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The role of magnetism in the biological functioning of hemoglobin has been debated since its discovery by Pauling and Coryell in 1936. The hemoglobin molecule contains four heme groups each having a porphyrin layer with a Fe ion at the center. Here, we present combined density-functional theory and quantum Monte Carlo calculations for an effective model of Fe in a heme cluster. In comparison with these calculations, we analyze the experimental data on human adult hemoglobin (HbA) from the magnetic susceptibility, Mössbauer and magnetic circular dichroism (MCD) measurements. In both the deoxygenated (deoxy) and the oxygenated (oxy) cases, we show that local magnetic moments develop in the porphyrin layer with antiferromagnetic coupling to the Fe moment. Our calculations reproduce the magnetic susceptibility measurements on deoxy and oxy-HbA. For deoxy-HbA, we show that the anomalous MCD signal in the UV region is an experimental evidence for the presence of antiferromagnetic Fe-porphyrin correlations. The functional properties of hemoglobin such as the binding of O 2 , the Bohr effect and the cooperativity are explained based on the magnetic correlations. This analysis suggests that magnetism could be involved in the functioning of hemoglobin.

Malaria, caused by an intracellular protozoan parasite of the genus Plasmodium, is one of the most important infectious diseases worldwide. In 2017, a total of 219 millions cases were reported with 435,000 deaths related to malaria. A major complication of malaria infection is cerebral malaria (CM), characterized by enhanced blood-brain barrier permeability, leukocyte infiltration and/or activation, and neuronal dropout resulting in coma and death in significant numbers of individuals, especially children. Despite the high incidence and mortality, the pathogenesis of cerebral malaria is not well characterized. Hemozoin (HMZ) or "malaria pigment," a by-product of intraerythrocytic parasite-mediated hemoglobin catabolism, is released into the bloodstream after lysis of the host infected erythrocyte. The effects of HMZ on brain cells has not been studied due to the contamination/adhesion/aggregation of the HMZ with host and toxic parasitic factors. We now demonstrate that extracellular purified HMZ is taken up by human neurons and astrocytes, resulting in cellular dysfunction and toxicity. These findings contribute to a better understanding of the neuropathogenesis of CM and provide evidence that HMZ accumulation in the bloodstream could result in CNS compromise. Thus, alternative approaches to reducing circulating HMZ could serve as a potential treatment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Heme proteins are crucial for biological systems by performing diverse functions. Nature has evolved diverse approaches to fine-tune the structure and function of heme proteins, of which post-translational modification (PTM) is a primary method. As reviewed herein, a multitude of PTMs have been discovered for heme proteins in the last several decades, including heme-protein cross-links with heme side chains (Cys-heme, Tyr-heme and Asp/Glu-heme, etc) or porphyrin ring (Lys-heme and Tyr-heme, etc), heme modifications (sulfheme and nitriheme, etc), amino acids cross-links between two or among multiple residues (Cys-Cys, Tyr-His, Tyr-Cys, Met-Tyr-Trp, etc), and amino acids modifications by oxidation, nitration, phosphorylation and glycation, etc. With the development of research methods and advances in research techniques, deep insights have been obtained for the formation mechanisms of PTMs, as well as their effects on the structure and function of heme proteins. Moreover, some positive PTMs have been successfully applied to create artificial heme proteins with advanced functions, whereas some negative PTMs have been regulated by rational design of inhibitors. The tremendous progress, together with those ongoing, will make it possible to rationally control the diverse PTMs of heme proteins, especially those associated with human diseases, toward our desired goals for a better life., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Proteins carrying an iron-porphyrin (heme) cofactor are essential for biological O 2 management. The nature of Fe-O 2 bonding in hemoproteins is debated for decades. We used energy-sampling and rapid-scan X-ray Kβ emission and K-edge absorption spectroscopy as well as quantum chemistry to determine molecular and electronic structures of unligated (deoxy), CO-inhibited (carboxy), and O 2 -bound (oxy) hemes in myoglobin (MB) and hemoglobin (HB) solutions and in porphyrin compounds at 20-260 K. Similar metrical and spectral features revealed analogous heme sites in MB and HB and the absence of low-spin (LS) to high-spin (HS) conversion. Amplitudes of Kβ main-line emission spectra were directly related to the formal unpaired Fe(d) spin count, indicating HS Fe(II) in deoxy and LS Fe(II) in carboxy. For oxy, two unpaired Fe(d) spins and, thus by definition, an intermediate-spin iron center, were revealed by our static and kinetic X-ray data, as supported by (time-dependent) density functional theory and complete-active-space self-consistent-field calculations. The emerging Fe-O 2 bonding situation includes in essence a ferrous iron center, minor superoxide character of the noninnocent ligand, significant double-bond properties of the interaction, and three-center electron delocalization as in ozone. It resolves the apparently contradictory classical models of Pauling, Weiss, and McClure/Goddard into a unifying view of O 2 bonding, tuned toward reversible oxygen transport., Competing Interests: The authors declare no conflict of interest.

Thiosulfate formation and biodegradation processes link aerobic and anaerobic metabolism of cysteine. In these reactions, sulfite formed from thiosulfate is oxidized to sulfate while hydrogen sulfide is transformed into thiosulfate. These processes occurring mostly in mitochondria are described as a canonical hydrogen sulfide oxidation pathway. In this review, we discuss the current state of knowledge on the interactions between hydrogen sulfide and hemoglobin, myoglobin and neuroglobin and postulate that thiosulfate is a metabolically important product of this processes. Hydrogen sulfide oxidation by ferric hemoglobin, myoglobin and neuroglobin has been defined as a non-canonical hydrogen sulfide oxidation pathway. Until recently, it appeared that the goal of thiosulfate production was to delay irreversible oxidation of hydrogen sulfide to sulfate excreted in urine; while thiosulfate itself was only an intermediate, transient metabolite on the hydrogen sulfide oxidation pathway. In the light of data presented in this paper, it seems that thiosulfate is a molecule that plays a prominent role in the human body. Thus, we hope that all these findings will encourage further studies on the role of hemoproteins in the formation of this undoubtedly fascinating molecule and on the mechanisms responsible for its biological activity in the human body., Competing Interests: The authors declare no conflict of interest.

Classical all α-helical globins are present in all living organisms and are ordered in three lineages: (i) flavohemoglobins and single domain globins, (ii) protoglobins and globin coupled sensors and (iii) truncated hemoglobins, displaying the 3/3 or the 2/2 all α-helical fold. However, over the last two decades, all β-barrel and mixed α-helical-β-barrel heme-proteins displaying heme-based functional properties (e.g. ligand binding, transport and sensing) closely similar to those of all α-helical globins have been reported. Monomeric nitrophorins (NPs) and α1-microglobulin (α1-m), belonging to the lipocalin superfamily and nitrobindins (Nbs) represent prototypical heme-proteins displaying the all β-barrel and mixed α-helical-β-barrel folds. NPs are confined to the Reduviidae and Cimicidae families of Heteroptera, whereas α1-m and Nbs constitute heme-protein families spanning bacteria to Homo sapiens. The structural organization and the reactivity of the stable ferric solvent-exposed heme-Fe atom suggest that NPs and Nbs are devoted to NO transport, storage and sensing, whereas Hs-α1-m participates in heme metabolism. Here, the structural and functional properties of NPs and Nbs are reviewed in parallel with those of sperm whale myoglobin, which is generally taken as the prototype of monomeric globins.

