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Cooperativity is essential for the proper functioning of numerous proteins by allosteric interactions. Hemoglobin from Scapharca inaequivalvis (HbI) is a homodimeric protein that can serve as a minimal unit for studying cooperativity. We investigated the structural changes in HbI after carbon monoxide dissociation using time-resolved resonance Raman spectroscopy and observed structural rearrangements in the Fe–proximal histidine bond, the position of the heme in the pocket, and the hydrogen bonds between heme and interfacial water upon ligand dissociation. Some of the spectral changes were different from those observed for human adult hemoglobin due to differences in subunit assembly and quaternary changes. The structural rearrangements were similar for the singly and doubly dissociated species but occurred at different rates. The rates of the observed rearrangements indicated that they occurred synchronously with subunit rotation and are influenced by intersubunit coupling, which underlies the positive cooperativity of HbI. [ABSTRACT FROM AUTHOR]

In our efforts to develop inhibitors selective for neuronal nitric oxide synthase (nNOS) over endothelial nitric oxide synthase (eNOS), we found that nNOS can undergo conformational changes in response to inhibitor binding that does not readily occur in eNOS. One change involves movement of a conserved tyrosine, which hydrogen bonds to one of the heme propionates, but in the presence of an inhibitor, changes conformation, enabling part of the inhibitor to hydrogen bond with the heme propionate. This movement does not occur as readily in eNOS and may account for the reason why these inhibitors bind more tightly to nNOS. A second structural change occurs upon the binding of a second inhibitor molecule to nNOS, displacing the pterin cofactor. Binding of this second site inhibitor requires structural changes at the dimer interface, which also occurs more readily in nNOS than in eNOS. Here, we used a combination of crystallography, mutagenesis, and computational methods to better understand the structural basis for these differences in NOS inhibitor binding. Computational results show that a conserved tyrosine near the primary inhibitor binding site is anchored more tightly in eNOS than in nNOS, allowing for less flexibility of this residue. We also find that the inefficiency of eNOS to bind a second inhibitor molecule is likely due to the tighter dimer interface in eNOS compared with nNOS. This study provides a better understanding of how subtle structural differences in NOS isoforms can result in substantial dynamic differences that can be exploited in the development of isoform-selective inhibitors.

Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site. Together with structure-guided in vitro and in vivo experiments, we further demonstrate the existence of a functional link between heme binding by Dri1 (Domain related to iron 1, formerly ssr1698) and post-translational regulation of succinate dehydrogenase in the cyanobacterium Synechocystis, suggesting an iron-dependent regulatory link between photosynthesis and respiration. Given the ubiquity of proteins containing homologous domains and connections to heme metabolism across eukaryotes and prokaryotes, we propose that DRI (Domain Related to Iron; formerly DUF2470) functions at the molecular level as a heme-dependent regulatory domain.

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.

The cytochrome P450 homolog, TxtE, efficiently catalyzes the direct and regioselective aromatic nitration of the indolyl moiety of L‐tryptophan to 4‐nitro‐L‐tryptophan, using nitric oxide (NO) and dioxygen (O2) as co‐substrates. Pathways for such direct and selective nitration of heteroaromatic motifs present platforms for engineering new nitration biocatalysts for pharmacologically beneficial targets, among a medley of other pivotal industrial applications. Precise mechanistic details concerning this pathway are only weakly understood, albeit a heme iron(III)‐peroxynitrite active species has been postulated. To shed light on this unique reaction landscape, we investigated the indole nitration pathway of a series of biomimetic ferric heme superoxide mimics, [(Por)FeIII(O2−⋅)], in the presence of NO. Therein, our model systems gave rise to three distinct nitroindole products, including 4‐nitroindole, the product analogous to that obtained with TxtE. Moreover, 15N and 18O isotope labeling studies, along with meticulously designed control experiments lend credence to a heme peroxynitrite active nitrating agent, drawing close similarities to the tryptophan nitration mechanism of TxtE. All organic and inorganic reaction components have been fully characterized using spectroscopic methods. Theoretical investigation into several mechanistic possibilities deem a unique indolyl radical based reaction pathway as the most energetically favorable, products of which, are in excellent agreement with experimental findings. [ABSTRACT FROM AUTHOR]

Bifunctional catalase‐peroxidase (KatG) features a posttranslational methionine‐tyrosine‐tryptophan (MYW) crosslinked cofactor crucial for its catalase function, enabling pathogens to neutralize hydrogen peroxide during infection. We discovered the presence of indole nitrogen‐linked hydroperoxyl adduct (MYW‐OOH) in Mycobacterium tuberculosis KatG in the solution state under ambient conditions, suggesting its natural occurrence. By isolating predominantly MYW‐OOH‐containing KatG protein, we investigated the chemical stability and functional impact of MYW‐OOH. We discovered that MYW‐OOH inhibits catalase activity, presenting a unique temporary lock. Exposure to peroxide or increased temperature removes the hydroperoxyl adduct from the protein cofactor, converting MYW‐OOH to MYW and restoring the detoxifying ability of the enzyme against hydrogen peroxide. Thus, the N‐linked hydroperoxyl group is releasable. KatG with MYW‐OOH represents a catalase dormant, but primed, state of the enzyme. These findings provide insight into chemical strategies targeting the bifunctional enzyme KatG in pathogens, highlighting the role of N‐linked hydroperoxyl modifications in enzymatic function. [ABSTRACT FROM AUTHOR]

Ferroptosis is a cell death mechanism mediated by iron-dependent lipid peroxidation. Although ferroptosis has garnered attention as a cancer-suppressing mechanism, there are still limited markers available for identifying ferroptotic cells or assessing their sensitivity to ferroptosis. The study focused on biliverdin, an endogenous reducing substance in cells, and examined the dynamics of intracellular biliverdin during ferroptosis using a biliverdin-binding cyanobacteriochrome. It was found that intracellular biliverdin decreases during ferroptosis and that this decrease is specific to ferroptosis amongst different forms of cell death. Furthermore, the feasibility of predicting sensitivity to ferroptosis by measuring intracellular biliverdin was demonstrated using a ferroptosis model induced by the re-expression of the transcription factor BACH1. These findings provide further insight into ferroptosis research and are expected to contribute to the development of cancer therapies that exploit ferroptosis. [ABSTRACT FROM AUTHOR]

Azadirachta indica A. Juss (Meliaceae) (AI) and Curcuma longa L. (Zingiberaceae) (CL) are used for malaria treatment but their anti-glycolytic and host mitochondrial effects have not been studied. The AI stem-bark and CL rhizomes were extracted with methanol. Methanol extract of CL (Turmeric) was partitioned to yield methanol fraction (MF). Swiss mice infected with Plasmodium berghei (NK 65 strain) were treated with 200 and 400 mg/kg of AI and turmeric for seven days. Turmeric and MF (200 and 400 mg/kg) were combined with 400 mg/kg AI to treat mice infected with Plasmodium berghei (ANKA strain) for four days. Drug and infected controls mice were treated with artemether lumefantrine (10 mg/kg) and distilled water (10 mL/kg), respectively. Serum lactate dehydrogenase (LDH) and aldolase activities were determined. Liver mitochondria were obtained for mitochondrial permeability transition (mPT) pore opening and FoF1 ATPase assays. The curcumin content of turmeric was determined using HPLC while LD50 of Turmeric and AI was also determined. The AI, and its combination with turmeric decreased parasite load and increased chemosuppression in both sensitive and resistant studies while MF and its combinations with AI induced mPT pore opening. In the resistant experiment, AI + Turmeric 400 mg/kg decreased FoF1 ATPase, LDH and aldolase activities against the infected control. The LD50 values of both extracts were above 2000 mg/kg while the MF had the highest curcumin content. Antiplasmodial mechanisms of action of AI, CL and their combinations involve anti-glycolytic effects. Their composite formulations are more potent in malaria treatment. [ABSTRACT FROM AUTHOR]

