To the packing contacts, we have recently assigned a key role for stabilizing the HbO2tetramer, as the formation of the a1b1 or a2b2 contact greatly suppresses the haem oxidation, partic
Trang 1R E V I E W A R T I C L E
Human haemoglobin
A new paradigm for oxygen binding involving two types of ab contacts
Keiji Shikama1,2and Ariki Matsuoka3
1 Biological Institute, Graduate School of Life Sciences, Tohoku University, Sendai, Japan, 2 PHP Laboratory for Molecular Biology, Sendai, Japan; 3 Department of Biology, Fukushima Medical University, Fukushima, Japan
This review summarizes the most recent state of
haemo-globin (Hb) research based on the literature and our own
results In particular, an attempt is made to form a unified
picture for haemoglobin function by reconciling the
cooperative oxygen binding with the stabilization of the
bound dioxygen in aqueous solvent The HbA molecule
contains two types of ab contacts One type is the a1b2 or
a2b1 contacts, called sliding contacts, and these are strongly
associated with the cooperative binding of O2to the a2b2
tetramer The other type is the a1b1 or a2b2 contacts, called
packing contacts, but whose role in Hb function was not
clear until quite recently However, detailed pH-dependence
studies of the autoxidation rate of HbO2have revealed that
the a1b1 and a2b2 interfaces are used for controlling the
stability of the bound O2 When the a1b1 or a2b2 contact is
formed, the b chain is subjected to a conformational con-straint which causes the distal (E7) histidine to be tilted slightly away from the bound dioxygen, preventing the proton-catalysed nucleophilic displacement of O2 from the FeO2by an entering water molecule This is one of the most characteristic features of HbO2stability Finally we discuss the role of the a1b1 or a2b2 contacts by providing some examples of unstable haemoglobin mutants These patho-logical mutations are found mostly on the b chain, especially
in the a1b1 contact regions In this way, HbA seems to differentiate two types of ab contacts for its functional properties
Keywords: ab contacts; distal (E7) histidine; HbA; heme oxidation; oxygen binding
Two types of ab contacts in HbA
In haemoglobin (Hb) research, the central problem is
understanding the mechanism for the cooperative oxygen
binding to the a2b2tetramer For human HbA, the a and b
chains contain 141 and 146 amino acid residues,
respect-ively, and a representative set of the successive
oxygen-binding constants is given in terms of Torr)1as follows:
K1¼ 0.0188, K2¼ 0.0566, K3¼ 0.407 and K4¼ 4.28 in
0.1MBis/Tris buffer containing 0.1MKCl at pH 7.4 and
25C [1] In this reaction, major differences have been
found between deoxyhaemoglobin and oxyhaemoglobin by
comparing their X-ray crystal structures (e.g [2–6]) These
include a movement of the iron atom into the haem plane
with a simultaneous change in the orientation of the
proximal (F8) histidine, a rotation of the a1b1 dimer relative
to the other a2b2 dimer about an axis P by 12–15 degrees, and a translation of the one dimer relative to the other along the P axis by approximately 1 A˚ The latter two changes are accompanied by sequential breaking of the so-called salt bridges by C-terminal residues Incidentally, the P is taken
as an axis which is perpendicular to the dyads of both the liganded and unliganded Hbmolecules
As illustrated in Fig 1, there are two types of ab contacts
in the Hbmolecule One is the a1b1 (or a2b2) contact involving B, G, and H helices and the GH corner, and the other is the a1b2 (or a2b1) contact involving mainly helices
C and G and the FG corner [3,7] When HbA goes from the deoxy to the oxy form, the a1b2 and a2b1 contacts undergo the principal changes associated with cooperative oxygen binding, so that these are named the sliding contacts As a result of the relative rotation of the a1b1 and a2b2 dimers, the gap between the b chains becomes too small to accommodate 2,3-diphosphoglyceric acid (DPG) that serves
to reduce the oxygen affinity of HbA At the a1b1 and a2b2 interfaces, on the other hand, negligible changes are found insofar as the crystal structure has been examined These are called the packing contacts accordingly, but their role in haemoglobin function was not clear for a very long time
To the packing contacts, we have recently assigned a key role for stabilizing the HbO2tetramer, as the formation of the a1b1 or a2b2 contact greatly suppresses the haem oxidation, particularly of the b chain at acidic pH values [8,9] Based on a nucleophilic displacement of O2 from the FeO2 centre, kinetic analyses of HbO2 oxidation were carried out with special focus on the proton-catalysed
Correspondence to K Shikama, PHP Laboratory for Molecular
Biology, Nakayama-Yoshinari 1-16-8, Sendai 989-3203, Japan.
E-mail: shikama@mail.cc.tohoku.ac.jp
Abbreviations: Hb, haemoglobin; DPG, 2,3-diphosphoglyceric acid.
Dedication: This review is dedicated to Max F Perutz
(19 May 1914–6 February 2002), who laid the foundation for an entire
field of haemoglobin research According to a kind suggestion made
by one of the referees, it should be added that Perutz once called
haemoglobin a honorary enzyme Both haemoglobin and myoglobin
are actually antienzymes, because they prevent the undesired
electron transfer from Fe(II) to the bound O 2 as far as possible in
aqueous solution.
