Functional Adaptation and Its Molecular Basis in Vertebrate

Respiratory Physiology & Neurobiology 144 (2004) 141159
Functional adaptation and its molecular basis in vertebrate

hemoglobins, neuroglobins and cytoglobins
Roy E. Weber , Angela Fago
Department of Zoophysiology, Institute of Biological Sciences, University of Aarhus,
C.F. Mllers Alle 131, DK-8000 Aarhus C, Denmark
Accepted 14 April 2004
Abstract
Hemoglobin (Hb), the paradigm for allosteric proteins through decades, has gained renaissance in recent years following
discovery of globins or their genes in all living organisms and in all tissues of higher animals, and of new members of the
globin family, such as neuroglobins, Ngb, found predominantly in neural and nerve tissues and cytoglobins, Cygb, that has
unprecedented nuclear location. The recent progresses in this field have been prompted by the development of sophisticated
techniques to probe molecular structure and functions, which have revealed novel functions, such as the scavenging and release
of vasoactive nitric oxide and the regulation of cellular metabolism. This review deals with the functional adaptations and the
underlying molecular mechanisms in globins and presents case examples of molecular adaptations encountered in vertebrates
and agnathans.
2004 Elsevier B.V. All rights reserved.
Keywords: Adaptation; Vertebrate globins; Hemoglobin; Allosteric interactions; Oxygen-binding; Neuroglobin; Cytoglobin; Functional
adaptation
1. Vertebrate globins in evolutionary

perspective
Abbreviations: Hb, hemoglobin; Mb, myoglobin; Ngb, neuroglobin; Cgb, cytoglobin; P50 , oxygen tension at half-saturation;
n50 , Hills cooperativity coefficient at half-saturation; KT and KR ,
O2 association equilibrium constants of the Hb in the deoxygenated
(tense) and oxygenated (relaxed) states, respectively; cdB3, cytoplasmic domain of band 3 membrane protein; IPP, inositol pentaphosphate; trout-P, chicken-P and human-P, 10-mer peptides corresponding to N-termini of cdB3 of the named species; DPG,2,3,
diphosphoglycerate; GTP, guanosine triphosphate; NTP, nucleoside
triphosphates; IHP, inositol hexaphosphate
Corresponding author. Tel.: +45 8942 2599; fax: +45 8619 4186.
E-mail address: roy.weber@biology.au.dk (R.E. Weber).
1569-9048/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.resp.2004.04.018
One hundred years after the discovery of the Bohr

effect [the decrease in Hb-O2 affinity by CO2 and
pH decrease] (Bohr et al., 1904), Hb and myoglobin
(Mb) that transport O2 in blood and store it in muscles remain the most intensively studied proteins as
regards function and its structural correlates. Recent
studies indicate that globins or their genes occur in
all organisms, including archeobacteria, protozoa and
plants (Weber and Vinogradov, 2001) and probably in
142
R.E. Weber, A. Fago / Respiratory Physiology & Neurobiology 144 (2004) 141159
all tissues in higher animals (Trent and Hargrove, 2002;

Burmester et al., 2002). Despite the enormous diversity in their primary and quaternary structures (amino
acid sequences and aggregation states) globin proteins
exhibit a characteristic tertiary structure (the globin
fold) suggesting a common ancestry. The ancestral
globin gene appears to have evolved 18,000 million
years ago, when O2 started to accumulate in the atmosphere suggesting that the proteins original function may have been to scavenge toxic O2 , CO and NO
gases (Hardison, 1999). The evolutionary, proteomerelated implication is that globins provide opportunity
to trace structurefunction relations in a single protein family throughout the five Kingdoms of living
organisms.
The tetrameric and monomeric vertebrate globins
stand in contrast with the enormous variation in
structure and function encountered in non-vertebrate
globins. Microorganism globins form three families:
(a) chimeric flavoproteins, where heme-carrying globin
domains are linked to oxido-reductive FAD-dependent
domains, (b) truncated Hbs with short polypeptide
chains, and (c) bacterial Hbs (Weber and Vinogradov,
2001; Wajcman and Kiger, 2002). Plant Hbs comprise
symbiotic Hbs (legHbs) from root nodules of leguminous plants that harbour symbiotic nitrogen-fixing
bacteria, as well as non-symbiotic Hbs, that may be
involved in several metabolic pathways (ArredondoPeter et al., 1998). Invertebrate Hbs illustrate phenomenal structural and functional diversity varying
from single-chain monomers with molecular masses
of 11.2 kDa, to multisubunit, multidomain crustacean
Hbs, complex extracellular (3600 kDa) annelid Hbs
where each molecule consists of 144 O2 binding globin
chains and a number of heme-free linker chains, and
include even larger (12 000 kDa) complexes found in
some bivalve molluscs. These proteins serve a wide
range of functions apart from transporting and storing O2 , such as controlling in vivo O2 levels, protection against sulphide, and enzymatic (oxidase and
peroxidase-like and superoxide dismutase) activities
(Weber and Vinogradov, 2001).
Following recent discoveries four types of globins
differing in tissue distribution and molecular structure are now recognized in vertebrates: Hb, Mb, neuroglobin (Ngb) and cytoglobin (Cygb, initially also
known as histoglobin). Compared to Ngb that is predominantly expressed in the nervous tissues (brain and
retinal tissues (Burmester et al., 2000), Cygb appears

to occur in all tissue types (Trent and Hargrove, 2002;
Burmester et al., 2002; Pesce et al., 2002) and to have
an unprecedented nuclear localization (Geuens et al.,
2003).
This treatise focuses on the basic structural and O2 binding properties of vertebrate and agnathan globins
also dealing with novel functions (including NO scavenging/release and regulation of red cell metabolism)
that emerged in recent years, and describes salient casestudy adaptations encountered in the different classes
that necessarily cannot be comprehensive.
2. Basic structural and functional properties

