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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., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The nonconventional yeast Kluyveromyces marxianus has potential for industrial production, but the lack of advanced synthetic biology tools for precise engineering hinders its rapid development. Here, we introduce a CRISPR-Cas9-mediated multilocus integration method for assembling multiple exogenous genes. Using SlugCas9-HF, a high-fidelity Cas9 nuclease, we enhance gene editing precision. Specific genomic loci predisposed to efficient integration and expression of heterologous genes are identified and combined with a set of paired CRISPR-Cas9 expression plasmids and donor plasmids to establish a CRISPR-based biosynthesis toolkit. This toolkit enables genome integration of large gene modules over 12 kb and achieves simultaneous quadruple-locus integration in a single step with 20% efficiency. As a proof-of-concept, we apply the toolkit to screen for gene combinations that promote heme production, revealing the importance of HEM4Km and HEM12Sc. This CRISPR-based toolkit simplifies the reconstruction of complex pathways in K. marxianus, broadening its application in synthetic biology., (© 2024. The Author(s).)

The development of a heme-responsive biosensor for dynamic pathway regulation in eukaryotes has never been reported, posing a challenge for achieving the efficient synthesis of multifunctional hemoproteins and maintaining intracellular heme homeostasis. Herein, a biosensor containing a newly identified heme-responsive promoter, CRISPR/dCas9, and a degradation tag N-degron was designed and optimized to fine-tune heme biosynthesis in the efficient heme-supplying Pichia pastoris P1H9 chassis. After identifying literature-reported promoters insensitive to heme, the endogenous heme-responsive promoters were mined by transcriptomics, and an optimal biosensor was screened from different combinations of regulatory elements. The dynamic regulation pattern of the biosensor was validated by the transcriptional fluctuations of the HEM2 gene involved in heme biosynthesis and the subsequent responsive changes in intracellular heme titers. We demonstrate the efficiency of this regulatory system by improving the production of high-active porcine myoglobin and soy hemoglobin, which can be used to develop artificial meat and artificial metalloenzymes. Moreover, these findings can offer valuable strategies for the synthesis of other hemoproteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Acute hepatic porphyria (AHP) is a group of four rare inherited diseases, each resulting from a deficiency in a distinct enzyme in the heme biosynthetic pathway. Characterized by acute neurovisceral symptoms that may mimic other medical and psychiatric conditions, lack of recognition of the disease often leads to a delay in diagnosis and initiation of effective treatment. Biochemical testing for pathway intermediates that accumulate when the disease is active forms the basis for screening and establishing a diagnosis. Subsequent genetic analysis identifies the pathogenic variant, supporting screening of family members and genetic counseling. Management of AHP involves avoidance of known exogenous and hormonal triggers, symptomatic treatment, and prevention of recurrent attacks. Here we describe six case studies from our own real-world experience to highlight current recommendations and challenges associated with the diagnosis and long-term management of the disease., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In plastids, conversion of light energy into ATP relies on cytochrome f, a key electron carrier with a heme covalently attached to a CXXCH motif. Covalent heme attachment requires reduction of the disulfide-bonded CXXCH by CCS5 and CCS4. CCS5 receives electrons from the oxidoreductase CCDA, while CCS4 is a protein of unknown function. In Chlamydomonas reinhardtii, loss of CCS4 or CCS5 yields a partial cytochrome f assembly defect. Here, we report that the ccs4ccs5 double mutant displays a synthetic photosynthetic defect characterized by a complete loss of holocytochrome f assembly. This defect is chemically corrected by reducing agents, confirming the placement of CCS4 and CCS5 in a reducing pathway. CCS4-like proteins occur in the green lineage, and we show that HCF153, a distant ortholog from Arabidopsis thaliana, can substitute for Chlamydomonas CCS4. Dominant suppressor mutations mapping to the CCS4 gene were identified in photosynthetic revertants of the ccs4ccs5 mutants. The suppressor mutations yield changes in the stroma-facing domain of CCS4 that restore holocytochrome f assembly above the residual levels detected in ccs5. Because the CCDA protein accumulation is decreased specifically in the ccs4 mutant, we hypothesize the suppressor mutations enhance the supply of reducing power through CCDA in the absence of CCS5. We discuss the operation of a CCS5-dependent and a CCS5-independent pathway controlling the redox status of the heme-binding cysteines of apocytochrome f., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Due to their unique biochemical and spectroscopic properties, both heme and phycocyanobilin are widely applied in the medical and food industries. Synechocystis sp. PCC 6803 contains both heme and phycocyanin, and is capable of synthesizing phycocyanin using heme as a precursor. The aim of this study was to uncover viable metabolic targets in the porphyrin pathway from Synechocystis sp. PCC 6803 to promote the accumulation of heme and phycocyanin in the recombinant strains of microalgae. A total of 10 genes related to heme synthesis pathway derived from Synechococcus elongatus PCC 7942 and 12 genes related to endogenous heme synthesis were individually overexpressed in strain PCC 6803. The growth rate and pigment content (heme, phycocyanin, chlorophyll a and carotenoids) of 22 recombinant algal strains were characterized. Quantitative real-time PCR technology was used to investigate the molecular mechanisms underlying the changes in physiological indicators in the recombinant algal strains. Among the 22 mutant strains, the mutant overexpressing the haemoglobin gene ( glbN ) of strain PCC 6803 had the highest heme content, which was 2.5 times higher than the wild type; the mutant overexpressing the gene of strain PCC 7942 ( hemF ) had the highest phycocyanin content, which was 4.57 times higher than the wild type. Overall, the results suggest that genes in the porphyrin pathway could significantly affect the heme and phycocyanin content in strain PCC 6803. Our study provides novel crucial targets for promoting the accumulation of heme and phycocyanin in cyanobacteria.

By exploiting versatile P450 enzymes, whole-cell biocatalysis can be performed to synthesize valuable compounds in Escherichia coli. However, the insufficient supply of heme limits the whole-cell P450 biocatalytic activity. Here a strategy for improving intracellular heme biosynthesis to enhance the catalytic efficiencies of P450s is reported. After comparing the effects of improving heme transport and biosynthesis on P450 activities, intracellular heme biosynthesis is optimized through the integrated expression of necessary synthetic genes at proper ratios and the assembly of rate-limiting enzymes using DNA-guided scaffolds. The intracellular heme level is fine-tuned by the combined use of mutated heme-sensitive biosensors and small regulatory RNA systems. The catalytic efficiencies of three different P450s, BM3, sca-2, and CYP105D7, are enhanced through fine-tuning heme biosynthesis for the synthesis of hydroquinone, pravastatin, and 7,3',4'-trihydroxyisoflavone as example products of chemical intermediate, drug, and natural product, respectively. This strategy of fine-tuned heme biosynthesis will be generally useful for developing whole-cell biocatalysts involving hemoproteins., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Heme (iron-protoporphyrin IX) is an essential but potentially toxic cellular cofactor. While most organisms are heme prototrophs, many microorganisms can utilize environmental heme as iron source. The pathogenic yeast Candida albicans can utilize host heme in the iron-poor host environment, using an extracellular cascade of soluble and anchored hemophores, and plasma membrane ferric reductase-like proteins. To gain additional insight into the C. albicans heme uptake pathway, we performed an unbiased genetic selection for mutants resistant to the toxic heme analog Ga3+-protoporphyrin IX at neutral pH, and a secondary screen for inability to utilize heme as iron source. Among the mutants isolated were the genes of the pH-responsive RIM pathway, and a zinc finger transcription factor related to S. cerevisiae HAP1. In the presence of hemin in the medium, C. albicans HAP1 is induced, the Hap1 protein is stabilized and Hap1-GFP localizes to the nucleus. In the hap1 mutant, cytoplasmic heme levels are elevated, while influx of extracellular heme is lower. Gene expression analysis indicated that in the presence of extracellular hemin, Hap1 activates the heme oxygenase HMX1, which breaks down excess cytoplasmic heme, while at the same time it also activates all the known heme uptake genes. These results indicate that Hap1 is a heme-responsive transcription factor that plays a role both in cytoplasmic heme homeostasis and in utilization of extracellular heme. The induction of heme uptake genes by C. albicans Hap1 under iron satiety indicates that preferential utilization of host heme can be a dietary strategy in a heme prototroph., Competing Interests: The authors have declared that no competing interests exist.