Previously, some bacteria were shown to harbour enzymes capable of catalysing the oxidative cleavage of the double bond of t -anethole and related compounds. The cofactor dependence of these enzymes remained enigmatic due to a lack of biochemical information. We report on catalytic and structural details of a representative of this group of oxidative enzymes: t -anethole oxygenase from Stenotrophomonas maltophilia (TAO Sm). The bacterial enzyme could be recombinantly expressed and purified, enabling a detailed biochemical study that has settled the dispute on its cofactor dependence. We have established that TAO Sm contains a tightly bound b-type heme and merely depends on dioxygen for catalysis. It was found to accept t -anethole, isoeugenol and O-methyl isoeugenol as substrates, all being converted into the corresponding aromatic aldehydes without the need of any cofactor regeneration. The elucidated crystal structure of TAO Sm has revealed that it contains a unique active site architecture that is conserved for this distinct class of heme-containing bacterial oxygenases. Similar to other hemoproteins, TAO Sm has a histidine (His121) as proximal ligand. Yet, unique for TAOs, an arginine (Arg89) is located at the distal axial position. Site directed mutagenesis confirmed crucial roles for these heme-liganding residues and other residues that form the substrate binding pocket. In conclusion, the results reported here reveal a new class of bacterial heme-containing oxygenases that can be used for the cleavage of alkene double bonds, analogous to ozonolysis in organic chemistry. • Identification of a new class of heme-containing enzymes. • Cofactor-independent biocatalytic C C cleavage. • Crystal structure of a trans-anethole oxygenase. • Novel active site architecture of a heme-containing enzyme. [ABSTRACT FROM AUTHOR]

Massive intravascular hemolysis occurs not unfrequently in many clinical conditions. Breakdown of erythrocytes promotes the accumulation of heme-derivates in the kidney, increasing oxidative stress and cell death, thus promoting acute kidney injury (AKI). NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species (ROS) in the kidney, however it is unknown the role of Nox4 in hemolysis and whether inhibition of this enzyme may protect from heme-mediated injury. To answer these questions, we elicited intravascular hemolysis in wild type and Nox4 knockout mice. We also evaluated whether nephrotoxic effects of heme may be reduced by using Nox4 siRNA and pharmacologic inhibition with GKT137831, a Nox4 inhibitor, both in vivo and in cultured renal cells. Our results showed that induction of massive hemolysis elicited AKI characterized by loss of renal function, morphological alterations of the tubular epithelium and podocytes, oxidative stress, inflammation, mitochondrial dysfunction, blockade of autophagy and cell death. These pathological effects were significantly prevented in Nox4-deficient mice and in animals treated with GKT137831. In vitro studies showed that Nox4 disruption by specific siRNAs or Nox4 inhibitors declined heme-mediated ROS production and cell death. Our data identify Nox4 as a key enzyme involved in intravascular hemolysis-induced AKI. Thus, Nox4 inhibition may be a potential therapeutic approach to prevent renal damage in patients with severe hemolytic crisis. [Display omitted] • Genetic deletion of Nox4 reduces acute renal injury during massive intravascular hemolysis. • Nox4 participates in hemolysis-mediated oxidative stress, mitochondrial dysfunction and inflammation. • Pharmacological Nox4 inhibition reduces renal damage associated to intravascular hemolysis. • Specific Nox4 targeting in podocytes and tubular cells declines heme-mediated oxidative stress, inflammation and cell death. [ABSTRACT FROM AUTHOR]

The monomeric heme protein myoglobin (Mb) is aberrantly expressed in approximately 40 % of breast tumors. Mb expression is associated with better patient prognosis, yet the molecular mechanisms underlying this effect are unclear. In muscle, Mb's heme moiety confers oxygen storage and delivery. However, prior studies demonstrate that low levels of Mb in cancer cells preclude this function. Several studies propose a fatty acid binding function for Mb via lysine residue K46. Because cancer cells can upregulate fatty acid oxidation (FAO) to fuel cell migration, we tested whether Mb-mediated fatty acid binding modulates FAO and migration. We demonstrate that stable expression of human Mb in MDA-MB-231 breast cancer cells decreases cell migration and FAO. Site-directed mutagenesis of Mb K46 disrupted fatty acid binding but did not improve FAO or migration. Conversely, cells expressing Apo-Mb (with disrupted heme binding) did not show impaired FAO or migration rates, suggesting Mb attenuates FAO and migration via a heme-dependent mechanism rather than through fatty acid binding. Mb's heme-dependent oxidant generation dysregulates migratory gene expression, which is reversed by catalase treatment. Collectively, these data demonstrate that Mb's heme-dependent oxidant production decreases breast cancer cell migration, prompting therapeutic strategies to modulate oxidant production and Mb in tumors. Mb-dependent oxidant generation (but not fatty acid binding) dysregulates mitochondrial respiration and migratory gene expression, leading to decreased cell migration. Created with BioRender. [Display omitted] • Myoglobin (Mb) expression in MDA-MB-231 breast cancer cells slows migration. • Mb expression decreases mitochondrial respiration and fatty acid oxidation. • Mb-dependent fatty acid binding does not regulate cell migration or respiration. • Mb-dependent oxidant generation decreases mitochondrial metabolism and migration. • Mb-derived oxidants dysregulate migratory gene expression. [ABSTRACT FROM AUTHOR]

This study investigates hemoglobin (Hb)‐induced kidney injury and the protective role of the ApoHemoglobin‐Haptoglobin (ApoHb‐Hp) complex against heme and Hb damage. Hb facilitates oxygen (O2) delivery but poses challenges outside red blood cells (RBCs) due to toxic Hb and heme mechanisms. These are managed by binding to serum proteins like Haptoglobin (Hp) and Hemopexin (Hpx). During hemolysis, depletion of Hp and Hpx leaves tissues vulnerable to Hb and heme. To address this, we developed the ApoHb‐Hp complex, based on Apohemoglobin, which is produced by removing heme from Hb, conjugated with Hp. This complex acts as a dual scavenger for Hb and heme, preventing tissue damage. Our findings demonstrate that ApoHb‐Hp significantly protects MPC5 podocytes from Hb‐induced damage. Fluorescent staining showed a higher percentage of nephrin‐positive cells in the ApoHb‐Hp group, and MTT assays revealed enhanced cell viability compared to Hb alone. Additionally, ApoHb‐Hp reduced reactive oxygen species (ROS) production, with the Hb group exhibiting significantly elevated ROS levels. The ApoHb‐Hp complex mitigated the depletion of protective mechanisms, as shown by significant increases in superoxide dismutase (SOD) and glutathione (GSH). Moreover, ApoHb‐Hp treatment reduced the activation of the NLRP3 inflammasome signaling pathway and inflammatory cytokines IL‐1β and IL‐18. These findings underscore the therapeutic potential of ApoHb‐Hp in mitigating Hb‐induced renal damage by preserving podocyte viability and reducing oxidative stress. Overall, ApoHb‐Hp maintained protective mechanisms depleted otherwise by Hb. These findings highlight ApoHb‐Hp's potential as a therapeutic agent against Hb‐induced renal damage, offering insights into its mechanisms and implications for treating conditions involving hemolysis. [ABSTRACT FROM AUTHOR]

Tryptophan-2,3-dioxygenase (TDO2) and indoleamine-2,3-dioxygenase (IDO1) are structurally distinct heme enzymes that catalyze the conversion of L-tryptophan to N-formyl-kynurenine, and play important roles in metabolism, inflammation, and tumor immune surveillance. The enzymes can adopt an inactive, heme-free (apo) state or an active, heme-containing (holo) state, with the balance between them varying dynamically according to biological conditions. Inhibitors of holo-TDO2 are known but, despite several advantages of the heme-free state as a drug target, no inhibitors of apo-TDO2 have been reported. We describe the discovery of the first apo-TDO2 binding inhibitors, to our knowledge, and their inhibition of cellular TDO2 activity at low nanomolar concentrations. The crystal structure of a potent, small molecule inhibitor bound to apo-TDO2 reveals its detailed binding interactions within the large, hydrophobic heme binding pocket of the active site. [ABSTRACT FROM AUTHOR]

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. Elucidation of heme binding affinity and artemisinin activation by the Plasmodium falciparum Kelch13 protein variants. [ABSTRACT FROM AUTHOR]

