From the Departments of Physics and ¶ Biomedical
Sciences, University of Antwerp, B-2610 Antwerp, Belgium,
INSERM, Unite 473, Hôpital de Bicètre, F94275 Le
Kremlin- Bicètre, France, and the ** National Institute
for Materials Physics, POB MG-7 Magurele-Bucuresti, Romania
Received for publication, October 17, 2002, and in revised form, November 27, 2002
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ABSTRACT |
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Neuroglobin is a recently discovered member of
the globin superfamily. Combined electron paramagnetic resonance
and optical measurements show that, in Escherichia coli
cell cultures with low O2 concentration overexpressing
wild-type mouse recombinant neuroglobin, the heme protein is mainly in
a hexacoordinated deoxy ferrous form (F8His-Fe2+-E7His),
whereby for a small fraction of the protein the endogenous protein
ligand is replaced by NO. Analogous studies for mutated neuroglobin
(mutation of E7-His to Leu, Val, or Gln) reveal the predominant
presence of the nitrosyl ferrous form. After sonication of the cells
wild-type neuroglobin oxidizes rapidly to the hexacoordinated ferric
form, whereas NO ligation initially protects the mutants from
oxidation. Flash photolysis studies of wild-type neuroglobin and its E7
mutants show high recombination rates (kon) and
low dissociation rates (koff) for NO,
indicating a high intrinsic affinity for this ligand similar to that of
other hemoglobins. Since the rate-limiting step in ligand combination
with the deoxy-hexacoordinated wild-type form involves the dissociation
of the protein ligand, NO binding is slower than for the related
mutants. Structural and kinetic characteristics of neuroglobin
and its mutants are analyzed. NO production in rapidly growing E. coli cell cultures is discussed.
Two new globins, neuroglobin
(Ngb)1 and cytoglobin (Cygb),
were recently added to the vertebrate globin family. Ngb is
predominantly expressed in the brain and in other nerve tissues whereas
Cygb is expressed in all tissues studied so far. Human Ngb and Cygb are
composed of 151 and 190 amino acids, respectively, the Cygb being ~40
residues longer than standard globins because of the presence of amino-
and carboxyl-terminal extensions of ~20 residues each. Although both
globins display the structural determinants of the globin fold (1),
they share little sequence identity with vertebrate hemoglobin (Hb) and
myoglobin (Mb) (2-7). In contrast with Mb, which is expressed in
muscle tissue in the millimolar range, Ngb and Cygb are expressed at a
much lower level (micromolar range) (2, 3).
Ngb and Cygb are hexacoordinated (hx), either in their ferrous or
ferric forms, having the distal HisE7 as the internal ligand (3, 8, 9,
10). Flash photolysis studies of Ngb, at normal temperature, show high
recombination (kon) and low dissociation (koff) rates for O2 and CO,
suggesting a high intrinsic affinity for both ligands. However, since
the rate-limiting step in ligand binding to the ferrous deoxy-hx form
involves dissociation of the distal HisE7 residue, ligand binding
in vivo is suggested to be low (P50 = 1 Torr)
(6-9). The study of the ligand binding over a wide range of
temperature reveals the presence of multiple, intrinsically
heterogeneous distal heme pocket conformations in Ngb-CO (11). Distal
heme pocket heterogeneity is also observed by Raman spectroscopy
(8).
Cytosolic hxHbs are also observed in bacteria (12), unicellular
eukaryotes (13), plants (14), and some invertebrates (15). It can be
extrapolated that hxHbs are universally spread over the living world
and thus may have essential function(s) in cell metabolism.
The physiological role of low expressed hxHb is not well understood.
Several functions have been suggested. First, these might be proteins
involved in O2 scavenging under hypoxic conditions and
supplying it for aerobic respiration (2, 6, 9). Second, they might
function as terminal oxidases by reducing NADH under micro-aerobic
conditions and enhancing as such the ATP production by glycolysis (16).
Third, they could be O2 sensor proteins activating other
proteins with regulatory function (11, 12). Fourth, they may display as
yet unknown enzymatic activities. Fifth, these might be involved in NO
metabolism as shown for Mb (17-18), some flavo-Hbs (19) and truncated
Hb (20), and the Ascaris (Nematoda) Hb (21).