Iron is the most abundant transition metal essential for numerous cellular processes. Although most mammalian cells acquire iron through transferrin receptors, molecular players of iron utilization under iron restriction are incompletely understood. To address this, we performed metabolism-focused CRISPRa gain-of-function screens, which revealed metabolic limitations under stress conditions. Iron restriction screens identified not only expected members of iron utilization pathways but also SLCO2B1, a poorly characterized membrane carrier. SLCO2B1 expression is sufficient to increase intracellular iron, bypass the essentiality of the transferrin receptor, and enable proliferation under iron restriction. Mechanistically, SLCO2B1 mediates heme analog import in cellular assays. Heme uptake by SLCO2B1 provides sufficient iron for proliferation through heme oxygenases. Notably, SLCO2B1 is predominantly expressed in microglia in the brain, and primary Slco2b1 -/- mouse microglia exhibit strong defects in heme analog import. Altogether, our work identifies SLCO2B1 as a microglia-enriched plasma membrane heme importer and provides a genetic platform to identify metabolic limitations under stress conditions., Competing Interests: Declaration of interests K.B. is a scientific advisor to Nanocare Pharmaceuticals and Barer Institute., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Heme is an oxygen carrier and a cofactor of both industrial enzymes and food additives. The intracellular level of free heme is low, which limits the synthesis of heme proteins. Therefore, increasing heme synthesis allows an increased production of heme proteins. Using the genome-scale metabolic model (GEM) Yeast8 for the yeast Saccharomyces cerevisiae , we identified fluxes potentially important to heme synthesis. With this model, in silico simulations highlighted 84 gene targets for balancing biomass and increasing heme production. Of those identified, 76 genes were individually deleted or overexpressed in experiments. Empirically, 40 genes individually increased heme production (up to threefold). Heme was increased by modifying target genes, which not only included the genes involved in heme biosynthesis, but also those involved in glycolysis, pyruvate, Fe-S clusters, glycine, and succinyl-coenzyme A (CoA) metabolism. Next, we developed an algorithmic method for predicting an optimal combination of these genes by using the enzyme-constrained extension of the Yeast8 model, ecYeast8. The computationally identified combination for enhanced heme production was evaluated using the heme ligand-binding biosensor (Heme-LBB). The positive targets were combined using CRISPR-Cas9 in the yeast strain (IMX581- HEM15-HEM14-HEM3-Δshm1-HEM2-Δhmx1-FET4-Δgcv2-HEM1-Δgcv1-HEM13 ), which produces 70-fold-higher levels of intracellular heme.

Cytochrome c peroxidase (Ccp1) is a mitochondrial heme-containing enzyme that has served for decades as a chemical model to explore the structure function relationship of heme enzymes. Unveiling the impact of its heme pocket residues on the structural behavior, the non-covalent interactions and consequently its peroxidase activity has been a matter of increasing interest. To further probe these roles, we conducted intensive all-atom molecular dynamics simulations on WT and nineteen in-silico generated Ccp1 variants followed by a detailed structural and energetic analysis of H 2 O 2 binding and pairwise interactions. Different structural analysis including RMSD, RMSF, radius of gyration and the number of Hydrogen bonds clearly demonstrate that none of the studied mutants induce a significant structural change relative to the WT behavior. In an excellent agreement with experimental observations, the structural change induced by all the studied mutant systems is found to be very localized only to their surrounding environment. The determined interaction energies between residues and Gibbs binding energies for the WT Ccp1 and the nineteen variants, helped to identify the precise effect of each mutated residues on both the binding of H 2 O 2 and the non-covalent interaction and thus the overall peroxidase activity. The roles of surrounding residues in adopting unique distinctive electronic feature by Ccp1 has been discerned. Our valuable findings have clarified the functions of various residues in Ccp1 and thereby provided novel atomistic insights into its function. Overall, due to the conserved residues of the heme-pocket amongst various peroxidases, the obtained remarks in this work are highly valuable., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Isolated sulfite oxidase deficiency (ISOD) is a rare recessive and infantile lethal metabolic disorder, which is caused by functional loss of sulfite oxidase (SO) due to mutations of the SUOX gene. SO is a mitochondrially localized molybdenum cofactor (Moco)- and heme-dependent enzyme, which catalyzes the vital oxidation of toxic sulfite to sulfate. Accumulation of sulfite and sulfite-related metabolites such as S-sulfocysteine (SSC) are drivers of severe neurodegeneration leading to early childhood death in the majority of ISOD patients. Full functionality of SO is dependent on correct insertion of the heme cofactor and Moco, which is controlled by a highly orchestrated maturation process. This maturation involves the translation in the cytosol, import into the intermembrane space (IMS) of mitochondria, cleavage of the mitochondrial targeting sequence, and insertion of both cofactors. Moco insertion has proven as the crucial step in this maturation process, which enables the correct folding of the homodimer and traps SO in the IMS. Here, we report on a novel ISOD patient presented at 17 months of age carrying the homozygous mutation NM_001032386.2 (SUOX):c.1097G > A, which results in the expression of SO variant R366H. Our studies show that histidine substitution of Arg366, which is involved in coordination of the Moco-phosphate, causes a severe reduction in Moco insertion efficacy in vitro and in vivo. Expression of R366H in HEK SUOX -/- cells mimics the phenotype of patient's fibroblasts, representing a loss of SO expression and specific activity. Our studies disclose a general paradigm for a kinetic defect in Moco insertion into SO caused by residues involved in Moco coordination resulting in the case of R366H in an attenuated form of ISOD., (© 2021 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Fur (ferric uptake regulator) is a transcription factor that regulates expression of downstream genes containing a specific Fe 2+ -binding sequence called the Fur box. In Vibrio cholerae, a Fur box is located upstream of the nik operon, which is responsible for nickel uptake, suggesting that its expression is regulated by Fur. However, there are no reports that Ni 2+ induces expression of Fur box genes. Accordingly, we here investigated whether Ni 2+ or Fe 2+ binds to Fur to regulate expression of the nik operon. We found that Fur binds to the Fur box in the presence of Fe 2+ with a dissociation constant (K d ) of 1.2 μM, whereas only non-specific binding was observed in the presence of Ni 2+ . Thus, Fur-mediated expression of the nik operon is dependent on Fe 2+ , but not Ni 2+ . Since most iron in cells exists as heme, we examined the effect of heme on the Fur box binding activity of V. cholerae Fur (VcFur). Addition of heme to the VcFur-Fur box complex induced dissociation of VcFur from the Fur box, indicating that expression of the V. cholerae nik operon is regulated by both iron and heme. Furthermore, VCA1098, a nik operon-encoded protein, bound heme with a K d of 1.3 μM. Collectively, our results suggest that the V. cholerae nik operon is involved not only in nickel uptake but also in heme uptake, and depends on iron and heme concentrations within bacteria., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The capacity of haem to transfer electrons, bind diatomic gases, and catalyse various biochemical reactions makes it one of the essential biomolecules on Earth and one that was likely used by the earliest forms of cellular life. Since the description of haem biosynthesis, our understanding of this multi-step pathway has been almost exclusively derived from a handful of model organisms from narrow taxonomic contexts. Recent advances in genome sequencing and functional studies of diverse and previously neglected groups have led to discoveries of alternative routes of haem biosynthesis that deviate from the 'classical' pathway. In this review, we take an evolutionarily broad approach to illuminate the remarkable diversity and adaptability of haem synthesis, from prokaryotes to eukaryotes, showing the range of strategies that organisms employ to obtain and utilise haem. In particular, the complex evolutionary histories of eukaryotes that involve multiple endosymbioses and horizontal gene transfers are reflected in the mosaic origin of numerous metabolic pathways with haem biosynthesis being a striking case. We show how different evolutionary trajectories and distinct life strategies resulted in pronounced tensions and differences in the spatial organisation of the haem biosynthesis pathway, in some cases leading to a complete loss of a haem-synthesis capacity and, rarely, even loss of a requirement for haem altogether., (© 2021 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.)