The virulence of many pathogens depends upon their ability to cope with immune-generated nitric oxide (NO·). In Escherichia coli, the major NO· detoxification systems are Hmp, an NO· dioxygenase (NOD), and NorV, an NO· reductase (NOR). It is well established that Hmp is the dominant system under aerobic conditions, whereas NorV dominates anaerobic conditions; however, the quantitative contributions of these systems under the physiologically relevant microaerobic regime remain ill defined. Here, we investigated NO· detoxification in environments ranging from 0 to 50 μM O2, and discovered a regime in which E. coli NO· defenses were severely compromised, as well as conditions that exhibited oscillations in the concentration of NO·. Using an integrated computational and experimental approach, E. coli NO· detoxification was found to be extremely impaired at low O2 due to a combination of its inhibitory effects on NorV, Hmp, and translational activities, whereas oscillations were found to result from a kinetic competition for O2 between Hmp and respiratory cytochromes. Because at least 777 different bacterial species contain the genetic requirements of this stress response oscillator, we hypothesize that such oscillatory behavior could be a widespread phenomenon. In support of this hypothesis,Pseudomonas aeruginosa, whose respiratory and NO· response networks differ considerably from those of E. coli, was found to exhibit analogous oscillations in low O2 environments. This work provides insight into how bacterial NO· defenses function under the low O2 conditions that are likely to be encountered within host environments.

Plasmodium falciparum (P. falciparum)-induced effects on the phenotype of human dendritic cells (DC) could contribute to poor induction of long-lasting protective immunity against malaria. DC ability to present antigens to naïve T cells, thus initiating adaptive immune responses depends on complex switches in chemokine receptors, production of soluble mediators and expression of molecules enabling antigen-presentation and maturation. To examine the cellular basis of these processes in the context of malaria, we performed detailed analysis of early events following exposure of human monocyte-derived DC to natural hemozoin (nHZ) and the synthetic analog of its heme core, β-hematin. DC exposed to either molecule produced high levels of the inflammatory chemokine MCP-1, showed continuous high expression of the inflammatory chemokine receptor CCR5, no upregulation of the lymphoid homing receptor CCR7 and no cytoskeletal actin redistribution with loss of podosomes. DC partially matured as indicated by increased expression of major histocompatibility complex (MHC) class II and CD86 following nHZ and β-hematin exposure, however there was a lack in expression of the maturation marker CD83 following nHZ but not β-hematin exposure. Overall our data demonstrate that exposure to nHZ partially impairs the capacity of DC to mature, an effect in part differential to β-hematin., (© 2015 John Wiley & Sons Ltd.)

The genome of the cyanobacterium Nostoc sp. PCC7120 carries three genes (all4978, all7016, and alr7522) encoding putative heme-binding GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) proteins that were annotated as transcriptional regulators. They are composed of an N-terminal cofactor domain and a C-terminal helix-turn-helix motif. All4978 showed the highest affinity for protoheme binding. The heme binding capability of All7016 was moderate, and Alr7522 did not bind heme at all. The "as isolated" form of All4978, identified by Soret band (λmax = 427 nm), was assigned by electronic absorption, EPR, and resonance Raman spectroscopy as a hexa-coordinated low spin Fe(III) heme with a distal cysteine ligand (absorption of δ-band around 360 nm). The protoheme cofactor is noncovalently incorporated. Reduction of the heme could be accomplished by chemically using sodium dithionite and electrospectrochemically; this latter method yielded remarkably low midpoint potentials of -445 and -453 mV (following Soret and α-band absorption changes, respectively). The reduced form of the heme (Fe(II) state) binds both NO and CO. Cysteine coordination of the as isolated Fe(III) protein is unambiguous, but interestingly, the reduced heme instead displays spectral features indicative of histidine coordination. Cys-His ligand switches have been reported as putative signaling mechanisms in other heme-binding proteins; however, these novel cyanobacterial proteins are the first where such a ligand-switch mechanism has been observed in a GAF domain. DNA binding of the helix-turn-helix domain was investigated using a DNA sequence motif from its own promoter region. Formation of a protein-DNA complex preferentially formed in ferric state of the protein., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The role of infection in erythropoietic dysfunction is poorly understood. In children with P. falciparum malaria, the by-product of hemoglobin digestion in infected red cells (hemozoin) is associated with the severity of anemia which is independent of circulating levels of the inflammatory cytokine tumor necrosis alpha (TNF-α). To gain insight into the common and specific effects of TNF-α and hemozoin on erythropoiesis, we studied the gene expression profile of purified primary erythroid cultures exposed to either TNF-α (10 ng/ml) or to hemozoin (12.5 μg/ml heme units) for 24 hours. Perturbed gene function was assessed using co-annotation of associated gene ontologies and expression of selected genes representative of the profile observed was confirmed by real time PCR (rtPCR). The changes in gene expression induced by each agent were largely distinct; many of the genes significantly modulated by TNF-α were not affected by hemozoin. The genes modulated by TNF-α were significantly enriched for those encoding proteins involved in the control of type 1 interferon signalling and the immune response to viral infection. In contrast, genes induced by hemozoin were significantly enriched for functional roles in regulation of transcription and apoptosis. Further analyses by rtPCR revealed that hemozoin increases expression of transcription factors that form part of the integrated stress response which is accompanied by reduced expression of genes involved in DNA repair. This study confirms that hemozoin induces cellular stress on erythroblasts that is additional to and distinct from responses to inflammatory cytokines and identifies new genes that may be involved in the pathogenesis of severe malarial anemia. More generally the respective transcription profiles highlight the varied mechanisms through which erythropoiesis may be disrupted during infectious disease.

Despite recent advances in medical technology and a global effort to improve public health and hygiene, parasitic infections remain a major health and economic burden worldwide. The World Health Organization estimates that about 1/3 of the world's population is currently infected with a soil-transmitted helminth, and millions more suffer from diseases caused by protozoan parasites including Plasmodium, Trypanosoma, and Leishmania species. Due to the selective pressure applied by parasitic and other infections, animals have evolved an intricate immune system; however, the current worldwide prevalence of parasitic infections clearly indicates that these pathogens have adapted equally well. Thus, developing a better understanding of the host-parasite relationship, particularly by focusing on the host immune response and the mechanisms by which parasites evade this response, is a critical first step in mitigating the detrimental effects of parasitic diseases. Macrophages are critical contributors during the host response to protozoan parasites, and the success or failure of these cells often tips the balance in favor of the host or parasite. Herein, we briefly discuss macrophage biology and provide an update on our current understanding of how these cells recognize glycosylphosphatidylinositol anchors from protozoan parasites as well as malarial hemozoin.

Haeme-responsive gene (HRG)-1 encodes a 16-kDa transmembrane protein that is induced by insulin-like growth factor-1 (IGF-1) and associates with the vacuolar-(H(+)) ATPase (V-ATPase). We previously reported that HRG-1 is essential for V-ATPase activity in endosomal acidification and receptor trafficking. Here, we show that in highly invasive and migratory cancer cell lines, HRG-1 and the V-ATPase are co-expressed at the plasma membrane, whereas in less invasive cell lines and non-transformed cells HRG-1 over-expression remains confined to intracellular compartments. Stable suppression of HRG-1 in invasive breast cancer MDA-MB-231 cells decreases extracellular pH, cell growth, migration and invasion. Ectopic expression of HRG-1 in non-invasive MCF-7 cells enhances V-ATPase activity, lowers the extracellular pH and increases the pH-dependent activity of MMP2 and MMP9 matrix metalloproteinases. HRG-1 enhances trafficking of the glucose transporter-1 (GLUT-1) with a concomitant increase in glucose uptake and lactate production. HRG-1 also promotes trafficking of the insulin-like growth factor I receptor (IGF-1R), β1-integrin and IGF-1 signalling. Taken together, our findings indicate that HRG-1 expression at the plasma membrane enhances V-ATPase activity, drives glycolytic flux and facilitates cancer cell growth, migration and invasion. Thus, HRG-1 may represent a novel target for selectively disrupting V-ATPase activity and the metastatic potential of cancer cells.

