A DFT Study on the Relative Affinity for Oxygen

of the α and β Subunits of Hemoglobin

JEAN-DIDIER MARÉCHAL,1 FELIU MASERAS,2,3 AGUSTÍ LLEDÓS,3 LILIANE MOUAWAD,4 DAVID PERAHIA1
1
Institut de Biochimie et de Biophysique Moléculaire et Cellulaire, Université Paris-Sud, Bât. 430,
94105 Orsay Cedex, France
2
Institute of Chemical Research of Catalonia (ICIQ), 43007 Tarragona, Catalonia, Spain
3
Unitat de Química Física, Edifici Cn, Universitat Autònoma de Barcelona, 08193 Bellaterra,
Catalonia, Spain
4
Laboratoire de Biophysique Moléculaire, Institut Curie, Université Paris-Sud, Bât. 112,
91405 Orsay Cedex, France

Received 30 November 2005; Accepted 28 February 2006

DOI 10.1002/jcc.20427
Published online in Wiley InterScience (www.interscience.wiley.com).

Abstract: DFT calculations are carried out on computational models of the active center of the α and β subunits of
hemoglobin in both its oxygenated (R) and deoxygenated (T) states. The computational models are defined by the full
heme group, including all porphyrin substituents, and the four amino acids closer to it. The role of the protein environment
is introduced by freezing the position of the α carbon atom of each of the four amino acids to the positions they have in the
available PDB structures. Oxygen affinity is then evaluated by computing the energy difference between the optimized
structures of the oxygenated and deoxygenated forms of each model. The results indicate a higher affinity of the α subunits
over the β ones. Analysis of the computed structures points out to the strength of the hydrogen bond between the distal
histidine and the oxygen molecule as a key factor in discriminating the different systems.

© 2006 Wiley Periodicals, Inc. J Comput Chem 27: 1446–1453, 2006

Key words: density functional theory; hemoglobin; heme group; oxygen affinity; cooperativity

Introduction
Oxygen transport is a fundamental process in most pluricellular
organisms. In vertebrates, this role is undertaken by hemoglobin
(Hb), a protein that picks up oxygen from the lungs and takes it
to the tissues. It is one of the fundamental components of blood,
and is responsible for its red color. The fixation center of oxygen
in hemoglobin is the heme group. The heme group is composed
by an iron atom and a porphyrin, a polycyclic planar ligand with
four coordination sites. The heme group fixes oxygen through its
attachment to iron.
Heme groups are common in biochemical systems. In oxygen
transporters, like hemoglobin and myoglobin, the metal is in its
ferrous oxidation state. In oxidation systems like cytochromes, iron
is usually in its ferric oxidation state. The chemistry of the heme
group is extensively studied, and an increasingly clear picture of
its main characteristics is emerging thanks to the contributions of
the experimental study of biological and biomimetical systems.1
A very significant effort has also been invested in the computational
study of its features using quantum chemistry. This has allowed to
clarify general aspects of the binding of dioxygen and other small
molecules to heme,2–5 the dynamics of movement around heme,6, 7
the role of the axial histidine by cysteine ligand,8, 9 and the nature of
intermediates and other mechanistic features of the catalytic cycle
of cytochrome P450 and related enzymes.10–12
One of the main biological features of hemoglobin is the coopera-
tivity behavior.13 There are four heme groups in a single hemoglobin
protein, and their affinity for oxygen depends on the oxygenation
state of the whole protein: the affinity increases with the number
of oxygen molecules fixed. That is, it is easier to bind the fourth
oxygen molecule in Hb(O2 )3 than to bind the first one in Hb. This
phenomenon has key biological consequences, because it makes
the saturation curve of the protein vary sharply in response to the
Correspondence to: F. Maseras; e-mail: fmaseras@iciq.es
Contract/grant sponsor: Training and Mobility of Research Program of the
European Union; contract/grant number: ERBFMBICT97-056
Contract/grant sponsor: Spanish MCYT and FEDER; contract/grant
numbers: CTQ2005.090000-CO1-01, CTQ2005-090000-CO1-02
Contract/grant sponsor: ICIQ Foundation
This article contains supplementary material available via the Internet at
http://www.interscience.wiley.com/jpages/0006-3525/suppmat

© 2006 Wiley Periodicals, Inc.

