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INTRODUCTION |
Endothelium-derived relaxant factor
(EDRF)1 or endogenously
derived nitric oxide (NO) (1) is formed enzymatically from
L-arginine (2) and is an important mediator in a variety of
physiologically relevant processes (3, 4). One important regulatory
role is that of endothelium-dependent relaxation of
vascular smooth muscle (5, 6), which affects vascular tone and
ultimately circulating blood pressure (7). However, despite numerous
studies describing the biological activity of EDRF/NO, there is no
consensus regarding the biological half-life of NO (8, 9) or possible stabilization of NO by reaction with carrier molecule(s) in
vivo that may result in a prolonged half-life with or without
preservation of NO bioactivity (10, 11).
Among the biological targets of NO, low molecular weight reduced
thiols, protein sulfhydryls (12-14), and heme and nonheme iron can be
nitrosated (15-17). S-Nitrosothiols exhibit characteristic electronic maxima in the regions 330-350 and 530-550 nm (12), whereas
heme-nitrosyl complexes can be readily detected directly by low
temperature EPR spectroscopy (18, 19). The decomposition of
S-nitrosothiols to regenerate NO is proposed to be a
mechanism whereby NO may be either translocated or prevented from
diffusing away from the site of synthesis (8).
Myoglobin is present in relatively high concentration in skeletal and
cardiac muscle (2.1-6 mg/g of wet tissue (Refs. 20-22)), and
myoglobin of identical sequence has recently been identified in human
smooth muscle cells (23). Intracellular concentrations of ferric or
metmyoglobin are strictly controlled in vivo by a "metmyoglobin reductase" that maintains the heme iron in the
reduced form and able to bind dioxygen (24). Similar to oxyhemoglobin (25), oxymyoglobin (oxy-Mb) is able to oxidize NO to yield nitrate (NO3
) (26), and this process has been
proposed as a mechanism to regulate NO in skeletal and cardiac muscle
(9) and vascular smooth muscle (27).
The amino acid sequence of human Mb is unique among mammalian
myoglobins in that it possesses a cysteine residue, Cys110
(28). Recently, we investigated the reaction of recombinant human Mb
and hydrogen peroxide and showed that Cys110 is readily
oxidized to a thiyl radical that promotes formation of a
disulfide-linked dimeric Mb product (29). This reactivity of the
sulfhydryl led us to hypothesize that human Mb may also react with NO
to yield an S-nitrosothiol group at Cys110 of
human Mb. If substantiated, such reactivity would endow Mb with the
unique combination of abilities to preserve NO bioactivity through
formation of a S-nitrosothiol group and to regulate NO in vivo by either formation of a nitrosyl Mb complex or
oxidizing NO to NO3
. In the present
work, we have studied the reaction of human Mb and its C110A variant to
evaluate the relative reactivities of the thiol group at
Cys110 and the heme iron in the in vitro
reaction of wild-type human Mb with NO through the combined use of EPR,
Fourier transform infrared (FTIR), electronic absorbance spectroscopy,
and electrospray mass spectrometry.
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EXPERIMENTAL PROCEDURES |
Materials
2,2'-Dithiodipyridine (DTP), horse heart Mb, reduced glutathione
(GSH), iodoacetamide, trypsin (type III, 10,200 units/mg of protein),
urea, EDTA, 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO),
Tris buffer, and diethylenetriamine pentaacetic acid (DTPA) were
obtained from Sigma.
Diethylammonium(z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DeaNO,
260 ~ 6500 M
1 cm
1)
was from Cayman Chemicals (Ann Arbor, MI). Electronic absorbance spectra of solutions of DeaNO at pH 10 and 7.4 indicated a single maximum at A260 nm consistent with that
reported by the manufacturer, so the reagent was used without further
purification. Concentrated stock solutions of DeaNO (up to 100 mM in 1 mM NaOH) were prepared immediately
prior to use. NaNO2 (purity 97%) was from Eastman Kodak
Co. Tryptone and yeast extract were from Becton Dickinson (Sparks, MD).
