Tài liệu Báo cáo khoa học: The swinging movement of the distal histidine residue and the autoxidation reaction for midge larval hemoglobins docx

The swinging movement of the distal histidine residue
and the autoxidation reaction for midge larval hemoglobins
Satoshi Kamimura
1
, Ariki Matsuoka
2
, Kiyohiro Imai
3
and Keiji Shikama
1,4
1
Biological Institute, Graduate School of Life Sciences, Tohoku University, Sendai, Japan;
2
Fukushima Medical University,
Fukushima, Japan;
3
Laboratory of Nanobiology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan;
4
PHP Laboratory for Molecular Biology, Nakayama-Yoshinari, Sendai, Japan
Some insects have a globin exclusively in their fast-growing
larval stage. This is the case in the 4th-instar larva of Toku-
nagayusurika akamusi, a common midge found in Japan.
In the polymorphic hemoglobin comprised of 11 separable
components, hemoglobin VII (Ta-VII Hb) was of particular
interest. When its ferric met-form was exposed to pH 5.0
from 7.2, the distal histidine was found to swing away from
the E7 position. As a result, the iron(III) was converted from
a hexacoordinate to a pentacoordinate form by a concom-
itant loss of the axial water ligand. The corresponding
spectral changes in the Soret band were therefore followed
by stopped-flow and rapid-scan techniques, and the

observed first-order rate constants of k
out
¼ 25 s
)1
and
k
in
¼ 128 s
)1
were obtained for the outward and inward
movements, respectively, of the distal histidine residue in
0.1
M
buffer at 25 °C. For O
2
affinity, Ta-VII Hb showed a
value of P
50
¼ 1.7 Torr at pH 7.4, accompanied with a
remarkable Bohr effect (dH
+
¼ )0.58) almost equal to that
of mammalian hemoglobins. We have also investigated the
stability property of Ta-VII HbO
2
in terms of the autoxi-
dation rate over a wide range of pH from 4 to 11. The
resulting pH-dependence curve was compared with those of
another component Ta-V HbO
2

and sperm whale MbO
2
,
and described based on a nucleophilic displacement mech-
anism. In light of the O
2
binding affinity, Bohr effect and
considerable stability of the bound O
2
against acidic
autoxidation, we conclude that T. akamusi Hb VII can play
an important role in O
2
transport and storage as the major
component in the larval hemolymph.
Keywords: Insect (midge) Hb; distal (E7) histidine; pH
jump; swinging movement; heme oxidation.
In previous papers, we reported that the hemoglobin from
the 4th-instar larva of Tokunagayusurika akamusi,acom-
mon midge (Diptera) found in eutrophic lakes in Japan, is
comprised of as many as 11 separable components (IA, IB,
II, III, IV, V, VIA, VIB, VII, VIII and IX) on a DEAE-
cellulose column, and found that these can be classified into
two groups on the basis of their presence or absence of the
distal (E7) histidine residue. For instance, hemoglobin VII
consists of 150 amino-acid residues and contains the usual
distal histidine at position 64, whereas component V replaces
it by an isoleucine at position 66 [1]. This is certainly one of
the unique characters of T. akamusi hemoglobins, as all the
Chironomus hemoglobins have a distal histidine residue.

Among the T. akamusi hemoglobins, component VII was of
particular interest. When its ferric met-form was placed in
acidic pH range, its Soret peak was considerably blue-shifted
and accompanied by a marked decrease in intensity,
indicative of the hemoglobin being converted into a structure
quite similar to that of Aplysia (sea hare) myoglobin lacking
the usual distal histidine residue. The pH-dependent Soret
magnetic circular dichroism (CD) spectra also revealed that
hemoglobin VII is in an equilibrium between a hexacoor-
dinate and a pentacoordinate structure for its ferric heme
iron [2]. We attributed this to a transition of an iron-ligated
water molecule that is hydrogen-bonded to the distal
histidine, to a water-free iron with the histidine swung away
from its E7 position.
In the present paper, we describe the swinging movement
of the distal histidine residue in T. akamusi hemoglobin VII
followed by stopped-flow rapid mixing techniques. To
demonstrate any unusual character of midge larval hemo-
globins, we also examine the autoxidation rate over a wide
range of pH, as well as the oxygen equilibrium parameters,
in 0.1
M
buffer at 25 °C. These examinations will provide us
with new insights about the unique characters of midge
larval hemoglobins and also the biochemical properties of
heme proteins in general.
Materials and methods
Chemicals
Butyl-Toyopearl (650
M

) was a product of Tosoh (Tokyo).
CM-cellulose (CM-32) and DEAE-cellulose (DE-32) were
purchased from Whatman. Mes, Mops, Taps, Caps and
Tris for buffer systems, and all other chemicals, were of
reagent grade from Wako Pure Chemicals (Osaka). Solu-
tions were made with deionized and glass-distilled water.
Preparation of midge larval hemoglobin components
As described previously [1], the hemoglobin from the 4th-
instar larva of T. akamusi is comprised of as many as 11
Correspondence to K. Shikama, PHP Laboratory for Molecular
Biology, Nakayama-Yoshinari 1-16-8, Sendai 989-3203, Japan.
E-mail:
(Received 13 September 2002, revised 28 January 2003,
accepted 4 February 2003)
Eur. J. Biochem. 270, 1424–1433 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03498.x
separable components on a DEAE-cellulose column. Of
these, hemoglobin VII, a major component, was isolated as
follows. Frozen larvae (20 g in each experiment) were
thawed quickly and homogenized with Polytron (Kinema-
tica, Switzerland) in a 3-volume (v/w) of 50 m
M
Tris/HCl
buffer (pH 8.4). After insoluble materials had been removed
by centrifugation, the extract was fractionated with ammo-
nium sulfate between 55% and 100% saturation. The
hemoglobin was centrifuged down at 30 000 g for 30 min,
dissolved in a minimum volume of 50 m
M
Tris/HCl buffer
(pH 8.4), and subjected to a Butyl-Toyopearl column

