sign in sign up
<<
Home , Globin, Hemoglobin, Hemoglobinemia, Hemoprotein, Metmyoglobin, Myoglobin

Heme, , (Haem, Haemoglobin: UK)

Oxygen binding and new functions

1 Myoglobin O2 Storage

2 You need O2 because of continuous swimming. You don’t need O2. More myoglobin. Less myoglobin.

3 Protoporphyrin IX, b Heme a is a form of heme found A flat and planar structure. In a and a3. -. Itself Toxic O2 , H2O2 are formed. 4 c with .

5 6 Globular have no systematic structures. There may be single chains, two or more chains which interact in the usual ways or there may be portions of the chains with helical Myoglobinis a small, 17 kDa, monomeric, O2- binding haemoprotein that typically occurs in structures, pleated structures, or completely cardiac and aerobic of random structures. Globular proteins are . The storage abundant in relatively spherical in shape as the name muscles. Acts as the heme Fe(II) complex. implies. Common globular proteins include egg Alpha (a) -helical form. , hemoglobin, myoglobin, insulin, Myo – muscle from Greek in , and many .7 His E7 Distal side

Heme iron

His F8 Proximal side

Myoglobin has a strong affinity for oxygen Heme is located in the hydrophobic site. when it is in the , and where the High oxygen affinity. pressure is around 100 torr. When it reaches His (E7) is the oxygen . the tissues, where it's around 20 torr, the affinity for oxygen is still quite high. This The oxygen binding curve for Myoglobin makes myoglobin less efficient of an oxygen forms an asymptotic shape, which shows a transporter than hemoglobin, which loses it's simple graph that rises sharply then levels affinity for oxygen as the pressure goes down off as it reaches the maximum saturation. and releases the oxygen into the tissues. The half-saturation, the point at which half Myoglobin's strong affinity for oxygen means of the myoglobin is binded to oxygen, is that it keeps the oxygen binded to itself reached at 2 torr which is relatively low instead of releasing it into the tissues. compared to 26 torr for hemoglobin. 8 Distal side

Proximal side

Hemoglobin, whose role is to transport oxygen in the blood, comprises 4 sub-units, each very similar to a myoglobin . Hemo – blood from Greek 9 One model is the interconversion of the hemoglobin between two states—the T (tense) and the R (relaxed) conformations—of the molecule. The R state has higher affinity for

oxygen. Under conditions where pO2 is high (such as in the lungs), the R state is favored; in

conditions where pO2 is low (as in exercising muscle), the T state is favored.

+ Hill plot log[Y/(1-Y)] = nlog(pO2) - nlogP50 Myoglobin slope n = 1 n: Hill coefficient Hemoglobin slope n = 2.8

Because hemoglobin has four subunits, its binding of oxygen can reflect multiple equilibria:

Hb + O2 = Hb-O2, Hb-O2 + O2 = Hb-(O2)2, Hb-(O2)2 + O2 = Hb-(O2)3, Hb-(O2)3 + O2 = Hb-(O2)4

The equilibrium constants for these four O2 binding events are dependent on each other and on the solution conditions. The influence of one oxygen's binding on the binding of another oxygen is called a homotropic effect. Overall, this cooperative equilibrium binding makes the binding curve sigmoidal rather than hyperbolic, as Figure shows. The P50 of hemoglobin in blood cells is about 26 torr under normal physiological conditions. In the alveoli of the lungs, pO2 is about 100 torr, and close to 20 torr in the tissues. So you may expect hemoglobin to be about 80% loaded in the lungs and a little over 40% loaded with oxygen in the . In fact, 10 hemoglobin can be more O2-saturated in the lungs and less saturated in the capillaries. Christian Bohr (1855-1911) Higher , lower affinity of haemoglobin for oxygen thus curve shifts to the of Hemoglobin right, showing lower saturation percentage of haemoglobin to oxygen since carbon dioxide is in CO2 pressure affects the ability higher concentration compared to oxygen. of hemoglobin to carry O2. Carbon dioxide helps the haemoglobin to Low pH: High CO2, High pH: Low CO2 dissociates oxygen, and thus providing oxygen for the oxygen deprived tissues. 11

Carbonic anhydrase enhances the reaction to increase pH.

12 Sickle & resistance

In sickle cell hemoglobin (HbS) glutamic in position 6 (in beta chain) is mutated to . This change allows the deoxygenated form of the hemoglobin to stick to itself. 13 Figure 1. HO-1 Protects against Severe Malaria Using a mouse model for cerebral malaria, Ferreira et al. (2011) suggest a biochemical basis for the link between and severe malaria. The figure shows a molecular pathway that may explain why carriers of the , who are heterozygous for the that causes the disease (HbS), may have more resistance to severe malaria symptoms. Mice harboring the human HbS allele have elevated levels of free heme in the blood. Free heme is toxic and can cause oxidative damage, but its effects are suppressed by the upregulation of -1 (HO-1), which converts heme into the (CO) and and releases iron to bind to H chain (FtH). HO-1 expression is regulated by Nrf2. In individuals with the sickle cell trait, the elevated levels of carbon monoxide prior to infection may inhibit pathogenic CD8+ T cell immune responses and also prevent Cell 145, 335 (2011). oxidative tissue damage during severe malaria. 14 Hemazoin

Quinine, and other 4- aminoquinolines inhibit pigment formation, as b-Hematin well as the heme degradative processes, and is composed of b-hematin. thereby prevent the detoxification of heme. The Digestion of hemoglobin by free heme destabilizes the food vacuolar the malaria parasite produces membrane and other membranes and leads to this malaria pigment. the death of the parasite. 15

Intravascular results from the rupture or lysis of red blood cells within the circulation, i.e. the red cells are lysing in vivo. When the membrane of erythrocytes rupture, they release their hemoglobin into the plasma.