Electron paramagnetic resonance (EPR) has been used for more than
thirty years to analyze nitrosyl (NO) ligation to the heme group of
different hemeproteins (22-31). The NO radical bound to the central
Fe2+ ion in a hemeprotein forms a paramagnetic, low-spin
(S = 1/2) complex, which can be detected by EPR.
Furthermore, EPR techniques have been extensively used to study the
oxidized ferric state of different heme proteins (32-35).
Here we present an extensive EPR analysis of recombinant wild-type (wt)
and mutant mouse Ngb (mNgb) in its ferric and nitrosyl ferrous form in
comparison with those of recombinant sperm whale (sw) wt and mutant
Mbs. Furthermore, we give a detailed kinetic analysis of the NO binding
to wt mNgb and mutants.
Expression Cloning and Purification of Recombinant Wild Type and
Mutant Mouse Ngb--
Expression cloning and purification of wt and
mutant mouse Ngb was performed as described previously (9). Briefly,
the expression plasmids (cDNA of mNgb in pET3a) were transformed in Escherichia coli, strain BL21(DE3)pLysS, and grown at
25 °C in the presence of ampicilin, chloramphenicol, and
Substitution of the distal His (E7) by Leu, Val, or Gln, was performed
on the recombinant Ngb using the QuickChangeTM
site-directed mutagenesis method (Strategene). The recombinant mutant
Ngb was subsequently expressed and purified as wt Ngb.
Derivatization of Ngb--
Purified ferric Ngb was converted to
the ferrous form by addition of sodium dithionite. Excess reagent was
eliminated by size exclusion chromatography (Amersham Biosciences PD10
column) under CO atmosphere (AtmosBagTM, Aldrich) and using
CO-saturated buffer (50 mM Tris-HCl, pH 7.5). Replacement
of CO by NO was performed by incubating the ferrous Ngb-CO with 1 mM spermine nonoate
(N-[4-(1[3-aminopropyl]-2-hydroxy-2-nitrosohydrazino)butyl]-1,3-propanediamine) at room temperature for 60 min (36). Mb and Mb mutants were expressed
and purified as described previously (37).
Analytical Techniques--
Dissolved O2 was measured
with a Hanna HI 9141 oxygen meter (HANNA Instruments).
NO Optical Spectra--
Spectral measurements were made with an SLM
DW2000 spectrophotometer (Hewlett Packard). Under air, the samples (10 µM on a heme basis in 4 × 10-mm quartz cuvettes)
oxidize within an hour; this form was taken to be the ferric state. The
spectra of the Ngb directly within the E. coli cell culture
were measured with the DW2a spectrophotometer (Aminco) in the split
beam mode, ranging from 350 to 650 nm.
Electron Paramagnetic Resonance--
The EPR spectra were
recorded on a Bruker ESP300E spectrometer (microwave frequency 9.43 GHz) equipped with a gas-flow cryogenic system, allowing operation from
room temperature down to 2.5 K. All presented spectra were recorded
with a microwave power of 10 milliwatts, a modulation frequency of 100 kHz, and a modulation amplitude of 0.5 milliTesla. The magnetic
field was calibrated using a sample of DPPH (diphenylpicrylhydrazyl)
and an NMR Gaussmeter (Bruker ER 035 M).
The EPR spectra were simulated using the EasySpin program
(www.esr.ethz.ch/). The intensities of the spectra were determined by double-integration of the spectra after baseline correction. In
relating the spectral intensities to relative spin concentrations the
total protein concentration and volume were taken into account. Samples
for the EPR measurements were taken at different stages in the
isolation and purification process of the proteins.
Spectra and Ligand Binding Kinetics--
For all the kinetic
measurements the experimental conditions were 100 mM
potassium phosphate, pH 7.0. The typical sample concentration was 10 µM on a heme basis, and the measurements were performed in a 4 × 10-mm quartz cuvette at 298 K.
NO Binding Kinetics--
NO bimolecular recombination rates
(k NO Dissociation Kinetics--
Spectral measurements were made
with a HP8453 diode array spectrophotometer. Optical changes were
monitored in the Soret and visible regions.