(5 · 10 cm) equilibrated with ammonium sulfate of 40%
saturation at pH 8.4. The elution was then carried out with
a linear concentration gradient of ammonium sulfate from
40% to 20% saturation in 50 m
M
Tris/HCl buffer (pH 8.4).
In this procedure, components IV, VIA, VIB and VII were
still not fully separated from each other. To isolate
hemoglobin VII, the combined protein solution was there-
fore developed on a DEAE-cellulose column (2 · 7cm),
which had been equilibrated with 5 m
M
Tris/HCl buffer
(pH 8.4). In the elution with 25 m
M
Tris/HCl buffer
pH 7.9, a small peak containing components IV, VIA and
VIB appeared first, and a large major peak containing
component VII in the ferric met-form was the second. The
oxy-form of component VII was finally obtained by
changing the buffer to 50 m
M
at pH 7.9. All these
procedures were carried out at low temperature (0–4 °C)
as far as possible. In the same way, hemoglobin V was also
prepared as described previously [3]. Identification of each
component was made using PAGE, according to the
specifications of Riggs [4].
The concentration of T. akamusi hemoglobin was deter-
mined, after conversion into cyanomet-form, by using an

absorption coefficient of 10.6 m
M
)1
Æcm
)1
at 540 nm for
both components V and VII [1].
Stopped-flow and rapid-scan spectroscopy
Rapid scan experiments were carried out in an Otsuka
stopped-flow spectrophotometer (RA-2000) equipped with
a 10-mm light path cell and two sample reservoirs (3 mL). A
350-lL solution of ferric hemoglobin VII (10 l
M
heme) in
10 m
M
Tris/HCl buffer, pH 7.2, was mixed with an equal
volume of 0.2
M
Mes/NaOH buffer pH 5.0. The absorption
spectra were recorded over 350–450 nm by means of a rapid
scanning photodiode array capable of 512/8192 counts in
1/3 ms. Reaction temperature was controlled by a water
bath (Lauda NM-454 L) maintained to within ± 0.1 °C.
The pH of the reaction mixture was carefully checked,
before and after the run, with a Hitachi-Horiba pH meter
(model F-22). Data sets were saved on an attached
computer (DELL, OptiPLex GXM 5133) for further
analysis. Time courses of the absorbance changes recorded
at different wavelengths or different pH values were

simultaneously fitted to a series of exponentials so as to
treat the reaction as 1st-order processes.
Autoxidation rate measurements
According to our standard procedure, the rate of autox-
idation of oxyhemoglobin was measured in 0.1
M
buffer at
25 °C over a wide pH range (4–12) and in the presence of
1m
M
EDTA. For example, a 1-mL solution containing
0.2
M
appropriate buffer and 2 m
M
EDTA was placed in a
test tube and incubated in a water bath maintained at
25 (± 0.1) °C. The reaction was started by adding an
equal volume of fresh HbO
2
solution (40 l
M
), and the
changes in the absorption spectrum over 450–700 nm were
recorded on the same chart at measured intervals of time.
For the final state of each run, the hemoglobin was
completely converted to the ferric met-form by the
addition of potassium ferricyanide. The buffers used were
acetate, Mes, Mops, Taps, Caps and phosphate (pK
3

). The
pH of the reaction mixture was carefully checked, before
and after the run, with a Hitachi-Horiba pH meter (model
F-22).
Spectral measurements
Absorption spectra were recorded in a Hitachi two-
wavelength double-beam spectrophotometer (U-3210 or
U-3300) or in a Beckman spectrophotometer (DU-650),
each being equipped with a thermostatically controlled
(within ± 0.1 °C) cell holder. CD spectra were obtained in
a Jasco spectropolarimeter (J-720) equipped with a ther-
mostatically controlled cell holder. In the Soret region,
recordings were made with 10 l
M
hemoglobin (as heme) in
a 1-mm cell and at the scale setting of 0.002 degrees per cm
on the chart.
Oxygen equilibrium measurements
Oxygen equilibrium curves were obtained in 50 m
M
Tris/
HClbufferplus0.10
M
KCl at 25 °C by using the automatic
oxygenation apparatus developed by Imai [5]. The optical
absorption and oxygen partial pressure data were acquired
in real time on a microcomputer, and the results were stored
on disks for further analysis.
Results
Rapid-scan spectroscopy for the swinging movement

of the distal histidine residue in hemoglobin VII
Eleven separable components of T. akamusi hemoglobin
can be divided into two groups on the basis of the
presence or absence of the distal (E7) histidine residue,
which plays an important role in the stability of the bound
dioxygen. Among those components, hemoglobins V and
VII are the major ones in each different group, and make
up 15% and 30%, respectively, of the total hemoglobin
concentration in the 4th-instar larval hemolymph. Figure 1
represents the complete amino acid sequences of both
hemoglobins. As is clear, T. akamusi hemoglobin VII
(Ta-VII) consists of 150 amino-acid residues and contains
the usual distal histidine at position 64, whereas hemo-
globin V (Ta-V) replaces it by an isoleucine at position 66
in the 152 sequence. In addition, the B10 residues appear
to be Phe32 in Ta-V and Leu30 in Ta-VII, based on the
structure of Chironomus Hb.
When the aquomet-form of T. akamusi hemoglobin VII
was placed in an acidic pH range, the Soret peak was
considerably blue-shifted and accompanied by a marked
decrease in intensity, indicative of the protein being
Ó FEBS 2003 Swinging of the distal histidine in midge Hb (Eur. J. Biochem. 270) 1425
converted from a hexacoordinate to a pentacoordinate
structure. Using stopped-flow rapid mixing techniques, we
have therefore studied this intramolecular isomerization
reaction in 0.1
M
buffer at 25 °C. Figure 2 shows such time-
resolved Soret absorption spectra of ferric Ta-VII Hb, when
its 10 l