Apoptosis in macrophages is responsible for immune-depression and pathological effects during malaria. Phagocytosis of PRBC causes induction of apoptosis in macrophages through release of cytosolic factors from infected cells. Heme polymer or β-hematin causes dose-dependent death of macrophages with LC50 of 132 µg/ml and 182 µg/ml respectively. The toxicity of hemin or heme polymer was amplified several folds in the presence of non-toxic concentration of methemoglobin. β-hematin uptake in macrophage through phagocytosis is crucial for enhanced toxicological effects in the presence of methemoglobin. Higher accumulation of β-hematin is observed in macrophages treated with β-hematin along with methemoglobin. Light and scanning electron microscopic observations further confirm accumulation of β-hematin with cellular toxicity. Toxicological potentiation of pro-oxidant molecules toward macrophages depends on generation of H2O2 and independent to release of free iron from pro-oxidant molecules. Methemoglobin oxidizes β-hematin to form oxidized β-hematin (βH*) through single electron transfer mechanism. Pre-treatment of reaction mixture with spin-trap Phenyl-N-t-butyl-nitrone dose-dependently reverses the β-hematin toxicity, indicates crucial role of βH* generation with the toxicological potentiation. Acridine orange/ethidium bromide staining and DNA fragmentation analysis indicate that macrophage follows an oxidative stress dependent apoptotic pathway to cause death. In summary, current work highlights mutual co-operation between methemoglobin and different pro-oxidant molecules to enhance toxicity towards macrophages. Hence, methemoglobin peroxidase activity can be probed for subduing cellular toxicity of pro-oxidant molecules and it may in-turn make up for host immune response against the malaria parasite.

Background: Malaria is an extremely devastating disease that continues to affect millions of people each year. A distinctive attribute of malaria infected red blood cells is the presence of malarial pigment or the so-called hemozoin. Hemozoin is a biocrystal synthesized by Plasmodium and other blood-feeding parasites to avoid the toxicity of free heme derived from the digestion of hemoglobin during invasion of the erythrocytes., Scope of Review: Hemozoin is involved in several aspects of the pathology of the disease as well as in important processes such as the immunogenicity elicited. It is known that the once best antimalarial drug, chloroquine, exerted its effect through interference with the process of hemozoin formation. In the present review we explore what is known about hemozoin, from hemoglobin digestion, to its final structural analysis, to its physicochemical properties, its role in the disease and notions of the possible mechanisms that could kill the parasite by disrupting the synthesis or integrity of this remarkable crystal., Major Conclusions: The importance and peculiarities of this biocrystal have given researchers a cause to consider it as a target for new antimalarials and to use it through unconventional approaches for diagnostics and therapeutics against the disease., General Significance: Hemozoin plays an essential role in the biology of malarial disease. Innovative ideas could use all the existing data on the unique chemical and biophysical properties of this macromolecule to come up with new ways of combating malaria., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a deadly complication of malaria, and its pathophysiology is insufficiently understood. Both in humans and in murine models, MA-ARDS is characterized by marked pulmonary inflammation. We investigated the role of hemozoin in MA-ARDS in C57Bl/6 mice infected with Plasmodium berghei NK65, P. berghei ANKA, and P. chabaudi AS. By quantifying hemozoin in the lungs and measuring the disease parameters of MA-ARDS, we demonstrated a highly significant correlation between pulmonary hemozoin concentrations, lung weights, and alveolar edema. Histological analysis of the lungs demonstrated that hemozoin is localized in phagocytes and infected erythrocytes, and only occasionally in granulocytes. Species-specific differences in hemozoin production, as measured among individual schizonts, were associated with variations in pulmonary pathogenicity. Furthermore, both pulmonary hemozoin and lung pathology were correlated with the number of infiltrating inflammatory cells, an increased pulmonary expression of cytokines, chemokines, and enzymes, and concentrations of alveolar vascular endothelial growth factor. The causal relationship between hemozoin and inflammation was investigated by injecting P. falciparum-derived hemozoin intravenously into malaria-free mice. Hemozoin potently induced the pulmonary expression of proinflammatory chemokines (interferon-γ inducible protein-10/CXC-chemokine ligand (CXCL)10, monocyte chemotactic protein-1/CC-chemokine ligand 2, and keratinocyte-derived chemokine/CXCL1), cytokines (IL-1β, IL-6, IL-10, TNF, and transforming growth factor-β), and other inflammatory mediators (inducible nitric oxide synthase, heme oxygenase-1, nicotinamide adenine dinucleotide phosphate- oxidase-2, and intercellular adhesion molecule-1). Thus, hemozoin correlates with MA-ARDS and induces pulmonary inflammation.

Severe Plasmodium falciparum malaria evolves through the interplay among capillary sequestration of parasitized erythrocytes, deregulated inflammatory responses, and hemostasis dysfunction. After rupture, each parasitized erythrocyte releases not only infective merozoites, but also the digestive vacuole (DV), a membrane-bounded organelle containing the malaria pigment hemozoin. In the present study, we report that the intact organelle, but not isolated hemozoin, dually activates the alternative complement and the intrinsic clotting pathway. Procoagulant activity is destroyed by phospholipase C treatment, indicating a critical role of phospholipid head groups exposed at the DV surface. Intravenous injection of DVs caused alternative pathway complement consumption and provoked apathy and reduced nociceptive responses in rats. Ultrasonication destroyed complement-activating and procoagulant properties in vitro and rendered the DVs biologically inactive in vivo. Low-molecular-weight dextran sulfate blocked activation of both complement and coagulation and protected animals from the harmful effects of DV infusion. We surmise that in chronic malaria, complement activation by and opsonization of the DV may serve a useful function in directing hemozoin to phagocytic cells for safe disposal. However, when the waste disposal system of the host is overburdened, DVs may transform into a trigger of pathology and therefore represent a potential therapeutic target in severe malaria.

Nitric oxide (NO) is an inevitable product of life in an oxygen- and nitrogen-rich environment. This reactive diatomic molecule exhibits microbial cytotoxicity, in large part by facilitating nitrosative stress and inhibiting heme-containing proteins within the aerobic respiratory chain. Metabolism of NO is therefore essential for microbial life. In many bacteria, fungi, and protozoa, the evolutionarily ancient flavohemoglobin (flavoHb) converts NO and O(2) to inert nitrate (NO(3)(-)) and undergoes catalytic regeneration via flavin-dependent reduction. Since its identification, widespread efforts have characterized roles for flavoHb in microbial nitrosative stress protection. Subsequent genomic studies focused on flavoHb have elucidated the transcriptional machinery necessary for inducible NO protection, such as NsrR in Escherichia coli, as well as additional proteins that constitute a nitrosative stress protection program. As an alternative strategy, flavoHb has been heterologously employed in higher eukaryotic organisms such as plants and human tumors to probe the function(s) of endogenous NO signaling. Such an approach may also provide a therapeutic route to in vivo NO depletion. Here we focus on the molecular features of flavoHb, the hitherto characterized NO-sensitive transcriptional machinery responsible for its induction, the roles of flavoHb in resisting mammalian host defense systems, and heterologous applications of flavoHb in plant/mammalian systems (including human tumors), as well as unresolved questions surrounding this paradigmatic NO-consuming enzyme., (Copyright © 2012 Elsevier Inc. All rights reserved.)