(Received 5 June 2003, revised 29 July 2003,
accepted 13 August 2003)
Trang 2process performed by the distal (E7) histidine residue Such
examinations seem to be of primary importance, not only
for a full understanding of the molecular mechanism of
haemoglobin autoxidation, but also for planning new
molecular designs for synthetic oxygen carriers that are
highly resistant to haem oxidation under physiological
conditions Finally, we revisit haemoglobin function as seen
from the two different types of ab contacts, and try to
reconcile cooperative oxygen binding with stabilization of
the bound dioxygen With respect to this, we also give
possible implications for the unstable haemoglobin mutants
leading to the formation of Heinz bodies in red blood cells,
resulting in haemolytic anaemia
constituent chains
Biphasic nature of the autoxidation reaction
The reversible and stable binding of molecular oxygen with
the haem iron(II) is the basis of haemoglobin function Even
in air-saturated buffers, however, HbA is oxidized easily
from the oxygenated form (HbO2) to the ferric(III)
met-form (metHb) with generation of the superoxide anion
[10,11] as follows:
HbO2 !kobs
where kobs represents the first-order rate constant
observed at a given pH value in terms of the constituent
chains This autoxidation reaction can be monitored by
the spectral changes with time, after fresh HbO2 was
placed in 0.1Mbuffer containing 1 mMEDTA at 35C
The spectra evolved to the final state of each run, which
was identified as the usual ferric met-form, with a set of
isosbestic points Consequently, the process was
fol-lowed by a plot of experimental data as _ln([HbO2]t/
[HbO2]0) vs time t, where the ratio of HbO2
concentra-tion after time t to that at time t¼ 0 can be obtained by
the absorbance changes at 576 nm for the a-peak of
human HbO2
Fig 2 shows such examples of the first-order plot for the
autoxidation reaction of human HbO2at two different pH
values At pH 6.2, HbA exhibited a biphasic curve that can
be described by the first-order kinetics containing two rate
constants as follows:
½HbO2t
½HbO20¼ P expðkf tÞ þ ð1 PÞ expðks tÞ
ð2Þ
In this equation, a fast first-order rate constant kf is attributed to the a chains and a slow rate constant ksis for the b chains in the HbO2tetramer P is the molar fraction
of the rapidly reacting haems This conclusion is based on the rapid chain separation experiment of partially (30%) oxidized HbO2on polyacrylamide gel [8,12]
By iterative least-squares procedures inserting various values for kf and ks into Eqn (2), the best fit to the experimental data was obtained as a function of time t In these computations, the value of P was also allowed to vary across a large range from 0.40 to 0.60 [8,9] In this way, the following parameters were established at pH 6.2: kf¼ 0.82 (± 0.03)· 10)1h)1, ks¼ 0.13 (± 0.01) · 10)1h)1, and
P¼ 0.52 (± 0.04) in 0.1MMes buffer at 35C At pH 9.2,
on the other hand, the reaction was described completely by
a single first-order rate constant of 0.99 (± 0.02)· 10)2h)1 (i.e kf¼ kswith P¼ 0.50) in 0.1MCaps buffer at 35C
We have also studied the effect of DPG on the autoxidation rate of HbA at 35C DPG was added to stripped HbO2
(0.13 mM) at molar excesses of 5, 14 and 24, b ut this allosteric effector offered no significant effect on either kfor
k values at pH 6.5 and 8.5 [13]
Fig 2 First-order plots for the autoxidation reaction of human HbO 2 in 0.1 M buffer at 35 °C Each curve was obtained by a least-squares fitting to the experimental points, based on Eqn (2) At pH 6.2, HbA showed a biphasic autoxidation curve containing two rate constants, k f
and k s , respectively At pH 9.2, however, the reaction was mono-phasic Redrawn from Yasuda et al [9].
Fig 1 Schematic diagram of HbA tetramer showing the two different
types of ab contacts HbA has a molecular dyad axis (which is
per-pendicular to the plane of the figure) relating the a1b1 dimer to the
a2b2 dimer.
