Critical Review

Reactivity of the Human Hemoglobin ‘‘Dark

Side’’

Paolo Ascenzi1,2*

Loris Leboffe2

Fabio Polticelli3,4

1Laboratorio Interdipartimentale di Microscopia Elettronica, Universit�aRoma Tre, Roma, Italy2Istituto Nazionale di Biostrutture e Biosistemi, Roma, Italy3Dipartimento di Biologia, Universit�a Roma Tre, Roma, Italy4Istituto Nazionale di Fisica Nucleare, Sezione di Roma Tre, Roma, Italy

Abstract

Summary: Ligand binding to the heme distal side is a para-

digm 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) a-

chains complexed with the a-hemoglobin stabilizing protein

and to ferric human hemoglobin b-chains, respectively. Strik-

ingly, the role generally played by the proximal HisF8 residue

is played by the distal HisE7 side chain forming the trans axial

ligand of the heme–Fe atom. This: i) brings to light that Hb

may utilize both heme distal and proximal sides for ligand dis-

crimination, ii) draws attention to the nonequivalence of a-

and b-chains, and iii) highlights the possibility that partially

unfolded Hb derivatives may display transient ligand-binding

properties different from those of the native globin. VC 2013

IUBMB Life, 65(2):121–126, 2013.

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

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

binding properties

For long time, ligand binding to the heme distal side has beenconsidered as a paradigm of biochemistry, the fifth trans axialligand of the heme–Fe atom being the proximal His residue.Ligands bind to the heme center with very different values ofthermodynamic and kinetic parameters depending on the ligandchemistry, on the oxidation and coordination state of the heme-

Fe atom, and on amino-acid residues building up the hemepocket. Moreover, ligand binding to heme-proteins may be modu-lated by homotropic and heterotropic allosteric effectors (1–8).

Recently, the possibility that NO could bind to the proximalheme side of heme-proteins came to light as the proximalHisAFe axial bond can be lost upon NO binding to the distalheme coordination side (2,9). Among others, the cleavage ofthe proximal HisAFe axial bond occurs in the a-chains of fer-rous nitrosylated human hemoglobin upon binding of hetero-tropic effectors switching the quaternary transition toward theT-state (2). Remarkably, the cleavage of the proximal HisAFeaxial bond has been reported to represent the first step of NObinding to the proximal heme coordination side (10–12).

NO binds to the proximal heme side of a periplasmic classIIa c-type cytochrome c0 from the denitrifying bacterium Alcali-genes xylosoxidans, of horse heart cytochrome c (hhcytc) com-plexed with cardiolipin, and of mammalian-soluble guanylatecyclase (sGC), displacing the proximal His residue and inducingthe formation of a penta-coordinated heme–Fe–NO derivative(10–16). Notably, in hhcytc a transient heme–Fe–bis–NO com-plex precedes the formation of the penta-coordinated heme–Fe–NO species (10,11). In mammalian sGC, data highlighting aheme–Fe–bis–NO complex are lacking and a bis–NO transitioncomplex was postulated to indicate the fast binding of a NO

Abbreviations AHSP, a-hemoglobin stabilizing protein; Hb, hemoglobin;

HbONO, NO2�-bound ferric Hb; NaHbONO, HbONO displaying nitrated 2-

vinyl 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

VC 2013 International Union of Biochemistry and Molecular Biology, Inc.Volume 65, Number 2, February 2013, Pages 121-126*Address for correspondence to: Paolo Ascenzi, LaboratorioInterdipartimentale di Microscopia Elettronica, Universit�a Roma Tre, Viadella Vasca Navale 79, I-00146 Roma, Italy. Tel.: þ39-06-5733-3621; Fax:þ39-06-5733-6321. E-mail: ascenzi@uniroma3.it.Received 17 October 2012; accepted 26 November 2012DOI: 10.1002/iub.1121Published online 3 January 2013 in Wiley Online Library(wileyonlinelibrary.com)

IUBMB Life 121

molecule at the proximal heme-Fe side with the concomitantcleavage of the proximal His-Fe bond, with the subsequent dis-charge of the NO ligand bound to the distal heme-Fe side (12).

The hhcytc–cardiolipin complex could play either proapop-totic effects, catalyzing lipid peroxidation and the subsequenthhcytc release into the cytoplasm, or antiapoptotic actions,protecting the mitochondrion by scavenging reactive nitrogenand oxygen species and binding CO and NO that inhibit lipidperoxidation and hhcytc translocation (17).

