Critical Review

Reactivity of the Human Hemoglobin Dark

Side

Paolo Ascenzi1,2*
Loris Leboffe2
Fabio Polticelli3,4

1
Laboratorio Interdipartimentale di Microscopia Elettronica, Universita
Roma Tre, Roma, Italy
2
Istituto Nazionale di Biostrutture e Biosistemi, Roma, Italy
3
Dipartimento di Biologia, Universita Roma Tre, Roma, Italy
4
Istituto Nazionale di Fisica Nucleare, Sezione di Roma Tre, Roma, Italy

Abstract
Summary: Ligand binding to the heme distal side is a paradigm of biochemistry. However, X-ray crystallographic studies
highlighted the possibility that O2 and NO22 may bind to the
proximal heme side of ferrous human hemoglobin (Hb) achains complexed with the a-hemoglobin stabilizing protein
and to ferric human hemoglobin b-chains, respectively. Strikingly, the role generally played by the proximal HisF8 residue
is played by the distal HisE7 side chain forming the trans axial

ligand of the hemeFe atom. This: i) brings to light that Hb

may utilize both heme distal and proximal sides for ligand discrimination, ii) draws attention to the nonequivalence of aand b-chains, and iii) highlights the possibility that partially
unfolded Hb derivatives may display transient ligand-binding
C 2013
properties different from those of the native globin. V
IUBMB Life, 65(2):121126, 2013.

Keywords: human hemoglobin; proximal heme side; distal heme side;

ligand discrimination; a- and b-chain nonequivalence; transient ligandbinding properties

For long time, ligand binding to the heme distal side has been
considered as a paradigm of biochemistry, the fifth trans axial
ligand of the hemeFe atom being the proximal His residue.
Ligands bind to the heme center with very different values of
thermodynamic and kinetic parameters depending on the ligand
chemistry, on the oxidation and coordination state of the hemeAbbreviations AHSP, a-hemoglobin stabilizing protein; Hb, hemoglobin;
HbONO, NO2
-bound ferric Hb; NaHbONO, HbONO displaying nitrated 2vinyl heme group of aHb; NHbONOa, HbONO displaying NO2
-free bHb and
nitrated 2-vinyl heme group of aHb and bHb; HbONOd,p, HbONO displaying
NO2
-bound to the distal and proximal heme sides of aHb and bHb,
respectively; aHb, a-chains of Hb; aHbO2oxygenated aHb; aHbONO, aHb of
HbONO; aHbONOd,p, aHb of HbONOd,p; bHb, b-chains of Hb; bHbONO;
bHb of HbONO; bHbONOd,p, bHb of HbONOd,p; hhcytc, horse heart
cytochrome c; Mb, myoglobin; sGC, soluble guanylate cyclase
C 2013 International Union of Biochemistry and Molecular Biology, Inc.
V
Volume 65, Number 2, February 2013, Pages 121-126
*Address for correspondence to: Paolo Ascenzi, Laboratorio
Interdipartimentale di Microscopia Elettronica, Universita Roma Tre, Via
della Vasca Navale 79, I-00146 Roma, Italy. Tel.: 39-06-5733-3621; Fax:
39-06-5733-6321. E-mail: ascenzi@uniroma3.it.
Received 17 October 2012; accepted 26 November 2012
DOI: 10.1002/iub.1121
Published online 3 January 2013 in Wiley Online Library
(wileyonlinelibrary.com)

IUBMB Life

Fe atom, and on amino-acid residues building up the heme

pocket. Moreover, ligand binding to heme-proteins may be modulated by homotropic and heterotropic allosteric effectors (18).
Recently, the possibility that NO could bind to the proximal
heme side of heme-proteins came to light as the proximal
HisAFe axial bond can be lost upon NO binding to the distal
heme coordination side (2,9). Among others, the cleavage of
the proximal HisAFe axial bond occurs in the a-chains of ferrous nitrosylated human hemoglobin upon binding of heterotropic effectors switching the quaternary transition toward the
T-state (2). Remarkably, the cleavage of the proximal HisAFe
axial bond has been reported to represent the first step of NO
binding to the proximal heme coordination side (1012).
NO binds to the proximal heme side of a periplasmic class
IIa c-type cytochrome c0 from the denitrifying bacterium Alcaligenes xylosoxidans, of horse heart cytochrome c (hhcytc) complexed with cardiolipin, and of mammalian-soluble guanylate
cyclase (sGC), displacing the proximal His residue and inducing
the formation of a penta-coordinated hemeFeNO derivative
(1016). Notably, in hhcytc a transient hemeFebisNO complex precedes the formation of the penta-coordinated hemeFe
NO species (10,11). In mammalian sGC, data highlighting a
hemeFebisNO complex are lacking and a bisNO transition
complex was postulated to indicate the fast binding of a NO