M
solution in 10 m
M
Tris/HCl buffer, pH 7.2, was
mixed with an equal volume of 0.2
M
Mes/NaOH buffer,
pH 5.0, in the stopped-flow apparatus at 25 °C. After the
pH was decreased from 7.2 to 5.0, the spectra were recorded
on a computer every 3 ms over a 500-s period and a range
of 350–450 nm. In the three-dimensional display, the
spectrum scanned 0.05 s after mixing still retained the usual
Soret absorption with maximum centered at 407 nm,
characteristic of the sixcoordinate ferric species. After
0.80 s, however, the peak was dramatically shifted to
397 nm with a set of isosbestic points at 396 and 426 nm,
and accompanied by a marked decrease in intensity. All
these features indicate that the protein was converted
completely from a sixcoordinate form to a fivecoordinate
one without formation of any intermediate species [6–8].
For more detailed study of this pH-dependent conversion, it
would be much more informative to inspect some repre-
sentative spectra at selected pH values, just corresponding
to the two-dimensional spectra at selected times during the
course of the reaction. In a previous paper [2], we have
already reported such a series of spectral changes that
led to the spectroscopic titration curve with a midpoint pH
of 6.3.
However, these spectral changes of Ta-VII were not due
to acid denaturation of the protein, but were totally

reversible with pH. Indeed, its CD magnitude at 222 nm
exhibited a constant value of )16 500 (± 500) degÆcm
2
Æ
dmol
)1
over the pH range of 7.5–4.6 [2]. We have therefore
concluded that the observed Soret absorbance changes can
be attributed to a transition of an iron-ligated water
molecule that is hydrogen-bonded to the distal histidine
residue at position 64, to a water-free iron with the histidine
swung away from the E7 position. Figure 3 represents such
a swinging movement of the distal histidine residue in a very
schematic way. Unfortunately, we cannot indicate where the
distal histidine moves to in T. akamusi Hb VII, as the X-ray
crystal structure is not yet available. However, it is
interesting to note that this transformation reaction is
accompanied by a complete reversal of the sign of the Soret
CD signal. At pH 7.0, ferric Hb VII gave the CD spectrum
containing a weak but distinct negative Soret signal, as
shown in Fig. 4. At pH 5.0, on the other hand, the protein
exhibited a well-developed, positive CD lobe with maximum
centered at 406 nm. These findings strongly suggest that the
swinging movement of the distal histidine would exert some
Fig. 1. Amino acid sequences of T. akamusi Hb components V and VII. ThemarkersareusedtoindicatetheproximalF8-His(#),thedistalE7
residue (*), and the identical residues between both components (:). The B10 residues appear to be Phe32 in Ta-V and Leu30 in Ta-VII.
Fig. 2. Time-resolved Soret absorption spectra of T. akamusi Hb VII
after the pH-drop from 7.2 to 5.0 at 25 °C. The ferric met-species in
10 m
M

Tris buffer pH 7.2 was mixed with an equal volume of 0.2
M
Mes buffer pH 5.0 in the stopped-flow apparatus. The first spectrum
was for the Soret band scanned 0.05 s after mixing, while the last one is
for 0.8 s later. The final heme concentration was 4.1 l
M
at pH 5.0.
1426 S. Kamimura et al.(Eur. J. Biochem. 270) Ó FEBS 2003
effects on the amino-acid chromophores in the very vicinity
of the heme moiety, so as to change the optical rotatory
dispersion of the Soret band. No such pH-dependent
reversal of the CD signal (or the Cotton effect) was observed
in another component Ta-V, as well as in sperm whale
myoglobin.
In this conversion reaction, we have also found the
involvement of a single dissociable group (AH) having a
pK
a
¼ 6.3 at 25 °C. At a glance, this pK
a
value was likely
that of the distal histidine residue, whose protonation would
be associated with the rupture of hydrogen bonding to the
coordinated water molecule. In sperm whale aquomet-
myoglobin, however, no spectral change was observed in the
Soret peak until the heme moiety began to separate abruptly
due to the globin denaturation at pH values below 4.5.
From the effect of temperatures on the pK
a
value, the AH

group involved was found to have thermodynamic param-
eters characteristic for the ionization of a carboxyl group,
although its pK
a
value does not lie in the normal range [2].
Anyway, our next step was to clarify how rapidly the distal
histidine swings away from the E7 position in T. akamusi
Hb VII.
Stopped-flow measurements for the distal histidine
swinging in hemoglobin VII
As demonstrated previously [2], the Soret absorption
spectra of ferric Hb VII changed from a hexacoordinate
form (k
max
¼ 407 nm) to a pentacoordinate one
(k
max
¼ 397 nm) with a set of isosbestic points at 396,
426, 576 and 640 nm, indicative of no production of any
intermediate species between the two forms. To observe
the time courses of the transformation reaction of ferric
Hb VII more directly, for this paper we have studied the
absorbance changes by selected wavelengths such as 410,
407 and 380 nm. Figure 5 gives such an example for
the spectral track followed at 407 nm (Soret peak of the
ferric high-spin species) up to a full development of the
reaction. The pH-drop experiment was carried out at
25 °C by mixing a 10-l
M
ferric Hb solution in 0.01

