Critical Review

Functional Properties of Neuroglobin and Cytoglobin. Insights into theAncestral Physiological Roles of Globins

Angela Fago, Christian Hundahl, Hans Malte and Roy E. WeberDepartment of Zoophysiology, Institute of Biological Sciences, Aarhus University, Aarhus C, Denmark

Summary

Neuroglobin and cytoglobin are two recently discovered

vertebrate globins, which are expressed at low levels in neuronaltissues and in all tissues investigated so far, respectively. Based on

their amino acid sequences, these globins appear to be

phylogenetically ancient and to have mutated less during evolutionin comparison to the other vertebrate globins, myoglobin and

hemoglobin. As with some plant and bacterial globins, neuroglobin

and cytoglobin hemes are hexacoordinate in the absence of external

ligands, in that the heme iron atom coordinates both a proximal anda distal His residue. While the physiological role of hexacoordinate

globins is still largely unclear, neuroglobin appears to participate in

the cellular defence against hypoxia. We present the current

knowledge on the functional properties of neuroglobin andcytoglobin, and describe a mathematical model to evaluate the role

of mammalian retinal neuroglobin in supplying O2 supply to the

mitochondria. As shown, the model argues against a significant suchrole for neuroglobin, that more likely plays a role to scavenge

reactive oxygen and nitrogen species that are generated following

brain hypoxia. The O2 binding properties of cytoglobin, which is

upregulated upon hypoxia, are consistent with a role for this proteinin O2-requiring reactions, such as those catalysed by hydroxylases.

IUBMB Life, 56: 689–696, 2004

Keywords Neuroglobin; cytoglobin; oxygen.

INTRODUCTION

The recent discovery of neuroglobin (Ngb) (1) and

cytoglobin (Cygb) (2, 3) has unexpectedly extended the family

of globin proteins, which was long believed to consist

exclusively of hemoglobin (Hb) and myoglobin (Mb) –

probably the two most extensively studied proteins.

A major factor that may have impeded an earlier discovery

of Ngb and Cygb is their low levels of expression (mM range)

in the tissues where they have been identified, viz. brain and

retinal tissues for Ngb and all tissues so far investigated for

Cygb. Both proteins have subsequently been identified in

several vertebrates, where they appear to have a widespread

distribution.

Ngb and Cygb exhibit the same overall tertiary structure as

Hb and Mb, their polypeptide chains folding into a-helicalsegments that characterize the globin fold (4, 5). However, the

quaternary structures vary, Ngb and Mb are monomeric,

Cygb is dimeric, whereas vertebrate Hb is typically tetrameric,

consisting of two a and two b chains.

A fundamental character distinguishing Ngb and Cygb

from Hb and Mb is heme hexacoordination (6), since in the

absence of external ligands (such as O2) the sixth coordination

position of the heme in Ngb and Cygb (in either the ferrous or

the ferric form) is occupied by the distal His (7). This residue

thus has to be displaced by external ligands binding to the

heme. In deoxy (ferrous) Hb and Mb the hemes are

pentacoordinate and the iron is freely accessible to ligand

binding, whereas in the met (ferric) form it binds a water

molecule. Prior to the discovery of Ngb and Cygb in

vertebrates, hexacoordination was known to occur in globins

from some plants, invertebrates and bacterial strains.

What is the function of these hexacoordinate globins that

are expressed at such low concentration in neuronal or

other cells? Despite the increasingly large body of informa-

tion available on their structures solved by X-ray (5, 8, 9)

and NMR (10), and on the spectroscopic properties of these

proteins (11 – 14), the in vivo functional role of Ngb and

Cygb is only just becoming clear. Insight into the

physiological roles of these globins are of crucial importance

in appreciating the original function of globins, since Ngb

and, to a lesser extent, Cygb appear to be phylogenetically-

ancestral vertebrate globins (6). The amino acid sequences

of Ngb and especially Cygb have changed less during

evolution than those of Mb and Hb, which suggest

Received 15 October 2004; accepted 31 December 2004Address correspondence to: Angela Fago, Department of