Iron homeostasis is essential for both sides of the host-pathogen interface. Restricting access of iron slows bacterial growth while iron is also a necessary cofactor for host immunity. Haem oxygenase 1 (HMOX1) is a critical regulator of iron homeostasis that catalyses the liberation of iron during degradation of haem. It is also a stress-responsive protein that can be rapidly upregulated and confers protection to the host. Although a protective role of HMOX1 has been demonstrated in a variety of diseases, the role of HMOX1 in Mycobacterium tuberculosis infection is equivocal across experiments with different host-pathogen combinations. Here, we use the natural host-pathogen pairing of the zebrafish-Mycobacterium marinum infection platform to study the role of zebrafish haem oxygenase in mycobacterial infection. We identify zebrafish Hmox1a as the relevant functional paralog of mammalian HMOX1 and demonstrate a conserved role for Hmox1a in protecting the host from M. marinum infection. Using genetic and chemical tools, we show zebrafish Hmox1a protects the host against M. marinum infection by reducing infection-induced iron accumulation and ferrostatin-sensitive cell death., (© 2021 Federation of European Biochemical Societies.)

GAPDH, a heme chaperone, has been previously implicated in the incorporation of heme into iNOS and soluble guanylyl cyclase (sGC). Since sGC is critical for myoglobin (Mb) heme-maturation, we investigated the role of GAPDH in the maturation of this globin, as well as hemoglobins α, β, and γ. Utilizing cell culture systems, we found that overexpression of wild-type GAPDH increased, whereas GAPDH mutants H53A and K227A decreased, the heme content of Mb and Hbα and Hbβ. Overexpression of wild-type GAPDH fully recovered the heme-maturation inhibition observed with the GAPDH mutants. Partial rescue was observed by overexpression of sGCβ1 but not by overexpression of a sGCΔβ1 deletion mutant, which is unable to bind the sGCα1 subunit required to form the active sGCα1β1 complex. Wild type and mutant GAPDH was found to be associated in a complex with each of the globins and Hsp90. GAPDH at endogenous levels was found to be associated with Mb in differentiating C2C12 myoblasts, and with Hbγ or Hbα in differentiating HiDEP-1 erythroid progenitor cells. Knockdown of GAPDH in C2C12 cells suppressed Mb heme-maturation. GAPDH knockdown in K562 erythroleukemia cells suppressed Hbα and Hbγ heme-maturation as well as Hb dimerization. Globin heme incorporation was not only dependent on elevated sGCα1β1 heterodimer formation, but also influenced by iron provision and magnitude of expression of GAPDH, d-aminolevulinic acid, and FLVCR1b. Together, our data support an important role for GAPDH in the maturation of myoglobin and γ, β, and α hemoglobins., (© 2021 Federation of American Societies for Experimental Biology.)

Diamond-Blackfan anemia (DBA) is a rare genetic disorder in which patients present a scarcity of erythroid precursors in an otherwise normocellular bone marrow. Most, but not all, patients carry mutations in ribosomal proteins such as RPS19, suggesting that compromised mRNA translation and ribosomal stress are pathogenic mechanisms causing depletion of erythroid precursors. To gain further insight to disease mechanisms in DBA, we performed a custom short hairpin RNA (shRNA) based screen against 750 genes hypothesized to affect DBA pathophysiology. Among the hits were two shRNAs against the erythroid specific heme-regulated eIF2α kinase (HRI), which is a negative regulator of mRNA translation. This study shows that shRNA-mediated HRI silencing or loss of one HRI allele improves expansion of Rps19-deficient erythroid precursors, as well as improves the anemic phenotype in Rps19-deficient animals. We found that Rps19-deficient erythroblasts have elevated levels of unbound intracellular heme, which is normalized by HRI heterozygosity. Additionally, targeting elevated heme levels by treating cells with the heme scavenger alpha-1-microglobulin (A1M), increased proliferation of Rps19-deficient erythroid precursors and decreased heme levels in a disease-specific manner. HRI heterozygosity, but not A1M treatment, also decreased the elevated p53 activity observed in Rps19-deficient cells, indicating that p53 activation is caused by ribosomal stress and aberrant mRNA translation and not heme overload in Rps19-deficiency. Together, these findings suggest that targeting elevated heme levels is a promising new treatment strategy for DBA., (Copyright © 2021. Published by Elsevier Inc.)