Trang 3pH-Dependencies of the autoxidation rate
If the values of kfand ksare plotted against the pH of the
solution, we can obtain a pH profile for the stability of
HbO2 Fig 3 shows such profiles for both of the a and b
chains in the HbO2tetramer over the range pH 5–11, under
air-saturated conditions in 0.1M buffer at 35C In the
acidic range of pH 7–5, the logarithmic values of kf
increased very rapidly with increasing hydrogen ion
con-centration The values of ksalso increased with increasing
proton concentration but much less so than for kf Rather,
the ksvalues exhibited a rate saturation behaviour on the
acidic extreme In a plot of log(kobs) vs pH, its slope showed
a value of n¼)1 for kf, whereas a value close to n¼)0.6
was for ks In the basic side higher than pH 8, on the other
hand, practically no difference was observed between the
kfand ks values, indicative of the oxidation curve being
monophasic Nevertheless, it is also true that both graphs
depend strongly upon the pH of the solution, having a
parabolic part with a minimum rate appearing at pH 8.5
At this point, the most important questions have arisen as
to whether the constituent a and b chains each has its own
different stability, and, if not, what the origin is of
nonequivalence of the chains in haem oxidation In this
regard, it should be noted that such a chain heterogeneity of
HbO2 oxidation can be retained even in very diluted concentrations of haemoglobin [13] When human HbO2is placed in dilution, the tetrameric species is known to dissociate into ab dimers along the a1b2 or a2b1 interface,
so that the dimers produced are of the a1b1 or a2b2 type [14,15] Accordingly, these results strongly suggest that the formation of the a1b1 or a2b2 contact must be responsible for the remarkable stability of the b chain against the acidic autoxidation This was the next step to be clarified Stability property of the separated a and b chains
In separated a and b chain solutions, the protein is known
to exist in an equilibrium of a *) a2 and b *) b4
respectively Under our experimental conditions, the mono-meric form (87%) was predominant in the a chain, while the tetrameric form (99%) was predominant in the b chain This estimation was made on the basis of the results of McDonald et al [16] As for the tetrameric form of the b chain, Borgstahl et al [7] have reported the 1.8 A˚ structure with carbonmonoxy-b4 (COb4) derivative, and compared subunit–subunit contacts between three types of interfaces (a1b1, a1b2, and a1a2) of HbO2 and the corresponding COb4 interfaces As a result, they found that the b1b2 interface of the COb4 tetramer is less stable and more loosely packed than its a1b1 counterpart in HbO2 In particular, there are significant packing differences at the end of the B helix between these homologous interfaces; the
B helix–H helix contact region is spread apart by approxi-mately 1 A˚ in COb4relative to oxyHb Specifically, the b1b2 interface of the COb4 tetramer does not include close contacts between residues Pro-125 (H3) and Val-33 (B15), Gln-127 (H5) and 34 (B16), and Ala-128 (H6) and
Val-34 (B16) The side chain disorder also makes the centre of the b1b2 interface packed less tightly in the COb4tetramer Therefore, the b1b2 contact sites in the b4 tetramer are indeed different from the corresponding a1b1 contact sites
in the HbA tetramer
Anyway, we have revealed that over the wide range of
pH 5–10, the separated a and b chains are both oxidized much more rapidly than in the parent HbO2 tetramer Fig 4 represents such pH-dependencies of the observed rate constants, ka
obsand kobsb , for autoxidation of the isolated
a and b chains in 0.1Mbuffer at 35C It thus becomes evident that the b chain, when separated from the HbO2 tetramer, does not show any rate saturation behaviour at low pH Rather, its rate increased very rapidly with increasing hydrogen ion concentration, exhibiting a value close to n¼)1 for the slope against the acidic pH We can therefore conclude that the intrinsic oxidation rate is almost the same with the separated a and b chains, completely freed from the remarkable differences between them in the autoxidation reaction of the parent HbO2tetramer
FeO2bonding and its nucleophilic displacement of O2–
It has been widely accepted that HbA is much more resistant
to autoxidation than myoglobin However, it is now evident that the constituent a and b chains, once separated from the parent HbO, are oxidized more rapidly than most
Fig 3 Differential pH-dependencies of k f and k s for the autoxidation
reaction of human HbO 2 in 0.1 M buffer at 35 °C A pair of the
observed first-order rate constants, k f (s) and k s (d), was obtained by
a least-squares fitting to each of the oxidation curves at different pH
values In the acidic range of pH 7–5, the logarithmic plots of k f give a
slope of n ¼ )1 against the pH, b ut n ¼ )0.6 for k s Redrawn from
Tsuruga et al [8].
Trang 4mammalian oxymyoglobins Such enhancements in the
oxidation rate have been frequently attributed to the
increased concentration of the deoxygenated species in
HbO2or MbO2solution, since the deoxy form is certainly
the preferred target for many kinds of oxidants This simple
mechanism, however, cannot explain the above-mentioned
results for the separated a and b chains, because it has been
definitively established that both chains have a much higher
oxygen affinity with fewer deoxygenated species than the
parent HbO2tetramer In 0.1Mphosphate buffer at pH 7.0
and 30C, indeed, Tyuma et al [17] reported the P50values
of 1.00 Torr for the a chains and 0.45 Torr for the b chains,
whereas HbA showed P50¼ 16.59 Torr in the absence of
DPG
Certainly, dioxygen is a powerful oxidizing agent in
a triplet ground state, 3P
g, whose biradical electronic configuration is given by the following notation:
O2ðr1sÞ2ðr1sÞ2ðr2sÞ2ðr2sÞ2ðr2pzÞ2
ðp2pxÞ2ðp2pyÞ2ðp 2pxÞ1ðp 2pyÞ1ðr 2pzÞ0 ð3Þ
Dioxygen therefore has a very strong tendency to take
electrons from other substances and to make the
com-plete electron-pairing in its unoccupied orbitals This
property leads to the sequential production of the
so-called active oxygen species such as superoxide anion
(O2), peroxide anion (O¼2Þ and hydroxyl radical (HO•)
For O at 760 Torr
oxidation-reduction potential is + 0.81 V for the com-plete, four-equivalent reduction to water, showing a total free energy change of)74.7 kcalÆmol)1()312 kJÆmol)1) Nevertheless, the addition of the first electron to O2is an unfavourable, uphill process with a low redox potential
of e¢(O2/O2)¼)0.33 V [18] All of the steps subsequent
to water are downhill In this sense, molecular oxygen is
a rather poor one-electron acceptor, and this thermo-dynamic barrier to the first step seems to be the crucial ridge located between the stabilization and the activation
of dioxygen bound to the haemoproteins [19]
Using a value of + 0.150 V for the oxidation–reduction potential of human Hbat pH 7 and 30C [20], we may write the primary step for the autoxidation reaction of HbO2as follows:
In this scheme, the reaction from left to right is associated with a change in redox potential (De¢) of)0.48 V, which corresponds to a positive free energy change of + 11.0 kcalÆ mol)1(+46.0 kJÆmol)1) Accordingly, a considerable energy barrier accompanies the reduction of O2to O2 by
deoxy-Hb, so this one-electron transfer cannot occur spontane-ously In many respects, the spontaneous dissociation of O2 from the FeO2 centre is an energetically unfavourable process, so that there must be involved some specific mechanism that causes very rapid generation of O2 from HbO2, as formulated in Eqn (1), in aqueous solution Recently, Shikama [21] has carefully evaluated various mechanisms proposed so far for the autoxidation reaction
of myoglobin and haemoglobin, including the effects of pH, oxygen pressure, and subsequent side reactions with the
H2O2produced by the spontaneous dismutation of O2 As
a result, he concluded that a displacement mechanism is needed to make it possible to yield O2 so readily from the FeO2centre In essence, kinetic and thermodynamic studies
of the stability of mammalian oxymyoglobins have shown that the autoxidation reaction is not a simple, dissociative loss of O2 from MbO2 but is due to a nucleophilic displacement of O2 from MbO2by a water molecule or a hydroxyl ion that can enter the haem pocket from the surrounding solvent The iron is thus converted to the ferric met-form, and the water molecule or the hydroxyl ion remains bound to the Fe(III) at the sixth coordinate position so as to form aqua- or hydroxide-metMb Even the complicated pH-dependence for the autoxidation rate can thereby be explained primarily in terms of the following three types of displacement processes [19,21–24]:
Mb(II)(O2Þ þ H2O!k0
Mb(III)(OH2Þ þ O2 ð5Þ Mb(II)(O2Þ þ H2Oþ Hþ!kH
Mb(III)(OH2Þ þ HO2 ð6Þ Mb(II)(O2Þ þ OH!kOH
Mb(III)(OHÞ þ O2 ð7Þ
In these equations, k0 is the rate constant for the basal displacement by HO, k is the rate constant for the
Fig 4 pH profiles for the autoxidation rate of the separated a and b
chains in 0.1 M buffer at 35 °C Both of the computed curves were
obtained by a least-squares fitting to the experimental points over the
whole range of pH studied, based on Eqn (8) Redrawn from Tsuruga
et al [8].
ð4Þ
Trang 5proton-catalysed displacement by H2O, and kOH is the
rate constant for the displacement by OH– The extent of
contribution of these elementary processes to the
observed or overall autoxidation rate, kobsin Eqn (1),
can vary with the concentrations of H+ or OH– ion
Consequently, the autoxidation rate exhibits a very
strong parabolic dependence on pH The reductive
displacement of the bound dioxygen as O2 b y H2O
can proceed without any protonation, but it has been
clearly shown that the rate is enormously accelerated
with the proton assistance by a factor of 106per mole, as
formulated by Eqn (6) In this proton catalysis, the
distal histidine, which forms a hydrogen bond to the
bound dioxygen [25], appears to facilitate the effective
movement of a catalytic proton from the solvent to the
bound, polarized dioxygen via its imidazole ring and by
a proton-relay mechanism [21,24] In this way, such a
nucleophilic displacement mechanism has successfully
been applied to detailed pH-dependence studies of the kf
and ks values, both for the HbO2 tetramer and the
separated chains, over the wide range of pH 5–11 in
0.1Mbuffer at 35C [8]
Numerical analyses of the pH-dependence curves
In the autoxidation reaction, pH can affect the rate in many
different ways To work out definitely the kinetic and
thermodynamic parameters contributing to each kobsvs pH
profile, we have proposed some mechanistic models for each
case The rate equations derived therefrom were tested for
their fit to the experimental data with the aid of a computer
As a result, the pH-dependence curves for the autoxidation
rate of the separated a and b chains have been analysed
completely in terms of an acid-catalysed two-state model
[8] In this kinetic formulation, it is assumed that a single,
dissociable group, XH with pK1, is involved in the reaction
Consequently, there are two forms of the oxygenated chain,
represented by A and B, at molar fractions ofF and Y
(¼ 1 – F), respectively, which are in equilibrium with each
other but which differ in dissociation state for the group
XH These forms can be oxidized to the ferric met-form by
a nucleophilic displacement of O2 from the FeO2centre
by an entering water molecule or hydroxyl ion
By using the rate constants defined in the preceding
section, the observed first-order rate constant, ka
obsor kbobsin Eqn (1), can be reduced to:
kaobsðor kbobsÞ ¼ fkA
0½H2O þ kA
H½H2O½HþgðUÞ
þ fkB0½H2O þ kBH½H2O½Hþ þ kBOH½OHgðWÞ ð8Þ where
þ
½Hþ þ K1
and W¼ ð1 UÞ ¼ K1
½Hþ þ K1
ð9Þ
By iterative least-squares procedures inserting various values for K1, the adjustable parameter in Eqn (9), the best
fit to more than 60 experimental points was obtained for each of ka
obsand kobsb as a function of pH (see Fig 4) In this way, the rate constants and the acid dissociation constant involved in the autoxidation reaction of the separated a and
b chains were established in 0.1M buffer at 35C, as summarized in Table 1
These results clearly indicate that both a and b chains are inherently quite susceptible to haem oxidation over the whole range of pH studied For example, their k0Bvalues are even higher (by 2.5–4.5-fold) than that of bovine MbO2
(kB
0 ¼ 0.17 · 10)3h)1ÆM )1) in 0.1Mbuffer at 35C [26] It becomes also evident that the proton-catalysed processes with the rate constants kA and kB
H promote most of the autoxidation reaction of each chain, above the basal processes in water with the rate constants kA
0 and kB
0 In fact, the catalytic proton enhances the rate dramatically both in the separated a and b chains, by a factor of more than 106per mole for state A and state B as well In this proton catalysis, the distal histidine (the dissociable group
XH with pK1¼ 6.1), which is located at position 58 for the
a chain and at position 63 for the b chain, appears to participate by a proton-relay mechanism the same as in mammalian oxymyoglobins [21,24] Indeed, random and undirected access of a proton to the bound dioxygen cannot yield such an enzyme-like, catalytic effect on the acidic autoxidation of MbO2and HbO2as well
In the HbO2 tetramer, on the other hand, a marked difference was found between the a and b chains in the oxidation rate As seen in Fig 3, the values of kf(due to the
a chain) were suppressed considerably over the wide range
of pH 7–11, but its pH-dependence was quite similar in shape to that of the separated a chain By the same mechanism as described in Eqn (8) therefore, we can obtain the best fit to more than 75 experimental points of kfover the whole pH range as follows:
Table 1 Rate constants and acid dissociation constants obtained from the pH-dependence curves for the autoxidation rate of the separated a and b chains in 0.1 M buffer at 35 °C Taken from Tsuruga et al [8].