Zoophysiology, Institute of Biological Sciences, C. F. MøllersAlle, 131, Aarhus University, DK-8000 Aarhus C, Denmark.Tel: +45 8942 2591. Fax: +45 8619 4186.E-mail: angela.fago@biology.au.dk

IUBMBLife, 56(11–12): 689–696, November/December 2004

ISSN 1521-6543 print/ISSN 1521-6551 online # 2004 IUBMB

DOI: 10.1080/15216540500037299

conservation of a crucial, ancient biological function. As

pointed out in several studies, the reversible binding of O2

as known for Hb and Mb, where the iron atom retains its

Fe(II) state, may represent a secondary evolutionary

development, and globins may originally have evolved to

protect the cell against potentially damaging reactive

molecules – like O2 –when these gases started to accumulate

in the atmosphere (15). Such protective roles are still

fulfilled today by globins from several unicellular and

invertebrate organisms (15, 16). We here review the current

knowledge regarding the possible functional roles of Ngb

and Cygb based on recent studies of their ligand-binding

properties, and present a mathematical model to analyse the

potential roles of Ngb in facilitating the diffusion of O2 and

functioning as an internal O2 store.

NEUROGLOBIN AS A PROMOTER OF O2 SUPPLY

The first major contributions pertaining to the functional

role of Ngb came from Greenberg and coworkers (17, 18),

who demonstrated a link between the levels of Ngb expression

and neuronal survival following hypoxia. These studies align

with the hypothesis originally formulated in connection with

the discovery of Ngb, viz. that Ngb, similar to Mb, is involved

in reversible O2 binding to sustain mitochondrial respiration, a

function that becomes increasingly important during hypoxic/

ischemic episodes.

Initial studies on the O2 binding properties of human Ngb

indeed indicated a high O2 affinity, with a P50 (O2 tension at

half-saturation) of *1 torr at 378C (7), similar to that of Mb.

However, based on an extensive investigation of O2 binding

equilibria, we have recently shown that the O2 affinity of

human Ngb under physiological conditions of pH and

temperature is much lower (P50=7.5 torr) (19), whereby only

a small fraction of Ngb in nervous tissues would be saturated

with O2 under normal conditions. The affinity of mouse Ngb

at physiological temperature and pH may be even lower.

Assuming the same enthalpy of oxygenation as human Ngb

between 25 and 378C (19), the P50 for mouse Ngb at 378C and

pH 7.0 calculated from data at 258 (19) is * 20 torr. These

results argue against a role for mammalian Ngbs as an

intracellular O2 store. The lower P50 value that was originally

recorded for human Ngb was presumably due to in vitro

formation of an internal disulfide bridge in the protein (20),

which increases the affinity for O2 (see below). We have also

shown that, in contrast to Mb, the O2 affinity of Ngb is

markedly sensitive to changes in pH, and that, depending on

the temperature range, both normal and reverse Bohr effects

are present (indicating an increase or a decrease in the O2

affinity, respectively, with a pH increase) (19). Such allosteric

properties are uncommon in monomeric proteins and reflect

the ability of Ngb to undergo conformational changes that

affect heme reactivity. These primarily involve changes in the

orientation of the E helix and ultimately in the affinity of the

distal His for heme hexacoordination, as also evident from

kinetic and spectroscopic studies (11 – 13, 20).

The hypothesis that Ngb is involved in O2 supply to

mitochondria has been put forward in studies on its

localization in the mammalian retina. In the inner segment

of the retinal photoreceptors the local concentration of Ngb

was estimated to be *100 mM (21), a value that approaches

the high concentration of Mb (4 1 mM) found in muscle

cells. Interestingly the high Ngb concentration in the inner

segment coincides with the location of mitochondria (21). The

correspondence of the sites of high local Ngb concentration

(21) and sites of high O2 consumption (22) has been considered

as further evidence for the role of Ngb in O2 metabolism.

