Download Heme, Myoglobin, Hemoglobin

Heme, Myoglobin, Hemoglobin
(Haem, Haemoglobin: UK)
Heme proteins = heme + proteins
Transfer & storage
of gas molecules
Oxidation
O2 + e-
O2, NO
P450, NOS
Mb, Hb
Electron
transfer
Heme sensor
Gas sensor
eCytochrome c
Iron Protoporphyrin IX, heme b
Prosthetic group
A flat and planar structure
Heme:HRI
O2 , CO, NO: FixL, DOS
Myoglobin O2 Storage
You need O2 because
of continuous swimming.
You don’t need O2.
More myoglobin.
Less myoglobin.
5
Iron Protoporphyrin IX, heme b
Prosthetic group
A flat and planar structure.
Itself Toxic O2-., H2O2 are formed.
Heme a
Heme a is a form of heme found
In cytochromes a and a3.
Cytochrome c with heme c.
Myoglobinis a small, 17 kDa, monomeric, O2binding haemoprotein that typically occurs in
cardiac and aerobic skeletal muscle of
vertebrates.
The oxygen storage protein abundant in
muscles. Acts as the heme Fe(II) complex.
Alpha (a) -helical form.
Myo – muscle from Greek
Globular proteins 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
structures, pleated structures, or completely
random structures. Globular proteins are
relatively spherical in shape as the name
implies. Common globular proteins include egg
albumin, hemoglobin, myoglobin, insulin,
serum globulins in blood, and many enzymes.
His E7
Distal side
Heme iron
His F8
Proximal side
Heme is located in the hydrophobic site.
High oxygen affinity.
His (E7) is the oxygen binding site.
The oxygen binding curve for Myoglobin
forms an asymptotic shape, which shows a
simple graph that rises sharply then levels
off as it reaches the maximum saturation.
The half-saturation, the point at which half
of the myoglobin is binded to oxygen, is
reached at 2 torr which is relatively low
compared to 26 torr for hemoglobin.
Myoglobin has a strong affinity for oxygen
when it is in the lungs, and where the
pressure is around 100 torr. When it reaches
the tissues, where it's around 20 torr, the
affinity for oxygen is still quite high. This
makes myoglobin less efficient of an oxygen
transporter than hemoglobin, which loses it's
affinity for oxygen as the pressure goes down
and releases the oxygen into the tissues.
Myoglobin's strong affinity for oxygen means
that it keeps the oxygen binded to itself
instead of releasing it into the tissues.
Distal side
Proximal side
Hemoglobin, whose role is to transport oxygen in
the blood, comprises 4 sub-units, each very
similar to a myoglobin molecule.
Hemo – blood from Greek
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 red 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 tissue capillaries. In fact,
hemoglobin can be more O2-saturated in the lungs and less saturated in the capillaries.
Christian Bohr (1855-1911)
Bohr Effect of Hemoglobin
CO2 pressure affects the ability
of hemoglobin to carry O2.
Higher carbon dioxide concentration, lower affinity
of haemoglobin for oxygen thus curve shifts to the
right, showing lower saturation percentage of
haemoglobin to oxygen since carbon dioxide is in
higher concentration compared to oxygen.
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.
Carbonic anhydrase enhances
the reaction to increase pH.
Sickle cell anemia &
malaria resistance
In sickle cell hemoglobin (HbS) glutamic acid in
position 6 (in beta chain) is mutated to valine. This
change allows the deoxygenated form of the
hemoglobin to stick to itself.
Cell 145, 335 (2011).
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 sickle cell disease and severe malaria.
The figure shows a molecular pathway that may
explain why carriers of the sickle cell trait, who are
heterozygous for the mutation 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 heme oxygenase-1 (HO-1), which
converts heme into the antioxidant molecules
carbon monoxide (CO) and biliverdin and releases
iron to bind to ferritin 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
oxidative tissue damage during severe malaria.
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Hemazoin
b-Hematin
Hemozoin is composed of b-hematin.
Digestion of hemoglobin by
the malaria parasite produces
this malaria pigment.
Quinine, chloroquine and other 4aminoquinolines inhibit pigment formation, as
well as the heme degradative processes, and
thereby prevent the detoxification of heme. The
free heme destabilizes the food vacuolar
membrane and other membranes and leads to
the death of the parasite.
Intravascular hemolysis 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. Haptoglobin (an α-2 globulin
produced in the liver) 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
glomerulus (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 urine
sediment). Because hemoglobin concentrations >20 mg/dL
will cause visible discoloration of plasma (light pink to dark
red, depending on how much hemoglobin is present),
hemoglobinemia is often visible with intravascular hemolysis.