Although the pathological significance of myeloid cell heterogeneity is still poorly understood, new evidence indicates that distinct macrophage subsets are characterized by specific metabolic programs that influence disease onset and progression. Within this scenario, distinct subsets of macrophages, endowed with high rates of heme catabolism by the stress-responsive enzyme heme oxygenase-1 (HO-1), play critical roles in physiologic and pathological conditions. Of relevance, the substrates of HO-1 activity are the heme groups that derive from cellular catabolism and are converted into carbon monoxide (CO), biliverdin and Fe2+, which together elicit anti-apoptotic, anti-inflammatory activities and control oxidative damage. While high levels of expression of HO-1 enzyme by specialized macrophage populations (erythrophagocytes) guarantee the physiological disposal of senescent red blood cells (i.e. erythrocateresis), the action of HO-1 takes on pathological significance in various diseases, and abnormal CO metabolism has been observed in cancer, hematological diseases, hypertension, heart failure, inflammation, sepsis, neurodegeneration. Modulation of heme catabolism and CO production is therefore a feasible therapeutic opportunity in various diseases. In this review we discuss the role of HO-1 in different pathological contexts (i.e. cancer, infections, cardiovascular, immune-mediated and neurodegenerative diseases) and highlight new therapeutic perspectives on the modulation of the enzymatic activity of HO-1. [ABSTRACT FROM AUTHOR]

The heme-based direct oxygen sensor DosP degrades c-di-GMP, a second messenger nearly unique to bacteria. In stationary phase Escherichia coli, DosP is the most abundant c-di-GMP phosphodiesterase. Ligation of O2 to a heme-binding PAS domain (hPAS) of the protein enhances the phosphodiesterase through an allosteric mechanism that has remained elusive. We determine six structures of full-length DosP in its aerobic or anaerobic conformations, with or without c-di-GMP. DosP is an elongated dimer with the regulatory heme containing domain and phosphodiesterase separated by nearly 180 Å. In the absence of substrate, regardless of the heme status, DosP presents an equilibrium of two distinct conformations. Binding of substrate induces DosP to adopt a single, ON-state or OFF-state conformation depending on its heme status. Structural and biochemical studies of this multi-domain sensor and its mutants provide insights into signal regulation of second-messenger levels. The bacterial phosphodiesterase DosP uses heme-based sensing of oxygen gas to control levels of cyclic di-GMP. Here, the authors determine the structural and biochemical basis of this regulatory signal. [ABSTRACT FROM AUTHOR]

Pathogens must efficiently acquire nutrients from host tissue to proliferate, and strategies to block pathogen access therefore hold promise for disease control. In this study, we investigated whether heme biosynthesis is an effective target for ablating the virulence of the phytopathogenic fungus Ustilago maydis on maize plants. We first constructed conditional heme auxotrophs of the fungus by placing the heme biosynthesis gene hem12 encoding uroporphyrinogen decarboxylase (Urod) under the control of nitrogen or carbon source‐regulated promoters. These strains were heme auxotrophs under non‐permissive conditions and unable to cause disease in maize seedlings, thus demonstrating the inability of the fungus to acquire sufficient heme from host tissue to support proliferation. Subsequent experiments characterized the role of endocytosis in heme uptake, the susceptibility of the fungus to heme toxicity as well as the transcriptional response to exogenous heme. The latter RNA‐seq experiments identified a candidate ABC transporter with a role in the response to heme and xenobiotics. Given the importance of heme biosynthesis for U. maydis pathogenesis, we tested the ability of the well‐characterized herbicide BroadStar to influence disease. This herbicide contains the active ingredient flumioxazin, an inhibitor of Hem14 in the heme biosynthesis pathway, and we found that it was an effective antifungal agent for blocking disease in maize. Thus, repurposing herbicides for which resistant plants are available may be an effective strategy to control pathogens and achieve crop protection. [ABSTRACT FROM AUTHOR]

Heme, an iron-containing tetrapyrrole in hemoproteins, including: hemoglobin, myoglobin, catalase, cytochrome c, and cytochrome P450, plays critical physiological roles in different organisms. Heme-derived chemicals, such as biliverdin, bilirubin, and phycocyanobilin, are known for their antioxidant and anti-inflammatory properties and have shown great potential in fighting viruses and diseases. Therefore, more and more attention has been paid to the biosynthesis of hemoproteins and heme derivatives, which depends on the adequate heme supply in various microbial cell factories. The enhancement of endogenous biosynthesis and exogenous uptake can improve the intracellular heme supply, but the excess free heme is toxic to the cells. Therefore, based on the heme-responsive regulators, several sensitive biosensors were developed to fine-tune the intracellular levels of heme. In this review, recent advances in the: biosynthesis, acquisition, regulation, and upcycling of heme were summarized to provide a solid foundation for the efficient production and application of high-value-added hemoproteins and heme derivatives. [ABSTRACT FROM AUTHOR]

Purpose: Uveitis is a common, sight-threatening inflammatory ocular disease and is the main cause of blindness, which is caused by autoimmune response, infection, and injury. The contribution of 14-3-3ζ in uveitis remains obscure. This study aims to investigate the role of 14-3-3ζ in regulating ferroptosis in retinal inflammation and its contribution to uveitis. Methods: A lipopolysaccharide (LPS)-induced uveitis mouse model and BV-2 cell line were used to examine the effect of LPS stimulation on the expression of 14-3-3ζ and ferroptosis in microglia. The expression of heme oxygenase-1 (HO-1) was also analyzed to understand its role in promoting microglial ferroptosis. Results: We found that LPS stimulation increased the expression of 14-3-3ζ and promoted ferroptosis in microglia. Additionally, 14-3-3ζ was found to promote microglial ferroptosis by stabilizing the expression of HO-1. These findings suggest that the 14-3-3ζ/HO-1 axis plays a crucial role in promoting microglial ferroptosis in retinal inflammation. Conclusion: The study provides valuable insights into the mechanisms underlying uveitis and highlights the potential of the 14-3-3ζ/HO-1 axis as a therapeutic target for the disease. Further research in this area could lead to the development of preventive and therapeutic strategies for uveitis. [ABSTRACT FROM AUTHOR]

During fire accidents, 2 highly toxic components of fire smoke, carbon monoxide (CO) and hydrogen cyanide (HCN), are produced simultaneously, and more than 50% of fire-related deaths can be attributed to the inhalation of these toxic gases. Despite the different CO and HCN detoxification mechanisms, oxygen (O2) ventilation is currently the primary clinical treatment, and no alternative methods are available at present. In this review, the toxicological mechanisms of CO and HCN are investigated with an emphasis on the toxic effects of their combination. On the basis of these toxic mechanisms, the advantages and disadvantages of the current clinical detoxification methods are analyzed and discussed. We also summarize the latest advancements in the development of CO- and HCN-scavenging antidotes, highlighting the strategy of using synthetic iron porphyrins that have been synthesized mainly in our laboratory as water-soluble synthetic hemoglobin biomimetics. [ABSTRACT FROM AUTHOR]

Decades of research describe myriad redox enzymes that contain cofactors arranged in tightly packed chains facilitating rapid and controlled intra‐protein electron transfer. Many such enzymes participate in extracellular electron transfer (EET), a process which allows microorganisms to conserve energy in anoxic environments by exploiting mineral oxides and other extracellular substrates as terminal electron acceptors. In this work, we describe the properties of the triheme cytochrome PgcA from Geobacter sulfurreducens. PgcA has been shown to play an important role in EET but is unusual in containing three CXXCH heme binding motifs that are separated by repeated (PT)x motifs, suggested to enhance binding to mineral surfaces. Using a combination of structural, electrochemical, and biophysical techniques, we experimentally demonstrate that PgcA adopts numerous conformations stretching as far as 180 Å between the ends of domains I and III, without a tightly packed cofactor chain. Furthermore, we demonstrate a distinct role for its domain III as a mineral reductase that is recharged by domains I and II. These findings show PgcA to be the first of a new class of electron transfer proteins, with redox centers separated by some nanometers but tethered together by flexible linkers, facilitating electron transfer through a tethered diffusion mechanism rather than a fixed, closely packed electron transfer chain. [ABSTRACT FROM AUTHOR]