The hemoglobin (which is a tetramer) breaks down into hemoglobin dimers in plasma. (an α-2 produced in the ) binds the liberated free hemoglobin dimers. However, haptoglobin is readily saturated (this occurs at around a hemoglobin concentration of 150 mg/dL). If intravascular hemolysis continues, the hemoglobin dimers are in excess in plasma and are filtered readily through the (because they are < 20 kd in size). This will cause a hemoglobinuria and a positive reaction for heme protein on the dipstick (with no erythrocytes evident in the sediment). Because hemoglobin >20 mg/dL will cause visible discoloration of plasma (light pink to dark red, depending on how much hemoglobin is present), is often visible with intravascular hemolysis.

16 The hemoglobin dimers that remain in circulation are oxidized to , which disassociates into a free heme and chains. The oxidized free heme (met-heme) binds to (a β-globulin, Hpx) and the met- heme and hemopexin complex(met-heme/Hpx) is taken up by a receptor on hepatocytes and within the , liver and (only hepatocyte uptake is illustrated in the image above). Similarly, the hemoglobin/haptoglobin complex is taken up by hepatocytes and macrophages (to a lesser extent). Within these cells, the hemoglobin disassociates into heme and globin chains.

The are broken down to amino , which are then used for protein synthesis. The heme is oxidized by heme oxygenase forming biliverdin and releasing iron. The iron can be transferred to apotransferrin (the iron transport protein) in plasma or can be stored within cells as ferritin (i.e. the iron is bound to the , apoferritin). The remaining ring (biliverdin) is degraded to unconjugated by biliverdin reductase. If the hemoglobin/haptoglobin complex is internalized by macrophages, the unconjugated bilirubin is released into the plasma, where it binds to albumin (to render it water-soluble) and is taken up by hepatocytes through the haptoglobin receptor. Thus, with intravascular hemolysis, increases in bilirubin are usually due to unconjugated bilirubin (indirect) and are likely of (rather than hepatocyte) origin.

Note that it is unusual for intravascular hemolysis to occur alone, i.e. it is usually accompanied by extravascular hemolysis. This extravascular hemolysis is the likely source of most of the unconjugated bilirubin that is produced by macrophages in a . Because haptoglobin is consumed during intravascular hemolysis, serum values of this protein usually decline with intravascular hemolytic or when hemoglobin is liberated into plasma by artifactual lysis of red cells in vitro (e.g. freezing of red cells, old samples - see below).

Haptoglobin is a positive acute phase reactant and values will increase as part of the acute phase response (an evolutionary conserved innate response to inflammation, injury or infection). In fact, an increase in haptoglobin is one of the main reasons for the high α-2 peak seen in acute phase responses in serum electrophoresis.

Corticosteroids will also increase serum values of haptoglobin in dogs. 17

Nitric Oxide (NO)

• An important signalling and cytotoxic molecule in the cardiovascular, nervous, and immune systems.

• From to hypertension, cancer to drug addiction, stroke to intestinal motility, memory and learning disorders to septic , sunburn to anorexia, male function to tuberculosis.

• Louis J. Ignarro, Robert F. Furchgott, and Ferid Murad have been jointly awarded the 1998 Nobel Prize in or Medicine for their discoveries concerning " as a signaling molecule in the cardiovascular system."

18 (NOS)

Heme Fe(III)-S--Cys

19 Surprise Discovery in Blood: Hemoglobin Has Bigger Role The New York Times 1996

Dr. Stamler and his colleagues discovered the new role of hemoglobin through considering a paradox about the supply of nitric oxide in the tissues.

Trends in Molecular Medicine 15, 452 (2009).

The protected transport of nitric oxide (NO) by hemoglobin (Hb) links the metabolic activity of working tissue to the regulation of its local blood supply through hypoxic . This physiologic mechanism is allosterically coupled to the O2 saturation of Hb and involves the covalent binding of NO to a cysteine residue in the β-chain of Hb (Cys β93) to form S-nitrosohemoglobin (SNO-Hb). Subsequent S- transnitrosation, the transfer of NO groups to on the RBC membrane and then in the plasma, preserves NO vasodilator activity for delivery to the vascular endothelium. This SNO-Hb paradigm provides insight into the respiratory cycle and a new therapeutic focus for diseases involving abnormal microcirculatory . In addition, the formation of S-nitrosothiols in other proteins may regulate an array of physiological functions.