According to the ‘‘sliding scale rule’’, the multistep mecha-nism for NO binding to mammalian sGC substantially increasesthe gas ligand affinity and impairs totally O2 binding (18). TheNO/O2 selectivity by mammalian sGC is crucial under physio-logical conditions where the NO concentration is much lessthan that of O2 and NO dioxygenation is unwanted (19).

Remarkably, nitrosylation of the proximal heme–Fe side ofheme-proteins has been postulated to be relevant in severalfunctions including: i) discrimination between ligands (e.g.,NO, CO, and O2), ii) initiation of specific gas-dependent signal-ing pathways, and iii) selective scavenging of reactive nitrogenand oxygen species (10–19).

Interestingly, a ligand-binding pocket has been created onthe heme proximal side in quadruple mutants of porcine myo-globin (Mb) by site-directed mutagenesis, the proximal HisF8residue having been replaced by Phe. The affinity of CO andcyanide for the heme proximal coordination side of porcine Mbquadruple mutants is similar to that for the heme distal pocketof wild-type Mb; however, the polar nature of the heme proxi-mal pocket is at the root of the rapid oxidation of the heme–Featom 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 ferrichuman hemoglobin (Hb), respectively, are reviewed. This sug-gests that: i) Hb may utilize both heme distal and proximal sidesfor ligand discrimination, ii) draws attention to the nonequiva-lence of the a- and b-subunits, and iii) highlights the possibilitythat partially unfolded Hb derivatives may display transientreactivity properties different from those of the native protein.

O2 Binding TO a-Chains OF PartiallyUnfolded Ferrous Human Hb

Hb, the O2 carrying and delivery system in humans, is anallosterically modulated hetero-tetramer formed by two a- andtwo b-chains (aHb and bHb, respectively) (2). Free aHb is anunstable monomer prone to heme–Fe oxidation and precipita-tion, likely contributing to the pathophysiology of several blooddisorders including b-thalassemia. On the other hand, bHbforms a relatively stable homotetramer (21,22).

The molecular chaperone a-hemoglobin-stabilizing protein(AHSP) is an erythroid protein that binds the a-globin polypep-tide and several forms of aHb, in the absence of bHb, to main-tain the aHb structure, to avoid aHb precipitation, and to limitaHb pro-oxidant activity both in vitro and in vivo. When avail-

able, bHb binds more avidly to aHb than AHSP, displacingAHSP and forming the Hb tetramer (23–29).

AHSP and oxygenated aHb (aHbO2) form a 1:1 binary com-plex (23). Both proteins are exclusively in the a-helical confor-mation, AHSP adopting an elongated three-helix bundle andaHb comprising six a-helices. The interface of the AHSP–aHbO2

complex primarily involves four a-helices, two from each pro-tein. AHSP contacts G and H a-helices of aHbO2 (25,26).Remarkably, the G and H a-helices of aHb are the primarystructural elements that interact with bHb to form the a1–b1Hb complex. Superimposition of the AHSP–aHbO2 complexonto the a1–b1 Hb dimer reveals that segments of the a1 anda2 helices of AHSP superimpose with G and H a-helices of bHb,forming similar interactions with aHb (26). This partiallyexplains the reasons why binding of aHb by bHb dissociatesAHSP from the AHSP–aHb complex (26).

AHSP binds to aHbO2 on the opposite side of the hemepocket, inducing dramatic conformational changes in aHb, withthe entire F helix becoming flexible and disordered. Comparedto oxygenated Hb (30), the heme–Fe atom and the heme groupof the AHSP–aHbO2 complex slides over a distance of approxi-mately 3.1 Å. Accordingly, the amino acid residues that coordi-nate the heme–Fe group in the AHSP–aHbO2 complex undergosignificant rearrangements. In particular, the sixth coordinationposition of the heme–Fe atom is positioned facing the F a-helixof aHbO2. Strikingly, although the heme–Fe group of aHb isinvariably bound by the proximal His(87)F8 residue in moststructures of Hb (31) (Fig. 1, panel aHbO2), the distal His(58)E7residue, rather than the proximal His(87)F8 side chain, coordi-nates the heme–Fe atom, with a distance of 2.13 Å between theNe atom of the His(58)E7 residue and the ferrous heme–Featom. Moreover, the O2 molecule, rather than the proximalHis(87)F8 residue, represents the trans axial ligand, the distancebetween the O1 atom of O2 and the heme–Fe atom being 2.74 Å(25) (Fig. 1, panel AHSP–aHbO2). In other words, O2 binds to theproximal heme–Fe side of aHb complexed with AHSP, the distalHis(58)E7 residue being coordinated to the heme–Fe atom andrepresenting the ‘‘proximal heme residue’’ (25,31).