Heme, a near ubiquitous cofactor, is synthesized by most organisms. The essential step of insertion of iron into the porphyrin macrocycle is mediated by the enzyme ferrochelatase. Several ferrochelatases have been characterized, and it has been experimentally shown that a fraction of them contain [2Fe-2S] clusters. It has been suggested that all metazoan ferrochelatases have such clusters, but among bacteria, these clusters have been most commonly identified in Actinobacteria and a few other bacteria. Despite this, the function of the [2Fe-2S] cluster remains undefined. With the large number of sequenced genomes currently available, we comprehensively assessed the distribution of putative [2Fe-2S] clusters throughout the ferrochelatase protein family. We discovered that while rare within the bacterial ferrochelatase family, this cluster is prevalent in a subset of phyla. Of note is that genomic data show that the cluster is not common in Actinobacteria, as is currently thought based on the small number of actinobacterial ferrochelatases experimentally examined. With available physiological data for each genome included, we identified a correlation between the presence of the microbial cluster and aerobic metabolism. Additionally, our analysis suggests that Firmicute ferrochelatases are the most ancient and evolutionarily preceded the Alphaproteobacterial precursor to eukaryotic mitochondria. These findings shed light on distribution and evolution of the [2Fe-2S] cluster in ferrochelatases and will aid in determining the function of the cluster in heme synthesis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Progesterone receptor membrane component 1 (PGRMC1) is a heme-binding protein implicated in a wide range of cellular functions. We previously showed that PGRMC1 binds to cytochromes P450 in yeast and mammalian cells and supports their activity. Recently, the paralog PGRMC2 was shown to function as a heme chaperone. The extent of PGRMC1 function in cytochrome P450 biology and whether PGRMC1 is also a heme chaperone are unknown. Here, we examined the function of Pgrmc1 in mouse liver using a knockout model and found that Pgrmc1 binds and stabilizes a broad range of cytochromes P450 in a heme-independent manner. Proteomic and transcriptomic studies demonstrated that Pgrmc1 binds more than 13 cytochromes P450 and supports maintenance of cytochrome P450 protein levels posttranscriptionally. In vitro assays confirmed that Pgrmc1 KO livers exhibit reduced cytochrome P450 activity consistent with reduced enzyme levels. Mechanistic studies in cultured cells demonstrated that PGRMC1 stabilizes cytochromes P450 and that binding and stabilization do not require PGRMC1 binding to heme. Importantly, Pgrmc1-dependent stabilization of cytochromes P450 is physiologically relevant, as Pgrmc1 deletion protected mice from acetaminophen-induced liver injury. Finally, evaluation of Y113F mutant Pgrmc1, which lacks the axial heme iron-coordinating hydroxyl group, revealed that proper iron coordination is not required for heme binding, but is required for binding to ferrochelatase, the final enzyme in heme biosynthesis. PGRMC1 was recently identified as the causative mutation in X-linked isolated pediatric cataract formation. Together, these results demonstrate a heme-independent function for PGRMC1 in cytochrome P450 stability that may underlie clinical phenotypes., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Molluscs exhibit diverse shell colors. The molecular regulation of shell coloration is however not well understood. To investigate the connection of shell coloration with pigment synthesis, we analyzed the distribution of porphyrins, a widespread group of pigments in nature, in four Pacific oyster strains of different shell colors including black, orange, golden, and white. The porphyrin distribution was analyzed in oyster mantles and shells by fluorescence imaging and UV spectrophotometer. The results showed that red fluorescence emitted by porphyrins under the UV light was detected only on the nacre of the orange-shell strain and mantles of orange, black and white-shell strains. Extracts from newly deposit shell, nacre and mantle tissue from orange-shell specimens showed peaks in UV-vis spectra that are characteristic of porphyrins, but these were not observed for the other shell-color strains. In addition, genes of the haem synthetic pathway were isolated and characterized. Phylogenetic analysis of CgALAS, CgALAD, CgPBGD, CgUROS, and CgUROD provide further evidence for a conserved genetic pathway of haem synthesis during evolution. Differential expression of the haem genes expressed in mantle tissues support these findings and are consistent with porphyrins being produced by the orange strain only. Tissue in situ hybridization demonstrated the expression of these candidate genes at the outer fold of C. gigas mantles where shell is deposited. Our studies provide a better understanding of shell pigmentation in C. gigas and candidate genes for future mechanistic analysis of shell color formation in molluscs., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Heme's interaction with Toll-like receptor 4 (TLR4) does not fully explain the proinflammatory properties of this hemoglobin-derived molecule during intravascular hemolysis. The receptor for advanced glycation end products (RAGE) shares many features with TLR4 such as common ligands and proinflammatory, prothrombotic, and pro-oxidative signaling pathways, prompting us to study its involvement as a heme sensor. Stable RAGE-heme complexes with micromolar affinity were detected as heme-mediated RAGE oligomerization. The heme-binding site was located in the V domain of RAGE. This interaction was Fe 3+ -dependent and competitive with carboxymethyllysine, another RAGE ligand. We confirmed a strong basal gene expression of RAGE in mouse lungs. After intraperitoneal heme injection, pulmonary TNF-α, IL1β, and tissue factor gene expression levels increased in WT mice but were significantly lower in their RAGE -/- littermates. This may be related to the lower activation of ERK1/2 and Akt observed in the lungs of heme-treated, RAGE -/- mice. Overall, heme binds to RAGE with micromolar affinity and could promote proinflammatory and prothrombotic signaling in vivo, suggesting that this interaction could be implicated in heme-overload conditions., (© 2020 Federation of European Biochemical Societies.)

The early detection of blood in urine (hematuria) can play a crucial role in the treatment of serious diseases ( e . g ., infections, kidney disease, schistosomiasis, and cancer). Therefore, the development of low-cost portable biosensors for blood detection in urine has become necessary. Here, we designed an ultrasensitive whole-cell bacterial biosensor interfaced with an optoelectronic measurement module for heme detection in urine. Heme is a red blood cells (RBCs) component that is liberated from lysed cells. The bacterial biosensor includes Escherichia coli cells carrying a heme-sensitive synthetic promoter integrated with a luciferase reporter ( luxCDABE ) from Photorhabdus luminescens . To improve the bacterial biosensor performance, we re-engineered the genetic structure of luxCDABE operon by splitting it into two parts ( luxCDE and luxAB ). The luxCDE genes were regulated by the heme-sensitive promoter, and the luxAB genes were regulated by either constitutive or inducible promoters. We examined the genetic circuit's performance in synthetic urine diluent supplied with heme and in human urine supplied with lysed blood. Finally, we interfaced the bacterial biosensor with a light detection setup based on a commercial optical measurement single-photon avalanche photodiode (SPAD). The whole-cell biosensor was tested in human urine with lysed blood, demonstrating a low-cost, portable, and easy-to-use hematuria detection with an ON-to-OFF ratio of 6.5-fold for blood levels from 5 × 10 4 to 5 × 10 5 RBC per mL of human urine.

Plasma cells (PCs) play an important role in the adaptive immune system through a continuous production of antibodies. We have demonstrated that PC differentiation can be modeled in vitro using complex multistep culture systems reproducing sequential differentiation process occurring in vivo. Here we present a comprehensive, temporal program of gene expression data encompassing human PC differentiation (PCD) using RNA sequencing (RNA-seq). Our results reveal 6374 differentially expressed genes classified into four temporal gene expression patterns. A stringent pathway enrichment analysis of these gene clusters highlights known pathways but also pathways largely unknown in PCD, including the heme biosynthesis and the glutathione conjugation pathways. Additionally, our analysis revealed numerous novel transcriptional networks with significant stage-specific overexpression and potential importance in PCD, including BATF2, BHLHA15/MIST1, EZH2, WHSC1/MMSET, and BLM. We have experimentally validated a potent role for BLM in regulating cell survival and proliferation during human PCD. Taken together, this RNA-seq analysis of PCD temporal stages helped identify coexpressed gene modules with associated up/downregulated transcription regulator genes that could represent major regulatory nodes for human PC maturation. These data constitute a unique resource of human PCD gene expression programs in support of future studies for understanding the underlying mechanisms that control PCD.

The shell color of the Mollusca has attracted naturalists and collectors for hundreds of years, while the molecular pathways regulating pigment production and the pigments themselves remain poorly described. In this study, our aim was to identify the main pigments and their molecular pathways in the pearl oyster Pinctada margaritifera -the species displaying the broadest range of colors. Three inner shell colors were investigated-red, yellow, and green. To maximize phenotypic homogeneity, a controlled population approach combined with common garden conditioning was used. Comparative analysis of transcriptomes (RNA-seq) of P. margaritifera with different shell colors revealed the central role of the heme pathway, which is involved in the production of red (uroporphyrin and derivates), yellow (bilirubin), and green (biliverdin and cobalamin forms) pigments. In addition, the Raper-Mason, and purine metabolism pathways were shown to produce yellow pigments (pheomelanin and xanthine) and the black pigment eumelanin. The presence of these pigments in pigmented shell was validated by Raman spectroscopy. This method also highlighted that all the identified pathways and pigments are expressed ubiquitously and that the dominant color of the shell is due to the preferential expression of one pathway compared with another. These pathways could likely be extrapolated to many other organisms presenting broad chromatic variation.

Efficiently carrying out the oxygen reduction reaction (ORR) is critical for many applications in biology and chemistry, such as bioenergetics and fuel cells, respectively. In biology, this reaction is carried out by large, transmembrane oxidases such as heme-copper oxidases (HCOs) and cytochrome bd oxidases. Common to these oxidases is the presence of a glutamate residue next to the active site, but its precise role in regulating the oxidase activity remains unclear. To gain insight into its role, we herein report that incorporation of glutamate next to a designed heme-copper center in two biosynthetic models of HCOs improves O 2 binding affinity, facilitates protonation of reaction intermediates, and eliminates release of reactive oxygen species. High-resolution crystal structures of the models revealed extended, water-mediated hydrogen-bonding networks involving the glutamate. Electron paramagnetic resonance of the cryoreduced oxy-ferrous centers at cryogenic temperature followed by thermal annealing allowed observation of the key hydroperoxo intermediate that can be attributed to the hydrogen-bonding network. By demonstrating these important roles of glutamate in oxygen reduction biochemistry, this work offers deeper insights into its role in native oxidases, which may guide the design of more efficient artificial ORR enzymes or catalysts for applications such as fuel cells.