NaCl (purity 99.5%) was obtained from Fisher Scientific (Fair Lawn,
NJ). NO gas (purity 98.5%) was from Linde Union Carbide (Praxair,
Vancouver, Canada). Where required, buffers were prepared from either
glass-distilled water or glass-distilled water purified further by
passage through a Barnstead Nanopure system. All buffers were stored
over Chelex-100® (Bio-Rad) at 4 °C for at least 24 h to remove contaminating transition metals as verified by ascorbate
autoxidation analysis (30). Organic solvents and all other reagents
employed were of the highest quality available.
Preparation of Recombinant Proteins
Transformed bacteria (strain AR68) containing plasmids for the
wild-type recombinant human myoglobin and the C110A variant (31) were
obtained from Prof. S. G. Boxer. The procedures used here for the
expression and purification of the various proteins has been described
in detail elsewhere (29, 31). The respective recombinant myoglobins
were isolated as the corresponding metmyoglobin. When required,
proteins were concentrated by centrifugal ultrafiltration (Centriprep-10 concentrators, Millipore).
Samples of the various apo-Mbs were prepared by adjusting the pH of
corresponding solutions of holo-Mb in Tris buffer (5 mM, pH
7.4) to pH = 2.5 by addition of 1 M HCl at 4 °C.
Free heme was then removed from the mixture by repeated extraction
(three times) of the acidified fraction into ice-cold 2-butanone as
described previously (32). The acidified aqueous fraction was then
dialyzed extensively against the buffer of choice (2, 20, and 50 mM phosphate buffer, pH 7.4, containing 100 µM DTPA), and the concentration of apo-Mb was determined
from the A280 nm (
280 = 16 mM
1
cm
1). For all apoprotein preparations, no
other absorption maxima were detected over
A220-700 nm except for a residual Soret band
(<2% of the initial Soret intensity) that could not be eliminated by
further extraction into 2-butanone. All working solutions of apo-Mb
were prepared immediately before use to avoid inadvertent oxidation of
redox-sensitive amino acids (e.g. Cys110 in
wild-type human Mb).
Reduction of Ferric Myoglobin(s) to Ferrous Myoglobin(s)
Samples of metmyoglobin were reduced to the corresponding
ferrous protein with a 2-3-fold molar excess of
Fe(EDTA)2
(33) under an atmosphere of nitrogen. Oxygen
was removed from protein solutions either by gently purging solutions
of the ferric or ferrous holo-Mb (or where applicable apo-Mb) with
purified nitrogen gas that had previously passed through a
vanadium(II)/mercury-zinc amalgam (34) or by alternately freezing and
thawing the sample under vacuum with a dual manifold vacuum line.
Anaerobic reactants and reaction mixtures were transferred with
gas-tight syringes and placed in flasks fitted with rubber septa to
avoid oxygen contamination. In some instances, dioxygen (99.0% purity,
Praxair, Vancouver, Canada) was bubbled directly into samples of
ferrous Mb, and then excess Fe(EDTA)2
and
Fe(EDTA)
were removed by gel filtration (PD-10 G-25
column, Amersham Pharmacia Biotech, Uppsala, Sweden). Under these
conditions, proteins were isolated as the corresponding ferrous
oxy-Mb.
Preparation of S-Nitrosated and Heme-Nitrosyl Complexes
In this investigation, three sources of nitrosating reagents
were employed throughout to yield nitroso complexes at either sulfhydryl (S) or heme (iron) centers. Each of the sources
has been verified previously as established means of generating NO (35-38).
Method 1--
Solutions of GSH or apo-Mb (either modified with
iodoacetamide or not for the case of wild-type human apo-Mb) were
prepared in 0.5 M HCl, exposed to NaNO2
(NO:target sulfhydryl ~2 mol/mol) and the reaction mixture stirred
for 30 min at 20 °C in either the presence or absence of air. Acidic
solutions of NO2
yield NO gas in a
stoichiometric reaction (18). The pH of the reaction mixtures was then
raised to pH 7.4 by titration with phosphate buffer (final phosphate
concentration 2, 25, or 50 mM and containing DTPA (100 µM)) and the protein was eluted over two gel filtration
columns (PD-10) to remove excess nitrite/nitrate. As holo-Mb is
sensitive to acidic conditions (<pH ~ 5.5), nitrosation of
either ferric or ferrous holo-Mb was not performed by method 1 but by
one of the following techniques.