The concentrated sample was first equilibrated under pure nitrogen to
remove the oxygen. Then the sample was equilibrated with 0.009 atm of
NO before the reduction of the oxidized fraction with a slight excess
of sodium dithionite. Once the heme is bound to NO the samples were
kept on ice. The Ngb-NO samples were diluted into the optical cuvette
containing the buffered solution in the presence of 1 mM
sodium dithionite equilibrated under 1 atm of CO. The dithionite was
added because it readily consumes the NO after its dissociation, in
order to facilitate its replacement by the large excess of CO. As
expected the dissociation kinetics were extremely slow, and the
experimental error was, ±30%. To correct for baseline drifts, full
spectra were measured, and analysis was performed on the spectral differences.
NO Binding to Ngb--
Fig.
1A shows the EPR spectra of
E. coli cell cultures overexpressing recombinant wt Ngb and
recombinant mutant Ngb (E7-Val, E7-Gln, and E7-Leu). The E. coli cell cultures were induced at OD 0.8 and all grown under
identical conditions. The spectra were recorded directly after freezing
the same amounts of the living cell cultures. The spectra are dominated
by an orthorhombic EPR signal, which is typical for a six-coordinated
Fe2+·heme(NO) complex (22-30). Repeated experiments
revealed that for the same quantity of cells, the EPR intensity was
found to be a factor 4-6 higher for the cell cultures overexpressing
mutant Ngb than for those overexpressing wt Ngb (see Fig.
1A). The EPR spectra showed no clear signals corresponding
to ferric globins.
During expression, the available O2 is completely consumed
by the exponentially growing cells creating a micro-anaerobic
environment (Table I). This explains the
lack of Fe3+ globins in the EPR spectra. Nitrate
consumption and nitrite production is obvious. Formation of volatile
N-derivatives, including NO, is likely.
The optical spectrum recorded directly in the living E. coli
cell cultures overexpressing recombinant wt Ngb showed the typical features (
Fig. 1B shows the EPR spectra of a batch of E. coli cells overexpressed with Ngb or its mutants after three
freeze-thaw steps and sonication. Note that Fig. 1, A and
B show different samples taken from a large batch of cells
before and after sonication (see "Experimental Procedures"). The
absolute spectral intensities of Fig. 1, A versus
B should therefore not be compared; only the relative
intensities of the spectra within one figure are relevant. In the case
of the recombinant mutant Ngbs (Fig. 1B, f-h),
the EPR spectra are virtually identical to those of the corresponding cell cultures (Fig. 1A, b-d). No significant
contribution of ferric forms can be found. However, sonication of the
cells overexpressing wt Ngb results in strong EPR contributions of
Fe3+ complexes besides the spectrum of the NO-heme form
(Fig. 1e). As shown earlier (9), one of the Fe3+
forms (indicated with an asterisk in Fig. 1e) can
be ascribed to the hexacoordinated ferric state of Ngb
(His-Fe3+-His). Again, the contribution of the hx
His-Fe2+-NO form is found to be a factor 4-6 larger in the
case of the mutants than of the wt. These results demonstrate that NO
addition initially protects the Fe2+ center against
oxidation. The hexacoordinated His-Fe2+-His form is however
less stable against oxidation.
Fig. 2A shows the EPR spectra
of wt, E7-Val, E7-Gln, and E7-Leu Ngb proteins at pH 8.5 after
purification of the proteins. In all spectra, different signals due to
Fe3+ complexes (both low- and high-spin) can be discerned.
They result from the long O2 exposure during purification
and will be discussed in detail in the next section. In all spectra,
the contribution of the NO adduct of the Ngb proteins can still be
seen, but likely represents a small percentage of the total heme.
In order to prove that the EPR spectra observed in Fig. 1A
can indeed be ascribed to a nitroxide adduct of the Ngb proteins, the
carboxyl ferrous forms of the proteins were reacted with the nitrosating reagent, spermine nonoate. The corresponding EPR spectra for wt and E7-Leu Ngb are depicted in Fig.