M
Tris/HCl buffer (pH 7.2) with an equal volume of 0.2
M
Mes/NaOH buffer (pH 5.0). The dead time of the
apparatus was 3 ms, and the noise level was indicated
in the first place as the absorbance fluctuations for the
residual solution of the preceding run. Thus, the conver-
sion process could be displayed as a single exponential
decay over the whole course of the reaction. At the same
time, it was of importance to know whether the moved
residue could come back again to the E7 position with
pH. We have therefore carried out similar experiments at
25 °C. A 10-l
M
ferric Hb VII solution in 10 m
M
Mes/
NaOH buffer (pH 5.0) was mixed with an equal volume
of 0.2
M
Tris/HCl buffer (pH 7.2). As soon as the pH
was jumped from 5.0 to 7.2, the absorbance at 407 nm
increased exponentially with increasing appearance of the
hexacoordinate species.
In formulating the pH-induced swinging movement of
the distal histidine residue, at least six kinetic micro
constants will be required, as follows:
Fig. 3. A schematic representation for the pH-dependent swinging-out of
the distal histidine residue. By a concomitant loss of the axial water
molecule, T. akamusi Hb VII is transformed from a hexacoordinate to

a pentacoordinate (or vacant-type) species.
Fig. 4. CD spectra of T. akamusi ferric Hb VII at pH 7.0 and 5.0.
Reversal of the sign of the Soret signal occurred between pH 7.0
(continuous line) and 5.0 (broken line). Heme concentration was
10 l
M
each in 50 m
M
phosphate buffer.
ð1Þ
Ó FEBS 2003 Swinging of the distal histidine in midge Hb (Eur. J. Biochem. 270) 1427
where AH represents the dissociable group of the
regulatory amino acid residue (probably a carboxyl group
of the heme propionate in this case), and the asterisk is for
the unstable intermediate species for each of the two forms.
The equilibrium between the hexacoordinate[-AH]* species
and the pentacoordinate[-AH] form is not guaranteed,
because the swung-out histidine is found in a completely
different Soret CD environment. The a is the molar fraction
of the hexacoordinate form present at a given pH value. In
this reaction scheme, k
out
represents the apparent first-order
rate constant for the outward movement, while k
in
is for the
inward or swing-back movement of the distal histidine
residue, as the protonation and deprotonation processes for
the AH group involved would be too fast to become rate-
limiting in the swinging movement. Moreover, the Soret

absorption used here is completely silent in such protona-
tion and deprotonation processes of a carboxyl group of the
heme propionate, so that unstable intermediates, even if
present, could not be detected by our spectrophotometric
techniques.
The swinging process, irrespective of its direction, was
therefore followed by a plot of absorbance data at 407 nm
as –ln{(A
t
) A
1
)/(A
0
) A
1
)} vs. time t after mixing.
Figure 6 represents such first-order plots for the pH-
induced conversion reaction of ferric Hb VII, from a
hexacoordinate to a pentacoordinate form and vice versa,
in 0.1
M
buffer at 25 °C. In both semilog plots, data points
were taken from several different runs of the same
experiment and could easily be extended on the straight
line for at least three half-lives (although not shown in Fig. 6
but clear from Fig. 5). From the slope of each straight line,
we obtained the observed first-order rate constants of k
out
¼ 25 s
)1

for the swing-out movement and k
in
¼ 128 s
)1
for
the swing-in process, respectively, of the distal histidine
residue. Consequently, the swing-away movement takes
place with a half-life period of t
1/2
¼ 27 ms, this being less
rapid than the swing-back process with t
1/2
¼ 5ms. At
present, no mechanistic explanation can be given for this
rate difference between the two constants k
out
and k
in
,aswe
do not know yet exactly where the distal histidine moved to.
Nevertheless, it is true that its new position is in favor of
making the residue swing back again to the original E7
position more easily by the pH-jump.
In the pH-shift experiments to obtain the first-order rate
constant of the outward or inward movement, it was
essential to check carefully both the initial and final pH
values of the solution. As the reaction is started whether
from the pH-drop or jump, the protein adjusts itself to the
rapid change in pH to reach an equilibrium. Consequently,
the rate constant (k

out
or k
in
) observed at any intermediate
pH value for the conversion involves the reacting popula-
tion of the hexacoordinate or pentacoordinate form. Both
molar fractions can be deduced from the spectroscopic
titration curve as a function of pH, as described previously
[2]. When the pH-drop experiments were carried out, for
instance, from pH 7.2 to 6.5, or 6.0, or 5.5, each observed
rate constant was associated with each reacted fraction of
hexacoordinate species in such a manner as to lead to the
required rate constant, that is to say k
out
for the full
conversion reaction. The situation was the same with the
pH-jump experiments. Therefore, the practical way to reach
the intrinsic value of k
out
or k
in
was to measure the swinging
reaction by jumping the pH from one extreme to the other
so as to complete the isomerization reaction, irrespective of
the direction. We have found such extreme limits to be
Fig. 6. First-order plots for the pH-dependent transformation reaction
of ferric Hb VII in 0.1
M
buffer at 25 °C. In these plots, k
out

represents
the outward movement of the distal histidine from its E7 position,
while k
in
is for the inward or backward movement to the original
position.
Fig. 5. Time courses for the pH-induced transformation reaction of
ferric Hb VII followed by the absorbance changes at 407 nm. The
experimental conditions for mixing were the same as is in Fig. 2, and
the pH was jumped down from 7.2 to 5.0 at 25 °C. The final heme
concentration was 5 l
M
.
1428 S. Kamimura et al.(Eur. J. Biochem. 270) Ó FEBS 2003
sufficient at pH 7.2 and 5.0, respectively [2]. Certainly, four
pH units should be required to complete the pK-dependent
conversion of one form to the other up to a 99% level.
Unfortunately, our case is not so ideal. The midge ferric
hemoglobin VII is subjected to acid denaturation at pH
values < 4.5, and to the alkaline transition at pH values
> 7.5. In these situations, a pH shift of two units (between
pH 5 and 7) was a possible way for this material. In the
swinging reaction of the distal histidine residue, we also
assumed that the ligand water dissociation or association
step would be too fast to become rate-limiting.
Oxygen binding and stability properties of
T. akamusi
hemoglobins: comparison of components V and VII
Table 1 summarizes the oxygen equilibrium parameters of
T. akamusi hemoglobins V and VII at three different pH