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molecule at the proximal heme-Fe side with the concomitant

cleavage of the proximal His-Fe bond, with the subsequent discharge of the NO ligand bound to the distal heme-Fe side (12).
The hhcytccardiolipin complex could play either proapoptotic effects, catalyzing lipid peroxidation and the subsequent
hhcytc release into the cytoplasm, or antiapoptotic actions,
protecting the mitochondrion by scavenging reactive nitrogen
and oxygen species and binding CO and NO that inhibit lipid
peroxidation and hhcytc translocation (17).
According to the sliding scale rule, the multistep mechanism for NO binding to mammalian sGC substantially increases
the gas ligand affinity and impairs totally O2 binding (18). The
NO/O2 selectivity by mammalian sGC is crucial under physiological conditions where the NO concentration is much less
than that of O2 and NO dioxygenation is unwanted (19).
Remarkably, nitrosylation of the proximal hemeFe side of
heme-proteins has been postulated to be relevant in several
functions including: i) discrimination between ligands (e.g.,
NO, CO, and O2), ii) initiation of specific gas-dependent signaling pathways, and iii) selective scavenging of reactive nitrogen
and oxygen species (1019).
Interestingly, a ligand-binding pocket has been created on
the heme proximal side in quadruple mutants of porcine myoglobin (Mb) by site-directed mutagenesis, the proximal HisF8
residue having been replaced by Phe. The affinity of CO and
cyanide for the heme proximal coordination side of porcine Mb
quadruple mutants is similar to that for the heme distal pocket
of wild-type Mb; however, the polar nature of the heme proximal pocket is at the root of the rapid oxidation of the hemeFe
atom preventing reversible O2 binding (20).
Here, the structural bases for O2 and NO2
binding to the
proximal heme side of a- and b-chains of ferrous and ferric
human hemoglobin (Hb), respectively, are reviewed. This suggests that: i) Hb may utilize both heme distal and proximal sides
for ligand discrimination, ii) draws attention to the nonequivalence of the a- and b-subunits, and iii) highlights the possibility
that partially unfolded Hb derivatives may display transient
reactivity properties different from those of the native protein.

O2 Binding TO a-Chains OF Partially

Unfolded Ferrous Human Hb
Hb, the O2 carrying and delivery system in humans, is an
allosterically modulated hetero-tetramer formed by two a- and
two b-chains (aHb and bHb, respectively) (2). Free aHb is an
unstable monomer prone to hemeFe oxidation and precipitation, likely contributing to the pathophysiology of several blood
disorders including b-thalassemia. On the other hand, bHb
forms a relatively stable homotetramer (21,22).
The molecular chaperone a-hemoglobin-stabilizing protein
(AHSP) is an erythroid protein that binds the a-globin polypeptide and several forms of aHb, in the absence of bHb, to maintain the aHb structure, to avoid aHb precipitation, and to limit
aHb pro-oxidant activity both in vitro and in vivo. When avail-