Introduction: Pulmonary ischemia-reperfusion (IR) injury (IRI) plays a significant role in various lung disorders and is a key factor in the development of primary graft dysfunction following lung transplantation. Hemopexin (Hx) is the major serum scavenger protein for heme, which is a prooxidant and pro-inflammatory compound. In the current study, we hypothesized that Hx could confer beneficial effects in sterile inflammation induced by IR-mediated lung injury. Methods: To examine this hypothesis, we administered Hx in an experimental mouse model of unilateral lung IRI. Results: Our results demonstrate that treatment with Hx alleviated histopathological signs of inflammation in ischemic lungs, as evidenced by a reduction in the number of infiltrating neutrophils and decreased levels of perivascular edema. In addition, thrombotic vaso-occlusion in pulmonary blood vessels of IRI lungs was reduced by Hx. Immunohistochemical analysis revealed that Hx inhibited the up-regulation of heme oxygenase-1, an enzyme highly induced by heme, in ischemic lungs. Finally, Hx administration caused a decrease in the levels of circulating B- and CD8+ T-lymphocytes in the peripheral blood of mice with pulmonary IRI. Conclusion: These findings suggest that the serum heme scavenger protein Hx holds therapeutic promise in alleviating lung IRI-mediated sterile inflammation. Thus, Hx may represent a preemptive therapeutic approach in IR-related lung disorders such as primary graft dysfunction in lung transplantation. [ABSTRACT FROM AUTHOR]

Fibrosis, an aberrant reparative response to tissue injury, involves a disruption in the equilibrium between the synthesis and degradation of the extracellular matrix, leading to its excessive accumulation within normal tissues, and culminating in organ dysfunction. Manifesting in the terminal stages of nearly all chronic ailments, fibrosis carries a high mortality rate and poses a significant threat to human health. Heme oxygenase‐1 (HO‐1) emerges as an endogenous protective agent, mitigating tissue damage through its antioxidant, anti‐inflammatory, and antiapoptotic properties. Numerous studies have corroborated HO‐1's potential as a therapeutic target in anti‐fibrosis treatment. This review delves into the structural and functional attributes, and the upstream and downstream pathways of HO‐1. Additionally, the regulatory networks and mechanisms of HO‐1 in cells associated with fibrosis are elucidated. The role of HO‐1 in various fibrosis‐related diseases is also explored. Collectively, this comprehensive information serves as a foundation for future research and augments the viability of HO‐1 as a therapeutic target for fibrosis. [ABSTRACT FROM AUTHOR]

Introduction: Pulmonary ischemia-reperfusion (IR) injury (IRI) plays a significant role in various lung disorders and is a key factor in the development of primary graft dysfunction following lung transplantation. Hemopexin (Hx) is the major serum scavenger protein for heme, which is a prooxidant and pro-inflammatory compound. In the current study, we hypothesized that Hx could confer beneficial effects in sterile inflammation induced by IR-mediated lung injury. Methods: To examine this hypothesis, we administered Hx in an experimental mouse model of unilateral lung IRI. Results: Our results demonstrate that treatment with Hx alleviated histopathological signs of inflammation in ischemic lungs, as evidenced by a reduction in the number of infiltrating neutrophils and decreased levels of perivascular edema. In addition, thrombotic vaso-occlusion in pulmonary blood vessels of IRI lungs was reduced by Hx. Immunohistochemical analysis revealed that Hx inhibited the up-regulation of heme oxygenase-1, an enzyme highly induced by heme, in ischemic lungs. Finally, Hx administration caused a decrease in the levels of circulating B- and CD8+ T-lymphocytes in the peripheral blood of mice with pulmonary IRI. Conclusion: These findings suggest that the serum heme scavenger protein Hx holds therapeutic promise in alleviating lung IRI-mediated sterile inflammation. Thus, Hx may represent a preemptive therapeutic approach in IR-related lung disorders such as primary graft dysfunction in lung transplantation. [ABSTRACT FROM AUTHOR]

The iron-containing porphyrin heme is of high interest for the food industry for the production of artificial meat as well as for medical applications. Recently, the biotechnological platform strain Corynebacterium glutamicum has emerged as a promising host for animal-free heme production. Beyond engineering of complex heme biosynthetic pathways, improving heme export offers significant yet untapped potential for enhancing production strains. In this study, a growth-coupled biosensor was designed to impose a selection pressure on the increased expression of the hrtBA operon encoding an ABC-type heme exporter in C. glutamicum. For this purpose, the promoter region of the growth-regulating genes pfkA (phosphofructokinase) and aceE (pyruvate dehydrogenase) was replaced with that of PhrtB, creating biosensor strains with a selection pressure for hrtBA activation. Resulting sensor strains were used for plate-based selections and for a repetitive batch f(luorescent)ALE using a fully automated laboratory platform. Genome sequencing of isolated clones featuring increased hrtBA expression revealed three distinct mutational hotspots: (i) chrS, (ii) chrA, and (iii) cydD. Mutations in the genes of the ChrSA two-component system, which regulates hrtBA in response to heme levels, were identified as a promising target to enhance export activity. Furthermore, causal mutations within cydD, encoding an ABC-transporter essential for cytochrome bd oxidase assembly, were confirmed by the construction of a deletion mutant. Reversely engineered strains showed strongly increased hrtBA expression as well as increased cellular heme levels. These results further support the proposed role of CydDC as a heme transporter in bacteria. Mutations identified in this study therefore underline the potential of biosensor-based growth coupling and provide promising engineering targets to improve microbial heme production. [ABSTRACT FROM AUTHOR]

Tetrapyrroles such as heme, chlorophyll, and vitamin B12 are essential for various metabolic pathways. They derive from 5-aminolevulinic acid (5-ALA), which can be synthesized by a single enzyme (5-ALA synthase or AlaS, Shemin pathway) or by a two-enzyme pathway. The genomes of some bacteriophages from aquatic environments carry various tetrapyrrole biosynthesis genes. Here, we analyze available metagenomic datasets and identify alaS homologs (viral alaS, or valaS) in sequences corresponding to marine and freshwater phages. The genes are found individually or as part of complete or truncated three-gene loci encoding heme-catabolizing enzymes. Amino-acid sequence alignments and three-dimensional structure prediction support that the valaS sequences likely encode functional enzymes. Indeed, we demonstrate that is the case for a freshwater phage valaS sequence, as it can complement an Escherichia coli 5-ALA auxotroph, and an E. coli strain overexpressing the gene converts the typical AlaS substrates glycine and succinyl-CoA into 5-ALA. Thus, our work identifies valaS as an auxiliary metabolic gene in phage sequences from aquatic environments, further supporting the importance of tetrapyrrole metabolism in bacteriophage biology. Tetrapyrroles such as heme and chlorophyll, which are essential for various metabolic pathways, are synthesized from 5-aminolevulinic acid (5-ALA). Here, the authors identify functional 5-ALA synthase genes in bacteriophage sequences from aquatic environments, thus highlighting the importance of tetrapyrrole metabolism in phage biology. [ABSTRACT FROM AUTHOR]

The presence of chlorophenols in water poses a significant threat to human health and the environment. In response to this issue, a study was undertaken to evaluate the catalytic capabilities of chlorinated Heme towards common chlorophenols present in water, such as 2,4-dichlorophenol, 2,4,6-trichlorophenol, and pentachlorophenol. The study employed the B3LYP method, a sophisticated computational technique within density functional theory, to investigate the molecular interactions and transformations involved. It scrutinized structural parameters, Wiberg Bond Indices, which offer insights into the strength and nature of chemical bonds, along with spectroscopic data including infrared vibrational spectra, ultraviolet-visible absorption spectra, and molecular fluorescence spectra. Furthermore, the research analyzed molecular binding energies and orbital energy levels before and after the formation of complexes between Heme and the targeted chlorophenols. The findings indicate that Heme displays a notable activation characteristic towards these chlorophenols. This suggests that Heme could act as an effective catalyst in the degradation of chlorophenols in water, presenting a novel approach to water purification. The theoretical insights derived from this study are invaluable, potentially guiding the development of more efficient catalytic systems for treating chlorophenol-contaminated water, thereby reducing the environmental and health risks associated with these hazardous compounds. [ABSTRACT FROM AUTHOR]