Vertebrate Hbs are tetrameric consisting of two
and two chains that in humans consist of 141 and 146
amino acid residues, respectively. The globin fold of
each chain (Fig. 1) characteristically consists of seven
or eight -helices (conventionally labelled AH) linked
by non-helical segments (e.g. EF, FG) segments, and of
N-terminal and C-terminal extensions referred to NA
and HC. Each chain has a crevice, the heme pocket,
between the E and F helices that harbours an O2 binding heme group. Individual amino acid residues
are usually referred to by their helical positions and/or
sequential numbers starting from the amino termini.
Thus LysEF6(82) refers to lysine that forms the sixth
residue of the EF segment and the 82nd of the chain.
Invariant amino acids are HisF8 that anchors the heme
to the protein moiety at the proximal side of the heme
(thus known as the proximal His) and PheCD1 that
wedges the heme into its pocket (Fig. 1). O2 binds at
the distal side of the heme, where a distal HisE7 is
highly conserved.
The physiological properties of Hbs are a product of their intrinsic O2 -binding properties that are
determined by the protein structures, and the effects
of physico-chemical factors, e.g. pH, temperature and
the cofactor concentrations, that modulate O2 binding.
The functional adaptations depend on homotropic interactions (cooperativity between the O2 binding heme
groups that is basic to the sigmoid shape of the O2
equilibrium curve) and the generally inhibitory heterotropic ones between effector binding sites and the
hemes (Fig. 2). The latter is exemplified by the Bohr effect (decreased O2 affinity at low pH). The homotropic
143
Fig. 1. Left: Side view of the 2 2 -dimer where the packing contacts are shaded and the 1 2 and 2 1 sliding contacts are hatched [modified
after Dickerson and Geis (1983)]. Right: The heme positioned between the E and F helices showing proximal and distal histidines HisE7 and
HisF8, the invariant PheCD1 and the bound O2 molecule [modified after Pesce et al. (2002)].
Fig. 2. Upper panel: The oxygenation reaction of vertebrate Hb involves breakage of salt bridges and the liberation of allosterically bound
effectors, like protons, chloride and organic phosphates, cdB3 (the cytoplasmic domains of the erythrocytic membrane protein, band 3) and of
heat, whereby the tetrameric Hb molecule shifts from the tense (T, low-affinity) to the relaxed (R, high-affinity) conformation. Lower left panel:
O2 dissociation curves showing that allosteric effectors and increased temperatures decrease O2 affinity (increase P50 ) altering the O2 liberated
for a given difference between arterial (a) and venous (v) O2 tensions (indicated by vertical arrows). Lower right panel: As shown by the lower
and upper asymptotes of the Hill plot, the effectors decrease the O2 affinity of the T-state (KT ) without markedly affecting that of the R-state
(KR ) thereby increasing cooperativity at half-saturation (slope of the Hill plot around P50 ). The Root effect (decreased O2 carrying capacity at
low pH seen in some fish Hbs) is associated with apparent anti-cooperative interactions (slopes 1).
144
and heterotropic allosteric interactions increase the O2

capacitance (turnover) for a given O2 tension difference between the sites of O2 loading (gills/lungs) and
unloading (tissues).
As we shall see, important sites controlling O2
binding in vertebrate Hbs are the heme-protein contacts, the intersubunit interfaces (Fig. 1) that may affect changes in protein quaternary structure, and the
amino acid residues implicated in effector binding (protons, organic phosphates and chloride) that are localized mainly at the N- and C-termini and in the central
cavity.
2.1. Cooperativity
In transporting O2 , Hb needs to fulfil conflicting
tasks of binding O2 at the respiratory surfaces and
releasing it in the tissues. Tetrameric vertebrate Hbs
may assume two major conformations, the high-affinity
oxygenated R (relaxed) state that prevails in the lungs
or gills, and the low-affinity, deoxygenated T (tense)
state that is constrained by salt bridges and hydrogen bonds and is found predominantly in the tissues
(Fig. 2). The tetramer consists of two rigid dimeric
units (1 1 and 2 2 ). According to the model originally proposed by Perutz (1970) that still is largely
valid, O2 binding at the hemes in the T-state causes the
iron atom to change its spin state and to move into
the plane of the porphyrin ring of the heme group.
These movements that are transmitted via the proximal HisF8 to the F helix, weaken the salt bridges between the two dimers and induce the protein to switch to
the R-conformation state as the two dimers rotate
relative to each other (Arnone, 1974) (Perutz, 1979).
Major changes occur between helix C1 and the corner FG2 , a region that is commonly called the switch
interface (Fig. 1). The T R shift is basic to cooperativity, the progressive increase in O2 affinity upon
O2 binding that is reflected by the characteristic sigmoid shape of O2 equilibrium curves. Cooperativity is
conveniently expressed by the Hill coefficient, n50 , the
slope of the Hill plot (log[oxy]/[deoxy] versus log PO2 )
at 50% saturation. For human Hb n50 is typically close
to 2.8. Precise Hb-O2 equilibria that include extreme
(low and high) saturation values allow assessment of
the O2 association equilibrium constants of the T and
R-states (KT and KR , respectively), as well as the intrinsic Adair association constants for successive bind-
ing of the 4O2 molecules (k1 , k2 , k3 , and k4 ) to Hb