CO is a colorless and odorless gas produced by the incomplete combustion of hydrocarbons, both of natural and anthropogenic origin. Several microorganisms, including aerobic and anaerobic bacteria and anaerobic archaea, use exogenous CO as a source of carbon and energy for growth. On the other hand, eukaryotic organisms use endogenous CO, produced during heme degradation, as a neurotransmitter and as a signal molecule. CO sensors act as signal transducers by coupling a "regulatory" heme-binding domain to a "functional" signal transmitter. Although high CO concentrations inhibit generally heme-protein actions, low CO levels can influence several signaling pathways, including those regulated by soluble guanylate cyclase and/or mitogen-activated protein kinases. This review summarizes recent insights into CO metabolism, sensing, and signaling., (Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.)

Virtually all bacterial pathogens require iron to infect vertebrates. The most abundant source of iron within vertebrates is in the form of heme as a cofactor of hemoproteins. Many bacterial pathogens have elegant systems dedicated to the acquisition of heme from host hemoproteins. Once internalized, heme is either degraded to release free iron or used intact as a cofactor in catalases, cytochromes, and other bacterial hemoproteins. Paradoxically, the high redox potential of heme makes it a liability, as heme is toxic at high concentrations. Although a variety of mechanisms have been proposed to explain heme toxicity, the mechanisms by which heme kills bacteria are not well understood. Nonetheless, bacteria employ various strategies to protect against and eliminate heme toxicity. Factors involved in heme acquisition and detoxification have been found to contribute to virulence, underscoring the physiological relevance of heme stress during pathogenesis. Herein we describe the current understanding of the mechanisms of heme toxicity and how bacterial pathogens overcome the heme paradox during infection.

The malaria parasite pigment, hemozoin, is a crystal of ferriprotoporphyrin IX (FP-Fe(III)), a product of hemoglobin digestion. Hemozoin formation is essential for FP-Fe(III) detoxification in the parasite; it is the main target of quinoline antimalarials and can modulate immune and inflammation responses. To gain further insight into the likely mechanisms of crystal formation and hemozoin reactivity, we have reanalyzed the crystal structure data for beta-hematin and solved the crystal structure of Plasmodium falciparum hemozoin. The analysis reveals that the structures are very similar and highlights two previously unexplored modes of FP-Fe(III) self-association involving pi-pi interactions that may initiate crystal formation and help to stabilize the extended structure. Hemozoin can be considered to be a crystal composed of pi-pi dimers stabilized by iron-carboxylate linkages. As a result, it is predicted that two surfaces of the crystal would consist of pi-pi dimers with Fe(III) partly exposed to solvent and capable of undergoing redox reactions. Accordingly, we demonstrate that the crystal possesses both general peroxidase activity and the ability to cause lipid oxidation.

Background: Porphyromonas gingivalis is a major etiological agent of chronic periodontitis. The aim of this study was to examine the species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of the P. gingivalis heme-binding protein HmuY., Results: HmuY is a unique protein of P. gingivalis since only low amino-acid sequence homology has been found to proteins encoded in other species. It is exposed on the cell surface and highly abundant in the outer membrane of the cell, in outer-membrane vesicles, and is released into culture medium in a soluble form. The protein is produced constitutively at low levels in bacteria grown under high-iron/heme conditions and at higher levels in bacteria growing under the low-iron/heme conditions typical of dental plaque. HmuY is immunogenic and elicits high IgG antibody titers in rabbits. It is also engaged in homotypic biofilm formation by P. gingivalis. Anti-HmuY antibodies exhibit inhibitory activity against P. gingivalis growth and biofilm formation., Conclusions: Here it is demonstrated that HmuY may play a significant role not only in heme acquisition, but also in biofilm accumulation on abiotic surfaces. The data also suggest that HmuY, as a surface-exposed protein, would be available for recognition by the immune response during chronic periodontitis and the production of anti-HmuY antibodies may inhibit biofilm formation.

Together, Plasmodium falciparum (P. falciparum) and HIV-1 infections cause more than four million deaths a year. There is still limited information about the putative impact of the malaria pigment hemozoin (HZ) on the dissemination of HIV-1. As so, we propose a premise where HZ present in human dendritic cells (DCs) could modulate HIV-1 transfer to CD4(+) T cells. We report here that HZ promotes transmission of HIV-1 by immature monocyte-derived DCs (iMDDCs). Moreover, we noted that in the presence of HZ, iMDDCs were less permissive to productive HIV-1 infection. The HZ-dependent modulation of the interaction between iMDDCs and HIV-1 seems to be partly due to a decreased expression of CCR5 and also to the induction of a more mature phenotype as proven by microscopy and flow cytometry analyses. Therefore, exposure of iMDDCs to malaria pigments provokes their maturation rendering them more potent to trans-infect CD4(+) T cells with HIV-1.

Caulobacter crescentus integrates phospho-signaling pathways and transcription factor regulatory cascades to drive the cell cycle. Despite the essential role of the CckA histidine kinase in the control of cell cycle events, the factors that signal its activation at a specific time in the cell cycle have remained elusive. A conditional genetic screen for CckA mislocalization mutants, using automated fluorescence microscopy and an image processing platform, revealed that the essential DivL protein kinase promotes CckA localization, autophosphorylation, and activity at the new cell pole. The transient accumulation of DivL at the new cell pole, but not its kinase activity, is required for the localization and activation of CckA. Because DivL and CckA accumulate at the same cell pole after the initiation of DNA replication and were found to interact in vivo, we propose that DivL recruits CckA to the pole, thereby promoting its autophosphorylation and activity.

Iron is a trace element that is vital for life. It is a component of innumerable hemoproteins and many essential non-heme iron proteins that are involved in oxygen binding and metabolism and electron transfer. Nevertheless, iron can also be toxic to cells as it catalyses the production of oxygen radicals. Iron uptake, transport, storage and utilization are therefore strictly regulated to meet the body's iron needs and to avoid its potential toxicity. Any imbalance in iron homeostasis may lead to the development of pathological conditions associated with either iron overload or iron deficiency. In this paper, we review the current understanding of iron biology with a focus on erythroid iron demand. In addition, we will discuss molecular pathophysiology with implications for novel therapies of selected hereditary defects of iron homeostasis.

Hemin-binding protein 35 (HBP35) may be an essential protein for bacterial survival in evasion from environmental stress in Porphyromonas gingivalis. The anti-recombinant HBP35 antibody inhibits P. gingivalis hemagglutination. This study considered the role of this protein for hemagglutination and adherence to host cells using the HBP35-deficient mutant (MD774) derived from P. gingivalis FDC381. FDC381 had strong hemagglutination activity, whereas MD774 had no activity. Anti-130-kDa hemagglutinin antibody, mAb-Pg-vc, reacted mainly with the 43- and 49-kDa molecules in the membrane fraction. However, no proteins reacted in the MD774. The hemolytic activity in MD774 was much lower than that in FDC381. Anti-recombinant HBP35 antibody strongly inhibited the P. gingivalis FDC381 adherence to epithelial cells. In addition, MD774 exhibited a significant decrease in the adherence. The hydrophobicity of MD774 was equal to 19.4% of that of FDC381. SDS-PAGE profiling of the membrane fractions of both strains showed very different profiles. Taken together, these findings suggest that HBP35 plays a role, not only in hemin-binding, but also in multiple P. gingivalis binding to erythrocytes, and host epithelial gingival cells. In addition, this protein may directly and/or indirectly affect the virulence of this organism.