However, cortical neuronal cell cultures that do and do not

express Ngb show similar rates of O2 consumption (17).

We have analysed the potential role that Ngb may play in

promoting O2 supply to the outer retina in facilitating the

diffusion of O2 and as an O2 store, using a mathematical

model of retinal O2 supply. In this model we treat the outer

retina as a three-layered structure; a middle Ngb-laden layer

that consumes O2 and two adjacent layers that are devoid of

Ngb and do not consume O2 (Fig. 1). As shown, the profile of

the PO2 calculated from the model is remarkably similar to

that obtained by direct measurements (22). The potential role

of Ngb in promoting the steady state O2 supply by facilitated

diffusion is illustrated in Fig. 2 that shows the fractional

inhibition of the O2 metabolism as a function of Ngb

concentration in a situation where the O2 consumption is

high. As evident, Ngb improves O2 supply only at concentra-

tions exceeding *300 mM, whereas at 100 mM and below the

effect is barely detectable.

Fig. 3 illustrates the potential role of Ngb as an O2 store

after a stepwise increase in the O2 consumption (as when no

light falls on the retina). The presence of Ngb increases the

time it takes for a stepwise change in O2 consumption to lead

to a new degree of inhibition of metabolism. However, even

Ngb concentrations of 100 mM increase the elapsed time by

only *3-fold compared to that in the absence of Ngb (t0).Considering that t0 is approximately 0.3 sec, it seems unlikely

that Ngb should play a major role as an O2 store under

ischemic episodes that last longer than a few seconds.

However, it could participate in reducing the impact of

short-time fluctuations in local O2 demand, as during brief

dark exposures.

In view of the high autoxidation rate of Ngb in the presence

of O2, a functional role for this protein in O2 binding and

delivery presupposes the presence of a highly effective system

of reductases in vivo. The Fe2+7O2 complex of Ngb is

unstable in vitro, whereby formation of ferric heme upon air

exposure at room temperature occurs within a few minutes (7,

19). Ngb-mediated reversible O2 binding within neurons can

only be possible if an enzymatic met-globin reducing system is

present, similar to the met-Hb reductase system identified

within red blood cells (23) or to that used in in vitro O2

690 FAGO ET AL.

equilibrium measurements (19, 24). However, it appears

unlikely that Ngb, which has retained a relatively conserved

primary structure during evolution, should be able to bind O2

reversibly only by means of a metabolically-expensive

consumption of reducing equivalents. In human Ngb, the

single mutation, of the distal His to a Gln residue greatly

stabilizes the O2 complex and increases the O2 affinity (19),

properties that would be advantageous for O2 delivery within

cells. Remarkably, the distal His, a major determinant of the

high autoxidation rate, is invariant among Ngbs.

NEUROGLOBIN AS A REDOX THIOL-SENSOR

A common feature of vertebrate Ngbs is the presence of

two to three solvent-accessible reactive cysteine residues (19,

20). These are located at the level of the CD corner and D

helix (one or two cysteines) or in the G helix (one cysteine) of

the protein. Specifically, the Cys D5 is invariant, whereas Cys

CD4 or CD7 and Cys G19 are highly conserved (20).

Marden and coworkers (20) have shown that the formation

of a disulfide bridge between the two cysteines located at the

Figure 1. A simple three-layer model of the retina was used to assess the role of Ngb in facilitated O2 diffusion and as an O2

buffer when O2 consumption abruptly increases. As in the outer retina, an O2 consuming, Ngb-containing layer, is surrounded

by two layers that are devoid of Ngb and do not consume O2. An external and internal O2 supply is modeled by assuming

constant PO2 on the surface of these layers. The model assumes instantaneous equilibrium between free and O2-bound Ngb. The

differential equation for the partial pressure of O2 (P) results from equating the time-rate of change of the total O2 concentration

to the negative divergence of the total O2 flux minus the volume-specific O2 consumption (the continuity equation):