(Imai, 1982) (Fig. 3).
2.2. Allosteric effectors
The most important proton binding sites for the Bohr
effect (Table 1) are the last amino acid residues of the
subunits (HisHC3) and the N-terminal ones of the
subunits (Val NA1). In T-state human Hb, the imidazole ring of HisHC3 makes a salt bridge with the negatively charged Asp FG1, which increases its affinity
for protons. In the R-state these protons are released, as
the two residues move away from each other. Val NA1
similarly has higher proton affinity in the T-state upon
binding chloride ion.
The main erythrocytic phosphate effector in mammals, DPG, binds in the central cavity between the
chains, at Val NA1 of one chain, and at HisNA2,
LysEF6 and His H21 of both chains, whereas chloride binding predominantly occurs at two sites, one
between Val NA1 and Ser H14 and the other between LysEF6 and Val NA1, and CO2 binds to the
unprotonated N-terminal residues Val NA1 of both
chains (Arnone, 1974) (Perutz, 1983). Thus, substitutions at very few sites (notably the seven residues mentioned here) can drastically alter the sensitivity of Hb
to the major allosteric effectors. In ectothermic vertebrates and birds the main phosphate effector is ATP
and IPP, respectively. Many fish species also have GTP
that forms an additional hydrogen bond with Hb compared to ATP and thus imparts a greater allosteric effect
(Lykkeboe et al., 1975; Weber et al., 1976; Gronenborn
et al., 1984). Some air breathing teleosts additionally
have high erythrocytic DPG levels, as in the catfish Hoplosternum, and IPP, as in Arapaima gigas and lungfish
(Val, 2000), and frogs often use ATP and DPG.
2.2.1. Water as an allosteric effector
As implicit in the direct correlation between O2
affinity and the chemical potential of water, human
HbA binds 60 additional molecules upon the transition from the T to the R-state, which agrees with
the concomitant increase in its water accessible surface area (Colombo et al., 1992; Colombo and BonillaRodriguez, 1996). A recent study (Hundahl et al., 2003)
documents analogous effects (reflecting oxygenationlinked binding of 1731 water molecules) in the cathodic trout HbI and eel HbC, and larger, mutually sim-
145
Fig. 3. Bohr effect of tench (Tinca tinca) Hb, and (inset) the pH dependence of P50 and the Adair O2 association contants for binding the first,
second, third and fourth O2 molecules (k1 , k2 , k3 and k4 , where k1
= KT and k4
= KR ). Under erythrocytic pH conditions (7.47.6) pH decrease
lowers k1 k3 (the affinities of the partially oxygenated Hb molecules) more than that (k4 ) of the almost fully oxygenated Hb. As shown, the
major affinity shift (conformational change) that occurs after binding the second O2 molecule at high pH and is delayed to after binding the third
molecule at low pH, due to proton stabilization of the T-state. Analogous effects are induced by the autochthonous phosphate effectors (ATP
and GTP) [modified after Weber et al. (1987)].
Table 1
Functionally important amino acid residues in tetrameric Hb subunits
-Chain
Helical position
Residue number in human Hb
Effector bindinga
Mammals
Human
Cow
Vicunia
Elephant
Birds
Bar-headed goose HbA
Ruppels griffon HbA
Amphibians
T. peruvianus
Anodic fish Hbs
Trout HbIV
Eel HbA
Cathodic fish Hbs
Trout HbI
Eel HbC
a
b
c
d
-Chain
NA1
1
H Clb
H2
119
H14
131
NA1
1
P
NA2
2
P
D6
55
EF6
82
P Clc
F9
93
FG1
94
H21
143
P
HC3
146
H
Val
Val
Val
Val
Pro
Pro
Pro
Pro
Ser
Asn
Asn
Ser
Val
Val
Val
His
Met
His
Asn
Met
Met
Met
Leu
Lys
Lys
Lys
Lys
Cys
Cys
Lys
Cys
Asp
Asp
Asp
Asp
His
His
His
His
His
His
His
His
Val
Val
Ala
Pro
Ala
Ala
Val
Val
His
His
Leu
Ile
Lys
Lys
Cys
Cys
Asp
Asp
Arg
Arg
His
His
ac-Xd
Asp
Ala
Val
His
Ser
Lys
Ser
Glu
Lys
His
ac-Ser
ac-Ser
Pro
Pro
Gln
Ser
Val
Val
Asp
Glu
Met
Met
Lys
Lys
Ser
Ser
Glu
Glu
Arg
Arg
His
His
ac-Ser
ac-Ser
Pro
Ala
Ala
Ala
Val
Val
Glu
Glu
Met
Gln
Leu
Lys
Ala
Asn
Asn
Glu
Ser
Lys
Phe
Phe
Proton (H), chloride (Cl) and organic phosphate (P) binding sites.
Also interacts with 131-residue.
Also interacts with 1-residue.
Acetylated unidentified amino acid residue. All sequences are available from the Swiss protein database.
146
ilar ones (binding of 4243 molecules) in anodic trout,

eel and human Hbs. This supports the earlier observation that changes in the chemical potential of water
induced by adding glycerol reduces the O2 affinity of
carp Hb (Kwiatkowski and Noble, 1993) and indicates
that the reported lack of solvation effects in cathodic
trout HbI (Bellelli et al., 1993) was due to the secondary
effects of the osmolyte choice.
The view that only a few (of the 287 or so) amino
acid residues of vertebrate Hbs play key roles in the expression of functional properties aligns with the neutral
theory of molecular evolution (Kimura, 1979) that the
majority of amino acid substitutions are non-adaptive
and harmless. However the experience that many substitutions are required to transplant special characters
of fish and crocodilian Hbs (Root effect and bicarbonate sensitivity, respectively) into human HbA indicate
that protein function may be fine tuned by mutations
distant from the residues directly implicated (Naoi et
al., 2001).
2.3. Temperature sensitivity
Mandated by the exothermic nature of the oxygenation reaction, the O2 affinity of globins decrease with
increasing temperature; this effect may be adaptive in
increasing O2 unloading in parallel with increasing
metabolic rate, or maladaptive in hampering O2 loading under hypoxia. As illustrated below, endothermic
processes that counter the exothermy of this reaction
include the O2 -linked release of protons (the Bohr effect) and anions (cf. Fig. 2) and conformational changes
associated with oxygenation (Wyman et al., 1977;
Weber and Wells, 1989).
3. Environmental and physiological

adaptations case studies
Adaptive changes in Hbs functional properties may
be mandated by exogenous and endogenous factors
such as ambient hypoxia, respiratory organ dysfunction and increased tissue O2 demands. Aquatic habitats
readily become hypoxic due to the 30-fold lower O2
content in aerated water, and 250,000-fold lower O2
diffusion rates in water compared to air. The deep-sea
marine hydrothermal vents may provide the harshest
conditions experienced by metazoans: high pressures
and extreme variations in temperature and in the levels of O2 , H2 S, CO, heavy metals and arsenic compounds, all of which may influence Hb oxygenation.
Air breathers normally only experience hypoxia at extreme altitudes. Thus inhaled O2 tensions are approximately 92, 48 and 36 mm Hg for Andean camelids and
frogs living at 4 km above sea level, for the bar-headed
goose crossing the Himalayas and for Ruppells griffon
soaring at 11.3 km (compared to 155 mm Hg at sea
level).
3.1. Agnathans links between invertebrate and
vertebrate Hbs?
Agnatha that include lampreys and hagfishes, considered the most primitive living vertebrates and chordates, respectively, have peculiar Hb systems. In contrast to the tetrameric Hbs from higher vertebrates,
agnathan Hbs exist as monomers when oxygenated
and form complex patterns of dimers or tetramers
when deoxygenated (Rumen and Love, 1963; Fago and
Weber, 1995; Fago et al., 2001). This oxygen-linked
reversible aggregation that replaces the TR equilibrium of tetrameric Hbs is responsible for pseudocooperative O2 binding and for the Bohr effect.
In contrast to vertebrate Hbs, where the heme groups
are well separated and allosteric interactions are mediated by wide-ranging protein conformational changes,
the heme-bearing helices of dimeric agnathan Hbs are
in direct contact with each other, resembling intracellular invertebrates Hbs. The dimeric, deoxygenated form
of the sea lamprey Petromyzon marinus HbV (Heaslet
and Royer, 1999) and the hagfish Epatretus burgeri
F1 (Mito et al., 2002) have similar subunit interfaces
comprising the E helix and the AB corner, an arrangement that resembles that of the EF-dimer found in the
Hb from the mollusc Scapharca inaequivalvis (Royer
et al., 1989, 2001) rather than the subunit contacts of
tetrameric vertebrate Hbs.
The Bohr effect is pronounced in lampreys Hbs
(Nikinmaa, 1993) but usually weak in hagfish Hbs
(Fago and Weber, 1998). Unexpectedly, the Bohr effect of lamprey P. marinus HbV originates from the increased proton affinity of two negatively charged Glu
residues (in position 31 and 75 of each subunit), which
are brought close to each other in the dimeric, deoxygenated structure where they share a proton (Heaslet
and Royer, 1999). In the hagfish Myxine glutinosa, the
magnitude of the Bohr effect correlates with the presence of the same Glu residues (Fago et al., 2001).
Two distinctive features of hagfish Hbs are the allosteric binding of bicarbonate ions that decrease O2
affinity (Fago et al., 1999), and of water molecules
that, in contrast to vertebrates, decrease O2 affinity by
stabilizing the deoxygenated state of the Hb in solution and in intact erythrocytes (Muller et al., 2003).
Interestingly, a water cluster at the interface between
the two monomers has been identified in the deoxygenated, dimeric S. inaequivalvis Hb, that exhibits a
similar sensitivity to water activity as Myxine (Royer
et al., 1996). Whereas the physiological significance
of the water effect in the Hbs of hagfishes, that apparently have not experienced changes in water osmolality during evolution, may be disputable, the effect
of bicarbonate is clearly linked to the lack of the anion exchanger membrane protein (band 3) (Ellory et
al., 1987; Peters and Gros, 1998). Bicarbonate originating from the hydration of CO2 in erythrocytes thus
cannot pass into the plasma and instead acts as an allosteric cofactor facilitating O2 delivery as well as CO2
hydration (Fago et al., 1999). This scheme of linkage between CO2 and O2 transport is a unique among
chordates.
3.2. Fish
Inter- and intraspecic adaptations Hbs from
teleosts probably display the most extensive variation in intrinsic O2 affinities and cofactor sensitivity
amongst vertebrates. The variation reflects the large diversity in physico-chemical conditions that fishes are
exposed to, ranging from cold (1.9 C), O2 -laden polar waters to the warm, anoxic tropical waters. As a
corollary to this, the Antarctic icefish living in environments where ambient O2 tension is high and metabolic
O2 demand is low, are the only vertebrates completely
lacking Hb, and the closely related Gymnodraco acuticeps exceptionally has a single Hb without a Bohr
effect (Tamburrini et al., 1992).
As reviewed earlier (Weber, 1996, 2000; Jensen et
al., 1998a; Val, 2000), intraspecic adaptation of fish
Hb function to exogenous factors like hypoxia and temperature commonly occurs through changes in the concentration of erythrocytic effectors, particularly of GTP
that exerts a greater effect on Hb-O2 affinity than ATP
(see Section 2.2).
147
Interspecic adaptation (involving gene-based