Method 2--
Solutions of ferric or ferrous holo-Mb were
degassed with purified nitrogen (where appropriate) and then treated
with authentic NO gas in a dual manifold vacuum line. Prior to the
introduction of nitric oxide gas into the vacuum line, the NO was first
passed through an anaerobic column of solid KOH pellets at 5 p.s.i. and 20 °C and then introduced to the sample chamber. Protein
solutions were then stirred continuously for 20 min under an atmosphere of NO gas (39) (concentration of dissolved NO ~2.1 mM at
20 °C, based on solubility (X) of NO = 3.78 × 10
5 mol of NO/mol of H2O at the
prevailing atmospheric pressure). Finally, reaction mixtures were
removed from the gas line, and excess low molecular weight contaminants
were removed by gel filtration (PD-10) using columns pre-equilibrated
with appropriately degassed buffers.
Method 3--
Solutions of apo-Mb or ferric or ferrous holo-Mb
(each prepared in 2, 25, or 50 mM phosphate buffer, pH 7.4)
were treated with alkaline solutions (1 mM NaOH) of
concentrated DeaNO (at ratios of DeaNO:protein ~ 0-20 mol/mol).
Concentrated stock solutions of DeaNO were used to minimize hydroxide
addition to the buffered solutions and resulting changes in pH. Where
appropriate, stock reagents and solutions containing the target protein
were thoroughly degassed prior to mixing to yield anaerobic reaction
mixtures. Reaction mixtures were then purified by gel filtration
(PD-10) using columns that had been equilibrated with appropriately
degassed buffers.
Regardless of the method used to generate the nitrosated product, DTPA
(final concentration, 100 µM) was included in all
reaction mixtures containing sufhydryls to minimize the possibility of either transition metal-mediated oxidation of the protein sulfhydryl or
the metal-mediated decomposition of the corresponding
S-nitrosothiol (12, 37). Method 3 proved to be the most
convenient protocol for addition of precise amounts of NO to solutions
of Mb and so was used for all concentration-dependent
studies (see "Results").
EPR Spectroscopy
X-band EPR spectroscopy was carried out at 77 K with a Bruker
model ESP 300e spectrometer equipped with a Hewlett Packard microwave
frequency counter. For low temperature EPR, samples (250 µl) of
reaction mixture were placed into a 3-mm quartz cell (Wilmad, Buena,
NJ), snap-frozen in liquid nitrogen, and transferred to a finger Dewar
insert (77 K) for EPR analysis. The time required to removing the
sample from the reaction vial and freezing for EPR analyses was
consistently <10 s. The limit of detection of a stable nitroxide
(TEMPO) under identical conditions was determined to be ~50
nM. Unless stated otherwise, EPR spectra were obtained as
an average of five scans with sweep time of 84 s. Microwave power,
modulation amplitude, and scan range were as indicated in the legend to
the figure(s).
Electrospray Ionization Mass Spectrometry (ESI-MS)
ESI-MS was performed with a triple quadrupole mass spectrometer
described in detail elsewhere (40). Samples of Mb (± NO gas) were
exchanged into water, diluted to a final concentration of 10 µM in heme (with methanol/water 1:10, v/v) and then
infused continuously (flow rate of 1 µl/min) into the ion source at
20 °C. Experiments were performed without an internal standard.
Multiply charged gas phase proteins were generated by pneumatically
assisted ESI. In some experiments, the voltage difference between the
orifice and skimmer was set at +100-110 V, conditions under which mass peaks for apo-Mb dominate (29, 40). Under these conditions, however,
some minor contamination from intact holoprotein was detected.
Estimates of mass for the holo-Mbs were performed with a voltage
difference of 50-60 V to preserve the heme-protein complex and afford
charge state distributions largely for the holo-Mb form of the protein.
The ESI-MS system was mass-calibrated with solutions of CsI. Horse
heart Mb (predicted mass of apoprotein = 16,950 atomic mass units)
was employed as a standard to test the mass accuracy of the system
prior to use. Mass values were obtained by standard fitting analyses of
the various m/z distributions with "BioMultiView"
software (Applied Biosystems, Foster City, CA). Mass determinations
were performed for
2 independent protein preparations on different
days. Mass values reported here refer to the mean ± S.D. from
5 analyses.