3A, a and
b. As a comparison, the spectrum for the recombinant E7-Leu
mutant of swMb, treated in the same way, is shown (Fig.
3Ac). Apart from EPR signals due to Fe3+
complexes, which were also present in the control measurements of the
carboxyl ferrous proteins and which can be ascribed to an incomplete
reduction of the Fe3+ proteins with sodium dithionite,
clear signals similar to the ones observed in the E. coli
cell cultures are observed. Both at pH 7.5 and 8.5, the
Fe2+-NO concentration in E7-Leu Ngb was larger than the one
in wt Ngb (the relative concentration was determined on the basis of the protein concentration, volume, and EPR signal intensity). The
release of NO by spermine nonoate is controlled by the pH, whereby a
higher release occurs at lower pH. The difference in the EPR
intensities confirms the earlier observed differences (Fig.
1A) between the EPR spectra of E. coli cells
overexpressing wt Ngb and its mutants.
Fig. 3B shows the NO-related signal in detail for E7-Val Ngb
and E7-Leu Mb with the corresponding simulations. The simulations were
done assuming an admixture of two species of rhombic (type I) and axial
(type II) symmetry. The EPR parameters for type I are given in Table
II, the axial g tensor of type
II was for all cases taken as g
On the basis of extensive EPR and ENDOR studies, type I has been
assigned to a hexacoordinated structure with the nitroso-ligand coordinated in a bent end-on orientation with the proximal histidine F8
as second axial ligand (23, 26, 28, 39). In this configuration the
Fe-N(NO) bond does not coincide with the porphyrin plane normal (39).
Density functional computations suggest that the NO is oriented toward
a meso-C atom of the porphyrin ring (40). The nature of type II is
still controversial, despite the large amount of spectral data
available. Several authors have ascribed type II at low temperatures to
a species where the Fe-N(NO) bond aligns with the normal of the
porphyrin ligand (28, 26, 29, 39), although a topological isomer with
the Fe atom displaced "below" the porphyrin ligand toward HisF8 has
also been proposed (22). Recent density functional computations
indicate that the observed axial g tensor agrees either with
a bent end-on orientation of NO where the NO ligand eclipses one of the
equatorial Fe-Nporph bonds or with a partially dissociated
hexacoordinated complex (distance (Fe-N(Im) >0.25 nm) with a freely
rotating NO ligand (40). The latter may explain the changes in the
g tensor upon increase of the temperature, but at low
temperatures (<40 K), rotation of the NO ligand can be excluded. The
computations excluded the "Fe-displacement" model.
NO Affinity and Kinetics--
As for other heme proteins, NO binds
with a high affinity to Ngb. Flash photolysis of the NO-bound form gave
small signals on the µs timescale. We therefore injected Ngb-CO into
an optical cuvette prepared under a partial NO atmosphere; these
samples were immediately photodissociated to allow observation of the NO binding. As for other Hbs, the bimolecular association rate is high
(Table III), approaching that for a
diffusion-limited reaction.
The replacement of NO by CO allowed a measurement of the NO
dissociation rate, which was quite low. Both the on- and off-rates of
NO to pentacoordinated Ngb were similar for the series of mutants; thus
the affinity for the pentacoordinated form
Kpenta are similar. The main difference is
therefore the competitive binding of the distal histidine for the wt
Ngb. As for other external ligands, this will decrease the observed
affinity by nearly a factor of 1000, as determined by the relation for
competing ligands : K = Kpenta/(1+ KHis). This
large reduction in affinity can also be explained as if there were two
types of association by the external ligand: when the site is free
(pentacoordinated form), there is a high rate of binding, whereas if
the external ligand encounters the hexacoordinated form, it must wait
for the histidine to dissociate, and the overall ligand replacement
will occur on a much slower timescale.
Analysis of the EPR Data of the Ferric Heme Proteins--
The
ferric form of the wt Ngb has been characterized earlier by EPR (10).
The study revealed the simultaneous presence, in a wide range of pH
values, of two related structural forms. The dominant low-spin form
(>90%) (LS1 (Table IV), indicated in Fig. 1e with an asterisk) could be attributed to
a His-Fe3+-His configuration. The high-spin form (HS1
(Table IV), indicated with a double asterisk in Fig.