changes in Hbs structure and intrinsic O2 -binding
properties) is neatly illustrated in two closely related
Amazonian osteoglossids. Here the higher blood O2
affinity in the water-breathing Osteoglossum bicirrhosum than in the obligate air-breathing Arapaima
gigas reflects the affinity difference in the respective
purified (stripped) Hbs (Johansen et al., 1978).
Analogously, stripped Hb of the zoarcid Thermarces
cerberus that is endemic to hydrothermal vents shows
a markedly higher O2 affinity than that of Zoarces
viviparus from shallow temperate waters (Weber et al.,
2003). Interestingly, a high intrinsic O2 affinity in the
cathodic Hb from the anguillid Symenchelis parasitica
that frequents vent habitats for food correlates with
loss of chloride sensitivity (Weber et al., 2003), as
seen in Andean frog and vicunia Hbs (see Sections 3.3
and 3.7).
Most fish express multiple isoHb components.
Based on their electrophoretical mobility at pH >8.0
and functional characteristics, fish isoHbs can be
categorised as anodic, with relatively low O2 affinities
and pronounced Bohr and Root effects or cathodic,
with high affinities, a reverse Bohr effect in the
absence of organic phosphates and no Root effect.
Whereas some species like carp only possess anodic
Hbs as in other vertebrates, others like eels, salmonids
and catfishes have both.
Cathodic Hbs are highly sensitive to organic phosphates and thus are primarily implicated in phosphatemediated adaptations in blood O2 affinity to prevailing O2 tensions. A notable exception is the phosphateand pH-insensitive cathodic HbI from trout (Binotti et
al., 1971), where the main phosphate and proton binding residues have been eliminated (Table 1). In the absence of organic phosphates, cathodic Hbs commonly
display a characteristic reverse Bohr effect (O2 affinity increases with pH decrease) that is obliterated by
phosphates (Garlick et al., 1979; Fago et al., 1995;
Weber, 2000). In these Hbs the major Bohr group,
HisHC3, is typically replaced by Phe whereas (except
in salmonids like trout) the positively charged phosphate binding residues are conserved (Table 1). The
reverse Bohr effect appears to be due to electrostatic
repulsions between these positively charged residues
that at low pH destabilize the T-state and shift the allosteric equilibrium towards the high-affinity R-state
(Fago et al., 1995).
148
Due to their high O2 affinity and pH insensitivity

the cathodic fish Hbs are considered to be adaptive to
hypoxia and acidotic spells. The view that they serve as
circulating O2 reserves when anodic components loose
O2 binding capacity at low pH is countered by findings
that red cell pH does not fall but actually increases
in trout exposed to burst exercise due to activation of
the adrenergic Na+ (in)/H+ (out) exchanger; moreover,
the Root effect in fish correlates not only with the presence of swimbladders, eye rete chorioidea but also with
the red cell Na+ /H+ exchanger (Primmett et al., 1986;
Pelster and Decker, 2004).
The Root effect. This effect that is observed in nearly
all anodic teleost fish Hbs (reviewed by Pelster and
Weber, 1991) is instrumental in secreting O2 into the
swim bladder for buoyancy regulation and in supplying O2 to the avascularized, metabolically active retina.
From a molecular point of view it is due to an overstabilization of the T-state that blocks the transition to the R
conformation upon O2 binding. It is thus characterized
by Hill coefficient values close to or even below unity
(Fig. 2), the latter indicating functional heterogeneity
of the subunits (Noble et al., 1986).
The molecular mechanism of the Root effect has
puzzled scientists for decades and a number of stereochemical models have been proposed. The original
stereochemical model (Perutz and Brunori, 1982) suggesting a crucial role of Ser F9 was disproved by
site mutagenesis studies (Luisi et al., 1987). With the
elucidation of the crystallographic structures of the
Hbs from spot (Leiostomus xanthurus) (Mylvaganam
et al., 1996) and from the Antarctic fish Pagothenia
bernacchii (Ito et al., 1995) and Trematomus newnesi
(Mazzarella et al., 1999) it became increasingly clear
that the Root effect derives from a concerted action
of different residues located in various regions of the
Hb molecule. We have proposed that the Root effect
requires: (1) a specific environment of the switch interface, that favours the T-state conformation at low pH
and does not change to the R-state upon O2 binding, (2)
HisHC3 and GluFG1 as major Bohr groups, and (3)
conservation of the positively charged residues in the
central cavity where organic phosphates bind (Fago et
al., 1997). This model is supported by the observation
that fish Hbs showing changes in at least one of these
three regions do not show the Root effect.
A recent solution of the crystal structure of tuna
Hb (J.R.H. Tame, personal communication) reveals a
novel low pH, T-state salt bridge between HisE13 and