 Relative Affinity for Oxygen of the Hemoglobin Subunits
oxygen concentration. The protein thus loads, or unloads, the four
oxygen molecules in response to slight changes, and this feature
greatly improves its ability as transporter. There are a variety of
theories aimed at explaining this cooperativity behavior,14 although
its intepretation at the microscopic level remains controversial.15, 16
Hemoglobin is a tetramer composed of two α and two β subunits,
each of them containing one heme attached to the subunit by a cor-
dination bond between iron and histidine F8 (also called proximal
histidine). The residues laying on the opposite side of the heme con-
stitute the distal pocket. The key of the cooperativity mechanism is
the transfer of information between the heme groups, which is trig-
gered by the motion of the iron atom from the out-of-plane position
in the absence of oxygen (pentacoordinate system) to the in-plane
position in the presence of oxygen (hexacoordinate system). The
understanding of this cooperativity requires the introduction of the
whole protein for its computational study, and is a current subject of
research with the joint use of molecular mechanics and molecular
dynamics.17–19 The present study addresses a more modest, yet rel-
evant, issue, which is the affinity that each subunit has for oxygen in
the structure it adopts in both the deoxygenated (T) and oxygenated
(R) forms of the protein. Direct measurement of this affinity is not
possible, and a number of indirect approaches have been proposed.
There is no consensus yet on the relative affinities,20 although some
experiments suggest a higher affinity for the α subunit.21–23 Our
tools are DFT calculations with a large basis set on a model defined
by the heme group, the oxygen molecule, and the amino acids closer
to the active center. The geometrical constraints associated to the
Figure 1. The model system used in the calculations, with the labeling of the atoms.
Journal of Computational Chemistry
1447
protein chain are introduced by freezing the positions of the Cα
atoms of the amino acids considered to the positions they have in
the X-ray PDB structures of both the T and R forms of the protein.
Computational Details
Density functional calculations with the BP8624, 25 and B3LYP
functionals26, 27 were performed with the Jaguar program.28 Two
different basis sets were used. Most calculations were carried out
with basis set I, which corresponds to that labeled as LACVP** in
the program. This implies an effective core potential replacing the
10 innermost electrons of iron.29 The basis set is valence double-ζ
for all atoms,29–31 and has a polarization shell for all atoms differ-
ent from iron.31 An additional set of calculations was carried out
with the larger basis set II, which is that labeled as LACV3P**++
in the program. This is a valence triple-ζ basis set.28, 32 It includes
diffuse functions for all atoms,33 and it retains the same polariza-
tion shells of basis set I.31 For the oxygenated systems, basis set I
contains 1342 basis functions, and basis set II, 2052.
Geometry optimizations were carried out with the BP86 func-
tional and basis set I (BP86/I description). Most of the discussion on
energetics is nevertheless based on single-point calculations with the
B3LYP functional on the optimized BP86 geometries. The problem
of the absolute energetics of the oxygen fixation is a difficult one
from a computational view because of the involvement of different
spin states. The energy comparison between different spin states
DOI 10.1002/jcc
1448 Maréchal et al. • Vol. 27, No. 12
Figure 2. Real (left) and model (right) form of the histidine amino acid. Atoms in parenthesis are frozen
in the calculation.
is a challenge for DFT methods,34, 35 and B3LYP calculations on
BP86 geometries seem to provide a reasonable balance.36 As will
be shown below, the BP86 results suggest larger binding energies,
but the trends between the different systems considered, which is
the key issue in this article, are practically unchanged. The calcula-
tions used the unrestricted formalism for the quintet deoxygenated
state, with the same ground state described in previous work.37 Spin
contamination was always small. For the singlet oxygenated state,
a restricted formalism was used.
Most of the energies presented are potential energies in the gas
phase. An additional set of calculations was carried out introducing
environmental effects through a continuum calculation with Jaguar’s
Poisson–Boltzmann solver.28 The dielectric constant is set to 4.0 and
the probe radius to 1.40. Zero-point energies and entropic contribu-
tions have not been evaluated. The second derivative calculations
would be very time consuming, and we think it is reasonable to
assume that the additional contributions would be similar for all
computed systems. In almost all cases, geometry optimizations were