1e) could be ascribed either to a hexacoordinated
His-Fe3+-H2O form or to a pentacoordinated
His-Fe3+. The EPR spectra strongly suggested that an
equilibrium exists between a low-spin species in which the E7 His
residue is in a distal imide coordination at the iron atom, in a
slightly tilted His-Fe3+-His structure and a
high-spin species, favored at low pH, in which the E7 His residue
swings out from the heme pocket possibly allowing a water molecule to
bind in the distal position. The equilibrium lies strongly on the side
of the low-spin species. Ferrous Ngb was earlier found to remain
hexacoordinated over a wide range of pH values (9). The fact that the
distal histidine of Ngb can bind to the heme iron in both ferric and
ferrous states is consistent with the distal histidine residing closer
to the heme than seen in mammalian Hbs and Mbs, but similar to other non-mammalian Hbs and cytochromes. Indeed, comparison with the EPR
spectrum of wt swMb (Fig. 2e) shows us that Mb is
predominantly in the aquomet form (HS5, Table IV) with a small
contribution of a low-spin Fe3+ species (LS4, Table IV),
which can be attributed to a His-Fe3+-OH
The signals marked by a triple asterisk in Fig.
1e, which can also be observed in the EPR spectra in Fig.
2A can be attributed to non-heme ferric residues (42) and
will not be discussed further.
The EPR spectra of the ferric Ngb E7-mutants are markedly different
from those of ferric wt Ngb (Fig. 2B). As expected the low-spin complex LS1 is not found in the mutants, confirming that this
low-spin complex can indeed be ascribed to an hx
His-Fe3+-His structure. Besides the contribution of the
ferrous nitrosyl heme form, EPR signals due to different high-spin (HS)
and low-spin (LS) ferric complexes can be observed in the spectra of
the Ngb mutants (Fig. 2A, b-d). This contrasts
clearly with the EPR spectra of the corresponding mutants in swMb (Fig.
2, f-h). These spectra consist of only the high-spin form.
For E7-Gln Mb, HS8 can be attributed to the aquomet form, whereby HS6
and HS7 of E7-Leu and E7-Val Mb can be related to a five-coordinate
heme iron (33) (Table IV and Supplemental Data, Fig. S3). Ikeda-Saito
et al. (33) showed that large spectral changes can occur in
the EPR spectra of E7-Leu, E7-Val, and E7-Gln Mb upon change of the
buffer. Analogous effects were observed for the Ngb mutants under study (see Supplemental Data). No buffer effect was detected for wt Ngb
(Supplemental Data, Fig. S4A) or for wt Mb (33). Although we
have no plausible explanation for the buffer effect, it seems that the
histidine residue at position E7 protects the heme pocket from
influences of the buffer molecules. In Table IV only those ferric
complexes of the Ngb mutants are mentioned that are observed regardless
of the buffer system. All mutants showed EPR signals due to a high-spin
FeIII complex (HS2, HS3, HS4). Since the EPR spectra were
found to be pH-independent and no clear signal of
His-Fe3+-OH Structural and Kinetic Analysis--
The EPR data and NO binding
kinetics clearly show a different behavior of the wt Ngb
versus its mutants E7-Leu, E7-Val, and E7-Gln. Fig.
4 shows schematically the reaction
pathways under anaerobic conditions in the presence of NO (E. coli cell cultures) and under aerobic conditions in the absence of
NO. As determined by EPR and optical spectroscopy, the wt Ngb is in the
rapidly growing E. coli cells predominantly in the
hexacoordinated His(F8)-Fe2+-His(E7) configuration, whereas
all mutants occur in the hx His(F8)-Fe2+-NO form. This
observation agrees with the fact that the NO affinity is about a factor
1000 lower for the wt Ngb than for the Ngb mutants (Table III). This
results from the competition between NO and the distal histidine since
the intrinsic NO affinities to the pentacoordinated form are comparable
for the wt and mutant Ngbs. However, the NO affinity is so high that
the difference in values between wt Ngb and mutants (nM
versus pM) probably does not matter. It is more likely that in hx wt Ngb the rate of NO binding is slower than in the
mutants, and thus other NO consuming reactions can compete with wt Ngb
for the ligand.
