Vol. 88, No. 2, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BlOCHEMlCAL
May 28, 1979
Pages
713-721
SPIN STATE EQUILIBRIA IN SOYBEAN FERRIC LEGHEMOGLOBIN AND ITS COMPLEXES WITH FORMATE AND ACETATE
School
J. Trewhella and P.E. Wright,* of Chemistry, University of Sydney, N.S.W. 2006, Australia and
C.A. Appleby, Division of Plant Industry, Commonwealth Scientific and Industrial Research Organisation, Canberra, A.C.T. 2601, Australia Received
April
7, 1979 SUMMARY
The interactions of fluoride, acetate and formate with soybean ferric leghemoglobin &have been investigated by ‘H NMR spectroscopy. In the presence of fluoride or acetate leghemoglobin is locked into a high spin ferric conformation whilst the formate complex exists as an equilibrium Both formate and acetate ligate mixture of high and low spin states. directly to the iron and the different magnetic properties of the complexes are attributed to steric constraints within the heme pocket. Leghemoglobins transport
structure
2.8 .8 resolution
acids
(3)
brium
mixture
molecular
show that differences
* Author
(I),
of spin
basis
for
including
states these
in the
and other
and for-mate iron
ferric
spin
to whom correspondence
(6).
protein
properties
fluoride,
state
iron
in these
should
nodules
(2).
long
to exist to gain
chain
an insight
on the interaction
acetate ligands
and formate. and discuss
carboxylate
to
Despite in many carboxylic
as an equili-
we have undertaken here
(1).
determined
from myoglobin
to bind
In order
are both
has been
differs
We report
a with
lupin
in oxygen
root
of myoglobin
ability
(4,5).
leghemoglobin
acetate
to that
its
of the
of leghemoglobin
ferric
similar
function
legume
from
leghemoglobin
and the tendency
NMR studies soybean
of leghemoglobin
resemblance,
properties
which
nitrogen-fixing
and is very
structural
of its
hemeproteins
in Rhizobium-infected,
The tertiary
this
are monomeric
into
the
detailed of We unexpected
complexes.
be addressed. 0006-291X/79/100713-09$01.00/0
713
Copyright All righrs
@ 1979
by Academic. Press. Inc. in anyformreserved.
ofreproducrion
BIOCHEMICAL
Vol. 88, No. 2, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS
AND METHODS
Soybean ferric leghemoglobin a was purified by a previously described procedure (7). For NMR measurements the protein was repeatedly dialysed against phosphate buffer (lOmM, pH 7.0) in D20. The carboxylate complexes were prepared by addition of sodium deuteroacetate (28-fold excess) or sodium formate (lo-fold excess) to ferric leghemoglobin. The pH was adjusted to 6.5 by careful addition of DC1 or NaOD. Proton NMR spectra were recorded using a Bruker HX-270 spectrometer. Dioxan was used as an internal standard but all peaks are referred to TSS (trimethylsilylpropanesulphonic acid). Optical difference spectra were recorded using a Cary 14 spect rophot omet er . RESULTS AND DISCUSSION The NMR spectrum shown in fig.
of ferric The absence
l(a),
resonances
has been
broadening
due to relatively
and low spin heme iron ration
which
Their
broadness
relaxation other
is
characteristic
resolved
binds
(4,s).
In the
into
resonate
hemoglobin
group
between
Ligation
high
of fluoride
high
spin
ferric
configu-
group
protons.
slow
iron
fluoride
unpaired
complex
spectrum
electron
spin
of leghemoglobin
has been reported
spin to the
from the heme methyl
in the
deuteroacetate been
of leghemoglobin
and
for
confirmed
iron for
like
configuration. as those
in which
myoglobin
clearly
atom in leghemoglobin. the acetate
714
a high
complex
spin
well62.9,
61.0
fluoride,
abnormal group
indicates This of lupin
is
group
B-subunits ligated
direct
and locks
The heme methyl
of the
a carboxylate
Thus the NMR spectrum to the
at 65.9,
deuteroacetate,
ferric
same region
to form
deuteroacetate
are observed
Clearly
spin
(10)
leghemoglobin
resonances
l(c)).
a high
M Milwaukee
atom (11).
recently
to
50 ppm
to ferric
spectrum
ppm at 30°C (fig.
protons
attributed
near
A similar
reversibly
heme methyl
leghemoglobin
iron
(8).
group
of resonances
to the of the
loss-‘)
a fully
set
and is
is
(9).