AspE16 that is broken in the R-state with the release
of one proton, and binding of an additional proton to the
T-state between a pair of carboxyl groups, AspG11
and AspG32 that may account for up to half of the
Root effect in the Hb from the Antarctic fish P. bernacchii (Ito et al., 1995). Together with earlier demonstrated importance of the -chain C-terminal His, these
results confirm that the Root effect may be brought
about by different mutations in fish (Yokoyama et al.,
2004) or involvement of several residues. Supporting
the concept that several evolutionary pathways have
resulted in the expression of Root effect Hbs, a recent
study (Bonaventura et al., in press) provides evidence
for large steric components that act in concert with the
quaternary shifts between relaxed and tense conformations to regulate ligand affinity in Root effect as well as
non-Root effect Hbs. Final proof of a Root effect must
await its transplantation in a non-Root-effect Hb.
Temperature sensitivity adaptations. Tunas and
lamnid sharks have core temperatures that exceed ambient values by up to 20 and 810 C, respectively, and
are the only fish that have (independently) developed
endothermy based on countercurrent heat exchangers,
rete mirabile. The P50 values of blood and Hb of bluefin
and skipjack tuna are virtually temperature independent (Rossi-Fanelli et al., 1960; Brill and Bushnell,
1991) implying that the Hb will not liberate heat upon
O2 binding in the gills, which will contribute to maintaining warm bodies. In bluefin tuna Hb temperature independence at half-oxygenation ensues from a normal
temperature effect at low O2 saturations and a reverse
one at high saturations, due to endothermic dissociation
of a large number of Bohr protons (Ikeda-Saito et al.,
1983). In the shark Lamna nasus the normal temperature effect at half-saturation is reversed in the presence
of ATP, which may be expected to reduce the O2
tension gradient (and thus O2 transfer) between arterial
and venous blood in the countercurrent exchangers
(Larsen et al., 2003).
In trout HbI the low temperature sensitivity of P50
found for CO binding is attributed to endothermic conformational changes occurring within the subunits in
the T-state that reverses the temperature sensitivity at
low saturation (Wyman et al., 1977). A similar effect
for O2 binding is seen in cathodic cutthroat trout Hb
(Southard et al., 1986). In other Hbs the decreased
temperature effect associated with proton binding ap-
149
pears to be manifested over the full O2 saturation range

(Jensen and Weber, 1987).
positive charges between the chains (Weber et al.,

2002; Tame, 2003).
3.3. Amphibians water to air transition
3.4. Non-crocodilian reptilians
The ontogenetic development of amphibians epitomizes the evolutionary transition from water to air and
reflects adaptation to increased O2 availability. Accordingly blood O2 affinities in amphibians decrease as air
breathing takes on importance (Lenfant and Johansen,
1967) and in the transition from tadpole to adult. Ontogenetic changes are illustrated in the bullfrog where
the tadpole development is associated with the successive proliferation of different populations of red cells
whose Hbs exhibit progressively lower O2 affinities
(Watt and Riggs, 1975). The major tadpole Hb (III)
lacks a Bohr effect (which maintains a high-affinity
at low pH) due to acetylation of NA1 residues and
substitution of AspFG1 (Watt et al., 1980) that otherwise forms the H-bond with the -chain C-terminal
His accounting for most of the Bohr effect. In the adult,
two of the major isoHbs (B and C) aggregate to form
a trimer of tetramers (BC2 ) that exhibits a lower O2
affinity than either component at low saturation (Tam
and Riggs, 1984). The resulting biphasic cooperativity
(that increases sharply at half-saturation) will favour
extraction of lung O2 reserves during dives (Maginniss
et al., 1980) if indeed the isoHbs occur in the same
erythrocytes.
Another interesting case is water-breathing Andean
frogs living in lakes and streams at 3.8 km altitude.
Telmatobius culeus from Lake Titicaca has poorly developed lungs and high blood O2 affinity and O2 carrying capacity compared to sea level anurans (Hutchison
et al., 1976) and bobs in hypoxic water to ventilate
its oversized vacularized skin. The major Hb of T. peruvianus combines a high O2 affinity with an almost
complete obliteration of chloride sensitivity. This novel
mode of altitude adaptation is attributable to modification at two -chain chloride binding sites (cf. Table 1):
acetylation of the NH2 terminal residues and replacement of polar Ser at position H14 by non-polar Ala
(Weber et al., 2002). Together with data on Hbs from
vicunia (below) and human embryonic Hbs (Zheng et
al., 1999) this indicates that chloride binds at discrete
sites and thus conflicts with the view (Perutz et al.,
1994) that chloride functions through general electrostatic effects within the protein by decreasing excess
Snake and turtle Hbs commonly exhibit high pH and

phosphate sensitivities, markedly saturation-dependent
cooperativities and distinct isoHb functional heterogeneity all indicative of pronounced O2 transport regulatory capacities.
Turtles like many birds express two main isoHbs,
HbA and HbD, that have the same chain but differD
D
ent -chains (A
2 2 and 2 2 ). The -chains may be
products of embryonic genes with persistent expression
in adults (Rucknagel et al., 1984). The two Hbs differ
markedly in their sensitivity to phosphates and CO2
(Reischl et al., 1984; Torsoni et al., 1997) indicating
division of labour. In Trachemys scripta a functional
interaction between the Hbs is witnessed by a higher
O2 affinity in the isolated isoHbs than in 1:1 mixtures
(Frische, 2000) in the absence as well as the presence
of organic phosphates. Physiological relevance of this
interaction follows from the unimodal distribution of
O2 affinities of individual red cells indicating that the
components concur in the same cells (Frische et al.,
2001).
Several snake Hbs exhibit unusual properties, being
dimeric when oxygenated and tetrameric when deoxygenated. Their Bohr effect, cooperativity and tetramerization are greatly enhanced by ATP (Ogo et al., 1984;
Oyama et al., 1993; Bonafe et al., 1999). In Helicops
modestus Hb, ATP-induced tetramerization correlates
with two -chain amino acid substitutions that delete
two negative charges at the 1 2 interface (Bonafe et
al., 1999). Molecular adaptation to habitat is indicated
by a higher intrinsic Hb-O2 affinity and smaller Bohr
factor in sluggish aquatic Helicops modestus than in
active terrestrial Liophis miliaris (Ogo et al., 1979).
3.5. Those dreadful crocodilians
Crocodiles (and hagfish) appear to be unique in
that their Hbs are sensitive to bicarbonate ions rather
than organic phosphates (Bauer et al., 1981). The effect may play a role in unloading O2 during the postprandial alkaline tide when blood [HCO3 ] peaks
and [Cl ] falls due to anion exchanges across the gut
wall (Weber and White, 1986). The bicarbonate effect
150
in crocodilian Hb was considered to result from three