The protein from Arabidopsis thaliana gene locus At1g79260.1 is comprised of 166-residues and is of previously unknown function. Initial structural studies by the Center for Eukaryotic Structural Genomics (CESG) suggested that this protein might bind heme, and consequently, the crystal structures of apo and heme-bound forms were solved to near atomic resolution of 1.32 A and 1.36 A, respectively. The rate of hemin loss from the protein was measured to be 3.6 x 10(-5) s(-1), demonstrating that it binds heme specifically and with high affinity. The protein forms a compact 10-stranded beta-barrel that is structurally similar to the lipocalins and fatty acid binding proteins (FABPs). One group of lipocalins, the nitrophorins (NP), are heme proteins involved in nitric oxide (NO) transport and show both sequence and structural similarity to the protein from At1g79260.1 and two human homologues, all of which contain a proximal histidine capable of coordinating a heme iron. Rapid-mixing and laser photolysis techniques were used to determine the rate constants for carbon monoxide (CO) binding to the ferrous form of the protein (k'(CO) = 0.23 microM(-1) s(-1), k(CO) = 0.050 s(-1)) and NO binding to the ferric form (k'(NO) = 1.2 microM(-1) s(-1), k(NO) = 73 s(-1)). Based on both structural and functional similarity to the nitrophorins, we have named the protein nitrobindin and hypothesized that it plays a role in NO transport. However, one of the two human homologs of nitrobindin contains a THAP domain, implying a possible role in apoptosis. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

We identified a novel extracellular heme-binding protein and named it neuferricin. The recombinant mouse neuferricin produced in High Five cells was secreted efficiently into the culture medium. Mouse neuferricin mRNA was expressed mainly in the brain at the embryo stage and gradually increased during development. At postnatal stage, it was widely expressed in the brain, heart, adrenal gland, and kidney. Mouse neuferricin has 263 amino acids. It has a cytochrome b5-like heme/steroid-binding domain and appeared to bind hemin because neuferricin solution, but not a solution of neuferricinDeltaHBD (a mutant lacking the heme-binding domain), was tinged with brown and had an absorbance peak at 402 nm. In addition, the experiment with anti-neuferricin antibody using heme-affinity chromatography proved that the endogenous neuferricin detected in the culture medium of Neuro2a cells was associated with hemin. Inhibition of endogenous neuferricin by RNA interference excessively promoted cell survival and proliferation and suppressed neurite outgrowth during the induction of differentiation in Neuro2a cells. Addition of recombinant mouse neuferricin, but not neuferricinDeltaHBD, suppressed survival of Neuro2a cells and rescued from the effects of neuferricin RNAi. In primary cultured mouse neural precursor cells, recombinant mouse neuferricin exhibited the ability to promote neurogenesis. The identification of neuferricin, a novel extracellular heme-binding protein with cytochrome b5-like heme/steroid-binding domain and its neurogenic activity, provide new insights not only into brain development but also the function of heme-binding proteins as extracellular signal transmitters.

In areas of holoendemic Plasmodium falciparum transmission, severe malarial anemia (SMA) is a leading cause of pediatric morbidity and mortality. Although many soluble mediators regulate erythropoiesis, it is unclear how these factors contribute to development of SMA. Investigation of novel genes dysregulated in response to malarial pigment (hemozoin [PfHz]) revealed that stem cell growth factor (SCGF; also called C-type lectin domain family member 11A [CLEC11A]), a hematopoietic growth factor important for development of erythroid and myeloid progenitors, was one of the most differentially expressed genes. Additional experiments with cultured peripheral blood mononuclear cells (PBMCs) demonstrated that PfHz decreased SCGF/CLEC11A transcriptional expression in a time-dependent manner. Circulating SCGF levels were then determined for Kenyan children (n = 90; aged 3 to 36 months) presenting at a rural hospital with various severities of malarial anemia. SCGF levels in circulation (P = 0.001) and in cultured PBMCs (P = 0.004) were suppressed in children with SMA. Circulating SCGF also correlated positively with hemoglobin levels (r = 0.241; P = 0.022) and the reticulocyte production index (RPI) (r = 0.280; P = 0.029). In addition, SCGF was decreased in children with reduced erythropoiesis (RPI of <2) (P < 0.001) and in children with elevated levels of naturally acquired monocytic PfHz (P = 0.019). Thus, phagocytosis of PfHz promotes a decrease in SCGF gene products, which may contribute to reduced erythropoiesis in children with SMA.

Background: Characteristic symptoms of malaria include recurrent fever attacks and neurodegeneration, signs that are also found in patients with a hyperactive Nalp3 inflammasome. Plasmodium species produce a crystal called hemozoin that is generated by detoxification of heme after hemoglobin degradation in infected red blood cells. Thus, we hypothesized that hemozoin could activate the Nalp3 inflammasome, due to its particulate nature reminiscent of other inflammasome-activating agents., Methodology/principal Findings: We found that hemozoin acts as a proinflammatory danger signal that activates the Nalp3 inflammasome, causing the release of IL-1beta. Similar to other Nalp3-activating particles, hemozoin activity is blocked by inhibiting phagocytosis, K(+) efflux and NADPH oxidase. In vivo, intraperitoneal injection of hemozoin results in acute peritonitis, which is impaired in Nalp3-, caspase-1- and IL-1R-deficient mice. Likewise, the pathogenesis of cerebral malaria is dampened in Nalp3-deficient mice infected with Plasmodium berghei sporozoites, while parasitemia remains unchanged., Significance/conclusions: The potent pro-inflammatory effect of hemozoin through inflammasome activation may possibly be implicated in plasmodium-associated pathologies such as cerebral malaria.

The intraerythrocytic parasite Plasmodium -- the causative agent of malaria -- produces an inorganic crystal called hemozoin (Hz) during the heme detoxification process, which is released into the circulation during erythrocyte lysis. Hz is rapidly ingested by phagocytes and induces the production of several pro-inflammatory mediators such as interleukin-1beta (IL-1beta). However, the mechanism regulating Hz recognition and IL-1beta maturation has not been identified. Here, we show that Hz induces IL-1beta production. Using knockout mice, we showed that Hz-induced IL-1beta and inflammation are dependent on NOD-like receptor containing pyrin domain 3 (NLRP3), ASC and caspase-1, but not NLRC4 (NLR containing CARD domain). Furthermore, the absence of NLRP3 or IL-1beta augmented survival to malaria caused by P. chabaudi adami DS. Although much has been discovered regarding the NLRP3 inflammasome induction, the mechanism whereby this intracellular multimolecular complex is activated remains unclear. We further demonstrate, using pharmacological and genetic intervention, that the tyrosine kinases Syk and Lyn play a critical role in activation of this inflammasome. These findings not only identify one way by which the immune system is alerted to malarial infection but also are one of the first to suggest a role for tyrosine kinase signaling pathways in regulation of the NLRP3 inflammasome.