Acetate complex
into
arise
attributed
(ca. -
temperatures.
region
heme methyl
(6)
exchange
to a broad
probably
hemeproteins
fluoride
slow
rise
low field
of hyperfine-shifted
leghemoglobin
(5) and gives
in the
by us previously
at ambient
atom locks
l(b))
54.9
reported
states
(fig.
leghemoglobin
of
to the
ligation
conclusion leghemoglobin
of
has by
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
L L
a
A.-
I
I
80
I
I
60
,
I
40
.L
__-I---
20
0
PPm Figure
X-ray
1.
270 MHz proton NMR spectra at 25’C of (a) ferric soybean leghemoglobin a (2.6 mM, pH 7.0). (b) Leghemoglobin fluoride (42 mM fluoride, pH 6.5). (c) Leghemoglobin deuteroacetate (27 mM deuteroacetate, pH 6.5).
diffraction
(12).
hy-perfine-shifted deuteroacetate exhibit
a normal
is
The observed
heme methyl
group
in
the high
spin
Curie
law
(l/T)
temperature resonances
ferric temperature
715
dependence confirms
state
(fig.
dependence
that 2).
of the leghemoglobin The resonances
and shift
upfield
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
716
Vol. 88, No. 2, 1979
BlOCHEMlCAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1
620
AA
T
=O.l
1
, ,
/
493 1 ,--
/
, -\
\
,’ 3’
\
, \
, ‘\
540 1 . -’
I
I
I
,
I
/
,
I
,
,-
\
i
,
S
Ferric
Lba
’
minus
Ferric
Acetate Lba
\
I
‘__.-___--
,,,’
Ferric
Lba
minus
Ferric
Formate Lba
T
631 450
500
600
550
650
700
Optical difference spectra for acetate and formate complexes of soybean leghemoglobin (50 uM in 50 mMMES/NaOHbuffer, pH 5.30 at 20°C). (a) Spectrum of leghemoglobin acetate (total acetate concentration is 250 mM)versus leghemoglobin. (b) Spectrum of leghemoglobin for-mate (total formate concentration is 250 mM) versus leghemoglobin.
Figure 3.
towards the positions at which they would resonate in a diamagnetic hemeprotein
(ca. 3 ppm). -
It is clear from the optical interacts
difference
spectra of fig.
with leghemoglobin in a rather different
other straight
chain aliphatic
carboxylates).
peaks at 620 nm and 493 nm and the ferric
3 that formate
manner to acetate (and
The charge transfer
band
hemochrometrough at 540 nm are
in accord with formation of a high spin complex when ferric
leghemoglobin
combines with acetate at 20°C. On reaction with formate, however, the appearance of ferric
hemochromepeaks at 535 and 562 nm and charge transfer
band troughs at 502 and 637 nm in the difference ferric
spectrum suggests that
leghemoglobin adopts a more low spin structure.
717
Vol. 88, No. 2, 1979
Further
BIOCHEMICAL
information
leghemoglobin
resonances
the
heme methyl
four
higher
fields
than
the Curie
law since
ture,
i.e.,
other
two resonances
an increase
is
indication
increase
Similar
of the
slow
high
(13) .
spin
in marked
loss-‘)
Exchange
and low are
the origin
between scale.
spin
contrast
formate,
to shift
of the spin
leghemoglobin
state
equilibria.
in leghemoglobin
formate
probably
‘A and ‘T energy
states,
constrained
field nitrogen
produced
by formate,
donor
atoms.