122

able, bHb binds more avidly to aHb than AHSP, displacing

AHSP and forming the Hb tetramer (2329).
AHSP and oxygenated aHb (aHbO2) form a 1:1 binary complex (23). Both proteins are exclusively in the a-helical conformation, AHSP adopting an elongated three-helix bundle and
aHb comprising six a-helices. The interface of the AHSPaHbO2
complex primarily involves four a-helices, two from each protein. AHSP contacts G and H a-helices of aHbO2 (25,26).
Remarkably, the G and H a-helices of aHb are the primary
structural elements that interact with bHb to form the a1b1
Hb complex. Superimposition of the AHSPaHbO2 complex
onto the a1b1 Hb dimer reveals that segments of the a1 and
a2 helices of AHSP superimpose with G and H a-helices of bHb,
forming similar interactions with aHb (26). This partially
explains the reasons why binding of aHb by bHb dissociates
AHSP from the AHSPaHb complex (26).
AHSP binds to aHbO2 on the opposite side of the heme
pocket, inducing dramatic conformational changes in aHb, with
the entire F helix becoming flexible and disordered. Compared
to oxygenated Hb (30), the hemeFe atom and the heme group
of the AHSPaHbO2 complex slides over a distance of approximately 3.1 . Accordingly, the amino acid residues that coordinate the hemeFe group in the AHSPaHbO2 complex undergo
significant rearrangements. In particular, the sixth coordination
position of the hemeFe atom is positioned facing the F a-helix
of aHbO2. Strikingly, although the hemeFe group of aHb is
invariably bound by the proximal His(87)F8 residue in most
structures of Hb (31) (Fig. 1, panel aHbO2), the distal His(58)E7
residue, rather than the proximal His(87)F8 side chain, coordinates the hemeFe atom, with a distance of 2.13 between the
Ne atom of the His(58)E7 residue and the ferrous hemeFe
atom. Moreover, the O2 molecule, rather than the proximal
His(87)F8 residue, represents the trans axial ligand, the distance
between the O1 atom of O2 and the hemeFe atom being 2.74
(25) (Fig. 1, panel AHSPaHbO2). In other words, O2 binds to the
proximal hemeFe side of aHb complexed with AHSP, the distal
His(58)E7 residue being coordinated to the hemeFe atom and
representing the proximal heme residue (25,31).
In the AHSPaHbO2 complex, the O2 molecule is solvent
exposed, resulting in a fast hemeFe oxidation rate. In the
resulting AHSP-bound ferric aHb, the proximal His(87)F8 residue of aHb serves as the trans ligand for the sixth coordination
position of the hemeFe atom, the distal His(58)E7 residue
representing the fifth coordination ligand (26). Thus, AHSP
binding to aHbO2 facilitates the oxidation of the hemeFe atom
and sequesters the oxidized heme in the hexa-coordinated
state. This inhibits heme loss from aHb and redox chemistry
catalysis, thus preventing cell damage (25,26).

Nonequivalent NO22 Recognition by

a- and b-Chains of Ferric Human Hb
Ferrous human Hb is pivotal for O2 transport; in contrast, the
ferric derivative does not bind O2. Moreover, in Hb valency
hybrids ferric heme(s) shifts the R-to-T allosteric transition

Ligand Binding to the Heme Proximal Side of Human Hb

FIG 1

Three-dimensional structure of the heme-binding site

of ferrous oxygenated human Hb derivatives aHbO2
and AHSPaHb. In aHbO2, O2 binds to the heme distal side, the trans axial ligand of the hemeFe atom
being His(87)F8. In AHSPaHb, O2 binds to the heme
proximal side, the trans axial ligand of the hemeFe
atom being His(58)E7. PDB entries are 1HHO and
1YO1 (25,30). Both pictures have been drawn using
the UCSF Chimera package (49). [Color figure can be
viewed in the online issue, which is available at
wileyonlinelibrary.com.]

toward the high-affinity state impairing O2 release (2,8).

Therefore, factors that facilitate Hb oxidation, such as NO2
are
relevant from the health viewpoint (21,32). Remarkably, NO2
is
recommended in the therapeutic treatment of cyanide poisoning
in combination with amyl nitrite and thiosulfate. NO2
antidotal
properties were initially attributed to the induction of ferric Hb,
removing cyanide from cytochrome c oxidase, and later to a NOmediated hemodynamic effect (33). Notably, although the NO2

concentration in plasma ranges between 1.3 and 13 lM (34),
during the therapeutic treatment of cyanide poisoning the NO2

Ascenzi et al.

concentration can reach millimolar plasma concentrations and

10 mM levels at the site of the intravenous administration. However, as NO2
in whole blood is very rapidly oxidized to NO3