20 Chemical characterization of the smalles S-nitrosothiol, HSNO; Cellular cross-talk of H2S and S-nitrosothiols J. Am. Chem. Soc. 134, 12016 (2012) Dihydrogen recently emerged as a biological signaling molecule with important physiological roles and significant pharmacological potential. Chemically plausible explanations for its mechanisms of action have remained elusive, however. Here, we report that H2S reacts with S-nitrosothiols to form thionitrous acid (HSNO), the smallest S-nitrosothiol. These results demonstrate that, at the cellular level, HSNO can be metabolized to afford NO+, NO, and NO– , all of which have distinct physiological consequences of their own. We further show that HSNO can freely diffuse through membranes, facilitating transnitrosation of proteins such as hemoglobin. The data presented in this study explain some of the physiological effects ascribed to H2S, but, more broadly, introduce a new signaling molecule, HSNO, and suggest that it may play a key role in cellular regulation. 21 Figure 9. Red interactions with NO include (1) NO binding to the deoxygenated heme in oxygenated red blood cells, forming iron-nitrosyl hemoglobin (FeII–NO); (2) oxy- hemoglobin scavenging NO and transferring it to the β-globin Cys-93 residue to form SNOHb (NO transport); (3) hemoglobin deoxygenation and structural transitions from R (oxy) to T (deoxy) facilitating the release of NO; and (4) T-state Hb reacting with NO species and undergoing Hb nitrosylation. Note: In the R state, Cys β93 is enclosed in a hydrophobic pocket, and the heme pocket is more accessible. In the T state, Cys β93 is exposed to reactions, and the heme pocket is less accessible. 22 New roles of hemoglobin (and myoglobin) as reductants

23 Nature Chemical Biology 5, 865 (2009) Meeting Report 24

25 implicated in blood pact Nature Medicine 9, 1460 (2003)

- The emerging biology of the nirite anion NO2 Nature Chemical Biology 1, 308 (2005) 26

Nature Chemical Biology 3, 785 (2007)

27

The Reaction between and Oxyhemoglobin: A MECHANISTIC STUDY

J. Biol. Chem. 283, 9615 (2008) 28

FIGURE 10. Possible pathways for nitrite catalysis of the reductive nitrosylation and routes for formation of nitrosating species through ferric-nitrite complexes. The two proposed pathways for the nitrite catalysis of reductive nitrosylation lead to the transient formation of nitrosating species such asNO2 and N2O3 (nitrite outer/inner sphere electron transfer routes). Similar routes with the formation of a Fe2- NO2 intermediate that can react with NO have been proposed (nitric oxide-inner electron transfer route). Note that the routes where the inner sphere electron transfer occurs yield similar products and can hypothetically start from either Fe3-NO or Fe3-NO2 complexes. J. Biol. Chem. 287, 18262 (2012) 29 J. Am. Chem. Soc. 134, 13861 (2012)

30 J. Exp. Biol. 213, 2726 (2010). Fig. 1. Schematic drawing summarizing the distinct interactions of Mb and oxides as a function of O2 tension (A) and its functional consequences for mitochondrial (B) in cardiomyocytes. Under fully oxygenated conditions MbO2 acts mainly as a NO scavenger rapidly regenerated by the robust activity of metMb reductase, thereby acting as a molecular firewall, which protects mitochondrial respiration from NO inhibition (A left). With decreasing O2 supply (A right), Mb gets increasingly deoxygenated uncovering its nitrite reductase activity, thereby releasing NO in proximity of the mitochondria which results in a reversible inhibition of cytochromes (B). As a consequence, myocardial O2 consumption is reduced and cardiac contractility is dampened, representing an endogenous protecting mechanism for the heart under limited O2 supply. For a detailed discussion please refer to the text. Abbreviations: I, II, III, IV, V represent complex I–V of the ; ADP, adenosine diphosphate; ATP, ; C, oxidase; deoxyMb, deoxygenated myoglobin; NADH, nicotinamide adenine dinucleotide; MbNO, nitrosylmyoglobin; MbO2, oxygenated myoglobin; metMb, ; metMbNO, nitrosylmetmyoglobin; mPTP, mitochondrial permeability transition pore; NO, nitric oxide; ·O2−, ; Q, coenzyme Q.

31 J. Exp. Biol. 213, 2734 (2010) Fig. 1. Schematic diagram displaying the possible effects of myoglobin (Mb), ultimately modulating physiological functions and diseases. (A) The regulation of nitric oxide (NO•) by Mb could prevent or diminish the NO•-mediated inhibition of resulting in the protection of the mitochondrial respiratory chain. By contrast, the NO• scavenging might avoid the protective effect of NO• against infections with parasites. (B) An enhanced dioxygen (O2) supply and elimination of NO• by Mb could impair the angiogenesis, which would be beneficial in cancer and detrimental in the setting of hindlimb . (C) The highly oxidizing species ferryl Mb (MbFeIV=O, •MbFeIV=O) might be less harmful in myocardial ischemia/reperfusion (I/R) injury in comparison with renal failure depending on the occurrence of Mb nitrite reductase and peroxidase activity, which compensate for the deleterious effects of ferryl Mb. This would be enhanced by the reductive properties of NO• and nitrite (ATP,

Adenosine-59-triphosphate; H2O2, hydrogen peroxide; metMb, ferric myoglobin; NO2−, nitrite; NO3−, nitrate). 32 , Signal transduction The Fe(III) heme complex inhibits GDP-GTP exchange in G roteins by sequestering the GDP-bound Ga subunit. JBC 278, 36505 (2003)

Cytoglobin regulation Found in the nucleus of cells. JBC 278, 30417 (2003)