complete except for the frozen chain atoms. In the calculation of the
subunit β in the T-state, the dihedral angle of the acidic hydrogen
of one of the propionic substituents had to be frozen to avoid the
formation of a spurious hydrogen bond with the distal histidine.
Computational Model
The model system used in these calculations is shown in Figure 1. It
consists of the full heme group with all its substituents, and the four
closest amino acids to the iron atom, His E7, Val E11, Phe CD1 for
the distal side, and His F8 for the proximal side. The latter is bound
to the iron through its  nitrogen, and is trans to the coordination site
of dioxygen. Its introduction is mandatory in any simple representa-
tion of the active center of hemoglobin, and it has been so considered
Journal of Computational Chemistry
• Journal of Computational Chemistry
in most previous calculations on the system. The other three amino
acids are in the so-called distal side of the heme, where dioxy-
gen binds to iron. The importance of this distal side of hemoglobin
in dioxygen fixation is well established from experimental studies
on mutant systems,38 and from molecular dynamics calculations.17
The three amino acids that have been considered are the same in
both the α and β subunits. They are sensitive to oxygen fixation
because they change notably their positions in the X-ray structures
of the T and R forms of hemoglobin. Furthermore, it is well known
that the distal histidine E7 is able to form a hydrogen bond with
dioxygen, especially in the α chain.13
The structural features of the different subunits and states of
hemoglobin are introduced in the calculation by constraining the
coordinates of selected atoms to the values provided in the Protein
Data Bank (PDB) (www.rcsb.org). The structures taken from the
PDB are that with code 2HHB for the deoxygenated T form,39 and
that labeled as 1HHO for the oxygenated R form.40 The computa-
tional approach consisted of freezing the α carbons of the amino
acids to the position they have in the PDB structure. The peptidic
bonds connecting these α carbons to the rest of the protein chain
have been suppressed in our calculation, and their valence shell
has been saturated with two hydrogen atoms, which have also been
kept at the same orientation the C—C and the C—N bonds have
in the PDB structure. The three C—H bond distances were frozen
to 1.090 Å. This procedure is illustrated graphically in Figure 2
for the case of histidine. There are thus four frozen atoms for each
amino acid. The position for the rest of the atoms in each amino
acid, as well as the full heme group (except for the dihedral angle of
the propionate mentioned above) and the dioxygen, when present,
are completely optimized without any additional constraint in the
calculation. The propionate groups attached to the heme have been
protonated to keep them in the neutral form. We could not introduce
the amino acids neutralizing them in our calculation for a reason of
DOI 10.1002/jcc
 Relative Affinity for Oxygen of the Hemoglobin Subunits
size, and we assume that this aspect will not be critical for oxygen
affinity.
Available X-ray PDB structures for the two possible states, R
and T, and the two different subunits, α and β were considered.
This gives four different systems, labeled Rα, Rβ, Tα, and Tβ. The
oxygen affinity of each of these systems was evaluated by introduc-
ing additional calculations of “non-PDB” systems where dioxygen
was added for T states, or removed for R states. The oxygen binding
energy was estimated by using the simple equation:
E = Ehexa − (Epenta + EO2 ).
The geometry optimizations were carried out on a total of eight
structures: RαD, RαO, TαD, TαO, RβD, RβO, TβD, and TβO.
An additional pair of the calculations, in the presence and absence
of dioxygen, were carried out on a simplified system with no dis-
tal amino acids, just an imidazole as the proximal ligand and the
substituents of the porphyrin replaced by hydrogen atoms.
Energetics of Oxygen Affinity
The binding energies for dioxygen of each of the hemoglobin sub-
units computed with the different methods are collected in Table 1.
Comparison of the first two columns shows a large difference
between the B3LYP and BP86 binding energies. For BP86, the reac-
tion is strongly exothermic, with the products ca. 30 kcal/mol below
the reactants; while for B3LYP it is weakly endothermic, with the
products between 5 and 15 kcal/mol above the reactants. This large
difference between the two functionals is likely related to the known
DFT problem of the unbalanced description of systems of differ-
ent spin states. Functionals with pure DFT exchange like BP86 are
known to overstabilize low spin states, while hybrid functionals with
an exact exchange part are known to overstabilize high spin states.35
This problem is particularly visible in this specific system because