HupZ is an expected heme degrading enzyme in the heme acquisition and utilization pathway in Group A Streptococcus. The isolated HupZ protein containing a C-terminal V5-His 6 tag exhibits a weak heme degradation activity. Here, we revisited and characterized the HupZ-V5-His 6 protein via biochemical, mutagenesis, protein quaternary structure, UV-vis, EPR, and resonance Raman spectroscopies. The results show that the ferric heme-protein complex did not display an expected ferric EPR signal and that heme binding to HupZ triggered the formation of higher oligomeric states. We found that heme binding to HupZ was an O 2 -dependent process. The single histidine residue in the HupZ sequence, His111, did not bind to the ferric heme, nor was it involved with the weak heme-degradation activity. Our results do not favor the heme oxygenase assignment because of the slow binding of heme and the newly discovered association of the weak heme degradation activity with the His 6 -tag. Altogether, the data suggest that the protein binds heme by its His 6 -tag, resulting in a heme-induced higher-order oligomeric structure and heme stacking. This work emphasizes the importance of considering exogenous tags when interpreting experimental observations during the study of heme utilization proteins.

Nitric oxide (NO) binds to soluble guanylyl cyclase (sGC), activates it in a reduced oxidized heme iron state, and generates cyclic Guanosine Monophosphate (cGMP), which results in vasodilatation and inhibition of osteoclast activity. In inflammation, sGC is oxidized and becomes insensitive to NO. NO- and heme-independent activation of sGC requires protein expression of the α 1 - and β 1 -subunits. Inflammation of the periodontium induces the resorption of cementum by cementoclasts and the resorption of the alveolar bone by osteoclasts, which can lead to tooth loss. As the presence of sGC in cementoclasts is unknown, we investigated the α 1 - and β 1 -subunits of sGC in cementoclasts of healthy and inflamed human periodontium using double immunostaining for CD68 and cathepsin K and compared the findings with those of osteoclasts from the same sections. In comparison to cementoclasts in the healthy periodontium, cementoclasts under inflammatory conditions showed a decreased staining intensity for both α 1 - and β 1 -subunits of sGC, indicating reduced protein expression of these subunits. Therefore, pharmacological activation of sGC in inflamed periodontal tissues in an NO- and heme-independent manner could be considered as a new treatment strategy to inhibit cementum resorption.

Macrophages are main effectors of heme metabolism, increasing transiently in the liver during heightened disposal of damaged or senescent RBCs (sRBCs). Macrophages are also essential in defense against microbial threats, but pathological states of heme excess may be immunosuppressive. Herein, we uncovered a mechanism whereby an acute rise in sRBC disposal by macrophages led to an immunosuppressive phenotype after intrapulmonary Klebsiella pneumoniae infection characterized by increased extrapulmonary bacterial proliferation and reduced survival from sepsis in mice. The impaired immunity to K. pneumoniae during heightened sRBC disposal was independent of iron acquisition by bacterial siderophores, in that K. pneumoniae mutants lacking siderophore function recapitulated the findings observed with the WT strain. Rather, sRBC disposal induced a liver transcriptomic profile notable for suppression of Stat1 and IFN-related responses during K. pneumoniae sepsis. Excess heme handling by macrophages recapitulated STAT1 suppression during infection that required synergistic NRF1 and NRF2 activation but was independent of heme oxygenase-1 induction. Whereas iron was dispensable, the porphyrin moiety of heme was sufficient to mediate suppression of STAT1-dependent responses in human and mouse macrophages and promoted liver dissemination of K. pneumoniae in vivo. Thus, cellular heme metabolism dysfunction negatively regulated the STAT1 pathway, with implications in severe infection.

Previously, we showed that Rab5a and Rab5b differentially regulate fluid-phase and receptor-mediated endocytosis in Leishmania, respectively. To unequivocally demonstrate the role of Rab5b in hemoglobin endocytosis in Leishmania, we generated null-mutants of Rab5b parasites by sequentially replacing both copies of LdRab5b with the hygromycin and neomycin resistance gene cassettes. LdRab5b -/- null-mutant parasite was confirmed by qPCR analysis of genomic DNA using LdRab5b specific primers. LdRab5b -/- cells showed severe growth defect indicating essential function of LdRab5b in parasite. To characterize the role of Rab5b in Hb endocytosis in parasites, LdRab5b -/- cells were rescued by exogenous addition of hemin in growth medium. Our results showed that LdRab5b -/- cells are relatively smaller in size. Ultrastructural analysis revealed the presence of relatively enlarged flagellar pocket and bigger intracellular vesicles in these cells in comparison to control cells. Both promastigotes and amastigotes of Rab5b null-mutant parasites were unable to internalize Hb but fluid phase endocytosis of different markers was not affected. However, complementation of LdRab5b:WT in LdRab5b -/- cells (LdRab5b -/- :pRab5b:WT) rescued Hb internalization in these cells. Interestingly, LdRab5b -/- cells showed significantly less Hb-receptor on cell surface in comparison to control cells indicating a block in HbR trafficking. Finally, we showed that LdRab5b -/- parasites can infect the macrophages but are unable to survive after 96 h of infection in comparison to control cells. However, supplementation of hemin in the growth medium significantly rescued LdRab5b -/- Leishmania survival in macrophage indicating that LdRab5b function is essential for the acquisition of heme from internalized Hb for the survival of Leishmania., (Copyright © 2020. Published by Elsevier B.V.)

The Apicomplexa phylum groups important human and animal pathogens that cause severe diseases, encompassing malaria, toxoplasmosis, and cryptosporidiosis. In common with most organisms, apicomplexans rely on heme as cofactor for several enzymes, including cytochromes of the electron transport chain. This heme derives from de novo synthesis and/or the development of uptake mechanisms to scavenge heme from their host. Recent studies have revealed that heme synthesis is essential for Toxoplasma gondii tachyzoites, as well as for the mosquito and liver stages of Plasmodium spp. In contrast, the erythrocytic stages of the malaria parasites rely on scavenging heme from the host red blood cell. The unusual heme synthesis pathway in Apicomplexa spans three cellular compartments and comprises enzymes of distinct ancestral origin, providing promising drug targets. Remarkably given the requirement for heme, T. gondii can tolerate the loss of several heme synthesis enzymes at a high fitness cost, while the ferrochelatase is essential for survival. These findings indicate that T. gondii is capable of salvaging heme precursors from its host. Furthermore, heme is implicated in the activation of the key antimalarial drug artemisinin. Recent findings established that a reduction in heme availability corresponds to decreased sensitivity to artemisinin in T. gondii and Plasmodium falciparum, providing insights into the possible development of combination therapies to tackle apicomplexan parasites. This review describes the microeconomics of heme in Apicomplexa, from supply, either from de novo synthesis or scavenging, to demand by metabolic pathways, including the electron transport chain., (© 2020 Federation of European Biochemical Societies.)