Electronic Absorption Spectra
Electronic absorption spectra were obtained with a Cary 300 UV-visible spectrophotometer at 25 °C. Mb solutions were diluted into 50 mM sodium phosphate buffer (pH 7.4) prior to
analysis. Holo-Mb concentration was determined from the Soret maximum
(
408 nm ~ 188,000 M
1
cm
1), while apo-Mb concentration was
determined from the A280 nm (
280 nm ~ 16,000 M
1
cm
1). For the analyses of NO complexes,
various Mb solutions were first exposed to NO using one of methods
1-3. The reaction mixture was then diluted into the appropriate
degassed buffer, and the spectrum was recorded. By performing the
nitrosation reactions in 2 mM phosphate buffer (pH 7.4) and
subsequently diluting into 50 mM phosphate buffer (pH 7.4),
the yields of the respective S-nitrosothiol were optimized.
Under these conditions, steady state levels of
S-nitrosothiols were monitored in the various reaction
mixtures. In some experiments, wild-type human Mb was incubated with
iodoacetamide (thiol-blocking reagent:protein = 10 mol/mol) to
block the free thiol prior to reaction with NO. Carboxymethylation of
the protein thiol was confirmed by determining residual thiol content
before and after iodoacetamide treatment using DTP (molar ratio of
DTP:protein = 10 mol/mol) and monitoring the reaction at
A325 nm (29, 41). Concentrations of free
reduced thiol were estimated by comparison with GSH standards. Consistent with our previous report (29), modification with iodoacetamide decreased the free thiol in wild-type human Mb by >85%
(data not shown).
FTIR Measurements
FTIR spectra were recorded with a Perkin-Elmer System 2000 FTIR
spectrometer equipped with a liquid nitrogen-cooled MCT detector. The
IR cell consists of two CaF2 windows and a Teflon spacer
(25 µm). The temperature of the sample was maintained at 20 °C
with a water-jacketed cell holder and IR cells that have large contact surface to allow efficient thermal equilibration (Specac, Inc.). In
addition, the sample compartment was purged continuously with dry
nitrogen gas to minimize vapor absorption. Spectra were collected at an
OPD velocity of 2 cm/s on both sides of the center burst and averaged
over 500 scans. An apodization function was used to truncate the
spectra, and the spectral resolution was 2 cm
1. Background absorption from the empty
cavity was recorded under conditions identical to those used for the
protein samples. As a result of the weak S-H stretching vibration for
protein sulfhydryls in solution, relatively high concentrations of
holo- or apoprotein (5-6 mM) were necessary to obtain
useful data above the FTIR background for water.
Statistical Analyses
Statistical analyses were performed with Student's t
test available in Excel (Microsoft), and significant difference was
accepted at p < 0.05.
 |
RESULTS |
Electronic Spectroscopy of Myoglobins following Exposure to
NO--
Spectroscopic characteristics of a variety of metmyoglobins
and their corresponding ferrous nitrosyl-heme complexes are collected in Table I. The ferric form of horse
heart Mb, wild-type human Mb, and the C110A variant of human Mb each
exhibited similar Soret and visible maxima, as expected from the
similarity of the amino acid sequences for all mammalian Mb at the heme
binding pocket (28, 42). In addition, each ferric protein exhibited a
ASoret/A280 nm ratio > 5, consistent with the high purity of these preparations (not shown). In the absence of dioxygen, both the ferrous form of
wild-type human Mb and the C110A variant of human Mb readily reacted
with either authentic NO gas (method 2) or NO derived from the
decomposition of DeaNO (method 3) to yield ferrous nitrosyl-heme complexes of the corresponding protein (Table I). Visible bands measured for solutions of the various human nitrosyl Mb complexes were
similar to each other and distinct from those for the corresponding metmyoglobin. Furthermore, the visible bands for NO complexes of both
wild-type human Mb and the C110A variant were nearly identical to those
that are characteristic for the ferrous NO complex of horse heart Mb
(Table I). Importantly, no significant increase in absorbance was
detected between 330 and 350 nm for the ferrous nitrosyl-heme complexes
of the various Mb proteins (Table I).