or four mutations in the central cavity that converts the
phosphate binding site to two bicarbonate binding sites
(Perutz, 1983). However, studies involving the creation
of mixed (crocodile/human) tetrameric molecules and
site-directed mutagenesis revealed that as many as 12
mutations, including some in the chains, were required to introduce HCO3 sensitivity in human HbA
(Komiyama et al., 1995) supporting the view (Naoi
et al., 2001) that interactions in distant parts of Hb
molecules may affect the reactivity of specific ligand
binding sites. Interestingly, crocodilian Hbs do show
phosphate sensitivity at low Cl-levels as found in the
erythrocytes during the alkaline tide (Weber and White,
1994).
3.6. Birds champions in altitudinal hypoxia
The O2 binding properties of bird Hbs are an integrated part of wide-ranging pulmonary and cardiovascular adaptations (Scheid, 1990). Birds like turtles possess a major component, HbA and commonly also less
abundant HbD that shares the same -chain, whereby
functional heterogeneity is due to -chain amino acid
exchanges. Super-cooperativity (Hills n values > 4),
which is observed at high O2 saturation in chicken
blood and in the Hb in the presence of IHP, and ostensibly increases O2 delivery particularly to the flight muscles, is attributable to self-association of the tetrameric
Hbs, that, in the case of HbD, appears to occur at the
hydrophobic patch that involves D and E helices of the
subunits (Knapp et al., 1999).
A fascinating case is the bar-headed goose that
crosses the Himalayas at altitudes near 10 km and has a
higher blood O2 affinity than the closely related greylag
goose from lower altitudes (P50 = 29.7 and 39.5 mm
Hg, respectively at 37 C and pH 7.4). Its major Hb
shows one amino acid substitution that is not encountered in other birds or mammals, viz., ProH2 Ala,
which results in loss of a H-bond with D6 at the
1 1 interface that otherwise stabilizes the T-structure
(Perutz, 1983; Weber, 1995). Interestingly the same
bond is broken by the Leu D6 Ser substitution in
the high-altitude Andean goose. Recombinant human
Hbs with the same mutations (in the and chains,
respectively) as in these two geese engineered by sitedirected mutagenesis show increases in O2 affinity similar to those seen when comparing highland to lowland
geese, indicating that disruption of a single (H2-D6)

bond is basic to the increased blood O2 affinities found
in species from widely separated geographical regions.
This is moreover supported by the small difference in
bond energy between the deoxy states of the mutants
and native human Hbs (Jessen et al., 1991; Weber et
al., 1993). This study illustrates that the relatively small
changes between tightly packed complementary 1 1
(and 2 2 ) surfaces are energetically effective in increasing O2 affinity. Interestingly the ProH2/MetD6
intradimer contact is also altered in other high-oxygen
affinity Hbs, including fish cathodic Hbs (Table 1).
Another factor that may contribute to the high O2
affinity in bar-headed goose Hb is the lack of the salt
bridge between HisHC3 and AspFG1 that destabilizes the T-state and reduces the Bohr effect (Liang
et al., 2001). Additionally, the Hb shows a Ser
AlaH14 substitution that may increase affinity via decreased Cl interaction as in the Andean frog (see Section 3.3).
The African griffon Gyps rueppelli (recorded at a
record 11.3 km altitude) has four isoHbs (A, A , D and
D ) with differentiated O2 affinities that provides basis
for isoHb division of labour under different O2 tension regimes. The differentiation correlates with specific amino acid exchanges that variously stabilize and
destabilize the T and R structures in human Hb (Hiebl
et al., 1988; Weber et al., 1988).
3.7. Mammals the importance of NA2 residue
in altitude adaptation
The dichotomy in mammalians Hbs, that consist
of high-affinity Hbs with high phosphate sensitivities
(the majority) as well as low-affinity Hbs with small
or no phosphate effects (found in ruminants and cats)
(Bunn, 1971), is attributed primarily to exchanges at
position NA2. In the former group this is occupied
by a hydrophilic residue and in the latter by a large
hydrophobic residue, whose side chain points inwards
stabilizing the T-state of Hb and mimicking the effect
of phosphates (Perutz and Imai, 1980; Perutz, 1983).
In several ungulates Hbs NA1 is deleted and NA2
is Met, modifications that virtually abolish their phosphate sensitivities. The high blood O2 affinity of highaltitude Andean camelids like llama is analogously due
to the HisNA2 Asn replacement that diminishes
DPG interaction. In vicunia blood O2 affinity is raised
by an additional increment (Jurgens et al., 1988) associated with the Ala H13 Thr substitution, which introduces a polar group that may interfere with chloride
binding at neighbouring AsnH14 (Kleinschmidt et al.,
1986; Piccinini et al., 1990) (Table 1). Despite their
lowland habitats, elephants have a high blood O2 affinity that aligns with the inverse, scaling relationship
between blood O2 affinity and body weight in mammals. This is similarly ascribable to AsnNA2, which
reduces DPG affinity (Braunitzer et al., 1982) and thus
may have helped Hannibals elephants to cross the Alps
in 218 BC (Perutz, 1983).
4. New members of the globin family:

neuroglobin (Ngb) and cytoglobin (Cygb)
Ngb and Cygb are newly discovered vertebrate
globins. Whereas Ngb is cytosolic and expressed
mainly in the brain and in other nervous tissues like
the retina, Cygb is found in the nuclei and cytoplasm
of most cell types (Burmester et al., 2000, 2002; Trent
and Hargrove, 2002; Schmidt et al., 2003; Geuens et
al., 2003). In contrast to deoxygenated Hb and Mb,
where the heme group is coordinated only to the proximal HisF8 and the distal O2 -binding site at the heme is
free, in Ngb and Cygb heme is also bound to the distal
HisE7, which impedes access to O2 and other external
ligands (Dewilde et al., 2001; Pesce et al., 2002). Such
heme groups are referred to as hexacoordinate since
all of the six iron coordination positions are occupied
whereas the heme of deoxygenated Hb and Mb is
pentacoordinate.
Hexacoordinate hemes appear to be widespread
among invertebrate nerve Hbs and plant Hbs (Kundu et
al., 2003). Amino acid sequence analyses show greater
homologies to invertebrate nerve Hbs than to Hbs and
Mbs of the same species and suggest that Ngbs have an
ancient phylogenetic origin (Burmester et al., 2000).
Phylogenetic analyses indicate that the more recent
Cygbs may share common ancestry with vertebrate
Mbs, which possibly evolved to meet the special needs
of muscle cells (Burmester et al., 2002).
As for other hexacoordinate globins, the physiological role of Ngb and Cygb remains unclear, although
Ngb has been shown to have a protective effect on
neural tissue during acute hypoxia (Sun et al., 2001).
This may relate to its ability to function as a temporary
151
O2 reserve (Dewilde et al., 2001) in analogy to Mb and