Pseudomonas aeruginosa is an opportunistic pathogen requiring iron for its survival and virulence. P. aeruginosa can acquire iron from heme via the nonredundant heme assimilation system and Pseudomonas heme uptake (Phu) systems. Heme transported by either the heme assimilation system or Phu system is sequestered by the cytoplasmic protein PhuS. Furthermore, PhuS has been shown to specifically transfer heme to the iron-regulated heme oxygenase HemO. As the PhuS homolog ShuS from Shigella dysenteriae was observed to bind DNA as a function of its heme status, we sought to further determine if PhuS, in addition to its role in regulating heme flux through HemO, functions as a DNA-binding protein. Herein, through a combination of chromatin immunoprecipitation-PCR, EMSA, and fluorescence anisotropy, we show that apo-PhuS but not holo-PhuS binds upstream of the tandem iron-responsive sRNAs prrF1,F2. Previous studies have shown the PrrF sRNAs are required for sparing iron for essential proteins during iron starvation. Furthermore, under certain conditions, a heme-dependent read through of the prrF1 terminator yields the longer PrrH transcript. Quantitative PCR analysis of P. aeruginosa WT and ΔphuS strains shows that loss of PhuS abrogates the heme-dependent regulation of PrrF and PrrH levels. Taken together, our data show that PhuS, in addition to its role in extracellular heme metabolism, also functions as a transcriptional regulator by modulating PrrF and PrrH levels in response to heme. This dual function of PhuS is central to integrating extracellular heme utilization into the PrrF/PrrH sRNA regulatory network that is critical for P. aeruginosa adaptation and virulence within the host., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The cyclic tetrapyrrole heme is used as a prosthetic group in a broad variety of different proteins in almost all organisms. Often, it is essential for vital biochemical processes such as aerobic and anaerobic respiration as well as photosynthesis. In Nature, heme is made from the common tetrapyrrole precursor 5-aminolevulinic acid, and for a long time it was assumed that heme is biosynthesized by a single, common pathway in all organisms. However, although this is indeed the case in eukaryotes, heme biosynthesis is more diverse in the prokaryotic world, where two additional pathways exist. The final elucidation of the two 'alternative' heme biosynthesis routes operating in some bacteria and archaea was achieved within the last decade. This review summarizes the three different heme biosynthesis pathways with a special emphasis on the two 'new' prokaryotic routes., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Heme oxygenase-2 (HO2) and -1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. HO1 expression is transcriptionally regulated; however, HO2 expression is constitutive. How the cellular levels and activity of HO2 are regulated remains unclear. Here, we elucidate the mechanism of post-translational regulation of cellular HO2 levels by heme. We find that, under heme-deficient conditions, HO2 is destabilized and targeted for degradation, suggesting that heme plays a direct role in HO2 regulation. HO2 has three heme binding sites: one at its catalytic site and the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, an HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared with the WT HO2. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2's role in maintaining heme homeostasis, paving the way for future investigation into HO2's pathophysiological role in heme deficiency response., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Liu et al.)

The porphyrias are a family of metabolic disorders caused by defects in the activity of one of the enzymes in the heme biosynthetic pathway. Acute intermittent porphyria (AIP), caused by autosomal dominant mutations in the gene encoding hydroxymethylbilane synthase, can lead to hepatocyte overaccumulation and systemic distribution of the proximal porphyrin precursors, 5-aminolevulinic acid (ALA) and porphobilinogen (PBG). ALA and PBG are toxic to neurons and extrahepatic tissue and cause the neurovisceral clinical manifestations of AIP. Management of AIP includes awareness and avoidance of triggering factors, infusions of hemin for severe acute attacks, and, if indicated for chronic suppressive therapy, maintenance treatment with hemin or givosiran, a small interfering RNA molecule that antagonizes ALA synthase 1 transcripts. Erythropoietic protoporphyria (EPP) is most commonly caused by autosomal recessive mutations in the gene encoding ferrochelatase (FECH), the heme pathway terminal enzyme. FECH deficiency leads to erythrocyte overaccumulation and high plasma levels of lipophilic protoporphyrins that photoactivate in the skin, causing burning pain and erythema. Protoporphyrins excreted in the bile can cause gallstones, cholestasis, fibrosis, and ultimately liver failure. Management of EPP includes skin protection and afamelanotide, an α-melanocyte stimulating hormone analog that increases melanin pigment and reduces photoactivation. Liver transplantation may be necessary for severe EPP-induced liver complications. Because AIP and EPP arise from defects in the heme biosynthetic pathway, hematologists are often consulted to evaluate and manage suspected or proven porphyrias. A working knowledge of these disorders increases our confidence and effectiveness as consultants and medical providers., Competing Interests: Conflict-of-interest disclosure: No conflict of interest declared., (© 2020 by The American Society of Hematology.)

The β subunit substitutions, F41Y and K82D, in sickle cell hemoglobin (Hb) (βE6 V) provides significant resistance to oxidative stress by shielding βCys93 from the oxidizing ferryl heme. We evaluated the oxidative resistance of βCys93 to hydrogen peroxide (H 2 O 2 ) in α subunit mutations in βE6 V (at both the putative and lateral contact regions) that included (1) αH20Q/βE6 V; (2) αH50Q/βE6 V; (3) αH20Q/H50Q/βE6 V; (4) αH20R/βE6 V; and (5) αH20R/H50Q/βE6 V. Estimation by mass spectrometry of irreversible oxidation of βCys93 to cysteic acid (CA) was unchanged or moderately increased in the single mutants harboring a H20Q or H50Q substitution when compared to control (βE6 V). The introduction of Arg (R) singularly or in combination with Q enhanced the pseudoperoxidative cycle by slightly decreasing the ferryl in favor of ferrous and ferric species after treatment with H 2 O 2 . Higher rates for heme loss from the ferric forms of the Q species to the receptor high affinity recombinant apomyglobin were observed in contrast to the R mutants and control. Because of their improved solubility, a combination of Q and R substitutions together with mutations carrying redox active variants (F41Y/K82D) may provide dual antioxidant and antisickling targets in the design of gene therapy-based candidates.