@Ctot=@t ¼ �r � jtot � _m. The (numerical) solution to the differential equation yields the partial pressure of O2 as a function of

time and distance. Three examples of the steady-state profiles obtained at three different O2 consumption rates, 0.08, 0.10 and

0.16 nmol � cm73 � s71 (dotted, continuous and dot-dashed line, respectively) are shown in the graph. Symbols used: P, partial

pressure of O2; b, O2 capacitance coefficient (1.56 nmol � cm73� torr71); CNb, Ngb concentration; S, Ngb O2 saturation; DO2and

DNb, diffusion coefficients of O2 (2 � 1075 cm2 � s71) and Ngb (1.7 � 1076 cm2 � s71), respectively; P50, half-saturation O2 partial

pressure for human Ngb (7.5 torr); Pm50, half-saturation O2 partial pressure for mitochondrial respiration (0.35 torr); _m,

volume-specific O2 consumption; _m0, volume-specific O2 consumption at infinite O2 partial pressure.

691FUNCTIONAL PROPERTIES OF NEUROGLOBIN AND CYTOGLOBIN

CD7 and D5 positions increases the O2 affinity of human Ngb

up to 10-fold depending on the experimental conditions (19,

20). Their studies indicate the existence of an important link

between the redox state of these Cys residues and the reactivity

of heme towards external ligands. As discussed above, it

appears unlikely that changes between reduced (SH) and

oxidized (S-S) state of thiols may promote in vivo O2 delivery

during hypoxia, as originally proposed (20), also given the fact

that S-S bonds are typically found in extracellular rather than

intracellular proteins and that mouse Ngb, lacking cysteines in

the CD corner, cannot form intermolecular S-S bonds.

Nevertheless, the presence of reactive Cys residues in Ngb

may turn out to be important in other physiological contexts,

including cellular signalling involving S-nitrosation pathways

(25), which play an important role in cell survival upon

oxidative stress (26, 27). Investigations in this direction are on

the drawing board.

NEUROGLOBIN AS SCAVENGER OF REACTIVE OXYGENAND NITROGEN SPECIES

In cells, ‘hypoxia’ not only denotes ‘low oxygen’ but also

‘high reactive oxygen and nitrogen species’. A protective role

of Ngb during hypoxia/ischemia may thus relate to a function

in neutralizing such reactive oxidizing species that are a major

cause of cellular damage. The levels of free radicals and related

molecular species are normally tightly regulated. The well-

known nitric oxide (NO) is a widespread brain cellular

messenger produced by the neuronal isoform of nitric oxide

synthase (NOS). Following hypoxia NO level increases from

nanomolar to the low micromolar ranges, presumably because

of Ca2+ influx (28). Other reactive oxidating species including

superoxide also become more abundant during hypoxia. A

major fate of NO produced during hypoxia is that it reacts

with superoxide in the diffusion-limited reaction, forming

peroxynitrite (ONOO7), a potent oxidating and nitrating

agent, whose role in cellular damage during brain hypoxia has

been recognized (28). We have recently shown that in the

Fe2+7NO form Ngb reacts more rapidly with peroxynitrite

than Hb does (29). This scavenging property may protect the

neurons and contribute to their survival following hypoxic

episodes. Additionally, in contrast to Hb and Mb, the reaction

of met (Fe3+) Ngb with peroxynitrite or hydrogen peroxide

does not appear to generate the cytotoxic ferryl (Fe4+) species,

another feature that may contribute to cellular survival (29).

The chemistry of peroxynitrite, however, changes drasti-

cally in the presence of CO2, where other nitrating and S-

nitrosating reactions become dominant (30). It is not known

whether the reaction of Ngb with peroxynitrite becomes faster

or slower in the presence of CO2. Recent studies have shown

Figure 2. The total relative O2 deficit, as defined in this figure, measures the fractional inhibition of O2 metabolism assuming a

P50 for Ngb of 5 and 20 torr. This deficit is plotted as a function of Ngb concentration at an O2 consumption rate large enough

for Ngb to have potential importance (see Fig. 1). As evident, the contribution of Ngb to the steady-state O2 supply of the tissue

is evident only when CNb exceeds 100 mM, and even at a relatively high O2 affinity (5 torr) this effect is not of major significance

unless CNb exceeds *300 mM.