Hemoglobinuria
The hemoglobin dimers that remain in circulation are oxidized to methemoglobin, which disassociates into a free
heme and globin chains. The oxidized free heme (met-heme) binds to hemopexin (a β-globulin, Hpx) and the metheme and hemopexin complex(met-heme/Hpx) is taken up by a receptor on hepatocytes and macrophages within the
spleen, liver and bone marrow (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 globins are broken down to amino acids, 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 storage protein,
apoferritin). The remaining porphyrin ring (biliverdin) is degraded to unconjugated bilirubin 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 macrophage (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 hemolytic anemia. Because haptoglobin is consumed during intravascular hemolysis, serum
values of this protein usually decline with intravascular hemolytic anemias 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.
Nitric Oxide (NO)
• An important signalling and cytotoxic molecule in the
cardiovascular, nervous, and immune systems.
• From diabetes to hypertension, cancer to drug addiction,
stroke to intestinal motility, memory and learning disorders
to septic shock, 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
Physiology or Medicine for their discoveries concerning
"nitric oxide as a signaling molecule in the cardiovascular
system."
21
Nitric Oxide Synthase (NOS)
Heme Fe(III)-S--Cys
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 vasodilation. 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 Stransnitrosation, the transfer of NO groups to thiols 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 perfusion. In addition,
the formation of S-nitrosothiols in other proteins may regulate an array
of physiological functions.
Chemical characterization of the smalles S-nitrosothiol, HSNO; Cellular cross-talk of H2S and S-nitrosothiols
J. Am. Chem. Soc. 134, 12016 (2012)
Dihydrogen sulfide 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– species, 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 H 2S, but, more broadly, introduce
a new signaling molecule, HSNO, and suggest that it may play a key role in cellular redox regulation.
Figure 9. Red blood cell interactions with NO include (1) NO binding to the deoxygenated
heme in oxygenated red blood cells, forming iron-nitrosyl hemoglobin (FeII–NO); (2) oxyhemoglobin 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.
New roles of hemoglobin (and myoglobin) as reductants
26
Nature Chemical Biology 5, 865 (2009) Meeting Report
27
28
Ion implicated in blood pact Nature Medicine 9, 1460
(2003)
The emerging biology of the nirite anion NO2Nature Chemical Biology 1, 308 (2005)
29
Nature Chemical Biology 3, 785 (2007)
30
The Reaction
between Nitrite and
Oxyhemoglobin: A
MECHANISTIC
STUDY
J. Biol. Chem. 283, 9615 (2008)
31
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 Fe2NO2 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)
J. Am. Chem. Soc. 134, 13861 (2012)
J. Exp. Biol. 213, 2726 (2010). Fig. 1.
Schematic drawing summarizing the distinct interactions of Mb
and nitrogen oxides as a function of O2 tension (A) and its
functional consequences for mitochondrial respiration (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 electron transport
chain; ADP, adenosine diphosphate; ATP, adenosine
triphosphate; C, cytochrome c oxidase; deoxyMb,
deoxygenated myoglobin; NADH, nicotinamide adenine
dinucleotide; MbNO, nitrosylmyoglobin; MbO2, oxygenated
myoglobin; metMb, metmyoglobin; metMbNO,
nitrosylmetmyoglobin; mPTP, mitochondrial permeability
transition pore; NO, nitric oxide; ·O2−, superoxide; Q, coenzyme
Q.
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•) homeostasis by Mb could prevent or
diminish the NO•-mediated inhibition of
cytochrome c oxidase 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 ischemia. (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).
Neuroglobin,
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
Gene regulation
Found in the nucleus of vertebrate cells.
JBC 278, 30417 (2003)
J. Inorg. Biochem. 99, 110 (2005)
(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.
(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 O .
Wikipedia
Neuroglobin is a member of the vertebrate
globin family involved in cellular oxygen
homeostasis. It is an intracellular hemoprotein
expressed in the central and peripheral
nervous system, cerebrospinal fluid, retina and
endocrine tissues. Neuroglobin is a monomer
that reversibly binds oxygen with an affinity
higher than that of hemoglobin. It also
increases oxygen availability to brain tissue and
provides protection under hypoxic or ischemic
conditions, potentially limiting brain damage.
It is of ancient evolutionary origin, and is
homologous to nerve globins of
invertebrates.Recent research on Neuroglobin
presence confirmed that Human neuroglobin
protein in cerebrospinal fluid(CSF)PMC554085
ScienceDaily (Aug. 3, 2010) — A team of
scientists at the University of California,
Davis and the University of Auckland has
discovered that neuroglobin may protect
against Alzheimer's disease by preventing
brain neurons from dying in response to
natural stress. The team published the
results of their study in the April, 2010 issue
of Apoptosis.
Scientists have learned that neuroglobin
protects cells from stroke damage, amyloid
toxicity and injury due to lack of oxygen.
Neuroglobin occurs in various regions of
the brain and at particularly high levels in
brain cells called neurons. Scientists have
associated low levels of neuroglobin in
brain neurons with increased risk of
Alzheimer's disease. Recent studies have
hinted that neuroglobin protects cells by
maintaining the function of mitochondria
and regulating the concentration of
important chemicals in the cell.