Background: 5-Aminolevulinic acid dehydratase (ALAD) porphyria (ADP) is an ultrarare autosomal recessive disease, with only eight documented cases, all of whom were males. Although classified as an acute hepatic porphyria (AHP), induction of the rate limiting hepatic enzyme 5-aminolevulinic acid synthase-1 (ALAS1) has not been demonstrated, and the marrow may also contribute excess 5-aminolevulinic acid (ALA). Two patients have died and reported follow up for the others is limited, so the natural history of this disease is poorly understood and treatment experience limited., Methods: We report new molecular findings and update the clinical course and treatment of the sixth reported ADP patient, now 31 years old and the only known case in the Americas, and review published data regarding genotype-phenotype correlation and treatment., Results: Circulating hepatic 5-aminolevulinic acid synthase-1 (ALAS1) mRNA was elevated in this case, as in other AHPs. Gain of function mutation of erythroid specific ALAS2 - an X-linked modifying gene in some other porphyrias - was not found. Seven reported ADP cases had compound heterozygous ALAD mutations resulting in very low residual ALAD activity and symptoms early in life or adolescence. One adult with a germline ALAD mutant allele developed ADP in association with a clonal myeloproliferative disorder, polycythemia vera., Conclusions: Elevation in circulating hepatic ALAS1 and response to treatment with hemin indicate that the liver is an important source of excess ALA in ADP, although the marrow may also contribute. Intravenous hemin was effective in most reported cases for treatment and prevention of acute attacks of neurological symptoms., Competing Interests: Declaration of Competing Interest AP Lahiji and VM Sadagopa Ramanujam declare no conflicts. A Chan and A Simon are employees of Alnylam Pharmaceuticals. KE Anderson and RJ Desnick consult for Alnylam Pharmaceuticals, Recordati Rare Diseases and Mitsubishi Tanabe Pharma America. All authors had access to the data and a role in writing the manuscript., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Iron acquisition is challenging in most environments. As an alternative to elemental iron, organisms can take up iron-protoporphyrin IX, or heme. Heme can be found in decaying organic matter and is particularly prevalent in animal hosts. Fungi have evolved at least three distinct endocytosis-mediated heme uptake systems, which have been studied in detail in the organisms Candida albicans, Cryptococcus neoformans and Schizosaccharomyces pombe. Here we summarize the known molecular details of these three uptake systems that enable parasitic and saprophytic fungi to take advantage of external heme as either cellular iron or heme sources., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Oxygen is fundamental to the life of aerobic organisms and is not always available to Paracoccidioides cells. During the life cycle stages, reduced oxygen levels directly affect general metabolic processes and oxygen adaptation mechanisms may play a fundamental role on fungal ability to survive under such condition. Heme proteins can bind to oxygen and participate in important biological processes. Several fungi, including Paracoccidioides, express a heme-binding globin (fungoglobin - FglA) presumable to regulate fungal adaptation to hypoxia. However, the characterization of fungoglobin in Paracoccidioides spp. has not yet been performed. In this study, we predicted the structure of fungoglobin and determined its level of expression during hypoxic-mimetic conditions. Genomic screening revealed that the fungoglobin gene is conserved in all species of the Paracoccidioides genus. Molecular modeling showed biochemical and biophysical characteristics that support the hypothesis that FglA binds to the heme group and oxygen as well. The fungoglobin transcript and proteins are expressed at higher levels at the early treatment time, remaining elevated while oxygen is limited. A P. brasiliensis fglA knockdown strain depicted reduced growth in hypoxia indicating that this protein can be essential for growth at low oxygen. Biochemical analysis confirmed the binding of fungoglobin to heme. Initial analyzes were carried out to establish the relationship between FlglA and iron metabolism. The FglA transcript was up regulated in pulmonary infection, suggesting its potential role in the disease establishment. We believe that this study can contribute to the understanding of fungal biology and open new perspectives for scientific investigations., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cytochrome P450s are an important group of enzymes catalyzing hydroxylation, and epoxidations reactions. In this work we describe the characterization of the CinA-CinC fusion enzyme system of a previously reported P450 using genetically fused heme (CinA) and FMN (CinC) enzyme domains from Citrobacter braaki. We observed that mixing individually inactivated heme (-) with FMN (-) domain in the CinA-10aa linker - CinC fusion constructs results in recovered activity and the formation of (2S)-2β-hydroxy,1,8-cineole (174 µM), a similar amount when compared to the fully functional fusion protein (176 µM). We also studied the effect of the fusion linker length in the activity complementation assay. Our results suggests an intermolecular interaction between heme and FMN parts from different CinA-CinC fusion protein similar to proposed mechanisms for P450 BM3 on the other hand, linker length plays a crucial influence on the activity of the fusion constructs. However, complementation assays show that inactive constructs with shorter linker lengths have functional subunits, and that the lack of activity might be due to incorrect interaction between fused enzymes.

α 1 -microglobulin (A1M) is a small protein present in vertebrates including humans. It has several physiologically relevant properties, including binding of heme and radicals as well as enzymatic reduction, that are used in the protection of cells and tissue. Research has revealed that A1M can ameliorate heme and ROS-induced injuries in cell cultures, organs, explants and animal models. Recently, it was shown that A1M could reduce hemolysis in vitro, observed with several different types of insults and sources of RBCs. In addition, in a recently published study, it was observed that mice lacking A1M (A1M-KO) developed a macrocytic anemia phenotype. Altogether, this suggests that A1M may have a role in RBC development, stability and turnover. This opens up the possibility of utilizing A1M for therapeutic purposes in pathological conditions involving erythropoietic and hemolytic abnormalities. Here, we provide an overview of A1M and its potential therapeutic effect in the context of the following erythropoietic and hemolytic conditions: Diamond-Blackfan anemia (DBA), 5q-minus myelodysplastic syndrome (5q-MDS), blood transfusions (including storage), intraventricular hemorrhage (IVH), preeclampsia (PE) and atherosclerosis., Competing Interests: B.Å., M.G. and S.R.H. are co-founders and minority shareholders of Guard Therapeutics International AB (former A1M Pharma AB), which holds patent rights on medical uses of A1M.

Extrinsic black dental staining is an external dental discoloration of bacterial origin, considered a special form of dental plaque. Currently, there is no definitive therapeutic option for eliminating black stain. This study employed 16S rRNA metagenomics to analyze black stain and white-plaque samples from 27 adult volunteers. Study objectives were to: describe the microbial diversity of adult black stain samples; characterize their taxonomic profile; compare the microbiomes of black stain versus white-plaque from adult volunteers and propose a functional map of the black stain microbiome using PICRUSt2. The black stain microbiome was poorer in species diversity as compared to white-plaque. The five most abundant genera in black stain were Capnocytophaga, Leptotrichia, Fusobacterium, Corynebacterium and Streptococcus. Functional analysis of microbial species revealed conserved and consistent clustering of functional pathways within and between black stain and white-plaque microbiomes. We describe enrichment of heme biosynthetic pathways in black stain. Our results suggest that the dysbiosis in black stain resembles "orally healthy" communities. The increased abundance of heme biosynthetic pathways suggests that heme-dependent iron sequestration and subsequent metabolism are key for black stain formation. Further research should decipher the regulation of heme biosynthetic genes and characterize the temporal sequence leading to colonization and dysbiosis.

Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria; however, resistance threatens to undermine global control efforts. To broadly explore artemisinin susceptibility in apicomplexan parasites, we employ genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. Using a sublethal concentration of dihydroartemisinin (DHA), we uncover the putative transporter Tmem14c whose disruption increases DHA susceptibility. Screens performed under high doses of DHA provide evidence that mitochondrial metabolism can modulate resistance. We show that disrupting a top candidate from the screens, the mitochondrial protease DegP2, lowers porphyrin levels and decreases DHA susceptibility, without significantly altering parasite fitness in culture. Deleting the homologous gene in P. falciparum, PfDegP, similarly lowers heme levels and DHA susceptibility. These results expose the vulnerability of heme metabolism to genetic perturbations that can lead to increased survival in the presence of DHA.

Heme is an essential cofactor for many biological processes in aerobic organisms, which can synthesize it de novo through a conserved pathway. Trypanosoma cruzi , the etiological agent of Chagas disease, as well as other trypanosomatids relevant to human health, are heme auxotrophs, meaning they must import it from their mammalian hosts or insect vectors. However, how these species import and regulate heme levels is not fully defined yet. It is known that the membrane protein Tc HTE is involved in T. cruzi heme transport, although its specific role remains unclear. In the present work, we studied endogenous Tc HTE in the different life cycle stages of the parasite to gain insight into its function in heme transport and homeostasis. We have confirmed that Tc HTE is predominantly detected in replicative stages (epimastigote and amastigote), in which heme transport activity was previously validated. We also showed that in epimastigotes, Tc HTE protein and mRNA levels decrease in response to increments in heme concentration, confirming it as a member of the heme response gene family. Finally, we demonstrated that T. cruzi epimastigotes can sense intracellular heme by an unknown mechanism and regulate heme transport to adapt to changing conditions. Based on these results, we propose a model in which T. cruzi senses intracellular heme and regulates heme transport activity by adjusting the expression of Tc HTE. The elucidation and characterization of heme transport and homeostasis will contribute to a better understanding of a critical pathway for T. cruzi biology allowing the identification of novel and essential proteins., Competing Interests: Conflict of interest—The authors declare that they have no conflict of interest with the content of this article., (© 2020 Pagura et al.)