values and 25 °C. Overall oxygen affinity was expressed in
terms of the oxygen pressure to half saturate the protein, P
50
(Torr). Bohr coefficient was also calculated as dH
+
by the
difference in log(P
50
) between pH 6.4 and 8.4. At neutral
pH, both components have P
50
values quite similar to those
of mammalian myoglobins. At acidic pH, however, their O
2
affinities became lower, particularly in Ta-VII. As a result,
hemoglobin VII exhibited a remarkable Bohr effect almost
equal to that of human hemoglobin (dH
+
¼ )0.48). In this
sense, Hb VII can play a central role in oxygen supply as the
major component of the larval hemolymph.
It is in the ferrous form that hemoglobin or myoglobin
can bind molecular oxygen reversibly and carry out its
physiological function. Under air-saturated conditions,
however, the oxygenated form of hemoglobin or myoglobin
is easily oxidized to its ferric met-form with generation of
the superoxide anion as follows:
HbðIIÞðO
2
ÞÀ!

k
obs
meHbðIIIÞþO
À
2
ð2Þ
where k
obs
represents the observed first-order rate constant
at a given pH value [9]. Therefore, the rate of the
autoxidation reaction is written by:
Àd½HbO
2

dt
¼ k
obs
½HbO
2
ð3Þ
This process was followed by a plot of experimental data as
)ln([HbO
2
]
t
/[HbO
2
]
0
)vs.timet, where the ratio of HbO

2
concentration after time t to that at time t ¼ 0canbe
monitored by the absorbance changes at a-peak of the
oxygenated species (578 nm in the case of T. akamusi
HbO
2
). Figure 7 shows such an example for the spectral
changes with time in the autoxidation reaction of T. aka-
musi Hb VII in 0.1
M
Mops/NaOH buffer at pH 7.2 and
25 °C. The spectra evolved with a set of isosbestic points (at
525 and 593 nm) to the final state of the run, which was
identified as a typical acidic met-form. Inserted is the first-
order plot monitored at 578 nm to obtain the rate constant
of k
obs
¼ 0.28 · 10
)1
h
)1
from the slope of the straight line.
This autoxidation rate was several times higher than those
of sperm whale MbO
2
(k
obs
¼ 0.50 · 10
)2
h

)1
) and human
psoas MbO
2
(k
obs
¼ 0.83 · 10
)2
h
)1
), but several times
lower than that of Aplysia (sea mollusca) MbO
2
(k
obs
¼ 0.11 h
)1
) under the same conditions [10]. As the
ferric met-species thus produced cannot bind molecular
oxygen, the observed rate constant (k
obs
) of autoxidation
provides us with a useful measure of the stability of the
bound dioxygen.
In this way, if the values of k
obs
are plotted against the pH
of the solution, we can obtain a stability profile of HbO
2
or

MbO
2
in terms of the autoxidation rate as a function of pH.
Figure 8 represents two such profiles for T. akamusi
hemoglobins V and VII in 0.1
M
buffer at 25 °C, compared
with sperm whale MbO
2
as a reference. In sperm whale
MbO
2
, it is clear that the rate of autoxidation increases
rapidly with increasing hydrogen ion concentration, that a
minimum rate appears at pH 9.2, and that a further increase
occurs at higher pH values. On the other hand, Ta-V HbO
2
was quite susceptible to autoxidation over the whole range
of pH values studied. At pH 9.0, for instance, its rate was
Table 1. Oxygen equilibrium parameters of T. akamusi hemoglobins in
50 m
M
Tris buffer plus 0.1
M
KCl at 25 °C. Heme concentration:
60 l
M
.
Hb component pH P
50

(Torr) dH
+
Ta-V 8.4 0.57
7.4 0.79 )0.20
6.4 1.43
Ta-VII 8.4 0.63
7.4 1.7 )0.58
6.4 9.1
Fig. 7. Spectral changes with time for the autoxidation of T. akamusi
Hb VII in 0.1
M
Mops buffer at pH 7.2 and 25 °C. Scans were made at
3-h intervals after the fresh HbO
2
was placed in air-saturated buffer
and in the presence of 1 m
M
EDTA. Inserted is the first-order plot
monitored at 578 nm. HbO
2
concentration: 20 l
M
heme.
Ó FEBS 2003 Swinging of the distal histidine in midge Hb (Eur. J. Biochem. 270) 1429
25 times higher than that of sperm whale MbO
2
.Further-
more, its pH-dependence was unusual. The rate also
increased with increasing hydrogen ion concentration but
much less so than in sperm whale MbO

2
.Rather,Ta-V
HbO
2
exhibited a distinct rate-saturation below pH 6. This
strongly suggests that the mode of action of the proton is
quite different between the two proteins. In sperm whale
MbO
2
, the rate increases so rapidly at acidic pH that a value
close to n ¼ )1 is always found for the slope of log(k
obs
)vs.
pH. This is a definite indication of the involvement of a very
strong acid-catalysis performed by the distal histidine
residue [3,7,10–14]. In marked contrast to this, Ta-V
HbO
2
stands with a slope of n ¼ )0.4 for the acidic
autoxidation. In fact, this protein has an isoleucine at
position 66 in place of the usual distal histidine residue
(Fig. 1).
In the autoxidation reaction, pH can affect the rate in
many different ways. Recent kinetic and thermodynamic
studies of the stability of mammalian oxymyoglobins have
shown that the autoxidation reaction is not a simple,
dissociative loss of O
2
–
from MbO