(>95% in 1 h), the therapeutic treatment must be repeated if
symptoms of cyanide poisoning recur (33).
The complexity of the HbNO2
interaction depends on the
capability of NO2
i) to be reduced to NO, ii) to oxidize the ferrous hemeFe atom, iii) to bind to the ferric hemeFe atom, iv)
to participate to the formation of N2O3, v) to nitrate the ferric
heme, and vi) to denaturate Hb with the concomitant release
of the ferric heme (3542).
Although the crystallization conditions (e.g., the NO2
concentration was 0.16 M, and the soaking time of Hb crystals
with NO2
ranged between 10 min and 1 week) (42,43) are
very different from those of pathophysiological situations
(33,34), four reaction products following NO2
binding to ferric
and ferrous Hb have recently been characterized by X-ray
crystallography, highlighting the unusual hemeFeNO2
binding geometries (42,43).
In crystals of NO2
-bound ferric Hb (HbONO) (obtained by
soaking ferric Hb crystals at pH 6.8 with NO2
for 10 min at
room temperature), NO2
adopts the uncommon O-nitrito binding mode in both aHb and bHb. A near-identical structure of
HbONO with O-nitrito ligands was observed in crystals obtained
from the ferric HbNO2
solution. In aHb of HbONO (aHbONO),
the FeO distance and the FeN(His(87)F8) distance are both
2.0 . The NO2
O1 atom is within hydrogen bonding distance
(2.9 ) of the Ne atom of the distal His(58)E7 residue (Fig. 2,
panel aHbONO). In bHb of HbONO (bHbONO), the FeO distance
is 1.9 and the FeN(His(92)F8) distance is 2.0 . The nitrite
O1 and N atoms are within hydrogen bonding distance (2.9 and
3.2 , respectively) of the Ne atom of the distal His(63)E7 residue (Fig. 2, panel bHbONO). However, NO2
conformations in
aHbONO and bHbONO are different, reflecting subtle effects of
the distal HisE7 in orienting the NO2
ligand. In aHbONO, the
FeONO moiety is trans with a torsion angle of 174 , the O
NO angle being 110 . On the other hand, in bHbONO, the Fe
ONO complex is in a distorted cis-like conformation with a
torsion angle of
91 and an ONO angle of 113 . Moreover,
the terminal nitrite atom is directed away from the distal
His(63)E7 residue of bHbONO, making the shortest contact with
the Cc atom of distal Val(67)E11 (3.2 ) (43).
The reaction of ferric Hb crystals with NO2
at pH 6.8 for 16
h results in the formation of a red-green product, named
NaHbONO. The NO2
binding mode to the heme distal pocket of
both aHb and bHb is similar to that observed for HbONO. At variance with HbONO, the long-time incubation of ferric Hb crystals
with NO2
leads to the nitration of the 2-vinyl heme group in
aHb only. The covalent modification of the vinyl hemeFe substituent with NO2
is regiospecific, the 2-nitrovinyl moiety being
essentially coplanar with the adjacent pyrrole ring, suggesting
extended conjugation with this group (42).
The reaction of ferric Hb crystals with NO2
at pH 6.5 for
1 week results in the formation of a green crystalline product,
named NHbONOa. The bulk features of the heme-binding

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IUBMB LIFE

FIG 2

Three-dimensional structures of the heme-binding site of NO2
-bound ferric human Hb derivatives aHbONO, bHbONO,
aHbONOd,p, and bHbONOd,p. In aHbONO, bHbONO, and aHbONOd,p, NO2
binds to the heme distal side, the trans axial ligand
of the hemeFe atom being His(87)F8 in a-chains and His(92)F8 in b-chains. In bHbONOd,p, NO2
binds to the heme proximal
side, the trans axial ligand of the hemeFe atom being His(63)E7. PDB entries are 3D7O and 3ONZ (42,43). Both pictures have
been drawn using the UCSF Chimera package (49). [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]

pocket of aHb of NHbONOa are similar to those reported for

NaHbONO. However, the hemeFe group of bHb is also
nitrated at the 2-vinyl position. Moreover, the hemeFe atom
of bHb is penta-coordinated, as no electron density for the
NO2
ligand has been observed (42).
Crystallization of deoxygenated Hb in the presence of
excess NO2
at pH 7.0 gave, after 3 days, a product, named
HbONOd,p, showing unprecedented features in Hb structural
biology. The NO2
binding mode to aHb of HbONOd,p
(aHbONOd,p) (Fig. 2, panel aHbONOd,p) is closely similar to that
observed in HbONO, NaHbONO, and NHbONOa. In fact, NO2

binds to aHb in the trans O-binding mode and the O1 atom is
hydrogen bonded to the Ne atom of the distal His(58)E7 residue. Moreover, the 2-vinyl heme group in aHb is not nitrated
(42). In contrast, the bHb of HbONOd,p (bHbONOd,p) exhibit
major differences from Hb structures reported in the Protein
Data Bank (31). In fact, bHbONOd,p exhibits: i) a large lateral
heme sliding (4.8 ) toward the protein exterior; ii) heme
stabilization by the formation of the distal His(63)E7AFe bond
(2.3 ); iii) loss of the proximal His(92)F8AFe bond (the closest

