidative stress and mitochondrial dysfunction were increased, and renal injury was aggravated. When exogenous ETHE1 protein was applied to renal cells, the opposite changes were observed. We also found that ETHE1 knockdown increased the expression of NF-E2-related factor 2 (Nrf2), a vital oxidative stress regulator, and its downstream molecules heme oxygenase-1 (HO-1) and NADPH quinone oxidoreductase 1 (NQO1). Nrf2 knockout also aggravated DU-induced oxidative stress, mitochondrial dysfunction, and kidney damage. In conclusion, DU causes oxidative stress and antioxidant defense imbalance in renal cells through the ETHE1/Nrf2 pathway, further causing mitochondrial dysfunction and ultimately leading to nephrotoxicity.

A Curious Case of Methaemoglobinemia

The most frequent cause of raised methaemoglobin is from recreational drug use, with oxidation of the haem molecule leading to methaemoglobinaemia. Our case describes that of a man in his early thirties who presented with shortness of breath, hypoxia and methaemoglobinemia due to ingestion of impure cocaine mixed with local anaesthetic agents, a common cause of acquired methaemoglobinemia. This case report demonstrates the need for clinicians to consider methaemoglobinaemia as a differential diagnosis for patients presenting with hypoxia.

Surface bonding of MN4 macrocyclic metal complexes with pyridine-functionalized multi-walled carbon nanotubes for non-aqueous Li-O2 batteries

It is essential to develop bifunctional catalysts with high activity and stability for reversible oxygen reduction reactions (ORRs) and oxygen evolution reactions (OERs) in lithium-oxygen (Li-O2) batteries. In this work, pyridine (Py) functionalized multi-walled carbon nanotubes (MWCNTs) were prepared to immobilize various solid MN4 macrocyclic metal complexes (MN4-MC) as cathode electrocatalysts for Li-O2 batteries. Three types of MN4-MC molecules, including iron phthalocyanine (FePc), cobalt phthalocyanine (CoPc) and iron protoporphyrin IX (Heme) were examined to evaluate the influence of central metal atoms and ligand substituents found in MN4-MC molecules on the electrocatalytic performance of the study samples. The order of the ORR/OER catalytic activity of the bifunctional catalysts is FePc > Heme > CoPc. The central metal atom in FePc molecule has the highest occupied molecular orbital (HOMO) energy than the corresponding metal atoms in CoPc and Heme molecules. This made the molecule to have better dioxygen-binding ability and higher catalytic activity in the ORR process; it also made it to easily lose electrons that were oxidized in the OER process. This study proposed a simplified scheme of the electrode surface route to assist in understanding the diverse ORR/OER performances of MN4-MC. It is discovered that the positive core of the MN5 coordination sphere in MN4-MC/Py/MWCNTs composite is the primary active site that can influence the formation of MN5···O2* and MN5-LOOLi cluster in the ORR process. The interfacial electron could be easily delivered between MWCNTs and MN5 active site through the Py bridge. This facilitated the formation and decomposition of MN5-LOOLi species during the ORRs/OERs, leading to the enhancement of its catalytic performance. This work provides a new insight into the effects of the molecular structure and organization of MN4-MC on the catalytic activity of O2 electrodes in Li-O2 batteries.