We have previously leghemoglobin involves
motion
of the
and ferric
arises
from
to lie
in energy
crossing
is generally
from a protein distal
conformational
histidine
718
on and off
state
differences
in
interconversion
four
iron
between ligand
pyrrole very
exchange
change the
(~a.
by the
Such inter-system
spin
This
crossing
and the
the slower
the
rate
state
histidine
that
the
since
exchange
spin
of
leghemoglobin
temperature.
intersystem
close
complexes
is fast
and indicates
The rapid
to an
between
the proximal
suggested
arises
and imidazole
state (6)
of spin
conclusion.
changing
to the relativelyslowspin
in ferric
this
formate
with
This
leading
of interconversion
steadily
do
The temperature
with
acetate
of leghemoglobin
than
mixture
species.
the azide
The
temperature.
in temperature
in accord
The rate
states
observed
observed
is
have been made for
on the NMR time
resonances is
spectrum
4).
Such behaviour
increasing
spin
follow
tempera-
dependence
acetate.
an increase
does not
(fig.
of an equilibrium
of the high
optical
observations
myoglobin is
formate,
in the proportion
dependence
positions
with
of leg-
increasing
temperature
shift
of the existence
in leghemoglobin
with
from
at considerably
dependence
of leghemoglobin
of
well-
in the spectrum
downfield
much smaller
in hyperfine
exhibits
are observed
diamagnetic
complex
ppm at 30°C arising
temperature
two of them shift
for-mate
which
resonances
their
resonances
reflects
These
corresponding
exhibit
of the
and 38.6
4).
move away from their
the corresponding
39.3
(fig.
Further,
state
NMR spectrum
43.3,
groups the
acetate.
states
from the
at 45.1,
hemoglobin
clear
on the magnetic
was obtained
resolved
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
rapid in ferric
and probably atom (6).
(14).
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Why does the formate complex of leghemoglobin exist as an equilibrium mixture of high spin and low spin states at ambient temperatures whilst the acetate complex is fully
high spin? This behaviour is unique to
leghemoglobin, myoglobin fox-mate for example being a high spin ferric complex with NMRspectrum similar to that of leghemoglobin deuteroacetate The inherent ligand field
strengths of formate and acetate ligands are
expected to be very similar, globin should exhibit
(9).
in which case their
complexes with leghemo-
similar magnetic properties.
The observed differences
in leghemoglobin formate and leghemoglobin acetate can, however, be rationalised
if it is assumedthat steric
pocket interfere
constraints
with the binding of the bulkier
to a weaker ligand field this interpretation,
within the heme
acetate ligand,
and a high spin ferric
complex.
fonnate binds more strongly
leading
In support of
to ferric
leghemoglobin
= 0.23 mM, pH 5.3 and 20°C) than does acetate (Kdiss = 4.5 mM, (Kdiss pH 5.3 and 20°C) (15), which has an affinity for leghemoglobin similar to that of longer straight a-CHa group of aliphatic affinity
chain carboxylic carboxylic
for leghemoglobin.
acids (3).
acids substantially
Isobutyric
Substitution
at the
reduces their
acid binds only weakly to ferric
leghemoglobin (Kdiss ~a. 500 mMat pH 5.3 and ZO'C, as determined by the spectrophotometric to bind at all.
method described in (16))and lactic
acid appears not
Thus it appears that whilst the hemepocket of ferric
leghemoglobin is more open than that of ferric
myoglobin, allowing the
binding of long chain carboxylic
constraints
pocket are still
acids, steric
important and influence
within the
the nature of the metal-ligand
bond.
ACKNOWLEDGEMENTS We thank Mrs J. Wellington the preparation for financial
and Mrs L. Grinvalds for assistance with
of leghemoglobin and the Australian support and for the use of facilities
720
Research Grants Committee at the National NMRcentre.
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
REFERENCES 1.