Trang 6kf ¼ fkA0½H2O þ kAH½H2O½HþgðUÞ
þ fkB
0½H2O þ kB
H½H2O½Hþ þ kB
OH½OHgðWÞ ð10Þ Table 2 summarizes the rate constants and the acid
dissociation constant involved in the autoxidation
reac-tion of the a chain in the HbO2tetramer [8] From these
results, it is quite clear that the proton-catalysed
processes with the rate constants kA and kB
H are mainly responsible for the acidic oxidation of human HbO2 In
this proton catalysis, the distal histidine at position 58
should also participate as the dissociable group XH with
pK1¼ 6.2
In sharp contrast to the a chain, the autoxidation of the b
chain in the HbO2 tetramer exhibited a rate-saturation
behaviour below pH 5 Unfortunately, at more acidic pH
data points could not be obtained due to denaturation of the
protein By a simple two-state model, however, we have
reached the best fit to more than 80 values of ksover the
whole range of pH studied, in a quite acceptable way as seen
in Fig 3 In this mechanism, we assumed that a single,
dissociable group (XH with pK1) is also involved in the
reaction, but the proton-catalysed processes (with the rate
constants kAand kB
H) were totally omitted from Eqn (10) as follows:
ks¼ fkA0½H2OgðUÞ þ fkB0½H2O þ kBOH½OHgðWÞ
ð11Þ where the molar fractions ofF and Y for the states A
and B can be given by Eqn (9) According to the same
fitting procedures, the rate constants and the acid
dissociation constant involved in the autoxidation of
the b chain in the HbO2 tetramer were established in
0.1Mbuffer at 35C, as summarized in Table 2 also
In these kinetic analyses, one of the most remarkable
features is that in the HbO2tetramer, the b chain does not
show any proton-catalysed process that has the term of
kH[H2O][H+] containing the distal histidine as its catalytic
residue Instead, the b chain shows the involvement of a
dissociable group (XH) with pK1¼ 5.1 in 0.1M buffer at
35C For this group the most probable candidate would
also be the distal histidine at position 63 This residue
however, if compared to the corresponding His58 (with
pK1¼ 6.2) of the a chain, seems to be less accessible to
solvent protons, titrating at a lower pH by almost one
pH unit Moreover, this residue in the b chain would
probably be located a little more apart from the bound
O2 so as to lose its catalytic effect on the acidic autoxi-dation
Key role of the a1b1 contact in stabilizing
Tilting of the distal histidine residue in the b chain
As is evident from Fig 3, the remarkable stability of human HbO2can be ascribed mostly to the delayed oxidation of the
b chain in acidic pH range It is also evident that the b chain has obtained this stability by blocking out the proton catalysis (Eqn 6) from the acidic oxidation At this point, it should be emphasized that such a stability characteristic of the HbO2 tetramer can b e retained even in the low concentrations of haemoglobin corresponding to appreci-able dissociation into a1b1 or a2b2 dimers [13] The mechanism whereby the b chain acquires the enhanced stability in the HbO2tetramer must therefore be associated with the formation of the a1b1 or a2b2 contact These recent findings have led us to conclude that the packing contact produces in the b chain a conformational constraint whereby the distal (E7) histidine at position 63 is tilted away from the bound dioxygen, so as to prevent the acid-catalysed displacement of O2 from the FeO2centre by an entering water molecule
Similarly, Shaanan [27] reported the stereochemistry of the iron-dioxygen bond in human HbO2b y single-crystal X-ray analysis In the a chain, the distance between Neof His (E7) and the terminal oxygen atom (O-2) is found to
b e 2.7 A˚, and the geometry favours a similar hydrogen bond as in the case of sperm whale MbO2 [25] In the
b chain, however, Ne(or Ne2relative to Ce1) of His (E7)
is located further away from both O-2 and O-1 (3.4 and 3.2 A˚, respectively), indicating that the hydrogen bond, even if formed, must be very weak Recently, Lukin et al [28] claimed that a hydrogen bond is formed between O2
and the distal histidine in both a and b chains of human HbO2, as revealed by heteronuclear NMR spectra of the chain-selectively labelled samples In 0.1M phosphate buffer at pH 8.0 and 29C, the (He2, Ne2) cross-peaks of the distal histidyl residues were clearly observed as doublets in the (1H, 15N) spectrum of HbO2, at 1H chemical shifts of 4.79 p.p.m for b63His and 5.42 p.p.m for a58His These were taken as an indication that the
Table 2 Rate constants and acid dissociation constants obtained from the pH-dependence curves for the autoxidation rate of HbO 2 tetramer in 0.1 M
buffer at 35 °C Taken from Tsuruga et al [8].