The concentration of molecular oxygen (O(2)) began to increase in the Earth's atmosphere approximately two billion years ago. Its presence posed a threat to anaerobes but also offered opportunities for improved energy conservation via aerobic respiration. The ability to sense environmental O(2) thus became, and remains, important for many bacteria, both for protection and switching between anaerobic and aerobic respiration. Utilizing an iron-sulfur cluster as the sensor of O(2) exploits the ability of O(2) to oxidize the iron-sulfur cluster, ultimately resulting in cluster disassembly. When utilizing heme as the sensor, the capacity of O(2) to form a reversible Fe-O(2) bond or alternatively the oxidation of the heme iron atom itself is used to detect O(2) and switch regulators between active and inactive forms.

This study represents the identification of a single amino acid residue that has the major responsibility for the isomeric orientation of a heme b cofactor in a ferriheme protein. The insertion of hemin b into the asymmetric environment of a protein pocket facilitates two cofactor orientations, A and B, which is often called "heme rotational disorder". The proteins studied herein are nitrophorins, a class of ferriheme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus, in this case NP2 and NP7. NMR spectroscopy (pH* 5.5) of the imidazole complex of NP7 revealed solely the A orientation, whereas NP2 shows primarily the B orientation ( approximately 1:5 A:B). The glutamate 27 residue in NP7 is an obvious difference in the heme pocket compared to those of NP1-4, all of which present a valine residue [valine 24 (NP2 and NP3) or valine 25 (NP1 and NP4)] at the same position. Consequently, the mutant NP2(V24E) was prepared and shown to reverse the heme orientation to exclusively A, whereas NP7(E27V) revealed an approximately 1:3 A:B ratio. The reversal A <--> B following the change glutamine <--> valine was further indicated in circular dichroism (CD) spectroscopy with a positive (A) or negative (B) Deltaepsilon of the heme Soret band. Moreover, CD spectroscopy was applied to the mutant NP7(E27Q) and indicated mainly the A orientation, which allows us to conclude that the steric hindrance provided by the glutamate residue is responsible for the heme orientation rather then the carboxylate charge.

In vitro, human monocytes avidly ingest hemozoin (HZ) that modifies a number of monocyte functions. Inhibitory effects: inhibition of: PMA-elicited respiratory burst, ability to killing and repeat phagocytosis, activity of NADPH-oxidase and PKC, expression of ICAM-1, integrin-CD11c, MHC-class-II (IFN-gamma-mediated), differentiation to functional, antigen-presenting dendritic cells. Stimulatory effects: increase in phagocytosis-related respiratory burst and accumulation of lipoperoxidation products; induction of metalloproteinase-9 and pro-inflammatory cytokines and chemokines. Mechanism of action: HZ generates by nonenzymatic catalysis large amounts of lipoperoxidation products, such as monohydroxy derivatives of arachidonic (HETE) and linoleic (HODE) acid, and 4-hydroxynonenal (HNE). Several HZ effects were reproduced by supplementation with plausible concentrations of HETE or HNE, the first most likely via interaction with PPAR-receptors, the second via adduct or crosslinks formation with critical targets.

We used a rapid scanning stop-flow technique to study the kinetics of reduction of cytochrome P450 3A4 (CYP3A4) by the flavin domain of cytochrome P450-BM3 (BMR), which was shown to form a stoichiometric complex (K(D)=0.48 microM) with CYP3A4. In the absence of substrates only about 50% of CYP3A4 was able to accept electrons from BMR. Whereas the high-spin fraction was completely reducible, the reducibility of the low-spin fraction did not exceed 42%. Among four substrates tested (testosterone, 1-pyrenebutanol, bromocriptine, or alpha-naphthoflavone (ANF)) only ANF is capable of increasing the reducibility of the low-spin fraction to 75%. Our results demonstrate that the pool of CYP3A4 is heterogeneous, and not all P450 is competent for electron transfer in the complex with reductase. The increase in the reducibility of the enzyme in the presence of ANF may represent an important element of the mechanism of action of this activator.

Heme-based sensors are a recently discovered functional class of heme proteins that serve to detect physiological fluctuations in oxygen (O(2)), carbon monoxide (CO), or nitric oxide (NO). Many of these modular sensors detect heme ligands by coupling a histidine-protein kinase to a heme-binding domain. They typically bind O2, CO, and NO but respond only to one of these ligands. Usually, they are active in the ferrous unliganded state but are switched off by saturation with O2. The heme-binding domains of these kinases are quite varied. They may feature a PAS fold, as in the Bradyrhizobium japonicum and Sinorhizobium melitoti FixL proteins, or a GAF fold, as in the Mycobacterium tuberculosis DevS and DosT proteins. Alternative folds, such as HNOB (also H-NOX), have also been noted for such signal-transducing kinases, although these classes are less well studied. Histidine-protein kinases function in partnership with cognate response-regulator substrate(s): usually transcription factors that they activate by phosphorylation. For example, FixL proteins specifically phosphorylate their FixJ partners, and DevS and DosT proteins phosphorylate DevR in response to hypoxia. We present methods for purifying these sensors and their protein substrates, verifying the quality of the preparations, determining the K(d) values for binding of ligand and preparing sensors of known saturation, and measuring the rates of turnover (k(cat)) of the protein substrate by sensors of known heme status.

In the presence of H(2)O(2), heme proteins form active intermediates, which are able to oxidize exogenous molecules. Often these products are not stable compounds but reactive species on their own, such as organic radicals. They can both diffuse to the bulk of the solution or react with the protein that generated them. Here, we describe the self-modification underwent by heme proteins with globin-type fold, that is, myoglobin, hemoglobin, and neuroglobin when treated with NO(2) (-) or catechols in the presence of H(2)O(2). The reactive nitrogen species generated by NO(2) (-) give rise to nitration, oxidation, and/or crosslinking reactions between the proteins or their subunits. The quinones formed upon reaction with catechols easily modify Cys and His residues and eventually cause protein aggregation, which induces precipitation. The pattern of modifications undergone by the protein strongly depends on the nature of the protein and the reaction conditions.

Globin-coupled sensors (GCSs) are multiple-domain transducers, consisting of a regulatory globin-like heme-binding domain and a linked transducer domain(s). GCSs have been described in both Archaea and bacteria. They are generally assumed to bind O(2) (and perhaps other gaseous ligands) and to transmit a conformational change signal through the transducer domain in response to fluctuating O(2) levels. In this study, the heme-binding domain, AvGReg178, and the full protein, AvGReg of the Azotobacter vinelandii GCS, were cloned, expressed, and purified. After purification, the heme iron of AvGReg178 was found to bind O(2). This form was stable over many hours. In contrast, the predominant presence of a bis-histidine coordinate heme in ferric AvGReg was revealed. Differences in the heme pocket structure were also observed for the deoxygenated ferrous state of these proteins. The spectra showed that the deoxygenated ferrous derivatives of AvGReg178 and AvGReg are characterized by a penta-coordinate and hexa-coordinate heme iron, respectively. O(2) binding isotherms indicate that AvGReg178 and AvGReg show a high affinity for O(2) with P(50) values at 20 degrees C of 0.04 and 0.15 torr, respectively. Kinetics of CO binding indicate that AvGReg178 carbonylation conforms to a monophasic process, comparable with that of myoglobin, whereas AvGReg carbonylation conforms to a three-phasic reaction, as observed for several proteins with bis-histidine heme iron coordination. Besides sensing ligands, in vitro data suggest that AvGReg(178) may have a role in O(2)-mediated NO-detoxification, yielding metAvGReg(178) and nitrate.