33 J. Inorg. Biochem. 99, 110 (2005)

34 (a) As with Mb and many other Mb-type molecules, both novel globins could either store O2 or assist in the of O2 within the cell towards the mitochondria [7]. (b) Both globins could function as oxygen sensor proteins, which have been well-studied in [55]. Alternatively, they could be involved in other intracellular signalling pathways. (c) Ngb and Cygb might act as terminal oxidases, regenerating NAD+ to support glycolysis and sustain ATP production under hypoxic conditions, as proposed for maize hemoglobin [56]. (d) Both globins could be instrumental as scavengers of reactive oxygen or nitrogen species, which are produced, e.g., after reperfusion/re-oxygenation following ischemia. (e) As proven for Mb in mammalian muscle cells [10], they could possess dioxygenase activity, converting harmful excess NO into innocuous nitrate. 35 (f) Several cytoplasmatic enzymes use molecular O2 for chemical reactions, and globins like Ngb or Cygb could supply these other enzymes with adequate amounts of O2. ScienceDaily (Aug. 3, 2010) — A team of Wikipedia scientists at the University of California, Neuroglobin is a member of the vertebrate Davis and the University of Auckland has globin family involved in cellular oxygen discovered that neuroglobin may protect homeostasis. It is an intracellular against Alzheimer's disease by preventing neurons from dying in response to expressed in the central and peripheral natural stress. The team published the nervous system, cerebrospinal fluid, retina and results of their study in the April, 2010 issue endocrine tissues. Neuroglobin is a of Apoptosis. that reversibly binds oxygen with an affinity higher than that of hemoglobin. It also Scientists have learned that neuroglobin increases oxygen availability to brain tissue and protects cells from stroke damage, amyloid provides protection under hypoxic or ischemic toxicity and injury due to lack of oxygen. conditions, potentially limiting brain damage. Neuroglobin occurs in various regions of the brain and at particularly high levels in It is of ancient evolutionary origin, and is brain cells called neurons. Scientists have homologous to nerve globins of associated low levels of neuroglobin in .Recent research on Neuroglobin brain neurons with increased risk of presence confirmed that Human neuroglobin Alzheimer's disease. Recent studies have protein in cerebrospinal fluid(CSF)PMC554085 hinted that neuroglobin protects cells by maintaining the function of mitochondria and regulating the concentration of important chemicals in the cell.

36 PLOS one Dec. 2, 2011 Neuroglobin-Deficiency Exacerbates Hif1A and c-FOS Response, but Does Not Affect Neuronal Survival during Severe In Vivo Background Neuroglobin (Ngb), a neuron-specific globin that binds oxygen in vitro, has been proposed to play a key role in neuronal survival following hypoxic and ischemic insults in the brain. Here we address whether Ngb is required for neuronal survival following acute and prolonged hypoxia in mice genetically Ngb- deficient (Ngb-null). Further, to evaluate whether the lack of Ngb has an effect on hypoxia-dependent gene regulation, we performed a transcriptome-wide analysis of differential using Affymetrix Mouse Gene 1.0 ST arrays. Differential expression was estimated by a novel data analysis approach, which applies non-parametric statistical inference directly to probe level measurements. Principal Findings Ngb-null mice were born in expected ratios and were normal in overt appearance, home-cage behavior, reproduction and longevity. Ngb deficiency had no effect on the number of neurons, which stained positive for surrogate markers of endogenous Ngb-expressing neurons in the wild-type (wt) and Ngb-null mice after 48 hours hypoxia. However, an exacerbated hypoxia-dependent increase in the expression of c-FOS protein, an immediate early transcription factor reflecting neuronal activation, and increased expression of Hif1A mRNA were observed in Ngb-null mice. Large-scale gene expression analysis identified differential expression of the glycolytic pathway after acute hypoxia in Ngb-null mice, but not in the wts. Extensive hypoxia-dependent regulation of chromatin remodeling , mRNA processing and energy pathways was apparent in both genotypes. Significance According to these results, it appears unlikely that the loss of Ngb affects neuronal viability during hypoxia in vivo. Instead, Ngb-deficiency appears to enhance the hypoxia-dependent response of Hif1A and c-FOS protein while also altering the transcriptional regulation of the glycolytic pathway. Bioinformatic analysis of differential gene expression yielded novel predictions suggesting that chromatin remodeling and mRNA metabolism are among the key regulatory mechanisms when adapting37 to prolonged hypoxia.

Biochem. J. 443, 153 (2012)

Transcriptional regulation mechanisms of hypoxia-indued neuroglobin gene expression

Ngb (neuroglobin) has been identified as a novel endogenous neuroprotectant. However, little is known about the regulatory mechanisms of Ngb expression, especially under conditions of hypoxia. In the present study, we located the core proximal promoter of the mouse Ngb gene to a 554 bp segment, which harbours putative conserved NF-κB (nuclear factor κB)- and Egr1 (early growth-response factor 1) -binding sites. Overexpression and knockdown of transcription factors p65, p50, Egr1 or Sp1 (specificity protein 1) increased and decreased Ngb expression respectively. Experimental assessments with transfections of mutational Ngb gene promoter constructs, as well as EMSA (electrophoretic mobility- shift assay) and ChIP (chromatin immunoprecipitation) assays, demonstrated that NF-κB family members (p65, p50 and cRel), Egr1 and Sp1 bound in vitro and in vivo to the proximal promoter region of the Ngb gene. Moreover, a κB3 site was found as a pivotal cis-element responsible for hypoxia-induced Ngb promoter activity. NF-κB (p65) and Sp1 were also responsible for hypoxia-induced up-regulation of Ngb expression. Although there are no conserved HREs (hypoxia-response elements) in the promoter of the mouse Ngb gene, the results of the present study suggest that HIF-1α (hypoxia-inducible factor- 1α) is also involved in hypoxia-induced Ngb up-regulation. In conclusion, we have identified that NF-κB, Egr1 and Sp1 played important roles in the regulation of basal Ngb expression via specific interactions with the mouse Ngb promoter. NF-κB, Sp1 and HIF-1α contributed to the up-regulation of mouse Ngb gene expression under hypoxic conditions38 .