invertebrate nerve Hbs (Kraus and Colacino, 1986).
This hypothesis seems consistent with the relatively
high local levels of Ngb (0.1 mM) estimated in the
mitochondria-rich segment of retinal rods (Schmidt et
al., 2003), although the concentration levels reported
in brain cells are only in the -molar range (Burmester
et al., 2000). Our recent results show that human Ngb
may prevent extensive cellular damage by scavenging
reactive oxygen and nitrogen species (such as NO
and peroxynitrite) generated at high levels in response
to brain hypoxia (Herold et al., 2004). Interestingly,
in retinal cells Ngb is localized in close proximity
to mitochondria that are potential sites of O2 radical
generation. The role of Ngb as NO scavenger in brain
cells is of particular interest because NO competes
with O2 to reversibly bind cytochrome c oxidase,
thereby inhibiting mitochondrial respiration (Garry
and Mammen, 2003). Another recent study shows
that formation of an internal disulfide bond increases
O2 affinity in human Ngb and Cygb, suggesting
that these proteins may be sensitive to changes in
the redox state of the cell (Hamdane et al., 2003).
Although not yet fully understood, Ngb and Cygb
may serve other specific functions possibly related
to their different (cytosolic and nuclear) localizations
and molecular weights (Ngb is monomeric, Cygb is
dimeric).
5. Interactions between NO and Hb: a role in

vasodilation?
Hb binds and releases not only O2 , but also nitric
oxide (NO), which is now recognized to be pivotal for
an increasing number of key physiological responses,
such as vasodilation, neurotransmission and immune
defense (Moncada et al., 1991). The discovery in 1987
that the long-sought endothelium-derived relaxing factor that regulates vasodilation is indeed NO (Ignarro et
al., 1987), the ligand with the highest affinity for Hb
(K 1012 ) (Kharitonov et al., 1996) posed the puzzle
of how NO is able to target and activate the vascular
smooth muscle without being scavenged by Hb. Subsequent studies have shown that Hb enclosed in red blood
cells is a far less effective NO scavenger because of the
diffusion resistance of the red cell membrane (Huang
et al., 2001a) and the existence of an erythrocyte free-
152
zone near the NO-producing endothelium (Vaughn et

al., 1998).
An alternative fascinating hypothesis emerged in
1996, with the discovery that a small fraction of Hb
in the blood carries NO bound to the thiol groups
of CysF9 as S-nitroso adduct (SNO-Hb) (Jia et al.,
1996). Since reactivity of CysF9 in human Hb is controlled by the quaternary state of the protein, it was
proposed that the nitrosated form of this residue is sensitive to the O2 -linked, allosteric changes of Hb. Indeed, SNO-Hb concentration was originally found to
be lower in venous than in arterial blood, suggesting
that SNO-Hb may release NO when the O2 tension
decreases, thus increasing vasodilation and blood flow
when tissues become hypoxic (Jia et al., 1996; Stamler
et al., 1997; McMahon et al., 2000, 2002). According
to this model, the allosteric RT transition of Hb results in a concerted release of O2 and NO, whereby Hb
assumes a novel physiological function, transporting
vasoactive NO in the circulation (Fig. 4). Needless to
say, this hypothesis has strongly boosted research in
this field worldwide.
A fundamental assumption is that the Hb-NO reaction serves to preserve NO bioactivity rather than to
consume it. It has been proposed (Gow et al., 1999) that
SNO-Hb originates in the arterial circulation where NO
preferentially reacts with the minor fraction (<1%) of
deoxygenated rather than oxygenated hemes. In a subsequent, O2 -dependent step, NO was considered to migrate from the heme to the side chain of CysF9 where
it would bind as nitrosonium cation (NO+ ) or, in the
absence of O2 , dissociate to form metHb and nitroxyl
anion (NO ) (Gow et al., 1999; Gow and Stamler,
1998).
Fig. 4. Release of NO from oxygenated S-nitrosylated Hb in a

transnitrosation reaction with thiols (e.g. cysteine, glutathione) mediated by the R T conformational change occurring during O2
release, according to the model proposed by Stamler and coworkers
(Gow et al., 1999; Jia et al., 1996; Gow and Stamler, 1998).
Although protein S-nitrosylation is now recognized

as a crucial redox-sensitive mechanism of cellular signalling (Stamler et al., 2001) its physiological role in
human Hb remains highly controversial. Gladwin et
al. (2000) and Hobbs et al. (2002) reported low (nmolar rather than -molar) red cell SNO-Hb levels
in humans and no difference between arterial and venous concentrations, raising doubts about the physiological relevance of SNO-Hb. Moreover, recent studies
have shown that NO does not appear to react preferentially with vacant hemes in partially carboxylated
or oxygenated Hb (Huang et al., 2001b; Joshi et al.,
2002; Han et al., 2002) nor to migrate to thiols following repetitive oxygenationdeoxygenation cycles (Xu
et al., 2003). More recently, we have shown that the
changes in absorbance spectra of NO-Hb due to reversible shifts between 5-coordinate and 6-coordinate
NO-heme may in previous studies have been attributed
to formation of SNO-Hb or met Hb under aerobic
and anaerobic conditions, respectively (Fago et al.,
2003).
Recent studies (Nagababu et al., 2003; Cosby et al.,
2003) illustrate how the decrease in the oxygenation
level of red blood cells may increase vasodilation, without formation of SNO-Hb. Accurate chemiluminescence measurements demonstrate that deoxygenated
Hb is able to reduce nitrite, originating from the reaction between NO and O2 . The product of such a redox
reaction, an unstable metHb-NO complex is considered
to release vasoactive NO. Exposing aquatic animals to
ambient nitrite increases cardiac pumping and vasodilation, suggesting the existence of a similar pathway of
NO generation from nitrite (Jensen, 2003).
Although it is unclear whether it occurs in mammals,
the SNO-Hb respiratory cycle may be active in other
vertebrates. Hb from spot fish appears to show oxygenlinked S-nitrosylation at residue CysH16, suggesting that the conservation of CysF993 is not essential
(Bonaventura and Lance, 2001). The lower SNO-Hb
content in primate than in rodent blood is strongly correlated with a decrease in thiol reactivity (Rassaf et al.,
2003). Clearly, further studies are needed to establish
the biological significance of S-nitrosylation in vertebrate Hbs. Turtles that show exceptional resistance to
hypoxia and have high erythrocytic glutathione concentrations (2 mM) (Reischl, 1986) and up to 1620
cysteine residues per Hb tetramer seem excellent candidates for further investigations.
6. Red cell membrane protein interactions