In the AHSP–aHbO2 complex, the O2 molecule is solventexposed, resulting in a fast heme–Fe oxidation rate. In theresulting AHSP-bound ferric aHb, the proximal His(87)F8 resi-due of aHb serves as the trans ligand for the sixth coordinationposition of the heme–Fe atom, the distal His(58)E7 residuerepresenting the fifth coordination ligand (26). Thus, AHSPbinding to aHbO2 facilitates the oxidation of the heme–Fe atomand sequesters the oxidized heme in the hexa-coordinatedstate. This inhibits heme loss from aHb and redox chemistrycatalysis, 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, theferric derivative does not bind O2. Moreover, in Hb valencyhybrids ferric heme(s) shifts the R-to-T allosteric transition

IUBMB LIFE

122 Ligand Binding to the Heme Proximal Side of Human Hb

toward the high-affinity state impairing O2 release (2,8).Therefore, factors that facilitate Hb oxidation, such as NO2

� arerelevant from the health viewpoint (21,32). Remarkably, NO2

� isrecommended in the therapeutic treatment of cyanide poisoningin combination with amyl nitrite and thiosulfate. NO2

� antidotalproperties were initially attributed to the induction of ferric Hb,removing cyanide from cytochrome c oxidase, and later to a NO-mediated 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

�

concentration can reach millimolar plasma concentrations and10 mM levels at the site of the intravenous administration. How-ever, as NO2

� in whole blood is very rapidly oxidized to NO3�

(>95% in 1 h), the therapeutic treatment must be repeated ifsymptoms of cyanide poisoning recur (33).

The complexity of the Hb–NO2� interaction depends on the

capability of NO2� i) to be reduced to NO, ii) to oxidize the fer-

rous heme–Fe atom, iii) to bind to the ferric heme–Fe atom, iv)to participate to the formation of N2O3, v) to nitrate the ferricheme, and vi) to denaturate Hb with the concomitant releaseof the ferric heme (35–42).

Although the crystallization conditions (e.g., the NO2� con-

centration was 0.16 M, and the soaking time of Hb crystalswith NO2

� ranged between 10 min and 1 week) (42,43) arevery different from those of pathophysiological situations(33,34), four reaction products following NO2

� binding to ferricand ferrous Hb have recently been characterized by X-raycrystallography, highlighting the unusual heme–Fe–NO2

� bind-ing 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 bind-

ing mode in both aHb and bHb. A near-identical structure ofHbONO with O-nitrito ligands was observed in crystals obtainedfrom the ferric Hb–NO2

� solution. In aHb of HbONO (aHbONO),the Fe–O distance and the Fe–N(His(87)F8) distance are both2.0 Å. The NO2

� O1 atom is within hydrogen bonding distance(2.9 Å) of the Ne atom of the distal His(58)E7 residue (Fig. 2,panel aHbONO). In bHb of HbONO (bHbONO), the Fe–O distanceis 1.9 Å and the Fe–N(His(92)F8) distance is 2.0 Å. The nitriteO1 and N atoms are within hydrogen bonding distance (2.9 and3.2 Å, respectively) of the Ne atom of the distal His(63)E7 resi-due (Fig. 2, panel bHbONO). However, NO2

� conformations inaHbONO and bHbONO are different, reflecting subtle effects ofthe distal HisE7 in orienting the NO2

� ligand. In aHbONO, theFe–O–N–O moiety is trans with a torsion angle of 174�, the O–N–O angle being 110�. On the other hand, in bHbONO, the Fe–O–N–O complex is in a distorted cis-like conformation with atorsion angle of �91� and an O–N–O angle of 113�. Moreover,the terminal nitrite atom is directed away from the distalHis(63)E7 residue of bHbONO, making the shortest contact withthe Cc atom of distal Val(67)E11 (3.2 Å) (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, namedNaHbONO. The NO2

� binding mode to the heme distal pocket ofboth aHb and bHb is similar to that observed for HbONO. At var-iance with HbONO, the long-time incubation of ferric Hb crystalswith NO2

� leads to the nitration of the 2-vinyl heme group inaHb only. The covalent modification of the vinyl heme–Fe sub-stituent with NO2

� is regiospecific, the 2-nitrovinyl moiety beingessentially coplanar with the adjacent pyrrole ring, suggestingextended 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

Three-dimensional structure of the heme-binding site

of ferrous oxygenated human Hb derivatives aHbO2

and AHSP–aHb. In aHbO2, O2 binds to the heme dis-

tal side, the trans axial ligand of the heme–Fe atom

being His(87)F8. In AHSP–aHb, O2 binds to the heme

proximal side, the trans axial ligand of the heme–Fe

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.]