Wikipedia PLOS One Feb. 16, 2012 is the protein product Protection from Intracellular Oxidative Stress by of CYGB, a human and Cytoglobin in Normal and Cancerous Oesophageal Cells mammalian gene.[1] Cytoglobin is an intracellular globin of unknown function that is expressed Cytoglobin is a globin molecule mostly in cells of a myofibroblast lineage. Possible functions of cytoglobin include buffering of intracellular oxygen and detoxification of reactive ubiquitously expressed in all oxygen species. Previous work in our laboratory has demonstrated that tissues and most notably utilized cytoglobin affords protection from oxidant-induced DNA damage when in marine . It is thought over expressed in vitro, but the importance of this in more physiologically relevant models of disease is unknown. Cytoglobin is a candidate for the to be a method of protection tylosis with oesophageal cancer gene, and its expression is strongly down- under conditions of hypoxia. The regulated in non-cancerous oesophageal biopsies from patients with TOC predicted function of cytoglobin is compared with normal biopsies. Therefore, oesophageal cells provide an the transfer of oxygen from ideal experimental model to test our hypothesis that downregulation of [2] cytoglobin expression sensitises cells to the damaging effects of reactive to the brain. oxygen species, particularly oxidative DNA damage, and that this could Cytoglobin is a ubiquitously potentially contribute to the TOC phenotype. In the current study, we expressed hexacoordinate tested this hypothesis by manipulating cytoglobin expression in both normal and oesophageal cancer cell lines, which have normal hemoglobin that may facilitate physiological and no expression of cytoglobin respectively. Our results diffusion of oxygen through show that, in agreement with previous findings, over expression of tissues, scavenge nitric oxide or cytoglobin in cancer cell lines afforded protection from chemically- , or serve induced oxidative stress but this was only observed at non-physiological concentrations of cytoglobin. In addition, down regulation of cytoglobin in a protective function during normal oesophageal cells had no effect on their sensitivity to oxidative oxidative stress (Trent and stress as assessed by a number of end points. We therefore conclude that Hargrove, 2002).[ normal physiological concentrations of cytoglobin do not offer cytoprotection from reactive oxygen species, at least in the current experimental model. 39

J. Histohem. Cytochem. 56, 863 (2008) Neuroglobin and Cytoglobin Distribution in the Anterior Eye Segment: A Comparative Immunohistochemical Study

This study provides a detailed description of immunolocalization of two oxygen- binding proteins, neuroglobin (Ngb) and cytoglobin (Cygb), in the anterior segment of healthy human and canine eyes. Specific antibodies against Ngb and Cygb were used to examine their distribution patterns in anterior segment structures including the cornea, iris, trabecular meshwork, canal of Schlemm, ciliary body, and lens. Patterns of immunoreactivity (IR) were imaged with confocal scanning laser and conventional microscopy. Analysis of sectioned human and canine eyes showed Ngb and Cygb IR in the corneal epithelium and endothelium. In the iris, Ngb and Cygb IR was localized to the anterior border and the stroma, iridal sphincter, and dilator muscle. In the iridocorneal angle, Ngb and Cygb were detected in endothelial cells of the trabecular meshwork and canal of Schlemm in human. In the ciliary body, Ngb and Cygb IR was localized to the non-pigmented ciliary epithelium of the pars plana and pars plicata and in ciliary body musculature. Ngb and Cygb distribution was similar and colocalized within the same structures of healthy human and canine anterior eye segments. Based on their immunolocalization and previously reported biochemical features, we hypothesize that Ngb and Cygb may function as scavengers of reactive oxygen species. 40 J. Inorg. Biochem. 99, 110 (2005)

41 Ngb is a substantially divergent member of the globin family, displaying only 20–25% sequence identity to Mbs and Hbs [13, Fig. 2]. Ngb represents a typical Mb-type monomeric globin, which can bind O2 reversibly [13], [17] and [18]. In spite of its sequence differences, Ngb features the conserved globin fold consisting of the eight α- helices A-H, albeit with some peculiarities which reflect a pronounced adaptive potential of this basic globin structure (Fig. 3). The crystal structure of human NGB [19] has recently been solved, revealing the presence of unusual protein cavities which are not found as such in Hb and Mb and which may influence storage and diffusion paths inside the molecule. The most peculiar structural characteristic of Ngb is the so-called ‘hexa- coordinated’ binding scheme of the heme Fe atom in the ferrous (Fe2+) deoxy and in the ferric (Fe3+) states (Fig. 3). The crystallographic data have ultimately confirmed several types of spectroscopic analyses [13], [17], [20], [21], [22], [23] and [24], showing that in the absence of external , the at position 7 of the E helix (HisE7) binds the heme iron at its sixth, distal position. Thereby, any external gaseous ligand such as O2 or CO has to 42 compete with the internal His(E7) ligand for Fe binding. (a) As with Mb and many other Mb-type molecules, both novel globins could either store O2 or assist in the diffusion of O2 within the cell towards the mitochondria [7]. (b) Both globins could function as oxygen sensor proteins, which have been well-studied in bacteria [55]. Alternatively, they could be involved in other intracellular signalling pathways. (c) Ngb and Cygb might act as terminal oxidases, regenerating NAD+ to support glycolysis and sustain ATP production under hypoxic conditions, as proposed for maize hemoglobin [56]. (d) Both globins could be instrumental as scavengers of reactive oxygen or nitrogen species, which are produced, e.g., after reperfusion/re-oxygenation following ischemia. (e) As proven for Mb in mammalian muscle cells [10], they could possess dioxygenase activity, converting harmful excess NO into innocuous nitrate. 43 (f) Several cytoplasmatic enzymes use molecular O2 for chemical reactions, and globins like Ngb or Cygb could supply these other enzymes with adequate amounts of O2. J. Exp. Biol. 212, 1423 (2009).