2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Appleby, C.A. (1974): The Biology of Nitrogen Fixation (Quispel, A. ed.) pp, 521-554, North-Holland Publishing Co., Amsterdam. Vainshtein, B.K., Arutyunyan, E.G., Kuranova, I.P., Borisov, V.V., Sosfenov, N.I., Pavlovskii, A.G.. Srebenko, A.I., Konareva, N.V. and Nekrasov, Yu. V. (1977) Dokl. Akad. Nauk. SSSR233, 238-241 (English Edition: 233, 67). Ellfolk, N. (1961) Acta Chem. Stand. 15, 975-984. Ehrenberg, A. and Ellfolk, N. (1963) Acta Chem. Stand. II, S343-S347. Appleby, C.A., Blumberg, W.E., Peisach, J., Wittenberg, B.A. and Wittenberg, J.B. (1976) J. Biol. Chem. 251, 6090-6096. Wright, P.E. and Appleby, C.A. (1977) FEBSLett. z, 61-66. Appleby, C.A., Nicola, N.A., Hurrell, J.G.R. and Leach, S.J. (1975) Biochemistry 14, 4444-4450. Ollis, D.L., Eight, P.E., Pope, J. and Appleby, C.A., submitted for publication. Morishima, I., Neya, S., Ogawa, S. and Yonezawa, T. (1977) FEBSLett. 83, 148-150. Fung, L.W.-M., Minton, A.P., Lindstrom, T.R., Pisciotta, A.V. and Ho, C. (1977) Biochemistry 16, 1452-1462. Perutz, M.F., Pulsinelli, P.D. and Ranney, H.M. (1972) Nature New Biol. 237, 259-263. Vainshteln, B.K. and Arutyunyan, E. (1978) abstract, Int. Symp. Biomolecular Structure, Conformation, Function and Evolution, Madras, India. Morishima, I. and Iizuka, T. (1974) J. Amer. Chem. Sot. 96, 5279-5283. Binstead, R.A., Beattie, J.K., Dose, E.V., Tweedle, M.F. and Wilson, L.J. (1978) J. Amer. Chem. Sot. 100, 5609-5614. Appleby, C.A. and Wittenberg, J.B., manuscript in preparation. Appleby, C.A., Wittenberg, B.A. and Wittenberg, J.B. (1973) Proc. Natl. Acad. Sci. USA'0, 564-568.
721
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
BlOCHEMlCAL
May 28, 1979
Pages
713-721
SPIN STATE EQUILIBRIA IN SOYBEAN FERRIC LEGHEMOGLOBIN AND ITS COMPLEXES WITH FORMATE AND ACETATE
School
J. Trewhella and P.E. Wright,* of Chemistry, University of Sydney, N.S.W. 2006, Australia and
C.A. Appleby, Division of Plant Industry, Commonwealth Scientific and Industrial Research Organisation, Canberra, A.C.T. 2601, Australia Received
April
7, 1979 SUMMARY
The interactions of fluoride, acetate and formate with soybean ferric leghemoglobin &have been investigated by ‘H NMR spectroscopy. In the presence of fluoride or acetate leghemoglobin is locked into a high spin ferric conformation whilst the formate complex exists as an equilibrium Both formate and acetate ligate mixture of high and low spin states. directly to the iron and the different magnetic properties of the complexes are attributed to steric constraints within the heme pocket. Leghemoglobins transport
structure
2.8 .8 resolution
acids
(3)
brium
mixture
molecular
show that differences
* Author
(I),
of spin
basis
for
including
states these
in the
and other
and for-mate iron
ferric
spin
to whom correspondence
(6).
protein
properties
fluoride,
state
iron
in these
should
nodules
(2).
long
to exist to gain
chain
an insight
on the interaction
acetate ligands
and formate. and discuss
carboxylate
to
Despite in many carboxylic
as an equili-
we have undertaken here
(1).
determined
from myoglobin
to bind
In order
are both
has been
differs
We report
a with
lupin
in oxygen
root
of myoglobin
ability
(4,5).
leghemoglobin
acetate
to that
its
of the
of leghemoglobin
ferric
similar
function
legume
from
leghemoglobin
and the tendency
NMR studies soybean
of leghemoglobin
resemblance,
properties
which
nitrogen-fixing
and is very
structural
of its
hemeproteins
in Rhizobium-infected,
The tertiary
this
are monomeric
into
the
detailed of We unexpected
complexes.