Background: Acute Intermittent Porphyria (AIP) is a rare inherited autosomal dominant disorder of heme biosynthesis. Porphyria-associated kidney disease occurs in more than 50% of the patients with AIP, and end stage renal disease (ESRD) can be a devastating complication for AIP patients. The outcomes of AIP patients after kidney transplantation are poorly known., Methods: We examined the outcomes of 11 individuals with AIP, identified as kidney transplant recipients in the French Porphyria Center Registry., Results: AIP had been diagnosed on average 19 years before the diagnosis of ESRD except for one patient in whom the diagnosis of AIP had been made 5 years after the initiation of dialysis. Median follow-up after transplantation was 9 years. A patient died 2 months after transplantation from a cardiac arrest and a patient who received a donation after cardiac death experienced a primary non-function. No rejection episode and no noticeable adverse event occurred after transplantation. Serum creatinine was on average 117 μmol/l, and proteinuria <0.5 g/l in all patients at last follow up. All usually prescribed drugs after transplantation are authorized except for trimethoprim/sulfamethoxazole. Critically, acute porphyria attacks almost disappeared after kidney transplantation, and skin lesions resolved in all patients., Conclusion: Kidney transplantation is the treatment of choice for AIP patients with ESRD and dramatically reduces the disease activity., Competing Interests: Declaration of Competing Interest This research was unfunded and the authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Transferrin-bound iron (TBI), the physiological circulating iron form, is acquired by cells through the transferrin receptor (TfR1) by endocytosis. In erythroid cells, most of the acquired iron is incorporated into heme in the mitochondria. Cellular trafficking of heme is indispensable for erythropoiesis and many other essential biological processes. Comprehensive elucidation of molecular pathways governing and regulating cellular iron acquisition and heme trafficking is required to better understand physiological and pathological processes affecting erythropoiesis. Here, we report the first genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens in human erythroid cells to identify determinants of iron and heme uptake, as well as heme-mediated erythroid differentiation. We identified several candidate modulators of TBI acquisition including TfR1, indicating that our approach effectively revealed players mechanistically relevant to the process. Interestingly, components of the endocytic pathway were also revealed as potential determinants of transferrin acquisition. We deciphered a role for the vacuolar-type H+ - ATPase (V- ATPase) assembly factor coiled-coil domain containing 115 (CCDC115) in TBI uptake and validated this role in CCDC115 deficient K562 cells. Our screen in hemin-treated cells revealed perturbations leading to cellular adaptation to heme, including those corresponding to trafficking mechanisms and transcription factors potentiating erythroid differentiation. Pathway analysis indicated that endocytosis and vesicle acidification are key processes for heme trafficking in erythroid precursors. Furthermore, we provided evidence that CCDC115, which we identified as required for TBI uptake, is also involved in cellular heme distribution. This work demonstrates a previously unappreciated common intersection in trafficking of transferrin iron and heme in the endocytic pathway of erythroid cells., (© 2020 Wiley Periodicals LLC.)

Iron is essential for nearly all aerobic organisms. One source of iron in nature is in the form of heme. Due to its critical physiological importance as a cofactor for several enzymes, organisms have evolved various means to secure heme for their needs. In the case of heme prototrophs, these organisms possess a highly conserved eight-step biosynthetic pathway. Another means used by many organisms is to acquire heme from external sources. As opposed to the knowledge of enzymes responsible for heme biosynthesis, the nature of the players and mechanisms involved in the acquisition of exogenous heme is limited. This review focuses on a description of newly discovered proteins that have novel functions in heme assimilation in the model organism Schizosaccharomyces pombe. This tractable model allows the use of the power of genetics to selectively block heme biosynthesis, setting conditions to investigate the mechanisms by which external heme is taken up by the cells. Studies have revealed that S. pombe possesses two independent heme uptake systems that require Shu1 and Str3, respectively. Heme-bound iron is captured by Shu1 at the cell surface, triggering its internalization to the vacuole with the aid of ubiquitinated proteins and the ESCRT machinery. In the case of the plasma membrane transporter Str3, it promotes cellular heme import in cells lacking Shu1. The discovery of these two pathways may contribute to gain novel insights into the mechanisms whereby fungi assimilate heme, which is an essentially biological process for their ability to invade and colonize new niches.

Pseudomonas aeruginosa exhibits a high requirement for iron, which it can acquire via several mechanisms, including the acquisition and utilization of heme. The P. aeruginosa genome encodes two heme uptake systems, the heme assimilation system (Has) and the Pseudomonas heme utilization (Phu) system. Extracellular heme is sensed via the Has system, which encodes an extracytoplasmic function (ECF) σ factor system. Previous studies have shown that the transfer of heme from the extracellular hemophore HasAp to the outer membrane receptor HasR is required for activation of the σ factor HasI and upregulation of has operon expression. Here, employing site-directed mutagenesis, allelic exchange, quantitative PCR analyses, immunoblotting, and 13 C-heme uptake experiments, we delineated the differential contributions of the extracellular FRAP/PNPNL loop residue His-624 in HasR and of His-221 in its N-terminal plug domain required for heme capture to heme transport and signaling, respectively. Specifically, we show that substitution of the N-terminal plug His-221 disrupts both signaling and transport, leading to dysregulation of both the Has and Phu uptake systems. Our results are consistent with a model wherein heme release from HasAp to the N-terminal plug of HasR is required to initiate signaling, whereas His-624 is required for simultaneously closing off the heme transport channel from the extracellular medium and triggering heme transport. Our results provide critical insight into heme release, signaling, and transport in P. aeruginosa and suggest a functional link between the ECF σ factor and Phu heme uptake system., Competing Interests: Conflict of interest—The authors declare they have no conflict of interest with the contents of this article., (© 2020 Dent and Wilks.)

Isolated sulphite oxidase deficiency (iSOD) is an autosomal recessive inborn error in metabolism characterised by accumulation of sulphite, which leads to death in early infancy. Sulphite oxidase (SO) is encoded by the SUOX gene and forms a heme- and molybdenum-cofactor-dependent enzyme localised in the intermembrane space of mitochondria. Within SO, both cofactors are embedded in two separated domains, which are linked via a flexible 11 residue tether. The two-electron oxidation of sulphite to sulphate occurs at the molybdenum active site. From there, electrons are transferred via two intramolecular electron transfer steps (IETs) via the heme cofactor and to the physiologic electron acceptor cytochrome c. Previously, we reported nitrite and oxygen to serve as alternative electron acceptors at the Moco active site, thereby overcoming IET within SO. Here, we present evidence for these reactions to occur in an iSOD patient with an unusual mild disease representation. In the patient, a homozygous c.427C>A mutation within the SUOX gene leads to replacement of the highly conserved His143 to Asn. The affected His143 is one of two heme-iron-coordinating residues within SO. We demonstrate, that the H143N SO variant fails to bind heme in vivo leading to the elimination of SO-dependent cytochrome c reduction in mitochondria. We show, that sulphite oxidation at the Moco domain is unaffected in His143Asn SO variant and demonstrate that nitrite and oxygen are able to serve as electron acceptors for sulphite-derived electrons in cellulo. As result, the patient H143N SO variant retains residual sulphite oxidising activity thus ameliorating iSOD progression., (© 2020 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Heme and Fe-S clusters regulate a plethora of essential biological processes ranging from cellular respiration and cell metabolism to the maintenance of genome integrity. Mutations in genes involved in heme metabolism and Fe-S cluster biogenesis cause different forms of ataxia, like posterior column ataxia and retinitis pigmentosa (PCARP), Friedreich's ataxia (FRDA) and X-linked sideroblastic anemia with ataxia (XLSA/A). Despite great efforts in the elucidation of the molecular pathogenesis of these disorders several important questions still remain to be addressed. Starting with an overview of the biology of heme metabolism and Fe-S cluster biogenesis, the review discusses recent progress in the understanding of the molecular pathogenesis of PCARP, FRDA and XLSA/A, and highlights future line of research in the field. A better comprehension of the mechanisms leading to the degeneration of neural circuity responsible for balance and coordinated movement will be crucial for the therapeutic management of these patients.