Trang 7He2proton is stabilized against solvent–water exchange by
a hydrogen bond between the distal His and the O2ligand
in both a and b chains At the same time, they reported
that much wider separation of 1.17 p.p.m appears on the
He1 resonances of the two distal histidine residues,
showing that b63His is different from a58His in either
the orientation or distance or both, with respect to the
haem-bound dioxygen Such marked differences between
the two distal haem pockets must also be responsible for
our kinetic results of the a and b chains in the HbO2
tetramer
Figure 5 illustrates in a very schematic way the structure
of human HbO2, as seen in the a1b1 (or a2b2) contact
leading to the nonequivalence of the a and b chains The
four haem pockets are all exposed at the surface of
the molecule, so that each FeO2 centre is always subject
to the nucleophilic attack of an entering water molecule
or hydroxyl ion In the a chain, the distal histidine at
position 58 can stabilize the bound O2by hydrogen bond
formation Nevertheless, it is also true that this residue
participates, via its imidazole ring and by a proton-relay
mechanism, in facilitating the effective movement of a
catalytic proton from the solvent to the bound, polarized
dioxygen This proton-assisted nucleophilic displacement
of O2 from the FeO2centre by an entering water molecule,
that is an SN-2 type process with proton assistance [21,24],
can account for most of the autoxidation reaction at acidic
pH side In the b chain, on the other hand, the remarkable
stability is produced by the formation of the a1b1 and
a2b2 contacts, which give rise to a conformational
constraint whereby the distal histidine at position 63 is
tilted away from the bound O2 As a result, the constituent
b chains lose a proton-catalysed process and thus provide
the HbO2tetramer with the enhanced stability against the acidic oxidation
To understand more quantitatively the effect of the a1b1
or a2b2 contact on the haem oxidation, the next step was to construct the iron valency hybrid tetramers containing either the a or b chains in the ferric met-form, and to test their stability as compared with the native HbO2tetramer as well as the separated a and b chains
Further evidence from the iron valency hybrid haemoglobins
By mixing equivalent amounts of the separated a and b chains whose sulfhydryl groups were completely recovered,
we can prepare the reconstructed HbO2 and its valency hybrid tetramers such as (a3+)2(bO2)2and (aO2)2(b3+)2 To obtain the ferric met-form for each chain, the oxygenated species was oxidized by the addition of potassium ferri-cyanide The mixed chain solution containing either the a
or b chain in the ferric met-form was then applied to a CM-cellulose column to separate each hybrid tetramer from its unassociated chains [9]
When the iron valency hybrids are placed in air-saturated buffers, the oxygenated chains of each tetramer are oxi-dized easily to the ferric met-form Fig 6 represents such first-order plots to show wide differences in the oxidation rate of the b chain, when it exists as the separated (bO2)4, valency hybrid (a3+)2(bO2)2, and reconstructed HbO2 tetramers in 0.1MMes buffer at pH 6.2 and 35C In this way, the resulting rate constants for the a and b chains are compared between the native, separated, reconstructed, and valency hybrid haemoglobins at several pH values [9]
At pH 6.2, for instance, native HbO2 gives the rate constants of kf¼ 0.82 · 10)1h)1and ks¼ 0.13 · 10)1h)1
in its biphasic curve As listed in Table 3, almost the same oxidation rates were obtained for the reconstructed HbO2with a biphasic ratio of kf/ks¼ 6.1 Among those, the most remarkable effect was found on the b chain The separated b chain in itself undergoes quite rapid oxidation with a rate constant of kobs¼ 0.10 h)1, b ut this rate was dramatically suppressed up to ks¼ 0.14 · 10)1h)1 (by sevenfold) in the reconstructed HbO2, as is in native Hb O2 More importantly, such a retarded ks value could be maintained totally in the valency hybrid (a3+)2(bO2)2 tetramer
All of these features were essentially the same at other
pH values Certainly, the biphasic nature of the autoxi-dation rate of HbO2became much slower at pH 7.5, and even disappeared at pH 9.0 Nevertheless, the rate of oxidation of the separated b chain was markedly reduced
by up to 15-fold at pH 7.5 and up to 23-fold at pH 9.0 in the tetrameric haemoglobin, either it is native or recon-structed or even valency hybrid species The similar situation was also found in the a chain, but its effect on the stability of human HbO2was much less crucial than the b chain
It thus becomes evident that the b chain has acquired a remarkable resistance against the acidic oxidation in a manner of contacting with the a chain, no matter which valency the latter partner is in, the ferrous or the ferric state From these recent findings, we conclude that the packing contact produces a conformational constraint in the
Fig 5 Schematic representation of human oxyhaemoglobin as seen in
the a1b1 contact to produce tilting of the distal histidine in the b chain In
HbO 2 , the four haem pockets are all exposed at the surface of the
molecule By the formation of the a1b1 contact, the b chain is subject
to a structural constraint whereby the distal histidine at position 63 is
tilted slightly away from the bound O 2
Trang 8b chain, so that the proton-catalysed process performed by
the distal histidine residue disappears from its acidic
autoxidation Furthermore, spectral examinations have
disclosed that the formation of the a1b1 or a2b2 contact also protects the b chain from its haemichrome conversion
As a matter of fact, the oxidation product of the isolated