Fig. 2. Some postulated functions of neuroglobin.

Neuroglobin may support the supply of O2 to the electron transport chain in mitochondria (A), may detoxify reactive oxygen or nitrogen species (B), may

Neuroglobin convert NO to NO3– at high PO2 and NO2– to NO at low PO2 (C), act as a signal protein by inhibiting the dissociation of GDP from Gα (D) or prevent hypoxia- induced apoptosis via reduction of cytochrome c (E). 44 JBC 277, 871 (2002) Truncated : A New Family of Hemoglobins Widely Distributed in Bacteria, Unicellular , and Truncated hemoglobins (trHbs) constitute a family of small oxygen-binding heme proteins distributed in eubacteria, , , and plants, forming a distinct group within the hemoglobin (Hb) superfamily. They are nearly ubiquitous in the kingdom, occur in many aggressively pathogenic bacteria, and are held to be of very ancient origin. None have been detected in the genomes of or metazoa. Characteristically, trHbs occur at nano- to micromolar intracellular concentration, hinting at a possible role as catalytic proteins. Many trHbs display amino acid sequences 20–40 residues shorter than non-vertebrate hemoglobins to which they are scarcely related by sequence similarity. Crystal structures show that trHb tertiary structure is based on a 2-on-2 α-helical sandwich, which represents an unprecedented editing of the highly conserved globin fold. Moreover, an almost continuous hydrophobic tunnel, traversing the protein matrix from the molecular surface to the heme distal site, may provide a path for ligand diffusion to the heme. trHbs are phylogenetically distinct within the Hb superfamily.

The 2-on2 a-helical fold characterizes trHbs.

Heme coordination: 6-coordination, low spin, Tyr as the axial ligand.

Networks of hydrogen bonds stalibize the heme distal ligand.

Ligands can enter the distal heme pocket through a protein matrix tunnel. trHbs server diverse funcitons. 45

B9 B10 CD1 E7 E14 F8

FIG. 1. Phylogenetic tree showing the relationships among trHbs. The distance tree (minimum method) was constructed using the PAUP program (version 4.0b1). Bootstrap values were cal- culated from 1000 replicates. Important residues (B9, B10, CD1, E7, E14, and F8) with regard to coordination of the heme and the ligand binding residue properties are indicated. The sequences alignment used for the cladistic analysis is shown in Supplemental Material (Fig. 1). 46 JBC 277, 871 (2002). Fig. 2 A structural overlay of C. eugametos trHb (red ribbons) on sperm Mb (green), the latter taken as the prototype of the (non)-vertebrate globin fold. N and C termini are labeled for C. eugametos trHb. This and similar structural comparisons with other (non)-vertebrate Hbs or Mbs indicate that the match between 2-on-2 trHbs and 3-on-3 globins is limited to less than 60 Cα pairs, mostly located on the distal side of the heme (right in the figure). The main trHb α-helical segments are labeled according to the topological conventions defined for the 3-on-3 globin fold.

2-on-2: antiparallel helix pairs B/E and G/H N-terminal A helix is deleted. The whole CD-D region is trimmed to 3 residues. 47 JBC 277, 871 (2002).

Fig. 3

A view of the distal site cavity in M. tuberculosis oxy-trHbN in an orientation close to that of Fig. 2. The heme group is edge-on and the iron atom is shown as apurple sphere; the iron- coordinated O2 molecule is displayed in red. Distal site residues and the proximal histidine are labeled according to their topological sites. The CD segment and part of the B- and of the E-helices are displayed as cyan ribbons. Dashed lines highlight the hydrogen-bonded interactions stabilizing the ligand in the distal site. 48

PNAS 99, 5902 (2002) Truncated hemoglobin HbN protects Mycobacterium bovis from nitric oxide Mycobacterium tuberculosis, the causative agent of human tuberculosis, and Mycobacterium bovis each express two genes, glbN and glbO, encoding distantly related truncated hemoglobins (trHbs), trHbN and trHbO, respectively. trHbN detoxifies NO 20- fold more rapidly than myoglobin. These results establish a role for a trHb and demonstrate an NO-metabolizing activity in M. tuberculosis or M. bovis. trHbN thus might play an important role in persistence of mycobacterial infection by virtue of trHbN′s ability to detoxify NO.

JBC 282, 13627 (2007) Structural and Functional Properties of a Truncated Hemoglobin from a Food-borne Pathogen Campylobacter jejuni Campylobacter jejuni contains two hemoglobins, Cgb and Ctb. Cgb has been suggested to perform an NO detoxification reaction to protect the bacterium against NO attack. On the other hand, the physiological function of Ctb, a class III truncated hemoglobin, remains unclear. The extremely high oxygen affinity of Ctb makes it unlikely to function as an oxygen transporter; on the other hand, the distal heme environment of Ctb is surprisingly similar to that of cytochrome c peroxidase, suggesting a role of Ctb in performing a peroxidase or P450-type of oxygen chemistry.