After sonciation of the cells, the autoxidation of the Ngb mutants is
slow indicating that NO ligation initially protects the heme pocket
against oxidation. The on-rate for NO binding to pentacoordinate E7-Leu
NGB is comparable with those for CO and O2 binding
(kon(O2) = 7 × 108
M
It is interesting to note that all Ngb proteins under study show at
10-15 K the same ratio of type I versus type II ferrous nitrosyl-heme complexes
(nI/nII = 80/20%) as
found for NO-ligated Biological Implications--
(i) NO production in E. coli: during protein expression in E. coli under
traditional laboratory conditions (250 ml of medium in a 1-liter
Erlenmeyer; normal shaking), the metabolism of the cells gradually
shifts from aerobic to anaerobic respiration because of the complete
consumption of the initially available O2 by the increasing
cell density. Under conditions of low O2, nitrate reductase is induced, and E. coli will reduce nitrate to nitrite and
nitrite to NO using this enzyme. The concentrations of NO produced by E. coli in this way never exceed 300 µM
despite large excesses of nitrite and formate and is self-limiting
(45-48). The poisonous NO will be part of the mechanism for rapidly
shutting down the TCA cycle and terminal oxidase in semi-anaerobic
conditions (49). Excess NO will be detoxified by diverse systems
including flavohemoglobin and flavorubredoxin (50, 51). During the
expression of a hemoprotein at high concentration, NO will be scavenged
by the heme-iron resulting in ferrous nitrosyl spectra. The degree of
NO binding however will depend on the distal site conformation.
It is well documented that co-expression of Vitreoscilla Hb,
in E. coli, will increase bacterial protein expression
(52-54). This effect is mainly attributed to an increased
O2 supply to the bacteria by the presence of an oxygen
carrier. However, in the light of our observations, the reduction of
the nitrosoactive stress by the scavenging of NO by the heme iron of
the Vitreoscilla Hb and thus a prolonged aerobic metabolism
might be of more importance than the increased O2 supply
(54, 55).
(ii) Ngb function: the involvement of Hb and Mb in the NO metabolism
became clear only recently (17, 56). As a tissue hemoprotein, Mb
functions as a scavenger of bioactive NO in cardio-myocytes by the
reaction of MbO2 + NO to metMb + NO
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino-levulinic acid. After induction, the cells were grown
overnight. The cells were broken by freeze-thawing and sonication (9).
The Ngb was purified from the clarified cell lysate by ammonium sulfate
precipitation, DEAE-Sepharose ion exchange, and size exclusion chromatography.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
X-band EPR spectra of samples of
E. coli cell cultures overexpressing
recombinant wild-type and mutant mouse neuroglobin taken before
(A) and after (B) sonication.
E. coli cell cultures overexpressing recombinant wt Ngb
(a, e), recombinant E7-Val Ngb
(b, f), recombinant E7-Gln (c,
g), and recombinant E7-Leu (d, h). All
spectra were measured at 13 K and with the same receiver gain. The
spectra 1e-h are scaled in accordance with the amount of
sample available for each measurement. The spin state of
FeIII is indicated by: *, low spin; ***, high spin; **,
non-heme.
O2, NO2, and NO3
concentration during neuroglobin expression in E. coli
band: 560 nm,
band: 530 nm, Soret band: 426 nm) of a
hx globin (His-Fe2+-His) (9). When the cells are
overexpressing the mutant Ngb proteins, the spectra become typical for
hexacoordinated NO-heme proteins (
band: 571 nm,
band: 541 nm,
Soret band: 417 nm) (38) (see Supplemental Data, Fig. S1). This
assignment is corroborated by the comparison with the absorption
spectra of purified ferrous Ngb in the hx deoxyform and bound to the
external ligand NO (see Supplemental Data, Fig. S2). The combination of
the optical and EPR data demonstrates that within the E. coli cell, recombinant wt Ngb occurs predominantly in its deoxy
ferrous hexacoordinated form, whereby a small fraction of the
hexacoordinated nitrosyl ferrous form is found. All recombinant mutant
Ngbs under study are predominantly in the hx nitrosyl ferrous heme form.