be addressed. 0006-291X/79/100713-09$01.00/0
713
Copyright All righrs
@ 1979
by Academic. Press. Inc. in anyformreserved.
ofreproducrion
BIOCHEMICAL
Vol. 88, No. 2, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS
AND METHODS
Soybean ferric leghemoglobin a was purified by a previously described procedure (7). For NMR measurements the protein was repeatedly dialysed against phosphate buffer (lOmM, pH 7.0) in D20. The carboxylate complexes were prepared by addition of sodium deuteroacetate (28-fold excess) or sodium formate (lo-fold excess) to ferric leghemoglobin. The pH was adjusted to 6.5 by careful addition of DC1 or NaOD. Proton NMR spectra were recorded using a Bruker HX-270 spectrometer. Dioxan was used as an internal standard but all peaks are referred to TSS (trimethylsilylpropanesulphonic acid). Optical difference spectra were recorded using a Cary 14 spect rophot omet er . RESULTS AND DISCUSSION The NMR spectrum shown in fig.
of ferric The absence
l(a),
resonances
has been
broadening
due to relatively
and low spin heme iron ration
which
Their
broadness
relaxation other
is
characteristic
resolved
binds
(4,s).
In the
into
resonate
hemoglobin
group
between
Ligation
high
of fluoride
high
spin
ferric
configu-
group
protons.
slow
iron
fluoride
unpaired
complex
spectrum
electron
spin
of leghemoglobin
has been reported
spin to the
from the heme methyl
in the
deuteroacetate been
of leghemoglobin
and
for
confirmed
iron for
like
configuration. as those
in which
myoglobin
clearly
atom in leghemoglobin. the acetate
714
a high
complex
spin
well62.9,
61.0
fluoride,
abnormal group
indicates This of lupin
is
group
B-subunits ligated
direct
and locks
The heme methyl
of the
a carboxylate
Thus the NMR spectrum to the
at 65.9,
deuteroacetate,
ferric
same region
to form
deuteroacetate
are observed
Clearly
spin
(10)
leghemoglobin
resonances
l(c)).
a high
M Milwaukee
atom (11).
recently
to
50 ppm
to ferric
spectrum
ppm at 30°C (fig.
protons
attributed
near
A similar
reversibly
heme methyl
leghemoglobin
iron
(8).
group
of resonances
to the of the
loss-‘)
a fully
set
and is
is
(9).
Acetate complex
into
arise
attributed
(ca. -
temperatures.
region
heme methyl
(6)
exchange
to a broad
probably
hemeproteins
fluoride
slow
rise
low field
of hyperfine-shifted
leghemoglobin
(5) and gives
in the
by us previously
at ambient
atom locks
l(b))
54.9
reported
states
(fig.
leghemoglobin
of
to the
ligation
conclusion leghemoglobin
of
has by
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
L L
a
A.-
I
I
80
I
I
60
,
I
40
.L
__-I---
20
0
PPm Figure
X-ray
1.
270 MHz proton NMR spectra at 25’C of (a) ferric soybean leghemoglobin a (2.6 mM, pH 7.0). (b) Leghemoglobin fluoride (42 mM fluoride, pH 6.5). (c) Leghemoglobin deuteroacetate (27 mM deuteroacetate, pH 6.5).
diffraction
(12).
hy-perfine-shifted deuteroacetate exhibit
a normal
is
The observed
heme methyl
group
in
the high
spin
Curie
law
(l/T)
temperature resonances
ferric temperature
715
dependence confirms
state
(fig.
dependence
that 2).
of the leghemoglobin The resonances
and shift
upfield
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
716
Vol. 88, No. 2, 1979
BlOCHEMlCAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1
620
AA
T
=O.l
1
, ,
/
493 1 ,--
/
, -\
\
,’ 3’
\
, \
, ‘\
540 1 . -’
I
I
I
,
I
/
,
I
,
,-
\
i
,
S
Ferric
Lba
’
minus
Ferric
Acetate Lba
\
I
‘__.-___--
,,,’
Ferric
Lba
minus
Ferric
Formate Lba
T
631 450
500
600
550
650
700
Optical difference spectra for acetate and formate complexes of soybean leghemoglobin (50 uM in 50 mMMES/NaOHbuffer, pH 5.30 at 20°C). (a) Spectrum of leghemoglobin acetate (total acetate concentration is 250 mM)versus leghemoglobin. (b) Spectrum of leghemoglobin for-mate (total formate concentration is 250 mM) versus leghemoglobin.