PLOS one Dec. 2, 2011
Neuroglobin-Deficiency Exacerbates Hif1A and c-FOS Response, but Does Not Affect Neuronal Survival
during Severe Hypoxia 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 Ngbdeficient (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 gene expression 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 genes after acute hypoxia in Ngb-null mice,
but not in the wts. Extensive hypoxia-dependent regulation of chromatin remodeling , mRNA processing
and energy metabolism 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 adapting
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 mobilityshift 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 factor1α) 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 conditions.
Wikipedia
PLOS One Feb. 16, 2012
Cytoglobin is the protein product Protection from Intracellular Oxidative Stress by
of CYGB, a human and
Cytoglobin in Normal and Cancerous Oesophageal Cells
Cytoglobin is an intracellular globin of unknown function that is expressed
mammalian gene.[1]
mostly in cells of a myofibroblast lineage. Possible functions of cytoglobin
Cytoglobin is a globin molecule
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 mammals. 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 downunder 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
ideal experimental model to test our hypothesis that downregulation of
the transfer of oxygen from
cytoglobin expression sensitises cells to the damaging effects of reactive
arterial blood to the brain.[2]
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
tested this hypothesis by manipulating cytoglobin expression in both
expressed hexacoordinate
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
cytoglobin in cancer cell lines afforded protection from chemicallytissues, scavenge nitric oxide or
reactive oxygen species, 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
normal physiological concentrations of cytoglobin do not offer
Hargrove, 2002).[
cytoprotection from reactive oxygen species, at least in the current
experimental model.
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 oxygenbinding 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.
J. Inorg. Biochem. 99, 110 (2005)
Ngb is a substantially divergent member of the globin family, displaying only 20–25% amino acid 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 αhelices 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 ligand storage and
diffusion paths inside the molecule. The most peculiar structural characteristic of Ngb is the so-called ‘hexacoordinated’ 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 ligands, the histidine 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
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.
(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 O .
J. Exp. Biol. 212, 1423 (2009).
Neuroglobin
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
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 hypoxiainduced apoptosis via reduction of cytochrome c (E).
JBC 277, 871 (2002)
Truncated Hemoglobins: A New Family of Hemoglobins Widely Distributed in Bacteria,
Unicellular Eukaryotes, and Plants
Truncated hemoglobins (trHbs) constitute a family of small oxygen-binding heme proteins
distributed in eubacteria, cyanobacteria, protozoa, and plants, forming a distinct group within the
hemoglobin (Hb) superfamily. They are nearly ubiquitous in the plant kingdom, occur in many
aggressively pathogenic bacteria, and are held to be of very ancient origin. None have been
detected in the genomes of archaea 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.
B9 B10 CD1 E7 E14 F8
FIG. 1. Phylogenetic
tree showing the
relationships among
trHbs. The distance
tree (minimum
evolution method) was
constructed using the
PAUP program (version
4.0b1). Bootstrap
values were calculated 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).
JBC 277, 871 (2002).
Fig. 2
A structural overlay of C. eugametos trHb
(red ribbons) on sperm whale 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.
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 ironcoordinated 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.
PNAS 99, 5902 (2002)
Truncated hemoglobin HbN protects Mycobacterium bovis from nitric oxide
Mycobacterium tuberculosis, the causative agent of human tuberculosis, and
Mycobacteriumbovis each express two genes, glbN and glbO, encoding distantly related
truncated hemoglobins (trHbs), trHbN and trHbO, respectively. trHbN detoxifies NO 20fold 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.
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 Nterminal Hb coupled to a ferredoxin 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 algae, 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 invertebrate 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.
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.
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.
Fig. 3. Autoxidation of hemeprotein (HP), reaction with peroxides,
and catalysis of lipid oxidation.
HP–X represents heme-to-protein cross-linked species.
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 Riftia pachyptila 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).
57
Fig. 3. P50 values (log scale) for extracellular Hbs from vent and cold-seep marine polychaetes
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].
58
Vestimentiferan tubeworms are often the most commonly encountered metazoan
animals in hydrothermal vent (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
polychaete annelids [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 annelid Hbs, Riftia Hb is comprised of hemebearing globin chains as well as non-heme, structural chains called linkers [43]. The HBL
Hb from the earthworm 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]. 59
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 raytracer PoVRay (http://www.povray.org)
60
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,
earthworms, and leeches.
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 livers on or near the ground.
H2S binding to deep sea hemoglobin
H2S pKa 6.7
Kraus, D.W.; Wittenberg, J. B. Hemoglobins of the Lucina pectinata/bacteria symbiosis. 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 resonance 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
hydrogen sulfide with hemeproteins. Biochemistry 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.
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. …….
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: 1534015349; 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.