Background: Anadara granosa, commonly known as the blood clam, exhibits the unusual characteristic of having red blood among invertebrates. There is significant individual variation in blood color intensity among blood clams; individuals with vibrant red blood are deemed healthier and exhibit stronger stress resistance. However, the molecular basis underlying these red blood traits (RBTs) remains poorly understood. Results: In this study, we performed genome-wide association studies (GWAS) in a population of 300 A. granosa individuals, focusing on RBTs as measured by hemoglobin concentration (HC), total hemocyte count (THC), and heme concentration (HEME). Our analysis identified 18 single nucleotide polymorphisms (SNPs) correlated with RBTs, subsequently selected 117 candidate genes within a 100 kb flanking region of these SNPs, potentially involved in the RBTs of A. granosa. Moreover, we discovered two haplotype blocks specifically associated with THC and HEME. Further analysis revealed eight genes (Septin7, Hox5, Cbfa2t3, Avpr1b, Hhex, Eif2ak3, Glrk, and Rpl35a) that significantly influence RBTs. Notably, a heterozygous A/T mutation in the 3'UTR of Cbfa2t3 was found to promote blood cell proliferation. These genes suggest that the hematopoietic function plays a significant role in the variability of RBTs in A. granosa. Conclusions: Our findings reveal a conservation of the regulatory mechanisms of RBTs between blood clams and vertebrates. The results not only provide a scientific basis for selective breeding in blood clams, but also offer deeper insights into the evolutionary mechanisms of RBTs in invertebrates. [ABSTRACT FROM AUTHOR]

5-Aminolevulinic acid (5-ALA), a non-proteinogenic amino acid, is an intermediate in the biosynthesis of heme and exerts antiviral effects against feline coronavirus (FCoV); however, the underlying mechanisms remain unclear. In the biosynthesis of heme, 5-ALA is condensed and converted to protoporphyrin IX (PpIX), which is then transformed into heme by the insertion of ferrous iron. Previous research has suggested that the metabolites generated during heme biosynthesis contribute to the antiviral effects of 5-ALA. Therefore, the present study investigated the in vitro mechanisms responsible for the antiviral effects of 5-ALA. The results obtained revealed that 5-ALA and PpIX both effectively reduced the viral titer in the supernatant of FCoV-infected fcwf-4 cells. Moreover, PpIX exerted virucidal effects against FCoV. We also confirmed that 5-ALA increased PpIX levels in cells. While hemin induced heme oxygenase-1 gene expression, it did not reduce the viral titer in the supernatant. Sodium ferrous citrate decreased PpIX levels and suppressed the antiviral effects of 5-ALA. Collectively, these results suggest that the antiviral effects of 5-ALA against FCoV are dependent on PpIX. [ABSTRACT FROM AUTHOR]

Chlorosis in azaleas is characterized by an interveinal yellowing of leaves that is typically caused by a deficiency of iron. This condition is usually due to the inability of cells to properly acquire iron as a consequence of unfavorable conditions, such as an elevated pH, rather than insufficient iron levels. The causes and effects of chlorosis were found to have similarities with those pertaining to a recently presented hypothesis that describes a pathogenic process in Alzheimer disease. This hypothesis states that iron becomes sequestered (e.g., by amyloid β and tau), causing a functional deficiency of iron that disrupts biochemical processes leading to neurodegeneration. Additional mechanisms that contribute to iron becoming unavailable include iron-containing structures not undergoing proper recycling (e.g., disrupted mitophagy and altered ferritinophagy) and failure to successfully translocate iron from one compartment to another (e.g., due to impaired lysosomal acidification). Other contributors to a functional deficiency of iron in patients with Alzheimer disease include altered metabolism of heme or altered production of iron-containing proteins and their partners (e.g., subunits, upstream proteins). A review of the evidence supporting this hypothesis is presented. Also, parallels between the mechanisms underlying a functional iron-deficient state in Alzheimer disease and those occurring for chlorosis in plants are discussed. Finally, a model describing the generation of a functional iron deficiency in Alzheimer disease is put forward. [ABSTRACT FROM AUTHOR]

Periodontitis is a common oral bacterial infection characterized by inflammatory responses. Its high prevalence lowers the quality of life for individuals and increases the global economic and disease burden. As microorganisms in dental plaque are responsible for this oral disease, antibacterial drug treatments are effective strategies for preventing and treating periodontitis. In this study, we investigated the inhibitory effect of nicotinamide (NAM), a vitamin B3 derivative, on the growth and virulence of Porphyromonas gingivalis, a key member of the red complex. Our findings revealed that NAM inhibited bacterial growth and gingipain activities, which played a dominant role in protein hydrolysis and heme acquisition. NAM decreased hemagglutination and hemolysis abilities and changed hemin and hemoglobin binding capacities, controlling bacterial infection through a starvation strategy by blocking access to growth‐essential nutrients from the outside and reducing bacterial virulence. Several experiments in an animal model showed the effectiveness of NAM in preventing alveolar bone loss and reducing inflammatory cell infiltration, shedding light on its potential therapeutic applicability. [ABSTRACT FROM AUTHOR]

Humic acid (HA) is a typical refractory organic matter, so it is of great significance to investigate its effect on the performance of Anammox granular sludge. When the dosage of HA ≤ 50 mg/L, HA promotes the total nitrogen removal rate (NRR) to 1.45 kg/(m3·day). When HA was between 50 and 100 mg/L, the NRR of Anammox was stable. At this time, the adsorption of HA causes the sludge to gradually turn from red to brown, but the activities of heme and enzymes showed that its capacity was not affected. When HA levels reached 250 mg/L, the NRR dropped to 0.11 kg/(m3·day). Moderate HA levels promoted the release of extracellular polymeric substance (EPS), but excessive HA levels lead to a decrease in EPS concentrations. HA inhibited Anammox activity, which indirectly hindered the transmission of substrate and accumulated substrate toxicity. Although HA promoted the increase of heterotrophic microbial abundance in Anammox system, the microbial diversity decreased gradually. With the increase of HA concentration, the abundance of Candidatus_Brocadia , the main functional microorganism of Anammox system, decreased gradually, while the abundance of Candidatus_Kuenenia increased gradually. [ABSTRACT FROM AUTHOR]

Objective(s): Hepatic ischemia-reperfusion (HIR) is a severe process in pathophysiology that occurs clinically in hepatectomy, and hepatic transplantations. The present study aimed to investigate the effect of PKC θ deletion against HIR injury and elucidate its mechanism in pathophysiology. Materials and Methods: HIR injury was induced in wild-type and PKC θ deletion mice treated with or without heme. The ALT and AST levels were determined to evaluate liver function. HIR injury was observed via histological examination. Oxidative stress and inflammatory response markers, and their signaling pathways were detected. Results: The study found that PKC θ knockout decreased serum AST and ALT levels when compared to the WT mice. Furthermore, heme treatment significantly reduced the ALT and AST levels of the PKC θ deletion mice compared with the untreated PKC θ deletion mice. PKC θ deletion markedly elevated superoxide dismutase activity in the liver tissue, reduced malondialdehyde content in the tissue, and the serum TNF-α and IL-6 levels compared with the WT mice. Heme treatment was observed to elevate the activity of SOD and reduced MDA content and serum of TNF-α and IL 6 in the PKC θ deletion animals. Meanwhile, heme treatment increased HO-1 and Nrf 2 protein expression, and reduced the levels of TLR4, phosphorylated NF-κB, and IKB-α. Conclusion: These findings suggested that PKC θ deletion ameliorates HIR, and heme treatment further improves HIR, which is related to regulation of PKC θ deletion on Nrf 2/HO-1 and TLR4/ NF-κB/IKB α pathway. [ABSTRACT FROM AUTHOR]