of the large change of spin involved: the oxygenated complex, a sin-
glet, breaks down into the deoxygenated complex, a quintet, and free
oxygen, a triplet. The discrepancy between BP86 and B3LYP ener-
gies thus does not seem related to the interactions between dioxygen
and the distal residues. This was further proved by a calculation on
a more simple system where the distal residues are deleted, all por-
phyrin substituents are hydrogen atoms, and the proximal histidine
is replaced by an imidazole. In this simplified model, the binding
energy was −28.6 kcal/mol with the BP86 functional and +8.3
kcal/mol with B3LYP.
Table 1. Binding Energy (kcal/mol) of Dioxygen Obtained with Different
Computational Methods for the Different Subunits (α, β) and Crystal
Structures (R, T) of Hemoglobin.
BP86/I B3LYP/I B3LYP/I + SCRF B3LYP/II
Rα −33.4 +5.6 +4.9 +8.8
Tα −33.5 +6.1 +5.4 +8.7
Rβ −27.4 +11.2 +10.2 +13.9
Tβ −25.6 +13.3 +11.8 +15.8
Model −28.6 +8.3 +8.0 +11.3
Data for the model system, with no amino acids or pending chains are also
included.
Journal of Computational Chemistry
1449
There is no simple way to treat the relative stability of different
spin states within the DFT formalism, but a consensus seems to
emerge in that the correct value should be between those of B3LYP
and pure functionals like BP86, and is usually closer to B3LYP. This
would leave a reaction exothermic by a few kcal/mol, which agrees
with the expected affinity for oxygen.
Therefore, it is difficult to give an accurate value for the absolute
oxygen affinity from these calculations. However, the relative val-
ues between the different subunits are relatively independent from
the method, and one can use either functional to obtain a reliable
comparison between the different subunits, which is the main goal
of this work. In what follows, we are going to focus on the B3LYP
values.
B3LYP results are presented in three of the columns of Table 1.
The second and third column differ only in the presence of solvent
effects in the latter. Solvent effects are found to slightly favor the
dissociation reaction, by values between 0.3 and 1.5 kcal/mol. The
change does not however, alter, the relative ordering of the energies.
The use of basis set II, in the fourth column, however, introduces
some interesting nuances. The improvement of the basis set has a
quite homogeneous effect on all four systems considered. Dissoci-
ation is always more difficult with the larger basis set, by a value
between 2.5 and 3.2 kcal/mol. Because of the small differences
involved, this forces a substantial approach between the dioxygen
affinities for systems Rα and Tα, which for basis set II are only
separated by 0.1 kcal/mol.
Concerning biochemical implications, a first observation from
Table 1 is that the β subunit of the protein is able to discriminate
between the R and T forms in terms of oxygen affinity. Binding
is 1.9 kcal/mol stronger in the case of the R structure. This is not
surprising, because R is the crystal structure where the oxygen is
present, and the protein environment has logically arranged itself for
an optimal interaction. This difference for the β subunit is not very
large, but sufficient for a biological effect. This is not, however, the
case for the α subunit, where the B3LYP/II result is, in fact, that the
deoxygenated form has an affinity 0.1 kcal/mol higher. A difference
of 0.1 kcal/mol is certainly not significant, but the result certainly
points out to an inferior capacity to discriminate by the α subunit.
Another interesting analysis comes from the comparison of the
affinities for the α and β subunits. The affinity of the α fragment
is always larger than the β one with a difference of 5.1 kcal/mol
for the R state and 7.1 kcal/mol for the T state. These values may
be overestimated, but we think that they clearly indicate, based
on the available structures of native Hb, that the coordination of
oxygen to deoxygenated hemoglobin must start in the α subunits.
Interestingly, Scherlis and Estrín41 obtained a different conclusion
from a study where DFT single-point calculations where applied
on structures partially optimized with the semiempirical SAM1
method. We believe that the current calculations are more reliable
because of the more complete geometry optimization with a more
accurate method. Our results are, furthermore, in agreement with the
more extended experimental view.21–23 The values of oxygen affin-
ity presented in Table 1 for the different computed systems can, in
fact, be well accommodated in a proposal for the mechanism of the
binding of the four molecules of oxygen to the protein.
In the deoxygenated T form, the affinity of the α subunit is sig-
nificantly higher than that of the β subunit, and it is reasonable to
assume that the oxygen uptake will start in the α subunits of the
DOI 10.1002/jcc
1450 Maréchal et al. • Vol. 27, No. 12 • Journal of Computational Chemistry