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Fig. 2.
X-band EPR spectra of the purified ferric
forms of recombinant wild-type and mutant mouse neuroglobin
(A) and recombinant wild-type and mutant sperm whale
myoglobin (B). a, wt Ngb at pH 8.5 (Tris buffer); b, E7-Val Ngb at pH 8.5 (Tris buffer);
c, E7-Gln Ngb at pH 8.5 (Tris buffer); d, E7-Leu
Ngb at pH 8.5 (Tris buffer); e, wt swMb; f,
E7-Val swMb; g, E7-Gln swMb; h, E7-Leu swMb. All
spectra were measured at 13 K. All spectra are normalized to the same
height.
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Fig. 3.
X-band EPR spectra of the ferrous carboxyl
forms of recombinant mouse neuroglobin and sperm whale myoglobin after
treatment with spermine NONOate at pH 7.5 (A). a,
recombinant wt Ngb; b, recombinant E7-Leu Ngb; c,
recombinant E7-Leu swMb. Detailed analysis of the EPR spectra of the
Fe2+ heme(NO) complexes (B). e,
recombinant E7-Val Ngb; f, recombinant E7-Leu swMb.
Experiment, full line; simulation, dashed lines.
All spectra were measured at 13 K.
2.035, g
1.98. The parameters are typical for a
six-coordinated Fe2+(heme)NO structure (22-31). The EPR
spectra of the Ngb proteins could be simulated using a ratio of type I
over type II (nI/nII) equal to 80/20%, whereby this ratio was 95/5% for E7-Leu Mb.
Variations in the nI/nII
ratio have been reported earlier for different heme proteins (22,
26-30). Isolated NO-ligated
(
)-chains of Hb give nI/nII ratios of 80/20
(10/90) at temperatures below 30 K (26, 28). For Mb at low temperature,
nI/nII ratios varying
from 50/50% (28) to 70/30% (27) have been observed, depending on the
type of Mb. For all hemeproteins, type II is found to dominate at high temperatures (>180 K). Furthermore, subtle variations of the
g tensors of type I were detected upon temperature increase
(27, 30).
EPR parameters for the hexacoordinated nitrosyl ferroheme complexes
(Type I)
Rates of NO binding to Ngb and sperm whale myoglobin
form
that is only present at high pH (33) (see also Supplemental Data).
The g values of ferric low-spin (LS) and high-spin (HS) species
observed in frozen solutions of recombinant wild-type and mutant
neuroglobin and recombinant wild-type and mutant sperm whale
myoglobin
could be observed at high pH,
these high-spin features can be attributed to a five-coordinate heme
iron rather than to the aquomet form. Differences between the EPR
parameters of the high-spin forms of the Ngb mutants and the
corresponding swMb mutants may be related to the different position of
the E7 amino acid versus the heme. Pulse EPR measurements
will be undertaken to investigate this. Furthermore, a low-spin signal
(LS2, Table IV) can be found in all EPR spectra of the Ngb mutants. The
g values are similar to those observed for a
His-Fe3+-Tyr
ligation, as was found at high
pH for Chlamydomonas chloroplast Hb (43). However, the lack
of a tyrosine at B10 (B10-Phe) and the fact that the spectrum of LS2 is
pH independent over the pH range 5-10, excludes this possibility. Also
the observation of the low-spin species LS3 in E7-Leu Ngb remains
inexplicable at present. The g values are in accordance with
a ligation of a sulfur-containing endogenous ligand such as mercaptide
or mercaptoethanol (44). To further examine these low-spin species, pulse EPR measurements are planned in the future.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 4.
Reaction schemes for recombinant wild-type
(A) and mutant (B) mouse neuroglobin
inside and outside the E. coli
cells.