Periodontitis is one of the most prevalent inflammatory disease worldwide, which affects 11% of the global population and is a major cause of tooth loss. Recently, oxidative stress (OS) has been found to be the pivital pathophysiological mechanism of periodontitis, and overactivated OS will lead to inflammation, apoptosis, pyroptosis and alveolar bone resorption. Interestingly, heme oxygenase-1 (HO-1), a rate-limiting enzyme in heme degradation, can exert antioxidant activites through its products—carbon monoxide (CO), Fe2+, biliverdin and bilirubin in the inflammatory microenvironment, thus exhibiting anti-inflammatory, anti-apoptotic, anti-pyroptosis and bone homeostasis-regulating properties. In this review, particular focus is given to the role of HO-1 in periodontitis, including the spatial-temporal expression in periodental tissues and pathophysiological mechanisms of HO-1 in periodontitis, as well as the current therapeutic applications of HO-1 targeted drugs for periodontitis. This review aims to elucidate the potential applications of various HO-1 targeted drug therapy in the management of periodontitis, investigate the influence of diverse functional groups on HO-1 and periodontitis, and pave the way for the development of a new generation of therapeutics that will benefit patients suffering from periodontitis. [ABSTRACT FROM AUTHOR]

Heme is an essential prosthetic group that serves as a co-factor and a signaling molecule. Heme levels decline with age, and its deficiency is associated with multiple hallmarks of aging, including anemia, mitochondrial dysfunction, and oxidative stress. Dysregulation of heme homeostasis has been also implicated in aging in model organisms suggesting that heme may play an evolutionarily conserved role in controlling lifespan. However, the underlying mechanisms and whether heme homeostasis can be targeted to promote healthy aging remain unclear. Here, we used Saccharomyces cerevisiae as a model to investigate the role of heme in aging. For this, we have engineered a heme auxotrophic yeast strain expressing a plasma membrane-bound heme permease from Caenorhabditis elegans (ceHRG-4). This system can be used to control intracellular heme levels independently of the biosynthetic enzymes by manipulating heme concentration in the media. We observed that heme supplementation leads to a significant extension of yeast replicative lifespan. Our findings revealed that the effect of heme on lifespan is independent of the Hap4 transcription factor. Surprisingly, heme-supplemented cells had impaired growth on YPG medium, which requires mitochondrial respiration to be used, suggesting that these cells are respiratory deficient. Together, our results demonstrate that heme homeostasis is fundamentally important for aging biology, and manipulating heme levels can be used as a promising therapeutic target for promoting longevity. [ABSTRACT FROM AUTHOR]

Abstract Excessive intake of red meat may cause damage to colorectal tissue but not cause significant damage to the small intestine. However, the underlying mechanism is not clear. In this study, the effect of myoglobin extracted from red meat was explored on the intestinal barrier function of the mice, and its potential mechanism was elucidated through cell culture experiments. Exclusively high‐dose myoglobin (3.39%, equivalent to 450 g red meat per day for human) resulted in marked intestinal permeability with increased levels of serum endotoxin, diamine oxidase, and d‐lactate but reduced the mRNA levels of tight junction proteins and mucin 2 in the duodenal and colonic tissues. The diet also increased free iron and heme levels in the duodenal and colonic tissues, leading to higher level of oxidative stress and inflammatory response. Metabolomic analysis of colonic contents showed that exclusively high‐dose myoglobin altered the relative content of indole and its derivatives, phenolic compounds, and 5‐hydroxy‐l‐tryptophan by regulating tryptophan metabolism and kynurenine cycle and destroying intestinal homeostasis. The very high myoglobin hydrolysate induced oxidative stress and apoptosis more seriously in HT29 cells than in INT407 cells, which could be the main reason for more severe colon injury. Nevertheless, normal low‐dose intake (0.38% myoglobin, equivalent to 50 g per day for human) did not show the above‐mentioned harmful effects. The findings provided a risk assessment for the dosage of red meat intake and new insights into the relationship between red meat intake and intestinal health.

Abstract The iron-containing porphyrin heme is of high interest for the food industry for the production of artificial meat as well as for medical applications. Recently, the biotechnological platform strain Corynebacterium glutamicum has emerged as a promising host for animal-free heme production. Beyond engineering of complex heme biosynthetic pathways, improving heme export offers significant yet untapped potential for enhancing production strains. In this study, a growth-coupled biosensor was designed to impose a selection pressure on the increased expression of the hrtBA operon encoding an ABC-type heme exporter in C. glutamicum. For this purpose, the promoter region of the growth-regulating genes pfkA (phosphofructokinase) and aceE (pyruvate dehydrogenase) was replaced with that of P hrtB , creating biosensor strains with a selection pressure for hrtBA activation. Resulting sensor strains were used for plate-based selections and for a repetitive batch f(luorescent)ALE using a fully automated laboratory platform. Genome sequencing of isolated clones featuring increased hrtBA expression revealed three distinct mutational hotspots: (i) chrS, (ii) chrA, and (iii) cydD. Mutations in the genes of the ChrSA two-component system, which regulates hrtBA in response to heme levels, were identified as a promising target to enhance export activity. Furthermore, causal mutations within cydD, encoding an ABC-transporter essential for cytochrome bd oxidase assembly, were confirmed by the construction of a deletion mutant. Reversely engineered strains showed strongly increased hrtBA expression as well as increased cellular heme levels. These results further support the proposed role of CydDC as a heme transporter in bacteria. Mutations identified in this study therefore underline the potential of biosensor-based growth coupling and provide promising engineering targets to improve microbial heme production.

Abstract The presence of chlorophenols in water poses a significant threat to human health and the environment. In response to this issue, a study was undertaken to evaluate the catalytic capabilities of chlorinated Heme towards common chlorophenols present in water, such as 2,4-dichlorophenol, 2,4,6-trichlorophenol, and pentachlorophenol. The study employed the B3LYP method, a sophisticated computational technique within density functional theory, to investigate the molecular interactions and transformations involved. It scrutinized structural parameters, Wiberg Bond Indices, which offer insights into the strength and nature of chemical bonds, along with spectroscopic data including infrared vibrational spectra, ultraviolet-visible absorption spectra, and molecular fluorescence spectra. Furthermore, the research analyzed molecular binding energies and orbital energy levels before and after the formation of complexes between Heme and the targeted chlorophenols. The findings indicate that Heme displays a notable activation characteristic towards these chlorophenols. This suggests that Heme could act as an effective catalyst in the degradation of chlorophenols in water, presenting a novel approach to water purification. The theoretical insights derived from this study are invaluable, potentially guiding the development of more efficient catalytic systems for treating chlorophenol-contaminated water, thereby reducing the environmental and health risks associated with these hazardous compounds.

The P450 enzyme CYP121 from Mycobacterium tuberculosis catalyzes a carbon-carbon (C-C) bond coupling cyclization of the dityrosine substrate containing a diketopiperazine ring, cyclo(l-tyrosine-l-tyrosine) (cYY). An unusual high-spin (S = 5/2) ferric intermediate maximizes its population in less than 5 ms in the rapid freeze-quenching study of CYP121 during the shunt reaction with peracetic acid or hydrogen peroxide in acetic acid solution. We show that this intermediate can also be observed in the crystalline state by EPR spectroscopy. By developing an on-demand-rapid-mixing method for time-resolved serial femtosecond crystallography with X-ray free-electron laser (tr-SFX-XFEL) technology covering the millisecond time domain and without freezing, we structurally monitored the reaction in situ at room temperature. After a 200 ms peracetic acid reaction with the cocrystallized enzyme-substrate microcrystal slurry, a ferric-hydroperoxo intermediate is observed, and its structure is determined at 1.85 Å resolution. The structure shows a hydroperoxyl ligand between the heme and the native substrate, cYY. The oxygen atoms of the hydroperoxo are 2.5 and 3.2 Å from the iron ion. The end-on binding ligand adopts a near-side-on geometry and is weakly associated with the iron ion, causing the unusual high-spin state. This compound 0 intermediate, spectroscopically and structurally observed during the catalytic shunt pathway, reveals a unique binding mode that deviates from the end-on compound 0 intermediates in other heme enzymes. The hydroperoxyl ligand is only 2.9 Å from the bound cYY, suggesting an active oxidant role of the intermediate for direct substrate oxidation in the nonhydroxylation C-C bond coupling chemistry.