49 J. Biol. Chem. 283, 8773 (2008) Diversity of globin function: Enzymatic, transport, storage, and sensing

The availability of genomic information from the three kingdoms of life has altered substantially our view of the globin superfamily. It is now evident that Hbs,2 defined as hemeproteins comprising five to eight α-helices (A–H), with an invariant His at position F8 providing the proximal ligand to the heme iron, occur as three families in two structural classes (1). Within each family, the Hb can be either chimeric or SD. Historically, the first members of the two families that display the canonical 3/3 α-helical fold were chimeric: the FHbs in Escherichia coli and yeast discovered in ∼1990, consisting of an N- terminal Hb coupled to a reductase-like domain, and the GCSs reported in bacteria and Archaea a decade later, comprising an N-terminal Hb linked to variable gene regulatory domains. The third family of Hbs discovered concomitantly in , ciliates, and bacteria were the 2/2Hbs (“truncated” Hbs), which exhibit a 2/2 α-helical fold (see supplemental figure). More recently, SD globins have been discovered in the FHb-like and sensor Hb families that we have called SDFgbs and SDSgbs, respectively (2, 3). Fig. 1 shows diagrammatically the three Hb families and summarizes their distribution in bacteria and eukaryotes. A classification of Hbs is presented in the supplemental table. Only bacteria have representatives of all three families in chimeric and SD guise; the Archaea and eukaryotes lack FHbs and GCSs, respectively. On the basis of the higher sequence similarity to bacterial FHbs/SDFgbs than to GCSs and 2/2Hbs and the presence of FHbs/SDFgbs in unicellular eukaryotes, we have proposed that all eukaryotic Hbs, including vertebrate α/β-globins, Mbs, Ngbs, and Cygbs and all the and plant Hbs, emerged from one or more ancestral bacterial SDFgbs (2).

The variety of Hbs in bacteria makes it clear that the familiar O2 transport function of vertebrate Hbs is a relatively recent adaptation and that the early Hb functions must have been enzymatic and O2-sensing. In this review, we will not discuss O2 transport by animal (metazoan) Hbs; instead, we will focus on the reactions and functions of the FHbs/SDFgbs in the first five sections. The functions of the remaining two globin families will be discussed in the last two sections. 50

Fig. 1 Diagrammatic representation of the three globin families in bacteria, each comprising chimeric and SD globins, and their relationships to eukaryotic globins. The chimeric monooxygenase 2/2Hb2 in Frankia and Streptomyces is the only known chimeric 2/2Hb. Note that sensor Hbs are absent in eukaryotes and that the function of SDSgbs is unknown.

51 Fig. 2. Reactions at heme group H. Reaction 1, deoxygenation; reaction 2, oxygenation; reaction 3, NO dioxygenation; reaction 4, nitrosylation; reaction 5, NO reduction; reaction 6, O2 nitrosylation (heme denitrosylation); reaction 7, nitrite reduction; and reaction 8, MetHb reduction.

52 Fig. 3. Autoxidation of hemeprotein (HP), reaction with peroxides, and catalysis of lipid oxidation. HP–X represents heme-to-protein cross-linked species.

53 Fig. 1. (a) Sheet lava on the ridge axis on the East Pacific Rise (9° 50′ N). Notice the paucity of the fauna, typical of deep-sea habitats at these depths (2500 m). (b) The fish Thermarces cerberus near a mussel bed (Bathymodiolus thermophilus) on the East Pacific Rise (9° 50′ N). (c) Cluster of on the East Pacific Rise (9° 50′ N). (d) Branchipolynoe aff. seepensis in the mantle cavity of its commensal mussel B. azoricus (collected on the Mid-Atlantic Ridge, Lucky Strike site). Photos (a–c) Stéphane Hourdez/HOPE99/Ifremer and photo (d) Stéphane Hourdez (ATOS cruise).

54 Fig. 3. P50 values (log scale) for extracellular Hbs from vent and cold-seep marine compared to non-vent and non-cold seep species. Experimental conditions and References: Arenicola marina, pH 7.6, 20 °C [109]; Siboglinum ekmani, pH 6.5, 20 °C [110]; A. pompejana, pH 7.6, 20 °C [59], [61] and [62]; A. caudata, pH 7.6, 20 °C [62]; Pista pacifica, pH 7.0, 20 °C [111]; Marphysa sanguinea, pH 7.3, 20 °C [60]; Eunice aphroditis, pH 7.0, 20 °C [112]; Arenicola cristata, pH 7.7, 20 °C [109]; Abarenicola clarapedii, pH 7.43, 20 °C [109]; Eupolymnia crescentis, pH 5–7, 10 °C [60]; B. symmytilida Hbs C1 and C2, pH 7.5, 20 °C [73]; M. dendrobranchiata, pH 7.5, 20 °C [72]; R. pachyptila HBL and 400 kDa Hbs, pH 7.0, 30 °C [41]. 55 Vestimentiferan tubeworms are often the most commonly encountered metazoan animals in (Fig. 1c) and cold-seep communities. After a long debate on their taxonomic standing they are now considered to form a highly specialized family of [31], [32] and [33]. They lack a mouth, digestive tract and anus [34] and [35], and their nutritional needs are provided for by symbiotic sulfide-oxidizing bacteria hosted in the ‘trophosome’ organ found deep inside their body [35], [36], [37] and [38]. Although the first vestimentiferan was discovered in 1969 [39], their respiratory pigments were investigated only after the discovery of hydrothermal vents that harbour the giant tubeworm Riftia pachyptila. This worm possesses two Hbs in its vascular blood and another in its coelomic fluid [40] and [41]. In the blood, a typical hexagonal bilayer (HBL) Hb of ∼3.6 MDa (as typically encountered in vascular fluids of other annelids, Fig. 2) co-occurs with a 400 kDa Hb that is specific of tubeworms. The coelomic fluid contains another 400 kDa Hb, differing from the vascular one in its subunit composition [42]. Other tubeworms from hydrothermal vents and cold-seeps possess vascular and coelomic Hbs with quaternary structures similar to those of Hbs from Riftia[114]. As in other extracellular HBL Hbs, Riftia Hb is comprised of heme- bearing globin chains as well as non-heme, structural chains called linkers [43]. The HBL Hb from the Lumbricus terrestris has been crystallized and its structure solved [44]. The molecules consist of two superimposed rings that each consists of 6 hollow globular structures (also referred to as ‘twelfths’ or ‘submultiples’) that are held together by the linkers. The whole structure consists of 144 globin chains and 36 linkers. Unique amongst annelid extracellular Hbs, the vestimentiferan 400 kDa Hbs lack linkers and are comprised of solely of globin chains, assembled in a 24-chain globular structure [42]. 56 Fig. 2. Surface representations of A. pompejana hemoglobin calculated from 3D reconstruction volume obtained in cryoelectron microscopy [107]. The isosurfaces are viewed in (a) top and (b) side orientations. They were calculated with SIGMA [108] and rendered with the ray- tracer PoVRay (http://www.povray.org)