Table 2. Selected Geometrical Parameters (Å and Degrees) for the Computed Oxygenated
R Structures and Deoxygenated T Structures with BP86 Functional.

RαO RβO TαD TβD

Fe–Nim 2.082 (1.94) 2.100 (2.07) 2.172 (2.15) 2.170 (2.15)

Fe–Np a 2.018 (1.98) 2.015 (1.96) 2.064 (2.10) 2.089 (2.03)
Nim –Fe–Np a 88.7 (94.) 88.5 (87.) 98.0 (102.) 97.4 (99.)
Ca –Nim –Fe–Np1 −39.1 (−12.) −24.6 (−27.) −6.0 (−17.) −5.8 (−21.)
Fe–Oa 1.749 (1.66) 1.745 (1.86) — —
Oa –Ob 1.303 (1.22) 1.295 (1.24) — —
Fe–Oa –Ob 119.9 (153.) 121.0 (159.) — —
Nd –Hd 1.034 (1.05)b 1.021 (1.05)b — —
Hd –Oa 2.467 (2.03)b 2.616 (2.31)b — —
Hd –Ob 1.879 (1.69)b 2.177 (2.87)b — —

Experimental PDB parameters are indicated in parentheses (see atom labels in Fig. 1).
a Average value.
b Assuming N –H is 1.05 Å.
d d