[Display omitted] • Modification solves the problem of water solubility of hematin. • Multi-level structured nanoparticles were prepared using microfluidic technology. • Multi-level structured nanoparticles exhibit excellent chemiluminescence signals. • A chemiluminescence immunosensor was constructed for sensitive detection of CEA. The properties of nanomaterials are heavily influenced by their size effects. Utilizing the self-assembly principle offers a promising avenue for crafting innovative nanomaterials, yet controlling this process at the microscopic level presents significant challenges, hindering effective regulation of nanomaterial morphology. Microfluidic technology, however, offers precise control over fluid velocities within microchannels, enabling micro-level manipulation. In this study, we synthesized amphiphilic molecules HP (Hematin@NH 2 -PEG-COOH) through hematin modification, leveraging microfluidic techniques to encapsulate luminol within HP molecules, resulting in the formation of highly efficient chemiluminescence (CL) HPL (HP@Luminol) nanoparticles. The size effects and intricate multi-level structures achieved during encapsulation endowed these nanoparticles with enhanced catalytic capabilities for CL. Finally, we used HPL nanoparticles as luminescent markers to develop a CL immunosensor for the sensitive detection of the tumor marker carcinoembryonic antigen (CEA), achieving satisfactory results. This innovative approach not only expands the repertoire of nanomaterial design and synthesis but also offers a practical solution for sensitive biomarker detection. Overall, this research introduces a novel strategy for designing and fabricating advanced nanomaterials, underscoring the potential of microfluidic technology in nanoscience and biosensing applications. [ABSTRACT FROM AUTHOR]

Abstract Heme is an important cofactor and a regulatory molecule involved in various physiological processes in virtually all living cellular organisms, and it can also serve as the primary iron source for many bacteria, particularly pathogens. However, excess heme is cytotoxic to cells. In order to meet physiological needs while preventing deleterious effects, bacteria have evolved sophisticated cellular mechanisms to maintain heme homeostasis. Recent advances in technologies have shaped our understanding of the molecular mechanisms that govern the biological processes crucial to heme homeostasis, including synthesis, acquisition, utilization, degradation, trafficking, and efflux, as well as their regulation. Central to these mechanisms is the regulation of the heme, by the heme, and for the heme. In this review, we present state‐of‐the‐art findings covering the biochemical, physiological, and structural characterization of important, newly identified hemoproteins/systems involved in heme homeostasis.

Abstract Heme serves as a prosthetic group in hemoproteins, including subunits of the mammalian mitochondrial electron transfer chain. The first enzyme in vertebrate heme biosynthesis, 5-aminolevulinic acid synthase 1 (ALAS1), is ubiquitously expressed and essential for producing 5-aminolevulinic acid (ALA). We previously showed that Alas1 heterozygous mice at 20–35 weeks (aged-A1 +/−s) manifested impaired glucose metabolism, mitochondrial malformation in skeletal muscle, and reduced exercise tolerance, potentially linked to autophagy dysfunction. In this study, we investigated autophagy in A1 +/−s and a sarcopenic phenotype in A1 +/−s at 75–95 weeks (senile-A1 +/−s). Senile-A1 +/−s exhibited significantly reduced body and gastrocnemius muscle weight, and muscle strength, indicating an accelerated sarcopenic phenotype. Decreases in total LC3 and LC3-II protein and Map1lc3a mRNA levels were observed in aged-A1 +/−s under fasting conditions and in Alas1 knockdown myocyte-differentiated C2C12 cells (A1KD-C2C12s) cultured in high- or low-glucose medium. ALA treatment largely reversed these declines. Reduced AMP-activated protein kinase (AMPK) signaling was associated with decreased autophagy in aged-A1 +/−s and A1KD-C2C12s. AMPK modulation using AICAR (activator) and dorsomorphin (inhibitor) affected LC3 protein levels in an AMPK-dependent manner. Our findings suggest that heme deficiency contributes to accelerated sarcopenia-like defects and reduced autophagy in skeletal muscle, primarily due to decreased AMPK signaling.

This research proposal aims for the replacement of N2 molecule (instead of O2) as a final electron receptor in micro-organisms so that they can produce NH3, which is important industrially, instead of H2O. This idea is based on the hypothesis that replacement of the central atom in gas-carrying compounds in living organisms such as chlorophyll, heme and hemocyanin would replace the fixed gas. However, NH3 is an expensive product because it is produced using the Haber-Bosch reaction technique which requires high temperature and pressure. Therefore, it is predictable here to produce NH3 so simply in bioreactors which is a great economic benefit.

Heme and iron metabolic pathways are highly intertwined, both compounds being essential for key biological processes, yet becoming toxic if overabundant. Their concentrations are exquisitely regulated, including via dedicated two-component systems (TCSs) that sense signals and regulate adaptive responses. HemKR is a TCS present in both saprophytic and pathogenic Leptospira species, involved in the control of heme metabolism. However, the molecular means by which HemKR is switched on/off in a signal-dependent way, are still unknown. Moreover, a comprehensive list of HemKR-regulated genes, potentially overlapped with iron-responsive targets, is also missing. Using the saprophytic species Leptospira biflexa as a model, we now show that 5-aminolevulinic acid (ALA) triggers the shutdown of the HemKR pathway in live cells, and does so by stimulating the phosphatase activity of HemK towards phosphorylated HemR. Phospho~HemR dephosphorylation leads to differential expression of multiple genes, including of heme metabolism and transport systems. Besides the heme-biosynthetic genes hemA and the catabolic hmuO, which we had previously reported as phospho~HemR targets, we now extend the regulon identifying additional genes. Finally, we discover that HemR inactivation brings about an iron-deficit tolerant phenotype, synergistically with iron-responsive signaling systems. Future studies with pathogenic Leptospira will be able to confirm whether such tolerance to iron deprivation is conserved among Leptospira spp., in which case HemKR could play a vital role during infection where available iron is scarce. In sum, HemKR responds to abundance of porphyrin metabolites by shutting down and controlling heme homeostasis, while also contributing to integrate the regulation of heme and iron metabolism in the L. biflexa spirochete model. [ABSTRACT FROM AUTHOR]

Iron‐catalyzed C−H amination is emerging as an attractive and sustainable method to install amine functionalities into organic compounds. Amination of C(sp3)−H bonds is usually mediated by an iron‐nitrene intermediate via a Hydrogen Atom Abstraction/Radical Recombination mechanism reminiscent of biomimetic C−H oxidation. Accordingly, this transformation can be catalyzed by engineered iron enzymes, heme and nonheme iron complexes as well as iron salts, although it is often limited to intramolecular reactions and/or activated positions. Aromatic C(sp2)−H amination is mediated by addition of electrophilic iron nitrenes or protonated N‐radical intermediates (produced with Fe catalysts) to aromatic systems. Again, high selectivity is obtained via (pseudo) intramolecular reactions. From a mechanistic perspective, several iron nitrene intermediates have been isolated and characterized over the years in different ligand scaffolds and iron oxidation states. Structure‐activity correlations have been drawn only in few cases and point to a key role of the spin density on the nitrene ligand and of the iron oxidation state. This review describes the state of the art for homogeneous iron catalyzed C(sp3)−H and C(sp2)−H amination focusing on the last 5 years (2019–2023) from a mechanism‐driven catalyst design perspective. [ABSTRACT FROM AUTHOR]