2
but is due to a
nucleophilic displacement of O
2
–
from MbO
2
byawater
molecule or a hydroxyl ion that can enter the heme 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-profile for the autoxidation rate can
thereby be explained primarily in terms of the following
three types of displacement processes [7,10–14]:
MbðIIÞðO
2
ÞþH
2
O À!
k
0
MbðIIIÞðOH
2
ÞþO
À
2
ð4Þ
MbðIIÞðO

2
ÞþH
2
O þ H
þ
À!
k
H
MbðIIIÞðOH
2
ÞþHO
2
ð5Þ
MbðIIÞðO
2
ÞþOH
À
À!
k
OH
MbðIIIÞðOH
À
ÞþO
À
2
ð6Þ
In these equations, k
0
is the rate constant for the basal
displacement by H

2
O, k
H
is the rate constant for the proton-
catalyzed displacement by H
2
O, and k
OH
is the rate constant
for the displacement by OH
–
. The extent of the contribution
of these elementary processes to the observed or overall
autoxidation rate, k
obs
in Eqn (3), can vary with the
concentrations of H
+
and OH
–
ions. Consequently, the
stability of MbO
2
exhibits a very strong pH-dependence
having a parabolic part, as typically seen in sperm whale
myoglobin. To determine definitely the kinetic and thermo-
dynamic parameters contributing to each k
obs
vs. pH profile
therefore, we have proposed some mechanistic models for

each case. The rate equations derived from these were tested
for their fit to the experimental data with the aid of a
computer, according to our previous specifications [11,13].
Based on such a nucleophilic displacement mechanism,
the pH profile of sperm whale MbO
2
has already been
analyzed completely in terms of an Ôacid-catalyzed two-state
modelÕ [3,7,13]. In this model, it is assumed that a single,
dissociable group, AH with pK
1
, is involved in the reaction.
Consequently, there are two states of the MbO
2
, represented
by A and B, at molar fractions of a and b (¼ 1–a)
respectively, which are in equilibrium with each other but
which differ in dissociation state for the group AH. These
forms can be oxidized to metMb by displacement of O
2
–
from MbO
2
by an entering water molecule or hydroxyl ion.
Using the rate constants defined above, the observed rate
constant, k
obs
in Eqn (3), could be reduced to:
k
obs

¼ k
A
0
½H
2
Oþk
A
H
½H
2
O½H
þ

ÈÉ
ðaÞ
þ
È
k
B
0
½H
2
Oþk
B
H
½H
2
O½H
þ
þk

B
OH
½OH
À

É
ðbÞð7Þ
where
a ¼
½H
þ

½H
þ
þK
1
and
b ¼ð1 À aÞ¼
K
1
½H
þ
þK
1
ð8Þ
By iterative least-squares procedures inserting various
values for K
1
, the adjustable parameter in Eqn (8), the best
fit to the experimental values of k

obs
was obtained as a
function of pH (Fig. 8). In this way, the rate constants and
the acid dissociation constant involved in the autoxidation
reaction of sperm whale MbO
2
were established in 0.1
M
buffer at 25 °C, as already reported previously [3,7,10].
From those results, it has become evident that the proton-
catalyzed processes with the rate constants k
A
H
and k
B
H
Fig. 8. pH-profiles for the stability of T. akamusi oxyhemoglobins and
sperm whale MbO
2
in 0.1
M
buffer at 25 °C. The logarithmic values of
the observed first-order rate constant, k
obs
in h
)1
, for the autoxidation
reaction are plotted against the pH of the solution. The pH-profile of
sperm whale MbO
2

is taken from our previous paper [7,13]. Heme
concentration: 20 l
M
for midge; 50 l
M
for sperm whale.
1430 S. Kamimura et al.(Eur. J. Biochem. 270) Ó FEBS 2003
promote most of the autoxidation reaction of sperm whale
MbO
2
above the basal processes in water with the rate
constants k
A
0
and k
B
0
. The reductive displacement of the
bound dioxygen as O
2
–
by H
2
O can proceed without any
protonation, but it is clear that the rate is enormously
accelerated with the proton assistance, by a factor of
4.7 · 10
6
mol
)1

for state A and by 1.1 · 10
8
mol
)1
for state
B. In this proton-catalysis formulated by Eqn (5), the distal
E7 histidine (the dissociable group AH with pK
1
¼ 6.2),
which forms a hydrogen bond to the bound dioxygen [15],
appears to facilitate the effective movement of a catalytic
proton from the solvent to the bound, polarized dioxygen
via the imidazole ring by a proton-relay mechanism [10,14].
On the other hand, Ta-V HbO
2
was characterized by the
distinct rate-saturation behavior below pH 6 (Fig. 8). We
have therefore measured the rate at more than 70 different
pH values, from 4 to 12, and finally established the best fit to
the experimental values of k
obs
by a simple Ôtwo-state modelÕ
[3]. In this mechanism, we assumed that a single, dissociable
group (AH with pK
1
) is also involved in the reaction but in a
different way. As a result, the pH profile for the autoxida-
tion rate of this insect protein could be described by the
following equation:
k

obs
¼ k
A
0
½H
2
O
ÈÉ
ðaÞþ k
B
0
½H
2
Oþk
B
OH
½OH
À

ÈÉ
ðbÞð9Þ
Using the same fitting procedures, we have thus obtained
the rate constants and the acid dissociation constant
involved in the autoxidation reaction of Ta-V HbO
2
in
0.1
M
buffer at 25 °C, as reported in the previous paper [3].
In this kinetic formulation, one of the most remarkable

features was that Ta-V HbO
2
does not show any proton-
catalyzed process having the term of k
H
[H
2
O][H
+
], such as
the one that can play a dominant role in the autoxidation
reaction of most mammalian myoglobins or hemoglobins
having the usual distal histidine residue. Instead, Ta-V
HbO
2
contained a dissociable group (AH with pK
1
¼ 6.2)
that is responsible for a small rate increase at the acidic pH
side. To characterize thermodynamically this dissociable
group AH, we have investigated the effect of temperatures
on the K
1
value, by analyzing respective pH-profiles
obtained at three different temperatures. As a result,
the enthalpy change of practically zero was deduced from
the slope of the van’t Hoff plot. Thus, the resulting
thermodynamic parameters were: DG° ¼ 33.1 kJÆmol
)1
,