FIG 1

Ascenzi et al. 123

pocket of aHb of NHbONOa are similar to those reported forNaHbONO. However, the heme–Fe group of bHb is alsonitrated at the 2-vinyl position. Moreover, the heme–Fe atomof bHb is penta-coordinated, as no electron density for theNO2

� 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 structuralbiology. The NO2

� binding mode to aHb of HbONOd,p

(aHbONOd,p) (Fig. 2, panel aHbONOd,p) is closely similar to thatobserved in HbONO, NaHbONO, and NHbONOa. In fact, NO2

�

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

nonbonding N(His(92)F8) AC(shifted heme pyrrole) distancebeing 2.8 Å); and iv) most unexpectedly, the replacement ofthe proximal His(92)F8AFe bond by the FeANO2

� bond. TheFe–O (NO2

�) distance is 3.0 Å (Fig. 2, panel bHbONOd,p). Inother words, in the bHbONOd,p, the ‘‘inversion’’ of the proxi-mal-to-distal His heme side occurs. In fact, the exogenousligand (i.e., NO2

�) is bound to the heme–Fe atom at the classi-cally called heme proximal position instead of His(92)F8, andthe so-called distal His(63)E7 corresponds to the proximalheme–Fe atom axial ligand (42).

As a whole, the multiple binding modes of NO2� to and

aHb and bHb highlight the nonequivalent functional propertiesof Hb chains at least in the ferric form (42,43). Notably, nitritebinding to the Hb tetramer displays biphasic kinetics similar tothose observed for ligation of isolated aHb and bHb (35). More-over, the HbONOd,p species, in which the NO2

�-bound heme–Fe group of bHb is displaced �4.8 Å from its original position,may represent an intermediate of Hb unfolding. In fact, theheme displacement would lead to eventual heme removal andprotein destabilization (42).

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 heme–Fe 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 heme–Fe 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.]

FIG 2

IUBMB LIFE

124 Ligand Binding to the Heme Proximal Side of Human Hb

Conclusions

Hb, playing a pivotal role in life, is a classic paradigm for thestudy of protein structure–function relationships (1,2,8,21).The unusual O2 and NO2

� binding mode in AHSP–aHbO2 andbHbONOd,p, respectively, highlights new modulation mecha-nisms contributing significantly to the understanding of Hbhomeostatic 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 liganddiscrimination, as observed for binding of the exogenous O2 andthe endogenous His(87)F8 ligand. In fact, O2 binding to theheme proximal side of ferrous aHb of AHSP–aHbO2 leads to theheme–Fe atom oxidation followed by the formation of the inertferric bis-histidyl adduct (25). Moreover, NO2

� binding to theheme proximal side of ferric bHbONOd,p represents a uniquecase (42); in fact, anionic ligands most invariably bind to theheme distal side of ferric Hb (31). Furthermore, NO2

� binding toferric Hb highlights the ligand-binding nonequivalence of aHband bHb. In fact, in NHbONOa NO2

� binds only to aHb, and inHbONOd,p NO2

� binds to heme-distal and heme-proximal side ofaHb and bHb, respectively (42). The different binding mode ofNO2

� to aHb and bHb in HbONOd,p reflects the sliding movementof the heme toward the bHb exterior, representing an interme-diate of Hb unfolding (42). Ligand-dependent heme sliding maybe a general mechanism for ligand-binding modulation as alsoobserved for carbonylation of murine neuroglobin (44).

Finally, the possibility that partially unfolded Hb deriva-tives may display transient ligand-binding properties differentfrom those of the native globin and of the unfolded proteinmay represent a case of ‘‘chronosteric effects’’ (45–47).Remarkably, the Hb reactivity toward CO increases transientlyat low pH (<4), preceding the irreversible protein denatura-tion. This reflects the protonation of the Ne atom of the HisF8residue and the cleavage of the proximal axial heme–Fe–Nebond, representing the first step of protein unfolding. In turn,the low reactive penta-coordinated heme becomes tetra-coor-dinated and highly reactive, the heme–Fe atom shifting fromthe ‘‘out’’ to the ‘‘in’’ plane geometry. This highlights the cru-cial role of the interaction(s) of the heme on the proximal sidein accounting for the difference in the ligand reactivitybetween the two quaternary R and T conformations of Hb (48).

AcknowledgementsDr. Loris Leboffe is supported by a grant from the NationalInstitute of Biostructures and Biosystems (INBB) of Italy.

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IUBMB LIFE

126 Ligand Binding to the Heme Proximal Side of Human Hb