124

nonbonding N(His(92)F8) AC(shifted heme pyrrole) distance

being 2.8 ); and iv) most unexpectedly, the replacement of
the proximal His(92)F8AFe bond by the FeANO2
bond. The
FeO (NO2
) distance is 3.0 (Fig. 2, panel bHbONOd,p). In
other words, in the bHbONOd,p, the inversion of the proximal-to-distal His heme side occurs. In fact, the exogenous
ligand (i.e., NO2
) is bound to the hemeFe atom at the classically called heme proximal position instead of His(92)F8, and
the so-called distal His(63)E7 corresponds to the proximal
hemeFe atom axial ligand (42).
As a whole, the multiple binding modes of NO2
to and
aHb and bHb highlight the nonequivalent functional properties
of Hb chains at least in the ferric form (42,43). Notably, nitrite
binding to the Hb tetramer displays biphasic kinetics similar to
those observed for ligation of isolated aHb and bHb (35). Moreover, the HbONOd,p species, in which the NO2
-bound heme
Fe group of bHb is displaced 4.8 from its original position,
may represent an intermediate of Hb unfolding. In fact, the
heme displacement would lead to eventual heme removal and
protein destabilization (42).

Ligand Binding to the Heme Proximal Side of Human Hb

Conclusions
Hb, playing a pivotal role in life, is a classic paradigm for the
study of protein structurefunction relationships (1,2,8,21).
The unusual O2 and NO2
binding mode in AHSPaHbO2 and
bHbONOd,p, respectively, highlights new modulation mechanisms contributing significantly to the understanding of Hb
homeostatic regulation in living organisms (25,42).
Here are some lessons learned in looking at Hb reactivity.
Hb may utilize both heme distal and proximal sides for ligand
discrimination, as observed for binding of the exogenous O2 and
the endogenous His(87)F8 ligand. In fact, O2 binding to the
heme proximal side of ferrous aHb of AHSPaHbO2 leads to the
hemeFe atom oxidation followed by the formation of the inert
ferric bis-histidyl adduct (25). Moreover, NO2
binding to the
heme proximal side of ferric bHbONOd,p represents a unique
case (42); in fact, anionic ligands most invariably bind to the
heme distal side of ferric Hb (31). Furthermore, NO2
binding to
ferric Hb highlights the ligand-binding nonequivalence of aHb
and bHb. In fact, in NHbONOa NO2
binds only to aHb, and in
HbONOd,p NO2
binds to heme-distal and heme-proximal side of
aHb and bHb, respectively (42). The different binding mode of
NO2
to aHb and bHb in HbONOd,p reflects the sliding movement
of the heme toward the bHb exterior, representing an intermediate of Hb unfolding (42). Ligand-dependent heme sliding may
be a general mechanism for ligand-binding modulation as also
observed for carbonylation of murine neuroglobin (44).
Finally, the possibility that partially unfolded Hb derivatives may display transient ligand-binding properties different
from those of the native globin and of the unfolded protein
may represent a case of chronosteric effects (4547).
Remarkably, the Hb reactivity toward CO increases transiently
at low pH (<4), preceding the irreversible protein denaturation. This reflects the protonation of the Ne atom of the HisF8
residue and the cleavage of the proximal axial hemeFeNe
bond, representing the first step of protein unfolding. In turn,
the low reactive penta-coordinated heme becomes tetra-coordinated and highly reactive, the hemeFe atom shifting from
the out to the in plane geometry. This highlights the crucial role of the interaction(s) of the heme on the proximal side
in accounting for the difference in the ligand reactivity
between the two quaternary R and T conformations of Hb (48).

Acknowledgements
Dr. Loris Leboffe is supported by a grant from the National
Institute of Biostructures and Biosystems (INBB) of Italy.

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Ligand Binding to the Heme Proximal Side of Human Hb