DH° ¼ 0kJÆmol
)1
and DS° ¼ )111 JÆmol
)1
ÆK
)1
in 0.1
M
buffer at 25 °C [3]. Although its pK
a
value does not lie in the
normal range, these parameters are those expected for the
ionization of a carboxyl group, and we suggest that the most
probable candidate is a carboxyl group of the heme
propionates, just as in the previous case of Aplysia MbO
2
.
In fact, when the protoheme was esterified with methanol to
block its propionic acid side-chains, Aplysia myoglobin
completely lost such a rate increase, with pK
1
¼ 6.1 [7].
Along with this line of evidence, our great interest was in
the stability property of Ta-VII HbO
2
, as in its ferric met-
form the distal histidine is found to swing away from the E7
position on the acidic pH side. As demonstrated in Fig. 8,
the pH profile of Ta-VII HbO
2

showed an intermediate
character between sperm whale MbO
2
and Ta-V HbO
2
.
With increasing hydrogen ion concentration, the rate
increased rapidly as in sperm whale MbO
2
, but began to
deviate from the theoretical line having a slope of n ¼ )1,
and finally reached a saturation level below pH 5. Among
these unusual kinetic properties, of most interest was that
the rate deviation occurred with a midpoint pH of
approximately 6, this being very close to the pH value
(¼ 6.4) at which half of the conversion reaction from a
hexacoordinate to a pentacoordinate structure is completed
in the ferric met-form. At the present time, we unfortunately
failed to formulate such extremely complexed pH-profile of
Ta-VII HbO
2
by a simple equation, but we suggest strongly
that the swinging-away movement of the distal histidine
residue proceeds in the oxygenated form of Ta-VII, also,
and this would certainly contribute to the protection of
Ta-VII HbO
2
from an accelerated proton-catalysis in the
acidic autoxidation. For detecting the swinging movement
of the distal histidine residue, the Soret absorption spectro-

scopy is silent in the oxygenated form of Mb or Hb.
Therefore, another approach will be needed for more
detailed kinetics of the autoxidation reaction and histidine
swinging in Ta-VII HbO
2
, and this remains open to our
future study.
Discussion
Physiological properties of
T. akamusi
hemoglobins
Some insects have a globin in their fast-growing larval stage,
but lose it after metamorphosis in favor of the diffusion of
gaseous oxygen through hollow tracheal tubes. This is the
case in the midge (Diptera, Chironomidae), and the
Chironomidae is one of the largest insect families. Among
the midge hemoglobins, extensive work has been carried out
with several species of Chironominae, such as Chironomus
thummi thummi [16–19], and Chironomus thummi piger
[20,21].Indeed,theC. thummi thummi (CTT) Hb-III was the
first invertebrate Hb whose X-ray structure was determined
at high resolution. In its crystal structure, displaying the
common globin fold, the heme group is rotated by 180° and
the heme cavity in the deoxy form has an unusual open gate
conformation at pH 7.0, with the distal His able to swing
out of the cavity [16,17]. This Hb has therefore been the
subject of structural, spectral and functional studies [18,19].
On the other hand, T. akamusi, a common species found
in Japan, belongs to a different subfamily (Orthocladiinae)
from Chironominae, and its larva is quite unique in

morphology and ecological behavior. In the Chironomid
group, the young hatch from the colorless, transparent egg
as wormlike larvae. The larva grows through four instars
(stages separated by a molt) without change of shape. As for
T. akamusi, the Japanese word ÔakamusiÕ means blood-
worm, which comes from the fact that a large amount of
hemoglobin is synthesized into the hemolymph of the 4th-
instar larva. This small bloodworm (15–18 mm in length
and 1.5 mm in diameter) begins to burrow into polluted and
extremely hypoxic mud flats of lakes to have a long period
(more than half a year) of diapause. The burrow can reach
up to 80 cm in depth, and e
0
¢ ¼ 0 V in the oxidation-
reduction potential. After diapause, the matured and sex-
differentiated larva crawls up above the ground again, and
undergoes a pupal molt in which the shape alters com-
pletely. The brown pupa is encased in a cuticle, and the
Ó FEBS 2003 Swinging of the distal histidine in midge Hb (Eur. J. Biochem. 270) 1431
pupal stage terminates with a final or imaginal molt in
which the adult, winged midge emerges from the pupal case.
The life of the adult midge is restricted only to a one-month
period or so.
A stage-specific expression of T. akamusi hemoglobin
appears to be adaptive for the bloodworm to extend its
inhabitable environment. By burrowing deeply into lake
mud flats, the bloodworm can protect itself from fish, and
thus have a fairly long period of diapause in safety. In light
of such unique behavior of the 4th-instar larva of T. aka-
musi, it was of particular interest to investigate the molecular