protein when it is still in the T form. Then, because of the low affin-
ity of the β subunits in the T form, the transition from the T to the
R form should take place, and the second pair of oxygen molecules
would be loaded in the β subunits with the protein already in the
R form. Thus, the transition from T to R should be induced by the
oxygenation of the α subunits. This observation is in good agree-
ment with what was observed by NMR studies22 and molecular
dynamics simulations.17
The release of the four oxygen molecules from the oxygenated
hemoglobin would follow the opposite direction. The β subunits
would lose their oxygen molecules first because their affinity for
oxygen is relatively low even in the R structure. Of course, within our
simplified model, it is impossible to predict whether the transition
between R and T states should be coupled with oxygen uptake or
release, or if it would require oxygenation (or deoxygenation) of
both subunits of the same type to be induced.
Computed Structures for the “PDB” Systems
X-ray data for four of the computed systems, the oxygenated R
structure, and the deoxygenated T structure, have been reported in
the PDB, and the frozen atoms of our constrained geometry opti-
mizations have been, in fact, obtained from them. Accuracy of PDB
structures is, however, not perfect, and it has been shown that they
can be refined sometimes by DFT calculations.42 In what follows,
we critically evaluate the quality of our computed structures.
Table 2 collects computed values for selected geometrical para-
meters for structures RαO, RβO, TαD, and TβD. The overall
characteristics of the structures follow the trends that have been
repeatedly reported in previous theoretical calculations,37 and that
now seem to be generally accepted in the scientific community. The
coordination sphere of iron in the deoxygenated species TαD, and
TβD is that of the square pyramid, with the four porphyrin nitro-
gens occupying equatorial positions and the proximal histidine in
the axial site. The iron atom is out of the porpyrin plane, towards the
proximal side, as reflected by the average Nim –Fe–Np bond angles
of 98.0◦ and 97.4◦ The axial Fe–Nim distance is quite long (2.172
and 2.170 Å), significantly longer than the average equatorial Fe–Np
distances (2.064 and 2.089 Å). The oxygenated species RαO and
RβO (Fig. 3) are better described as octahedral complexes, with
the proximal histidine and the dioxygen ligand occupying trans
sites. The octahedral arrangement results in the atom center lying

Figure 3. Computed BP86 geometries of structures RαO (left) and RβO (right). Hydrogen atoms different
from Hd are omitted for clarity.