The toxic effect of high concentrations of CO gas in living organisms is coherently typified at biochemical levels by the high affinity of CO for hemoglobin and cytochromes, heme-dependent proteins that are indispensable for oxygen transport and mitochondrial respiration. However, the basal production of CO during heme degradation and the ability of heme oxygenase-1 (HO-1) to increase CO availability pose the question of how this gaseous molecule interacts with metal centers within the intracellular milieu to serve as one of the most unconventional signaling mediators. Emerging evidence indicates that the diverse and multifaceted beneficial effects exerted by "low concentrations" of CO cannot be explained solely by the activation of classic prototypic targets (i.e., guanylate cyclase/potassium channels) but entails the dynamic and concerted activation/inhibition of a group of CO-responsive proteins. As the complexity of the temporal and spatial action of CO is progressively being appreciated, this review aims to (a) highlight the current knowledge on certain metal-containing proteins that interact directly with CO; (b) analyze the latest notions on their functional role in response to CO; and finally (c) propose a rational view on the mode these CO targets may interrelate with and be regulated by the HO/CO pathway.

Nitrophorins (NPs) are a class of NO-transporting and histamine-sequestering heme b proteins that occur in the saliva of the bloodsucking insect Rhodnius prolixus. A detailed study of the newly described member, NP7, is presented herein. NO association constants for NP7 [KIII(eq)(NO)] reveal a drastic change when the pH is varied from 5.5 (reflecting the insect's saliva) to slightly above plasma pH (7.5) (>10(9) M-1 --> 4.0 x 10(6) M-1); thus, the protein promotes the storage of NO in the insect's saliva and its release inside the victim's tissues. In contrast to the other nitrophorins, NP1-4, histamine sequestering cannot be accomplished in vivo due to the low binding constant [KIII(eq)(histamine)] of 10(5) M-1 compared to the histamine concentration of 1-10 x 10(-9) M in the blood. A major part of this study deals with the N-terminus, 1Leu-Pro-Gly-Glu-Cys5 of NP7, which is not found in NP1-4. Since NP7 has not been isolated from the insects but was recognized in a cDNA library instead, the N-terminal site of signal peptidase cleavage upon protein secretion was predicted by the program SIGNALP [Andersen, J. F., Gudderra, N. P., Francischetti, I. M. B., Valenzuela, J. G., and Ribeiro, J. M. C. (2004) Biochemistry 43, 6987-6994]. In marked contrast to wild-type NP7, NP7(Delta1-3) exhibits a very high NO affinity at pH 7.5 [KIII(eq)(NO) approximately 10(9) M-1], suggesting that the release of NO in the plasma cannot efficiently be accomplished by the truncated form. Comparison of the reduction potentials of both constructs by spectroelectrochemistry revealed an average increase of +85 mV for various distal ligands bound to the heme iron when the 1Leu-Pro-Gly3 peptide was removed. However, 1H NMR and EPR spectroscopy show that the electronic properties of the FeIII cofactor are similar in both wild-type NP7 and NP7(Delta1-3). Further, thermal denaturation that revealed a higher stability of wild-type NP7 compared to NP7(Delta1-3), in combination with a homology model based on the NP2 crystal structure (rmsd = 0.39 A), suggests that interaction of the 1Leu-Pro-Gly3 peptide with the A-B and/or G-H loops is key for proper protein function.

Heme is a prosthetic group of various types of proteins, such as hemoglobin, myoglobin, cytochrome c, cytochrome p450, catalase and peroxidase. In addition, heme is involved in a variety of biological events by modulating the function or the state of hemoproteins. For example, protein synthesis is inhibited in erythroid cells under heme deficiency, as the consequence of the activation of heme-regulated inhibitor (HRI). Iron concentration in the cell is sensed and regulated by the heme-mediated oxidization and subsequent degradation of iron regulatory protein 2 (IRP2). Heme also binds to certain types of potassium channels, thereby inhibiting transmembrane K(+) currents. Importantly, heme determines its own fate; namely, heme regulates its synthesis and degradation through the feedback mechanisms, by which intracellular heme level is precisely maintained. Heme reduces heme synthesis by suppressing the expression of non-specific 5-aminolevulinate synthase (ALAS1) and stimulates heme breakdown by inducing heme oxygenase (HO)-1 expression. ALAS1 and HO-1 are the rate limiting enzymes in heme biosynthesis and catabolism, respectively. Accordingly, under the heme-rich condition, heme binds to cysteine-proline (CP) motifs of ALAS1 and those of transcriptional repressor Bach1, thereby leading to repression of mitochondrial transport of ALAS1 and induction of HO-1 transcription, respectively. Moreover, chemosensing functions of HO-2 containing CP motifs, another isozyme of HO, have been unveiled recently. In this review article, we summarize and update the pleiotropic effects of heme on various biological events and the regulatory network of heme biosynthesis and catabolism.

Escherichia coli flavohaemoglobin (Hmp) is the best-understood nitric oxide (NO) detoxifying protein and exhibits a robust dioxygenase activity, converting NO to nitrate ion with oxygen as co-substrate. Synthesis of Hmp via transcriptional regulation of hmp gene expression is an adaptive response to NO and related nitrosative stresses since Hmp levels are greatly elevated on exposure in vitro to these agents. Here we show that expression of hmp is greatly enhanced by NO but not by other haem ligands (azide, cyanide and carbon monoxide). Flavohaemoglobins of other pathogenic bacteria have been implicated in conferring resistance to NO in vitro and in macrophage-like cells but the role of the E. coli flavohaemoglobin has not been studied in macrophages. We therefore compared survival of wild-type K-12 E. coli cells and an isogenic hmp mutant after internalisation by human macrophages. Wild-type bacteria survived significantly better than the hmp mutant after incubation with macrophages, despite binding and internalisation rates being similar for both strains. Unexpectedly, however, when grown in MOPS minimal medium, in mixed cultures, more hmp mutant cells were recovered than wild-type. Significantly, an hmp mutant failed to exhibit swarming motility on soft agar and this phenotype was rescued by a plasmid-borne copy of the wild-type hmp(+) gene. Thus, although Hmp constitutes an important mechanism of protection from NO-mediated killing by human macrophages in the model E. coli strain K-12, and probably contributes to the survival of enteropathogenic E. coli during the intestinal inflammatory response, synthesis of Hmp in vitro may represent a selective disadvantage. The lack of swarming motility of the hmp mutant and its aflagellate state suggest that Hmp synthesis is a metabolic burden in the absence of NO-related stresses.

Infections caused by bacteria belonging to genus Mycobacterium are among the most challenging threats for human health. The ability of mycobacteria to persist in vivo in the presence of reactive nitrogen and oxygen species implies the presence in these bacteria of effective detoxification mechanisms. Mycobacterial truncated hemoglobins (trHbs) have recently been implicated in scavenging of reactive nitrogen species. Individual members from each trHb family (N, O, and P) can be present in the same mycobacterial species. The distinct features of the heme active site structure combined with different ligand binding properties and in vivo expression patterns of mycobacterial trHbs suggest that these globins may accomplish diverse functions. Here, recent genomic, structural and biochemical information on mycobacterial trHbs is reviewed, with the aim of providing further insights into the role of these globins in mycobacterial physiology.