The dependence of key erythrocytic functions such
as glycolytic rates on the state of Hb oxygenation indicates a role for Hb as O2 sensor governing red cell
glycolysis. The molecular mechanism is considered to
be competition between deoxyHb and at least three glycolytic enzymes for binding to the cytoplasmic domain
of the red cell membrane protein band 3 (cdB3), which
binds at the organic phosphate site of human Hb decreasing its O2 affinity (Walder et al., 1984). Band 3
is known to mediate electroneutral Cl /HCO3 anion
exchange across red cell membranes. Curiously, however, 10-mer peptides corresponding to trout cdB3 do
not affect O2 affinity of trout Hbs but decrease that of
human Hb (Jensen et al., 1998b; Weber, 2000).
A recent study (Weber and Voelter, 2004; Weber
et al., 2004) of the interactions of trout, chicken and
human peptides with corresponding and other vertebrate Hbs show that the interaction is based on charge
complementarity between the anionic peptide and the
cationic Hb binding site and that it is reduced by organic
phosphates that compete for the binding site, and intracellular divalent cations like Mg2+ that may neutralize
cdB3 (Fig. 5). It also indicates small Hb-cdB3 interactions in fish and amphibian erythrocytes, which have
153
predominantly aerobic metabolism and exhibit large

cellular pH variations, but pronounced ones in chicken
and especially human erythrocytes, where the small
pH variations are unlikely to be effective in regulating
glycolysis. The recently reported interaction between
cdB3 and SNO-Hb may also be implicated in targeting NO (Bonaventura et al., 2002). Since band 3 is the
erythrocytic membranes major organising centre, the
deoxygenation dependent cdB3-Hb reaction may influence a number of other functions that vary with Hb
oxygenation, such as adrenergic Na+ /H+ exchange and
K+ /Cl cotransport seen in red cells of fish (Borgese
et al., 1987; Nikinmaa and Jensen, 1992). However,
definitive evidence is often lacking.
7. Concluding remarks
Globins or their genes appear to occur all living
organisms. Apart from Hb and Mb, with pentacoordinated heme groups, vertebrates have hexacoordinate
Ngb and Cygb found in cytoplasm and nuclei of
tissue cells. Although the exact physiological roles of
Ngb and Cygb remain unclear, Ngb is known to have
a protective role during acute hypoxia. In addition
to transporting and storing O2 (functions that have
Fig. 5. Effects of a 10-mer peptide corresponding to the N-terminal cytoplasmic domain of human band 3 protein (cdB3) on O2 binding of
human Hb in the absence and presence of (7 mmol L1 ) MgCl2 at 25 C and pH 6.968 0.026; showing that anionic chloride anions and cdB3
decrease O2 affinity (increase P50 ), whereas MgCl2 inhibits the cdB3 effect, ostensibly by partially neutralizing cdB3 charge.
154
been intensively studied in circulating Hb and muscle

Mb) globins may also be implicated in transporting
vasoactive NO, scavenging reactive oxygen and
nitrogen species generated in mitochondria (curbing
cellular damage) and acting as transducers that link
erythrocyte glycolytic rate to oxygenation state.
The links between O2 binding properties and molecular structures are becoming increasingly clear in the
pentacoordinate as well as hexacoordinate globins. Key
adaptations in Hb structure and function involve predominantly amino acid substitutions at sites that form
interchain contacts or sliding contacts (as the molecule
shifts between the deoxygenated and oxygenated structures), or the relatively few binding sites for effectors
like protons, chloride, organic phosphates. The broad
array of physiological and ecological O2 transport requirements observed among vertebrates correlate with
specific residue configurations at one or more of these
sites. The involvement of few sites limits the options
available for molecular adaptation, and may be basic to
convergent adaptations seen in phylogenetically distant
species. This is illustrated in high-altitude bar-headed
and Andean goose Hbs, where high Hb-O2 affinity is
attributed to two amino acid substitutions that delete
the same 1 1 contact, and by Andean frog Hb whose
high blood O2 affinity is associated with an exchange
that deletes a chloride binding site also seen in barheaded goose Hb. Analogously, the NA2 substitution that reduces phosphate sensitivity in ruminant Hb
is also present in an embryonic mouse and adult ostrich Hbs. However, as evidenced by studies to transplant specific characters (Root effect of fish Hb and
HCO3 sensitivity of crocodile Hb) into human Hb,
the reactivity of specific ligand binding sites may be
dependent on substitutions in distant parts of the Hb
molecule.
Although they are only part of the broad range of
adaptations exhibited at the molecular, cellular and systemic levels of biological organisation, globin adaptations can critically govern survival under specific environmental conditions and physiological requirements.
Acknowledgments
Supported by grants from the Danish Natural Science Research Council, and the EU (contract no.
QLRT-201-01548).
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Respiratory Physiology & Neurobiology 144 (2004) 141159

Functional adaptation and its molecular basis in vertebrate

1. Vertebrate globins in evolutionary

One hundred years after the discovery of the Bohr

all tissues in higher animals (Trent and Hargrove, 2002;

retinal tissues (Burmester et al., 2000), Cygb appears

2. Basic structural and functional properties

and heterotropic allosteric interactions increase the O2

ing of the 4O2 molecules (k1 , k2 , k3 , and k4 ) to Hb

ilar ones (binding of 4243 molecules) in anodic trout,

3. Environmental and physiological

Interspecic adaptation (involving gene-based

Due to their high O2 affinity and pH insensitivity

novel low pH, T-state salt bridge between HisE13 and

pears to be manifested over the full O2 saturation range

positive charges between the chains (Weber et al.,

3.3. Amphibians water to air transition

3.4. Non-crocodilian reptilians

Snake and turtle Hbs commonly exhibit high pH and

in crocodilian Hb was considered to result from three

geese, indicating that disruption of a single (H2-D6)

4. New members of the globin family:

O2 reserve (Dewilde et al., 2001) in analogy to Mb and

5. Interactions between NO and Hb: a role in

zone near the NO-producing endothelium (Vaughn et

Fig. 4. Release of NO from oxygenated S-nitrosylated Hb in a

Although protein S-nitrosylation is now recognized

6. Red cell membrane protein interactions

predominantly aerobic metabolism and exhibit large

been intensively studied in circulating Hb and muscle

Bohr, C., Hasselbalch, K., Krogh, A., 1904. Uber

eared turtle Trachemys scripta. J. Appl. Physiol. 90, 1679

Jurgens, K.D., Pietschmann, M., Yamaguchi, K., Kleinschmidt, T.,

Perutz, M.F., 1979. Regulation of oxygen affinity of hemoglobin:

Watt, K.W., Maruyama, T., Riggs, A., 1980. Hemoglobins of the

Yokoyama, T., Chong, K.T., Miyazaki, G., Morimoto, H., Shih,

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