Figure 3.
towards the positions at which they would resonate in a diamagnetic hemeprotein
(ca. 3 ppm). -
It is clear from the optical interacts
difference
spectra of fig.
with leghemoglobin in a rather different
other straight
chain aliphatic
carboxylates).
peaks at 620 nm and 493 nm and the ferric
3 that formate
manner to acetate (and
The charge transfer
band
hemochrometrough at 540 nm are
in accord with formation of a high spin complex when ferric
leghemoglobin
combines with acetate at 20°C. On reaction with formate, however, the appearance of ferric
hemochromepeaks at 535 and 562 nm and charge transfer
band troughs at 502 and 637 nm in the difference ferric
spectrum suggests that
leghemoglobin adopts a more low spin structure.
717
Vol. 88, No. 2, 1979
Further
BIOCHEMICAL
information
leghemoglobin
resonances
the
heme methyl
four
higher
fields
than
the Curie
law since
ture,
i.e.,
other
two resonances
an increase
is
indication
increase
Similar
of the
slow
high
(13) .
spin
in marked
loss-‘)
Exchange
and low are
the origin
between scale.
spin
contrast
formate,
to shift
of the spin
leghemoglobin
state
equilibria.
in leghemoglobin
formate
probably
‘A and ‘T energy
states,
constrained
field nitrogen
produced
by formate,
donor
atoms.
We have previously leghemoglobin involves
motion
of the
and ferric
arises
from
to lie
in energy
crossing
is generally
from a protein distal
conformational
histidine
718
on and off
state
differences
in
interconversion
four
iron
between ligand
pyrrole very
exchange
change the
(~a.
by the
Such inter-system
spin
This
crossing
and the
the slower
the
rate
state
histidine
that
the
since
exchange
spin
of
leghemoglobin
temperature.
intersystem
close
complexes
is fast
and indicates
The rapid
to an
between
the proximal
suggested
arises
and imidazole
state (6)
of spin
conclusion.
changing
to the relativelyslowspin
in ferric
this
formate
with
This
leading
of interconversion
steadily
do
The temperature
with
acetate
of leghemoglobin
than
mixture
species.
the azide
The
temperature.
in temperature
in accord
The rate
states
observed
observed
is
have been made for
on the NMR time
resonances is
spectrum
4).
Such behaviour
increasing
spin
follow
tempera-
dependence
acetate.
an increase
does not
(fig.
of an equilibrium
of the high
optical
observations
myoglobin is
formate,
in the proportion
dependence
positions
with
of leg-
increasing
temperature
shift
of the existence
in leghemoglobin
with
from
at considerably
dependence
of leghemoglobin
of
well-
in the spectrum
downfield
much smaller
in hyperfine
exhibits
are observed
diamagnetic
complex
ppm at 30°C arising
temperature
two of them shift
for-mate
which
resonances
their
resonances
reflects
These
corresponding
exhibit
of the
and 38.6
4).
move away from their
the corresponding
39.3
(fig.
Further,
state
NMR spectrum
43.3,
groups the
acetate.
states
from the
at 45.1,
hemoglobin
clear
on the magnetic
was obtained
resolved
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
rapid in ferric
and probably atom (6).
(14).
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Why does the formate complex of leghemoglobin exist as an equilibrium mixture of high spin and low spin states at ambient temperatures whilst the acetate complex is fully
high spin? This behaviour is unique to
leghemoglobin, myoglobin fox-mate for example being a high spin ferric complex with NMRspectrum similar to that of leghemoglobin deuteroacetate The inherent ligand field
strengths of formate and acetate ligands are
expected to be very similar, globin should exhibit
(9).
in which case their
complexes with leghemo-
similar magnetic properties.