57 Seep verb to pass gradually or leak through or as if through small openings or a porous substance; ooze noun a small spring or place where water, oil, etc, has oozed through the ground. Related to Middle High German sifen, Swedish dialect sipa

Annelid A large phylum of segmented worms including ragworms, , and .

Littoral zone The part of a sea, lake or river that is close to the shore.

Terrestrial animal An animal that lives on land opposed to living in water, or sometimes an animal that on or near the ground. 58

H2S binding to deep sea hemoglobin H2S pKa 6.7

Kraus, D.W.; Wittenberg, J. B. Hemoglobins of the Lucina pectinata/bacteria . I. Molecular properties, kinetics and equilibria of reactions with ligands. J. Biol. Chem. 265: 16043-16053; 1990.

Kraus, D.W.; Wittenberg, J.B.; Jin-Fen, L.; Peisach, J. Hemoglobins of the Lucina pectinata/bateria symbiosis. II. An electron paramagnetic and optical spectral study of the ferric proteins. J. Biol. Chem. 265: 16054-16059; 1990.

Pietri, R.; Lewis, A.; Leoon, R.G.; Casanoba, G.; Kiger, L.; Yeh, S.R.; Ferdandez-Alberti, S.; Marden, M.C.; Cadilla, C.L.; Lopez-Garriga, J. Factors controlling the reactivity of with hemeproteins. 48: 4881-4891; 2009.

Bailly, X.; Vinogradov, S. The sulfide binding function of annelid hemoglobins: relic of an old biosystem? J. Inorg. Biochem. 99: 142-150; 2005.

Fast binding of HS- to heme Fe(III) complex of those hemoglobins.

The heme Fe(III)-SH complex is stable, different from human hemoglobins where the heme Fe(III)-SH complex quickly converts into heme Fe(II) complex.

The presence of free Cys residues might be associated with those results. 59 The loss of the hemoglobin H2S-binding function in annelids from sulfide-free habitats reveals molecular adaptation driven by Darwinian positive selection Proc. Nat. Acad. Sci., USA 100, 5885 (2003)

The hemoglobin of the deep-sea hydrothermal vent vestimentiferan Riftia pachyptila (annelid) is able to bind toxic hydrogen sulfide (H2S) to free cysteine residues and to transport it to fuel endosymbiotic sulfide-oxidising bacteria. The cysteine residues are conserved key amino acids in annelid globins living in sulfide-rich environments, but are absent in annelid globins from sulfide-free environments. …..performed to understand how the sulfide-binding function of hemoglobin appeared and has been maintained during the course of evolution. This study reveals that the sites occupied by freecysteine residues in annelids living in sulfide-rich environments and occupied by other amino acids in annelids from sulfide-free environments, have undergone positive selection in annelids from sulfide-free environments. We assumed that the high reactivity of cysteine residues became a disadvantage when H2S disappeared because free cysteines without their natural ligand had the capacity to interact with other blood components, disturb homeostasis, reduce fitness and thus could have been counterselected. ……. 60 Sulfhemoglobin

Berzofsky, J.A.; Peisach, J.; Blumber, W.E. Sulfheme proteins. I. Optical and magnetic properties of sulfmyoglobin and its derivatives. J. Biol. Chem. 246: 3367-3377; 1971.

Berzofsky, J.A.; Peisach, J.; Blumberg, W.E. Sulfheme proteins. II. The reversible oxygenation of ferrous sulfmyoglobin. J. Biol. Chem. 246: 7366-7372; 1971.

Andersson, L.A.; Loehr, T.M.; Lim, A.R.; Mauk, A.G. Sulfmyoglobin. Resonance Raman spectroscopic evidence for an iron-chlorin prosthetic group. J. Biol. Chem. 259: 15340- 15349; 1984.

Lloyd, E.; Mauk, A.G. Formation of sulphmyoglobin during expression of horse heart myoglobin in Escherichia coli. FEBS Lett. 340: 281-286; 1994.

Hb (or Mb) is treated with both H2O2 and H2S, the sulfur atom is incorporated into the heme iron complex and produces sulfheme complex.

61