Journal of Computational Chemistry DOI 10.1002/jcc

 Relative Affinity for Oxygen of the Hemoglobin Subunits 1451

very near the porpyrin plane, and this is reflected by bond angles Table 3. Selected Geometrical Parameters (Å and Degrees) for the
around the iron center close to 90◦ . This is the case for the aver- Computed “Non-PDB” Structures: Oxygenated T and Deoxygenated R.
age Nim –Fe–Np angles of 88.7◦ and 88.5◦ . Dioxygen binds to iron
through only one of its atoms, resulting in a quite short Fe–O dis- RαD RβD TαO TβO
tance of ca. 1.75 Å. The Fe–Oa –Ob bond angle is near 120◦ and
119.9◦ for RαO, and 121.0◦ for RβO. Fe–Nim 2.168 2.156 2.103 2.093
It is interesting to compare these computed values with the PDB Fe–Np a 2.087 2.089 2.018 2.020
data also included in Table 2. The overall trends are the same, but Nim –Fe–Np a 96.9 97.1 88.7 88.7
differences are significant. This is particularly the case for the coor- Ca –Nim –Fe–Np1 −38.2 −17.9 −25.0 −41.1
Fe–Oa — — 1.750 1.748
dination of the dioxygen molecule. The PDB values in RαO and
Oa –Ob — — 1.302 1.284
RβO for the Fe–Oa distances are 1.66 and 1.86 Å, respectively, Fe–Oa –Ob — — 121.1 121.3
which are 0.09 Å shorter for α and 0.12 Å longer for β than the Nd –Hd — — 1.033 1.017
corresponding computed values. The discrepancy is even larger for Hd –Oa — — 2.593 6.088
the Fe–Oa –Ob bond angle, which is experimentally reported around Hd –Ob — — 1.905 4.937
155◦ , while the computed values are near 120◦ . We think that these
a Average value.
discrepancies are essentially due to the intrinsic inaccuracy involved
in the complicated X-ray measurement of proteins, which can, in
fact, be corrected sometimes by accurate quantum chemistry cal-
in Table 2, essentially because they will be discussed together with
culations.42 Computed geometries around Fe are more similar for
those of the “non-PDB” structures in the next subsection.
RαO and RβO than they are in the PDB structures. Furthermore, the
computed structures are closer to those of the biomimetic systems,
where the X-ray measurements are much more accurate. “PDB” vs. “non-PDB” Structures
It is reasonable that the larger inaccuracies in the PDB structures
are in the position of dioxygen because different conformers exist This section presents the geometries of the so called “non-PDB”
associated to rotation around the Fe–Oa axis. In fact, for the partic- structures, emerging from the computational experiment resulting
ular case of RβO we computed three of them, and only the most of substracting dioxygen from the R PDB conformations and adding
stable one is reported in Table 2. The other two structures had ener- it to the T PDB structures. Selected parameters from the calculated
gies 1.4 and 1.6 kcal/mol above. We did not compute the transition geometries of RαD, RβD, TαO, and TβO are collected in Table 3.
states connecting them, but the energy barriers are likely to be low. The main features in the coordination sphere of iron follow the gen-
In this situation, disorder is likely to affect the X-ray diffraction, eral trends described above. Deoxygenated structures are square
because of movements in the diffraction time scale or because of pyramidal (Nim –Fe–Np values around 97◦ ), and oxygenated struc-
population of different conformers. This disorder will, in any case, tures are octahedral (Nim –Fe–Np values around 89◦ ). The dioxygen
hamper interpretation of the diffraction spectra, thus rendering data ligand is bound η1 to iron and bent (Fe–Oa –Ob around 121◦ ) in the
less accurate. oxygenated structures.
Not all discrepancies can be easily attributed to inaccuracies The joint consideration of the data in Tables 1 to 3 shows a good
in the PDB data. For instance, the differences between computed correlation between the affinity for oxygen of a given subunit and the
and PDB structures in the dihedral angle Ca –Nim –Fe–Np1 can be geometrical parameters describing the hydrogen bond between the
as large as 27.1◦ (−39.1◦ vs. −12.◦ ) in the case of RαO while distal histidine and dioxygen in its oxygenated form. From Table 1,
insignificant (−24.6◦ vs. −27.◦ ) in RβO. The variability of this the affinity of the different subunits for dioxygen follows the order
angle, which essentially measures the rotation of the porphyrin ring Rα  T α > Rβ > T β. A stronger hydrogen bond will be geo-
around the Fe–Nim axis, can mainly be attributed to the lack of metrically reflected in a lengthening of the Nd –Hd distance and a
amino acids in our model, which most likely impede the rotation in shortening of the Hd –Ob distance. This is precisely what is found in
the protein. We think that this particular parameter should have a Tables 2 and 3 for the series described above. The Nd –Hd is longer
minor effect on the oxygen affinity based on the low energy barriers for the subunits with the higher oxygen affinities, with values of
calculated for this rotation.8 It would furthermore be pointless to 1.034, 1.033, 1.021, and 1.017 Å for Rα, Tα, Rβ, and Tβ, respec-
seek perfect reproduction of the experimental structure, because tively. The corresponding values for the Hd –Ob distance are 1.879,
our computational scheme is based on the frozen PDB positions of 1.905, 2.177, and 4.937 Å, respectively. This particular hydrogen
a set of atoms, and we have just seen above that the accuracy of this bond plays a fundamental role on the relative oxygen affinity of
PDB data is not perfect to start with. the different subunits. Similar hydrogen bonds had been previously
Table 2 also contains the parameters related to the hydro- reported in related systems,41, 43 and have, in fact, been proposed
gen bond between the distal histidine and the dioxygen molecule, to be responsible for the discrimination between oxygen and car-
Nd –Hd , Hd –Oa , and Hd –Ob . In this case, the comparison is partic- bon monoxide in myoglobin,44–46 a protein similar to one subunit
ularly complicated because the hydrogen atoms are not located in (α or β) of hemoglobin.
the X-ray structure, and their PDB position is assigned by assum- Figure 4 presents the computed structures of the models for the
ing a N–H distance of 1.05 Å in the direction pointing away from TαO and TβO subunits. The geometry of TβO clearly shows that
the imidazole ring. The experimental relative distance to the oxy- this is the only oxygenated structure with no hydrogen bond between
gen atoms is furthermore affected by the uncertainty in the PDB the distal histidine and the dioxygen molecule, which is consistent
position of oxygen discussed above. These parameters are included with the fact that Tβ has the lowest affinity for oxygen. The joint