b chain was not for the usual ferric met-form but for its admixture with haemichrome In this way, the noticeable stability of human HbO2 depends largely upon the very unique property of the b chain on the a1b1 or a2b2 interface
Concluding remarks: a unified picture for Hb function
In HbA, the four haem pockets are all exposed at the surface of the molecule From the X-ray crystal structures (e.g [2–6]), however, it becomes apparent that the ligands) including O2 and CO to the ferrous form and
H2O, OH–, N3 and CN–to the ferric form) cannot gain access to the closed haem pockets of haemoglobin as in the case of myoglobin Karplus and McCammon [29] expressed this situation by the following passage in a satirical way If the structure of sperm whale myoglobin was so rigid that the rotations of side chains were impossible, an oxygen molecule might take many billions
of years to enter or leave the haem pocket across high energy kinetic barriers: the time would be much longer than a whale’s lifetime Consequently, the thermal fluctu-ations of side chain amino acid residues are essential for the penetration of ligands from the surrounding solvent through the globin matrix to the haem pocket [29–32] In this respect, much attention has been paid to the possible roles of the distal (E7) histidine residue in myoglobin and haemoglobin functions It has been suggested that it acts
as a gate [29] or a swinging door [33,34] for ligand entry into the haem pocket, and that it stabilizes the bound dioxygen by hydrogen-bond formation [25], as well as it stabilizes the axial water molecule of the ferric, high-spin species [35–37] Furthermore, the distal histidine via its imidazole ring participates in a proton-relay mechanism as
a catalytic residue for the acidic oxidation of MbO2 and HbO2 [8,21,24]
Fig 6 First-order plots to compare the autoxidation rate of the b chain
between three different haemoglobin derivatives in 0.1 M maleate buffer
at pH 6.2 and 35 °C Each curve was obtained by a least-squares fitting
to the experimental points, based on Eqn (2) The oxidation of the
separated b chains could be described by a single rate constant of
k obs ¼ 0.10 h)1in the presence of 20% (v/v) glycerol This inherent
rate was dramatically suppressed not only in the reconstructed HbO 2
but also in the valency hybrid (a 3+ ) 2 (bO 2 ) 2 as well Redrawn from
Yasuda et al [9].
Table 3 Comparison of the autoxidation rate constants between the whole, separated, reconstructed, and hybrid haemoglobins in 0.1 M buffer at
pH 6.2 and 35 °C Taken from Yasuda et al [9].
Trang 9To make clear the functional role of the distal histidine
residue in the autoxidation reaction, Brantley et al [38]
were the first to use systematically the site-directed
mutagenesis of sperm whale myoglobin They showed
that mutations of the distal His at position 64, such as
those of H64G, H64V, H64L and H64Q, caused dramatic
increases in the autoxidation rate At pH 7.0, for instance,
the H64V mutant MbO2 was oxidized 400 times more
rapidly than the wild-type (H64H) MbO2 Using these
mutant myoglobins, we have also carried out detailed
pH-dependence studies of the autoxidation rate over the
wide range of pH 5–12 in 0.1Mbuffer at 25C [39] The
resulting pH-profiles were then compared with those of the
corresponding myoglobins occurring in nature As a result,
if the distal (E7) histidine was replaced by other amino
acid residues, all such mutant oxymyoglobins were found
to contain no proton-catalysis in the autoxidation reaction
Their pH profiles could be formulated by the kinetic
equations lacking in the rate constants kA and kB
H
accordingly
Along with these lines of evidence, we have recently
proposed that the distal histidine can play a dual role in the
nucleophilic displacement of O2 from MbO2or HbO2[39]
One is in a proton-relay mechanism via the imidazole ring
at acidic pH Insofar as we have examined, such a
proton-catalysed process could never be observed in the
autoxi-dation reaction of myoglobins lacking the usual distal
histidine residue, no matter what the protein is, the
naturally occurring or the distal His mutant [39] As a
matter of fact, even if the distal residue is a histidine, it
cannot manifest any proton-catalysis when the residue is
tilted away from the precise E7 position This is just the
case we have described here for the b chain in the Hb O2
tetramer The other role of the distal histidine would be in
the maximum protection of the FeO2 centre against a
water molecule or a hydroxyl ion that can enter the haem
pocket from the surrounding solvent [38] This is relevant
to the considerable stability of MbO2 and HbO2 in the
neutral pH range In this way, the distal histidine provides
the delicate balance of catalytic and steric factors necessary
for controlling the reversible oxygen binding to myoglobin
and haemoglobin in aqueous solution
It is now clear that the constituent a and b chains, once
separated from the HbO2, are oxidized much more easily
than in the parent tetramer over the whole range of
pH 5–10 Moreover, their rates come to be almost equal to
each other and exhibit a very strong acid catalysis This
inherently high oxidation rate of each chain can be
suppressed dramatically by the formation of a1b1 (or
a2b2) contact In particular, the b chain provides a further
effect on the stability of HbO2by preventing the
proton-catalysed oxidation at acidic pH In order to explain such
unique properties of human HbO2, a nucleophilic
displace-ment mechanism has successfully been applied to detailed
pH-dependence studies of the autoxidation rate
As for the dimer and tetramer effects on haem
oxidation, probable explanations are as follows At basic