ObjectivesMethodResultsConclusionThis study explores the early identification of rheumatoid arthritis (RA) patients at elevated risk of progression. Haem-oxygenase-1 (HO-1) is a marker of oxidative stress in inflammation. Here, we investigate HO-1 as a biomarker of oxidative stress and its association with clinical disease activity and radiographic progression in RA.Baseline HO-1 was measured sequentially in plasma samples from patients with early rheumatoid arthritis (eRA) (n = 80). Disease Activity Score based on 28-joint count–C-reactive protein, Clinical Disease Activity Index, and total Sharp score were used to evaluate the disease course serially over 2 years. Paired plasma and synovial fluid samples were examined for HO-1 in active established rheumatoid arthritis (esRA) (n = 20). Plasma from healthy control subjects was also included (n = 35).Plasma HO-1 levels were increased in eRA {1373 pg/mL [interquartile range (IQR) 1110–2050]} and esRA [2034 pg/mL (IQR 1630–2923)] compared with controls [1064 pg/mL (IQR 869.5–1378)]. HO-1 plasma levels decreased with treatment. Baseline HO-1 correlated with disease activity and radiographic progression. A strong, linear correlation was found between synovial and plasma HO-1 levels (r = 0.75, p < 0.001).In eRA, plasma levels of HO-1 were increased and correlated with disease and radiographic progression. A baseline measurement of plasma HO-1 levels demonstrated superior performance to currently used clinical and serological disease markers in the prediction of radiographic progression. Plasma HO-1 may function as a first-in-class biomarker of synovial oxidative stress in RA. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Ferroptosis of neurons is a significant cause of brain injury following intracerebral hemorrhage (ICH). As an iron-containing compound in hemoglobin, heme contributes to nerve injury post-ICH. Melatonin has been shown to mitigate the effects of ICH, yet its specific functions remain largely elusive. In this study, we aimed to explore the roles and mechanisms of melatonin in heme-induced ferroptosis subsequent to ICH. MATERIALS AND METHODS: C57BL/6 mice were intracranially injected with heme and then treated with melatonin. Behavior tests [modified neurological severity score (mNSS), forelimb placing, and corner turn tests], H&E staining, Nissl staining, and Prussian blue staining were used to evaluate mouse brain tissue injury. In vitro, HT-22 cells were stimulated with heme and cell viability was determined by crystal violet staining. The iron contents were determined in heme-treated brains and cells, and the levels of 4-hydroxynonenal (4-HNE) and malonaldehyde (MDA) were assessed by ELISA. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to investigate the mRNA levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). Immunoblotting was used to analyze the protein expression of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), Nrf2, and HO-1. Finally, small interfering RNA (siRNA) was used to knock down Nrf2 in HT-22 cells. RESULTS: Melatonin treatment alleviated heme-induced injuries to neural function, as indicated by improved behavior in the mice. Moreover, melatonin decreased cell death and iron concentrations, increased MDA and 4-HNE levels, and reversed the decreases in GPX4, SLC7A11, Nrf2, and HO-1 induced by heme in vitro and in vivo. These results indicated that melatonin could improve the ferroptosis induced by heme. In addition, we found that Nrf2 knockdown attenuated the therapeutic effect of melatonin on neuronal ferroptosis induced by heme. CONCLUSIONS: In general, melatonin alleviates heme-induced ferroptosis by activating the Nrf2/HO-1 pathway, which implies that melatonin is a promising treatment for ferroptosis in ICH. [ABSTRACT FROM AUTHOR]

Monoxenous trypanosomatid Strigomonas culicis harbors an endosymbiotic bacterium, which enables the protozoa to survive without heme supplementation. The impact of H2O2 resistance and symbiont elimination on intracellular heme and Fe2+ availability was analyzed through a comparison of WT strain with both WT H2O2-resistant (WTR) and aposymbiotic (Apo) protozoa. The relative quantification of the heme biosynthetic pathway through label-free parallel reaction monitoring targeted mass spectrometry revealed that H2O2 resistance does not influence the abundance of tryptic peptides. However, the Apo strain showed increased coproporphyrinogen III oxidase and ferrochelatase levels. A putative ferrous iron transporter, homologous to LIT1 and TcIT from Leishmania major and Trypanosoma cruzi, was identified for the first time. Label-free parallel reaction monitoring targeted mass spectrometry also showed that S. culicis Iron Transporter (ScIT) increased 1.6- and 16.4-fold in WTR and Apo strains compared to WT. Accordingly, antibody-mediated blockage of ScIT decreased by 28.0% and 40.0% intracellular Fe2+concentration in both WTR and Apo strains, whereas no effect was detected in WT. In a heme-depleted medium, adding 10 μM hemin decreased ScIT transcript levels in Apo, whereas 10 μM PPIX, the substrate of ferrochelatase, increased intracellular Fe2+ concentration and ferric iron reduction. Overall, the data suggest mechanisms dependent on de novo heme synthesis (and its substrates) in the Apo strain to overcome reduced heme availability. Given the importance of heme and Fe2+ as cofactors in metabolic pathways, including oxidative phosphorylation and antioxidant systems, this study provides novel mechanistic insights associated with H2O2 resistance in S. culicis. [ABSTRACT FROM AUTHOR]

Bilirubin (BR) is an important ingredient of a valuable Chinese medicine, Calculus bovis. Over recent decades, increasing evidence has confirmed that BR offers health benefits in cardiovascular health, stroke, diabetes, and metabolic syndrome. However, BR is mainly produced by extraction from pig bile. In this study, we assembled an efficient pathway for BR production by metabolic engineering of Escherichia coli. First, heme oxygenase (HO1) and biliverdin reductase were co-expressed in E. coli. HPLC and LC–MS confirmed the accumulation of BR in the recombinant E. coli cells. To improve BR production, the catalytic abilities of HO1 from different species were investigated. In addition, the outermembrane-bound heme receptor (ChuA) and the enzymes involved in heme biosynthesis were overexpressed among which ChuA, 5-aminolevulinic acid dehydratase (HemB), protoporphyrin oxidase (HemG), and ferrochelatase (HemH) were found to enhance BR accumulation in E. coli. In addition, expression of ferredoxin (Fd) was shown to contribute to efficient conversion of heme to BR in E. coli. To increase supply of NADPH, isocitrate dehydrogenase (IDH), NAD kinase (nadK), NADP-specific glutamate dehydrogenase (gdhA), and glucose-6-phosphate 1-dehydrogenase (ZWF) were overexpressed and were found to enhance BR accumulation when these proteins were expressed with a low-copy plasmid pACYCduet-1. Modular optimization of the committed genes led to a titer of 17.2 mg/L in strain M1BHG. Finally, fed-batch fermentation was performed for the strains M1BHG and M1, resulting in accumulation of 75.5 mg/L and 25.8 mg/L of BR, respectively. This is the first report on biosynthesis of BR through metabolic engineering in a heterologous host. [ABSTRACT FROM AUTHOR]

Heme has attracted considerable attention due to its indispensable biological roles and applications in healthcare and artificial foods. The development and utilization of edible microorganisms instead of animals to produce heme is the most promising method to promote the large-scale industrial production and safe application of heme. However, the cytotoxicity of heme severely restricts its efficient synthesis by microorganisms, and the cytotoxic mechanism is not fully understood. In this study, the effect of heme toxicity on Saccharomyces cerevisiae was evaluated by enhancing its synthesis using metabolic engineering. The results showed that the accumulation of heme after the disruption of heme homeostasis caused serious impairments in cell growth and metabolism, as demonstrated by significantly poor growth, mitochondrial damage, cell deformations, and chapped cell surfaces, and these features which were further associated with substantially elevated reactive oxygen species (ROS) levels within the cell (mainly H 2 O 2 and superoxide anion radicals). To improve cellular tolerance to heme, 5 rounds of laboratory evolution were performed, increasing heme production by 7.3-fold and 4.2-fold in terms of the titer (38.9 mg/L) and specific production capacity (1.4 mg/L/OD 600), respectively. Based on comparative transcriptomic analyses, 32 genes were identified as candidates that can be modified to enhance heme production by more than 20% in S. cerevisiae. The combined overexpression of 5 genes (SPS22 , REE1 , PHO84 , HEM4 and CLB2) was shown to be an optimal method to enhance heme production. Therefore, a strain with enhanced heme tolerance and ROS quenching ability (R5-M) was developed that could generate 380.5 mg/L heme with a productivity of 4.2 mg/L/h in fed-batch fermentation, with S. cerevisiae strains being the highest producers reported to date. These findings highlight the importance of improving heme tolerance for the microbial production of heme and provide a solution for efficient heme production by engineered yeasts. • Heme is highly toxic to S. cerevisiae. • A significant increase in ROS is an important cause of heme cytotoxicity. • Yeast tolerance to heme can increase through laboratory evolution. • Thirty-two genes were identified as beneficial for the heme production in S. cerevisiae. • The strain with the highest heme yield to date in yeast was constructed by improving its heme tolerance. [ABSTRACT FROM AUTHOR]