692 FAGO ET AL.

that the reaction of Hb with peroxynitrite is indeed faster in

the presence of 1.2 mM CO2 (31).

A role of Ngb in the metabolism of NO is further supported

by the high concentration of Ngb found in retina, where

cGMP is a fundamental intermediate in the phototransduction

response. Synthesis of cGMP is catalyzed by the enzyme

guanylate cyclase, which is activated by NO. Interestingly,

guanylate cyclase has been detected at high levels in Purkinje

cells of the cerebellum and in the olfactory bulb (32), sites

where Ngb levels are also high (50).

Ca2+-dependent, neuronal NOS is moreover present in

several parts of the retina, including photoreceptors (33, 34),

and in Purkinje cells (35). Given the high affinity of globins

(36), including Ngb (37) for NO, one would expect Ngb to be

in the Fe2+7NO form inside NO-producing neurons. Clearly,

the present evidence for a protective role of Ngb against

nitrosative and oxidative stress deserves further investigation,

in view of the complex chemistry of NO and related products

(including peroxynitrite) under physiological and pathological

conditions.

NEUROGLOBIN AND ITS INTERACTIONS WITH OTHERPROTEINS

The interactions of Ngb with other intracellular proteins

have recently been investigated by Wakasugi et al. Using

surface plasmon resonance, these authors initially showed that

ferric, but not liganded ferrous (CO) Ngb, binds to the GDP-

bound form of G protein (Ga), which would inhibit the rate of

exchange of GDP for GTP, release Gbg and ultimately lead to

protection against neuronal death (38). As pointed by

Burmester and Hankeln (39), the amino acid sequence of

Ngb does not align with that of regulators of G proteins, as

erroneously was stated by the authors (38).

Nevertheless, a measurable interaction was detected, which

interestingly depended not only on the redox state of the heme,

but also on the presence of the internal S-S bond, which

suggests a role of the CD corner of Ngb in the interaction with

other proteins. By using surface plasmon resonance or two-

hybrid systems, other studies by the same authors have shown

that Ngb interacts with other proteins, such as cystatin C, a

cysteine proteinase inhibitor (40), and flotillin-1 (41). The in

vivo relevance of these protein – protein interactions is

presently unknown.

CYTOGLOBIN

Cygb, the toddler of the vertebrate globin family, was

discovered in 2002 in several vertebrate tissues, independently

by two groups of investigators (2, 3). It has not yet been

intensively investigated from a functional point of view. As in

Ngb, the heme in Cygb is hexacoordinate in the absence of

external ligands (6), but the Fe2+7O2 complex is much more

stable than in Ngb and autoxidation is negligible (14, 19).

Figure 3. The half-time for change in the O2 deficit (t) following a stepwise increase in O2 consumption (see insert) relative to that

attained at zero Ngb concentration (t0=0.31 sec) at a given Ngb concentration. Only at concentrations well above 100 mM, can

Ngb significantly delay the time required for O2 shortage to be manifested after an abrupt increase in O2 consumption.

693FUNCTIONAL PROPERTIES OF NEUROGLOBIN AND CYTOGLOBIN

Moreover, O2 affinity is rather high (*1 torr at 208C) (19) andsimilar to that of Mb. Also, O2 binding to Cygb is cooperative

(19), which could permit greater O2 loading and unloading

within a narrow range of low O2 tensions than in the case of

non-cooperative Ngb and Mb. Because of the unique dimeric

arrangement of Cygb (4), the mechanism of heme-heme

interaction in this globin is unknown.