The observed differences
in leghemoglobin formate and leghemoglobin acetate can, however, be rationalised
if it is assumedthat steric
pocket interfere
constraints
with the binding of the bulkier
to a weaker ligand field this interpretation,
within the heme
acetate ligand,
and a high spin ferric
complex.
fonnate binds more strongly
leading
In support of
to ferric
leghemoglobin
= 0.23 mM, pH 5.3 and 20°C) than does acetate (Kdiss = 4.5 mM, (Kdiss pH 5.3 and 20°C) (15), which has an affinity for leghemoglobin similar to that of longer straight a-CHa group of aliphatic affinity
chain carboxylic carboxylic
for leghemoglobin.
acids (3).
acids substantially
Isobutyric
Substitution
at the
reduces their
acid binds only weakly to ferric
leghemoglobin (Kdiss ~a. 500 mMat pH 5.3 and ZO'C, as determined by the spectrophotometric to bind at all.
method described in (16))and lactic
acid appears not
Thus it appears that whilst the hemepocket of ferric
leghemoglobin is more open than that of ferric
myoglobin, allowing the
binding of long chain carboxylic
constraints
pocket are still
acids, steric
important and influence
within the
the nature of the metal-ligand
bond.
ACKNOWLEDGEMENTS We thank Mrs J. Wellington the preparation for financial
and Mrs L. Grinvalds for assistance with
of leghemoglobin and the Australian support and for the use of facilities
720
Research Grants Committee at the National NMRcentre.
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
REFERENCES 1.
2.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Appleby, C.A. (1974): The Biology of Nitrogen Fixation (Quispel, A. ed.) pp, 521-554, North-Holland Publishing Co., Amsterdam. Vainshtein, B.K., Arutyunyan, E.G., Kuranova, I.P., Borisov, V.V., Sosfenov, N.I., Pavlovskii, A.G.. Srebenko, A.I., Konareva, N.V. and Nekrasov, Yu. V. (1977) Dokl. Akad. Nauk. SSSR233, 238-241 (English Edition: 233, 67). Ellfolk, N. (1961) Acta Chem. Stand. 15, 975-984. Ehrenberg, A. and Ellfolk, N. (1963) Acta Chem. Stand. II, S343-S347. Appleby, C.A., Blumberg, W.E., Peisach, J., Wittenberg, B.A. and Wittenberg, J.B. (1976) J. Biol. Chem. 251, 6090-6096. Wright, P.E. and Appleby, C.A. (1977) FEBSLett. z, 61-66. Appleby, C.A., Nicola, N.A., Hurrell, J.G.R. and Leach, S.J. (1975) Biochemistry 14, 4444-4450. Ollis, D.L., Eight, P.E., Pope, J. and Appleby, C.A., submitted for publication. Morishima, I., Neya, S., Ogawa, S. and Yonezawa, T. (1977) FEBSLett. 83, 148-150. Fung, L.W.-M., Minton, A.P., Lindstrom, T.R., Pisciotta, A.V. and Ho, C. (1977) Biochemistry 16, 1452-1462. Perutz, M.F., Pulsinelli, P.D. and Ranney, H.M. (1972) Nature New Biol. 237, 259-263. Vainshteln, B.K. and Arutyunyan, E. (1978) abstract, Int. Symp. Biomolecular Structure, Conformation, Function and Evolution, Madras, India. Morishima, I. and Iizuka, T. (1974) J. Amer. Chem. Sot. 96, 5279-5283. Binstead, R.A., Beattie, J.K., Dose, E.V., Tweedle, M.F. and Wilson, L.J. (1978) J. Amer. Chem. Sot. 100, 5609-5614. Appleby, C.A. and Wittenberg, J.B., manuscript in preparation. Appleby, C.A., Wittenberg, B.A. and Wittenberg, J.B. (1973) Proc. Natl. Acad. Sci. USA'0, 564-568.
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