pH, the separated a and b chains are both quite susceptible
to autoxidation Each haem pocket seems to be
consid-erably open to allow easier attack of the solvent hydroxyl
ion on the FeO2 centre As a result, there occurs a very
rapid formation of hydroxide-met species, its rate being
dependent directly upon the concentration of OH– ion When the a1b1 (or a2b2) contact is formed, accessibility of
OH–ion to the haem pocket would be greatly reduced by conformational constraints As OH– ion is one of the strongest nucleophiles in vivo, practically no rate difference could be observed between the a and b chains on the basic
pH side, so that the autoxidation curve would become monophasic regardless of the ab dimer and the HbO2 tetramer
On the acidic side from neutral pH, the displacing nucleophile is an entering water molecule and its concen-tration is always taken as 55.5M in aqueous solution Participation of the catalytic proton via the distal histidine residue should therefore be a most decisive factor in accelerating the displacing rate of O2 from FeO2 with
H2O This is just the case with the separated a and b chains, both exhibiting a very strong acid catalysis in their oxidation rate Once the a1b1 (or a2b2) contact is established, the
b chain is subjected to a conformational constraint whereby the distal histidine at position 63 is tilted away from the bound dioxygen so as to be free from the proton-catalysed displacement In this way, the b chain can acquire a remarkable resistance against the acidic autoxidation, and this is one of the most characteristic features of the HbO2 stability
In relevance to a clinical aspect, it is interesting to note that a quite large number of unstable haemoglobins have been reported so far in the medical literature [3,4,40] Many
of the mutants which occur at the a1b2 interface have altered oxygen affinity, but bulk of evidence suggests that the a1b1 interface is much more important in maintaining normal haemoglobin stability than is the a1b2 interface In fact, haemolytic anaemia is known to result from substitu-tions affecting the a1b1 interface or the haem pocket If such mutations occur, the haem iron will be more easily oxidized, and a sequence of events leads to the globin precipitation or Heinz body formation in red blood cells Typical examples
of such variants are: Tacoma [b30(B12)Argfi Ser], Abraham Lincoln [b32(B14)Leufi Pro], Castilla [b32 (B14)Leufi Arg], Philly [b35(C1)Tyr fi Phe], Peterbor-ough [b111(G13)Valfi Phe], Madrid [b115(G17)Ala fi Pro], Khartoum [b124(H2)Profi Arg], J Guantanamo [b128(H6)Alafi Asp], Leslie [b131(H9)Gln fi deleted] and so on Surprisingly, most of the pathological mutations are found on the b chain, especially in the a1b1 contact regions In these unstable haemoglobins, the a1b1 contact would become loose or disruptive due to many different causes including: the insertion of proline (Abraham Lincoln, Madrid), the substitution with a too-small amino acid side chain (Tacoma) or a too-large side chain (Peterborough), the introduction of a charged or very polar group (Castilla, Khartoum, J Guantanamo), and the deletion of amino acid residue (Leslie)
The transport and storage of molecular oxygen by haemoglobin and by myoglobin are essential to life The iron(II)-dioxygen bond in these haem proteins plays a vital role in their physiology It is in the ferrous form that haemoglobin or myoglobin can bind molecular oxygen reversibly and carry out its physiological function From known changes in valency of the haem iron, one can write the functional cycle of the haemoglobin molecule as follows:
Trang 10During reversible oxygen binding, the oxygenated form of
haemoglobin, as well as of myoglobin, is oxidized easily to
the ferric met-form with generation of the superoxide
anion The met-haemoglobin or met-myoglobin thus
produced cannot bind molecular oxygen and is therefore
physiologically inactive
In red blood cells and muscle tissues, however, an
NADH-cytochrome b5oxidoreductase is present which can
reduce metHbor metMbto the ferrous deoxy-species again
and thus prevent the continued accumulation of the ferric
met-form in situ The enzyme is called methaemoglobin
reductase [41] and metmyoglobin reductase [42],
respec-tively, and is known to have a FAD group that can accept
electrons from NADH As a matter of fact, a strong and
cyclic reduction of the iron(III) species by these enzymes is a
basis for the continuity of haemoglobin and myoglobin
functions in vivo, since the autoxidation reaction is inevitable
in nature for all oxygen-binding haem proteins [21,23,24], as
well as for all synthetic dioxygen carriers [43,44] In fact, it
is a matter of our experience that the metMbcontent in
myoglobin extracts from various muscle tissues is
com-monly about 40%, while the metHb content of freshly
drawn blood is usually maintained within 1–2% but by a
very strong reductive environment
In conclusion, human haemoglobin seems to differentiate
two types of ab contacts quite properly for its functional
properties The a1b2 or a2b1 contact is associated with the
cooperative oxygen binding, whereas the a1b1 or a2b2
contact is used for controlling the stability of the bound O2
We can thus form a unified picture for haemoglobin function
by closely integrating the cooperative and the stable binding
of molecular oxygen with iron(II) in aqueous solvent
Acknowledgements
The materials of our previous publications were used with permission
from Publishers including: American Society for Biochemistry and
Molecular Biology, Inc (J Biol Chem.) and Blackwell Publishing Ltd.
(Eur J Biochem.).
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