Journal of Computational Chemistry DOI 10.1002/jcc

1452 Maréchal et al. • Vol. 27, No. 12
Figure 4. Computed BP86 geometries of structures TαO (left) and TβO (right). Hydrogen atoms different
from Hd are omitted for clarity.
observation of Figures 3 and 4, presenting the four oxygenated struc-
tures, shows how the hydrogen bond always takes place with Ob , the
oxygen atom further away from iron. In all cases the Nd –Hd bond is
in a direction mostly perpendicular to the Fe–Oa –Ob plane. Ob could
actually get closer to Hd by rotating the dioxygen ca. 90◦ around
the Fe–Oa axis. This orientation would, in fact, be the electronically
preferred, as found in calculations where the distal histidine is left
unconstrained.4 However, our attempts to optimize it in the case of
RβO produced a structure with a higher energy. The strain intro-
duced by the geometrical constraints in the Cα atoms of both the
proximal and the distal histidine preclude this structure. Another
alternative conformer, with Ob pointing away from the distal histi-
dine, would favor a hydrogen bond between Hd and Oa . This was
also tested, and found to have a higher energy than the reported struc-
ture. Moreover, the binding of oxygen to the Tβ structure induces an
important shift of the porphyrin. This is due to steric hindrance of the
distal amino acids in the deoxygenated conformation. This is con-
sistent with observations made previously with molecular dynamics
simulations where it was shown that the porphyrin must be displaced
before binding of oxygen.17 The distal histidine thus plays a funda-
mental role both on the energetics and the structure of the dioxygen
binding to the subunits of hemoglobin. However, the role of the other
amino acids used in these calculations must not be overlooked. The
proximal histidine is essential, because it actually fixes the position
of the heme group in space. The role of the other two amino acids,
valine and phenylalanine, is not so obvious, but from the structure of
TαO in Figure 4 it seems that the distal histidine cannot reorientate
itself to move Hd closer to the dioxygen because of the blocking
presence of the valine amino acid.
Conclusions
DFT calculations on a model system of the active center of the two
subunits of hemoglobin give information on their relative oxygen
affinities. In first place, the calculations correctly predict a higher
affinity of the β subunits for oxygen in the R configuration than
in the T configuration, which gives a certain confirmation to the
validity of the computational model. The calculations also indicate
a higher affinity for dioxygen in the case of the α subunit, a result that
has been previously suggested from experimental data. From these
Journal of Computational Chemistry
• Journal of Computational Chemistry
results, a mechanism can be proposed where oxygen coordination
starts in the α subunits of the T form of hemoglobin, which once
oxygenated, can induce the transition from the T to the R form, and
hence, coordination to the β subunits takes place. Deoxygenation
would follow the opposite pathway, starting by the loss of oxygen
from the β subunits. Analysis of the computed structures indicates a
fundamental role of the distal histidine amino acid in the energetics
of oxygen binding, in particular through the formation of a hydrogen
bond with the oxygen atom further away from the metal center.
A further understanding of the behavior of the affinity for oxygen by
the α and β subunits, including the key cooperativity feature, would
require introduction of the full protein, and is out of the scope of the
current work.
Acknowledgment
J.-D.M. thanks the financial support of the Training and Mobil-
ity of Researchers program of the European Union under contract
No. ERBFMBICT97- 056. Part of the calculations were carried
out on the IDRIS Supercomputer Center of the CNRS (France)
and in CESCA of Barcelona (Spain). Financial support from the
Spanish MCyT and FEDER (Projects CTQ2005-090000-CO1-01,
CTQ2005-090000-CO1-02) and the ICIQ foundation is acknowl-
edged.
Supplementary Material Available
Cartesian coordinates and total energies for the 10 optimized
structures discussed in the text.
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Journal of Computational Chemistry DOI 10.1002/jcc