A fundamental challenge to the study of oxidative stress responses of Mycobacterium tuberculosis (Mtb) is to understand how the protective host molecules are sensed and relayed to control bacilli gene expression. The genetic response of Mtb to hypoxia and NO is controlled by the sensor kinases DosS and DosT and the response regulator DosR through activation of the dormancy/NO (Dos) regulon. However, the regulatory ligands of DosS and DosT and the mechanism of signal sensing were unknown. Here, we show that both DosS and DosT bind heme as a prosthetic group and that DosS is rapidly autooxidized to attain the met (Fe3+) form, whereas DosT exists in the O2-bound (oxy) form. EPR and UV-visible spectroscopy analysis showed that O2, NO, and CO are ligands of DosS and DosT. Importantly, we demonstrate that the oxidation or ligation state of the heme iron modulates DosS and DosT autokinase activity and that ferrous DosS, and deoxy DosT, show significantly increased autokinase activity compared with met DosS and oxy DosT. Our data provide direct proof that DosS functions as a redox sensor, whereas DosT functions as a hypoxia sensor, and that O2, NO, and CO are modulatory ligands of DosS and DosT. Finally, we identified a third potential dormancy signal, CO, that induces the Mtb Dos regulon. We conclude that Mtb has evolved finely tuned redox and hypoxia-mediated sensing strategies for detecting O2, NO, and CO. Data presented here establish a paradigm for understanding the mechanism of bacilli persistence.

Bradyrhizobium japonicum FixL is a modular oxygen sensor that directs adaptations to hypoxia by coupling the status of a heme-binding domain to a histidine-protein kinase activity. The oxygen-bound form is the "off-state". The unliganded form is the "on-state" active kinase that phosphorylates a transcription factor, FixJ. We have developed methods to optimize the kinase reactions of FixL and measure the turnover rates (kcat) for reactions catalyzed by highly inhibited states of this sensor at constant, precisely known oxygen saturations. The resulting oxygen dose-response curve shows that an in vitro system with FixL and the response regulator FixJ as its only proteins manifests such a sharp ligand response that, when the proportion of deoxy-FixL decreases less than 3-fold, the kinase activity drops over 50-fold, and by the time the deoxy-FixL declines just 8-fold, the activity is inhibited over 1100-fold. This response is entirely reversible and similar to that reported for the in vivo hypoxic induction of FixLJ-regulated genes. FixL binds oxygen noncooperatively. When complexed with FixJ, FixL is dimeric in both oxy and deoxy states. Therefore traditional models involving cooperative binding of ligand or robust allosteric regulation cannot account for the extremely nonlinear kinase response to the heme saturation. This response, however, can be explained by a form of enzyme hysteresis with the simple assumptions that (i) on association of oxygen with the heme, the kinase is rapidly switched off; (ii) after dissociation of oxygen, the kinase remains inhibited longer than the average time that it takes a deoxy-heme to encounter an oxygen molecule at most oxygen saturations.

FixL is a heme-based O2 sensor protein, which responds to low O2 concentrations by activating the transcriptional activator FixJ. Signal transduction is initiated by the dissociation of O2 from the sensor domain of FixL, resulting in protein conformational changes that are transmitted to a histidine kinase domain. To gain insight into the FixL sensing mechanism, we monitored changes in the protein's structure in the picosecond to millisecond time frame, following the dissociation of the ligand using time-resolved resonance Raman spectroscopy. This study presents the time-resolved resonance Raman spectra of Sinorhizobium meliloti FixL upon O2 dissociation, as well as upon CO dissociation. The FixL spectra show that there are three steps in the dynamic structural changes that result from ligand dissociation. Ligand-dependent structural dynamics are observed in the earliest step. On the basis of comparisons of these structural changes, a scheme for the signal transduction of FixL is proposed which supports the FG loop switch mechanism. Similar spectral changes were observed both for the sensor domain and for the full-length protein, although structural changes occurred faster with the former than with the latter. This difference in rate suggests that the structural changes occurring in the heme pocket are coupled to those of the kinase domain. The implications of these results for FixL's sensing mechanism are discussed.

The FixL protein of Bradyrhizobium japonicum is a dimeric oxygen sensor responsible for initiating regulation of transcription of genes encoding proteins involved in nitrogen fixation and oxidative stress. It consists of an N-terminal heme-bound PAS domain, denoted bjFixLH, and a C-terminal histidine kinase domain whose enzymatic activity depends on the ligation state of the heme. To investigate the molecular basis for this dependence and the dynamics associated with conversion between ligated and unligated states, we have conducted time-resolved Laue diffraction studies of CO recombination in bjFixLH. Time-dependent difference Fourier maps from 1 micros to 10 ms after photolysis of the heme-CO bond show movement of the side chain of Leu236 and the H and I beta-strands into the ligand binding pocket formerly occupied by CO. Long-range conformational changes are evident in the protein, driven by relaxation of steric interactions between the bound ligand and amino acid side chains and/or changes in heme stereochemistry. These structural changes fully reverse as CO rebinds to the heme. Spectroscopic measurements of CO recombination kinetics in bjFixLH crystals relate the behavior of crystalline bjFixLH to solution and provide a framework for our time-resolved crystallographic experiments. Analysis of the time-dependent difference Fourier maps by singular value decomposition reveals that only one significant singular value accounts for the data. Thus only two structural states are present, the photolyzed and the CO-bound states. The first left singular vector represents the difference in density between these two states and shows features common to difference maps calculated from the static CO and deoxy states. The first right singular vector represents the time course of this difference density and agrees well with the CO recombination kinetics measured spectroscopically. We refine the structure of the photolyzed state present in the early-microsecond time range and find that it does not differ significantly in conformation from static, deoxy bjFixLH. Thus, structural relaxation from CO-bound to deoxy bjFixLH is complete in less than 1 micros.

Bacteria use a number of mechanisms for coping with the toxic effects exerted by nitric oxide (NO) and its derivatives. Here we show that the flavohemoglobin encoded by the hmp gene has a vital role in an adaptive response to protect the soil bacterium Bacillus subtilis from nitrosative stress. We further show that nitrosative stress induced by the nitrosonium cation donor sodium nitroprusside (SNP) leads to deactivation of the transcriptional repressor NsrR, resulting in derepression of hmp. Nitrosative stress induces the sigma B-controlled general stress regulon. However, a sigB null mutant did not show increased sensitivity to SNP, suggesting that the sigma B-dependent stress proteins are involved in a nonspecific protection against stress whereas the Hmp flavohemoglobin plays a central role in detoxification. Mutations in the yjbIH operon, which encodes a truncated hemoglobin (YjbI) and a predicted 34-kDa cytosolic protein of unknown function (YjbH), rendered B. subtilis hypersensitive to SNP, suggesting roles in nitrosative stress management.

Hemozoin (HZ) is an insoluble crystal formed in the food vacuole of malaria parasites. HZ has been reported to induce inflammation by directly engaging Toll-like receptor (TLR) 9, an endosomal receptor. "Synthetic" HZ (beta-hematin), typically generated from partially purified extracts of bovine hemin, is structurally identical to natural HZ. When HPLC-purified hemin was used to synthesize the crystal, beta-hematin had no inflammatory activity. In contrast, natural HZ from Plasmodium falciparum cultures was a potent TLR9 inducer. Natural HZ bound recombinant TLR9 ectodomain, but not TLR2. Both TLR9 stimulation and TLR9 binding of HZ were abolished by nuclease treatment. PCR analysis demonstrated that natural HZ is coated with malarial but not human DNA. Purified malarial DNA activated TLR9 but only when DNA was targeted directly to the endosome with a transfection reagent. Stimulatory quantities of natural HZ contain <1 microg of malarial DNA; its potency in activating immune responses was even greater than transfecting malarial DNA. Thus, although the malarial genome is extremely AT-rich, its DNA is highly proinflammatory, with the potential to induce cytokinemia and fever during disease. However, its activity depends on being bound to HZ, which we propose amplifies the biological responses to malaria DNA by targeting it to a TLR9(+) intracellular compartment.