properties of the hemoglobin components isolated from the
larval hemolymph. The polymorphic forms of T. akamusi
hemoglobin would certainly be advantageous to the larval
life in O
2
transport and storage under the particular adverse
conditions.
Molecular properties of
T. akamusi
hemoglobins
The hemoglobin from Tokunagayusurika akamusi consists
of at least 11 components, which fall into two approxi-
mately equal groups; one (VIA, VIB, VII, VIII and IX)
having a distal histidine and the other (IA, IB, II, III, IV and
V) lacking in it [1]. We have therefore carried out a matrix
analysis to test the sequence homology of the major
components V and VII from T. akamusi, with other midge
hemoglobins including 10 components from C. thummi
thummi [16,18] and three components from C. thummi piger
[20,21]. As a result, component VII has a higher percentage
identity (40–48%) with the Chironomus Hb sequences rather
than does component V (26–27%). Because all the Chiro-
nomus hemoglobins have a distal histidine residue, the
appearance of component V-type globins may be very
specific to the genus Tokunagayusurika.
For the distal histidine swinging, Johnson et al.[22]
observed it in the structure of myoglobin-ethylisocyanide.
They described that when such a bulky ligand bound to
ferric sperm whale myoglobin, the distal (E7) histidine
swung up and away from the iron atom, just like a

swinging door, toward the protein surface. In sperm
whale myoglobin, Tian et al.[23]measuredtheonand
off rate-constants for O
2
-binding as a function of pH,
and reported a dramatic increase in the O
2
-dissociation
rate at low pH, where the imidazole side chain of the E7-
His becomes protonated, loses a hydrogen bond to the
bound O
2
, and moves outward on a microsecond
timescale. In T. akamusi Hb VII, a similar movement of
the distal histidine could occur in the ferric met-form but
on a millisecond timescale. Consequently, this pH-
dependent swinging is quite different from the distal His
movement controlling the on and off rate processes of
O
2
-binding in myoglobins and hemoglobins.
In oxygen equilibrium measurements, the most remark-
able result is that O
2
affinities of the two components are
almost the same at pH 8.4 and the E7-Ile hemoglobin V has
rather a higher affinity at lower pH. In all the recombinant
myoglobins reported so far, a His to Ile or Leu mutation at
the E7 position always causes a dramatic decrease in O
2

affinity, resulting in a very large increase (> 10 Torr) in P
50
value [24]. In this respect, it is possible that the B10-Phe at
position 32 is stabilizing the bound O
2
in hemoglobin V
(Fig. 1). Among the distal heme pocket residues, the B10 is
known to be very relevant for the O
2
-binding property, in
addition to the E7 residue [25].
In the autoxidation reaction, Brantley et al.[26]werethe
first to use site-directed mutagenesis of sperm whale
myoglobin to make clear the possible role(s) of the distal
histidine residue. They showed that mutations of the
distal His at position 64, such as those of His64fiGly,
His64fiVal, His64fiLeu and His64fiGln, caused dramatic
increases in the autoxidation rate, but the relative effects of
pH were the same with that for the wild-type (His64)
myoglobin if the absolute rates were normalized to pH 7.0.
However, our examinations for a dozen naturally occurring
myoglobins have shown that only the proteins having the
usual distal histidine can manifest a very strong proton-
catalysis in the autoxidation reaction [3,7]. Using some
typical His64 mutants of sperm whale myoglobin, we have
therefore carried out detailed pH-dependence studies of the
autoxidation rate over the wide range of pH 5–12 in 0.1
M
buffer at 25 °C. The resulting pH profiles were then
compared with those of the corresponding myoglobins

occurring in nature [3]. As a result, sperm whale MbO
2
(wild-type) is approximately 400 times more resistant to
autoxidation if compared with the His64fiVal and
His64fiGly mutant proteins at pH 7.0. Such a comparison
certainly leads us to conclude that the distal (E7) histidine
inhibits heme oxidation by obstructing easy access of a
water molecule to the FeO
2
center. This is true in a neutral
pH range. Nevertheless, it is also true that the rate of
autoxidation of sperm whale MbO
2
increases markedly, not
only with increasing hydrogen ion concentration but also
with increasing hydroxyl ion concentration, as shown in
Fig. 8.
Consequently, we have proposed that the distal histidine
can play a dual role in the nucleophilic displacement of O
2
–
from MbO
2
or HbO
2
. One is in a proton-relay mechanism
via its imidazole ring. Insofar as we have examined for more
than a dozen myoglobins, such a proton-catalyzed process
could never be observed in the autoxidation reaction of
myoglobins lacking the usual distal histidine residue, no

matter what the protein is, the naturally occurring or the
distal His mutant [3]. As a matter of fact, even if the distal
residue is a histidine, the protein cannot manifest any
proton catalysis if the residue is tilted away from the precise
E7 position. We have found this to be the case here, for the
autoxidation of T. akamusi hemoglobin VII below pH 6.0,
as well as for the b-chain in the autoxidation of the human
HbO
2
tetramer [11]. The other role of the distal histidine is
in the maximum protection of the FeO
2
center against a
water molecule or a hydroxyl ion that can enter the heme
pocket from the surrounding solvent [26]. 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 hemoglobin in protic, aqueous
solvent.
In light of the oxygen equilibrium parameters and
considerable resistance to acidic autoxidation, we conclude
that T. akamusi hemoglobin VII, the major component, can
play an important role in O
2
transport and storage against
the extremely acidic and hypoxic adversity. For the pH of
the larval hemolymph, there is no report of its direct
measurement, but there is a strong possibility that it drops to
around pH 4. Under anaerobic conditions, the end-products
1432 S. Kamimura et al.(Eur. J. Biochem. 270) Ó FEBS 2003

of carbohydrate metabolism by the Chironomus larva are
known to involve large amounts of lactic and succinic acids
[27]. Multiplicity of other components in the O
2
-binding
affinity, Bohr effect, and FeO
2
stability would also be
advantageous for the insect larva to overcome various
stringent circumstances.
Acknowledgements
This work was partly supported by grants-in-aid for Scientific Research
(07640896, 10440248 and 12640659) from the Ministry of Education,
Culture and Science of Japan.
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