The presence of Cygb in collagen-producing fibroblasts of

connective tissues, as well as in chondroblasts and osteoblasts,

suggests a role for this protein in the O2-requiring collagen

hydroxylation catalyzed by Pro-hydroxylase (42). However,

other related functions are plausible, since Cygb also is

expressed in many other tissues, including neurons, both in the

cytoplasm and in the nucleus (42). Interestingly, Asn- or Pro-

hydroxylases occur in the nucleus and cytoplasm, where under

normoxic conditions they mediate hydroxylation of hypoxia-

inducible factor a (HIF1a), which causes its degradation (43).

Cygb is upregulated under hypoxia (42); thus if Cygb provides

molecular O2 for hydroxylation of HIF1a, it would also

enhance the destabilization of HIF1a, ultimately leading to the

apparent paradox that the cell may fail to sense hypoxia.

Recent studies have shown that this may indeed be the case, as

NO-mediated inhibition of mitochondrial respiration during

hypoxia actually increases O2 availability for HIF1a hydro-

xylation (44). Future studies may verify whether an increase in

the extent of collagen or HIF-a hydroxylation occurs in the

presence of Cygb, elucidating the importance of Cygb in these

reactions.

A role of Cygb in other O2 requiring reactions, such as the

synthesis of NO mediated by NOS, has also been suggested

(42), but in the absence of data on the colocalization of NOS

and Cygb, this remains speculative. As with Ngb, a functional

role of Cygb in O2 supply to mitochondria can be excluded on

the basis of its low (micromolar) intracellular concentration.

CONCLUSIONS AND PERSPECTIVES

Whereas the in vivo function of Cygb is likely to relate to

reversible O2 binding, the physiological (or pathological) role

of Ngb remains a matter of debate. However, our analyses

indicate that a major function for vertebrate Ngb as O2 store

can be excluded. In contrast to this inference on vertebrate

Ngbs, specific nerve hemoglobins of invertebrates play

significant roles as O2 stores that can be exploited during

neuronal activity (45). However, the intracellular concentra-

tion of the invertebrate nerve hemoglobins is much higher – to

the extent that the nerves are visibly red. Moreover, the O2

affinity of invertebrate nerve Hbs is generally higher than that

of vertebrate Ngbs (50).

A role for Ngb in CO or NO sensing is moreover unlikely

given the unfavourably low partition coefficient between O2

and CO (7) or NO (37), which renders Ngb unable to

accurately discriminate between different ligands. Addition-

ally, the known O2, CO or NO sensors are multidomain

proteins, consisting of a sensing domain and a domain

involved in the signal transduction response (46). The proteins

that until now have been shown to interact with Ngb in vitro

are not normally involved in regulatory cascade responses as

would be expected if Ngb was involved in signal transduction

pathways. An alternative requirement would be oxygenation-

linked interactions with molecules that are implicated in

specific regulated processes – as exemplified by vertebrate Hbs

that may regulate glycolysis by competing with glycolytic

enzymes for binding to band 3 red cell membrane proteins

(47).

A more likely role for vertebrate Ngb appears to be cellular

detoxification of free radicals and related oxidizing species

(such as peroxynitrite) that are generated under conditions of

oxidative stress during hypoxia. Such a role is also suggested

by the presence in Ngb of invariant redox sensitive thiol

groups. Hypoxia, however, is not the only condition where

reactive oxygen and nitrogen species are generated. Interest-

ingly, the free radicals that are produced during hypoxia are

also produced during hyperoxia (i.e., high O2 levels), as shown

in recent studies on retina exposed to high O2 (48).

Remarkably, Purkinje cells of the cerebellum that are highly

sensitive to hypoxia also have high levels of Ngb. It is not

known at present whether Ngb may protect neurons against

hyperoxia. As most cells (at least in the brain) are exposed to

O2 tensions that are much lower than atmospheric conditions

(49), one can speculate that Ngb may originally have evolved

to take part to the cellular antioxidant defence as O2 levels in

the atmosphere started to increase (15). The discovery of Ngb

and Cygb has veritably provided ‘fresh blood for the

vertebrate globin family’ (6), and new inspiration for globin

researchers.

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