1 s
1,
kon(CO) = 2 × 108
M
1 s
1), but the off-rate is
considerably lower in the case of NO binding than for O2
binding (koff(O2) = 200 s
1,
koff(CO) not determined) (9), which explains the
observed protection against oxidation by NO. This protection is not
given by the distal histidine ligation, as becomes obvious from the appearance of the EPR spectra of the His(F8)-Fe3+-His(E7)
form immediately after sonication of the cells (Fig. 1e). This agrees
with the earlier determined high autoxidation rate of Ngb and with the
fact that the NO dissociation rate constant is about a factor 6000 lower as the histidine dissociation rate constant
(koff = 1.2 s
1) (9). Our earlier
EPR analysis of Ngb showed that ferric Ngb is predominantly in the
low-spin hexacoordinated form (Fig. 2a and Ref. 10). Besides
the expected EPR signal of the high-spin pentacoordinated ferric heme
complexes (HS2, HS3, HS4), unidentified signals of low-spin
Fe3+ complexes could be observed in the EPR spectra of the
purified Ngb mutants (Fig. 2A). Most of these signals were
found to be buffer-dependent indicating that mutation of
E7-His destabilizes the heme pocket.
Hb. An extensive proton ENDOR analysis of the
type I and type II forms in horse heart Mb at 10 K showed that for both
isomers the E7-His and E11-Val residues are present in the heme pocket and stabilize the bound NO (29). Using electron spin echo envelope modulation (ESEEM) spectroscopy, an interaction with the
N
nitrogen of the distal E7-His could be found for
NO-ligated heme proteins of type II, where this interaction was not
observed for type I (28). This seems to correlate with our observation
that at 13 K E7-Leu Mb is quasi in the pure type I form (95%), whereas this is only 50-70% for wt Mb (27, 28). The substitution of the
distal histidine has a significant influence on the formation of the
type II nitrosyl isomer in Mb. Furthermore, the stabilization of NO by
a hydrogen-bonded N
in the axial state II seems to agree
with x-ray crystal structure (41). Although type I and type II species
have been found in frozen solutions of NO-ligated tetraphenyl porphyrin
imidazole (26), the ENDOR and ESEEM data on Hbs and Mbs seem to
indicate that the variations of the NO binding geometry is in heme
proteins controlled by the heme's protein surrounding. The ENDOR data
on type I and type II suggest that the difference between the type I
and type II form lies not only in the deviation of the Fe-N(NO) axis
from the porphyrin normal, but also in a displacement of both distal
and proximal histidine versus the heme plane (30, 39).
Interestingly, mutation of the E7-His site in Ngb does not seem to
influence the nI/nII ratio. From the observation of the hexacoordinated His-Fe-His configuration for Ngb we assume that the distal histidine resides closer to the heme in Ngb than in mammalian Hbs and Mbs. The current observations seem to indicate that in this geometry E7-His can influence less the equilibrium between the two NO binding modes than
observed in Mb. This might also explain why the effect of the polarity
of the E7 amino acid on the NO association rate constants is less
pronounced for Ngb than for Mb (Table III).
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ACKNOWLEDGEMENTS |
---|
We thank M. L. Van Hauwaert (University of Antwerp) for technical assistance. R. Van Grieken and B. Meersman are kindly thanked for chemical analysis. John Olson is thanked for the critical reading of the article and his constructive contribution. T. Burmeister and T. Hankels are acknowledged for their collaboration.
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FOOTNOTES |
---|
* This work was supported in part by Fund for Scientific Research-Flanders (FWO) Grant G.0409.02 (to E. G.) and Grant QLRT-2001-01548 from the European Union.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains supplementary figures and data.
§ Postdoctoral fellow from the FWO. To whom correspondence should be addressed: Dept. of Physics, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium. Tel.: 0032-3-8202461; Fax: 0032-3-8202470; E-mail: Sabine.VanDoorslaer@ua.ac.be.
Supported by Grant G.0069.98 from the FWO.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M210617200
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ABBREVIATIONS |
---|
The abbreviations used are: Ngb, neuroglobin; Mb, myoglobin; Hb, hemoglobin; Cygb, cytoglobin; hx, hexacoordinated; wt, wild type; sw, sperm whale; EPR, electron paramagnetic resonance; ENDOR, electron nuclear double resonance; ESEEM, electron spin echo envelope modulation; LS, low spin; HS, high spin; n.d., not determined; NO, nitric oxide.
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