EPR of Myoglobins following Exposure to NO--
To verify
the formation of ferrous heme-NO complexes of the various Mb proteins,
EPR spectra were obtained at 77 K for samples from the reaction of
authentic NO gas (NO:protein ~ 4 mol/mol) and the ferrous form
of both horse heart Mb and wild-type human Mb performed under anaerobic
conditions (Fig. 1). Consistent with the
electronic spectra obtained for corresponding reaction mixtures, treatment of either ferrous horse heart Mb or ferrous wild-type human
Mb with NO gas in the absence of dioxygen (method 2) resulted in a
broad EPR absorption in the g ~ 2 region (Fig. 1,
A and D). Each EPR signal exhibited
characteristic components centered at g1 ~ 2.08, g2 ~ 2.01, and g3 ~ 1.98 that have been assigned previously as arising from the
nitrosyl-heme complex of Mb (18, 19). Therefore, the EPR signals
detected here were assigned to the ferrous nitrosyl-heme complex for
the horse and wild-type human Mb proteins, respectively. Similarly,
reaction of the ferrous form of either horse heart Mb or wild-type
human Mb with NO (from reaction with DeaNO, method 3) also gave the
corresponding ferrous nitrosyl-Mb (Fig. 1, B and
E), indicating that NO derived from the decomposition of the
DeaNO behaved in identical fashion to NO gas. Thus, the EPR data
obtained from the reaction of various Mb with NO were in complete
agreement with the optical data on the corresponding samples and
confirmed the formation of a nitrosyl-heme complex in wild-type human
Mb under anaerobic reaction conditions. Consecutive EPR spectra (77 K)
measured for up to 30 min using the same solution of nitrosyl-Mb
remained unchanged, consistent with the high affinity that NO shows for
heme iron (dissociation constant Kd ~ 10
5 M) (13) (data not shown).

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Fig. 1.
EPR analyses of heme iron-nitroso complexes
of horse heart- and wild-type human Mb. Ferrous nitrosyl complexes
of horse heart Mb (spectra A-C) and human
wild-type Mb (spectra D-F) were prepared from
their respective proteins (~0.5 mM protein in 2 mM phosphate buffer, pH 7.4) using either method 2 (A and D) or method 3 (B and
E) for anaerobic NO generation. After formation of the
nitrosyl-Mb complex, a sample of reaction mixture was removed for EPR
analyses at 77 K (see "Experimental Procedures"). In the presence
of air, neither ferrous horse heart Mb (C) or ferrous
wild-type human Mb (F) afforded EPR signals when exposed to
aerobic NO generated by method 3. EPR parameters were as follows:
microwave power, 5 milliwatts; modulation frequency, 100 kHz; and
modulation amplitude, 0.4 millitesla. Spectra are an average of five
scans with sweep time of 84 s. The data represent three
independent analyses using different Mb preparations. mT,
milliteslas.
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In contrast to the relative ease of formation of ferrous nitrosyl-Mb
complexes in the absence of dioxygen, aerobic treatment of oxy-Mb
solutions (horse heart or wild-type human Mb) with authentic NO gas
(method 1, ~4-fold excess) resulted in the conversion of oxy-Mb to
metmyoglobin as judged by characteristic changes to the electronic
absorbance spectra for these solutions (data not shown). Similarly,
addition of a relatively low mole excess of NO to oxy-Mb
(NO:protein ~ 2 mol/mol, method 3) also resulted in the
oxidation of oxy-Mb to metmyoglobin (data not shown). Nitrosyl-heme was
not detected by EPR spectroscopy upon aerobic exposure of either horse
heart or wild-type human metmyoglobin to NO generated by method 2 (data
not shown) or method 3 (Fig. 1, C and F). This latter observation contrasts with reports of Mb-NO formation in reactions of ferric myoglobin with NO under low oxygen tension (43,
44).
Characterization of S-Nitrosated Wild-type Human Mb--
The
results above suggested that aerobic treatment of wild-type human
oxy-Mb with NO yields only metmyoglobin without detectable generation
of nitrosyl-Mb as judged by optical absorbance analyses. Additionally,
aerobic reaction of metmyoglobin and NO (generated by method 2 or 3)
did not yield nitrosyl-Mb as monitored directly by EPR. Therefore, we
next investigated whether the apoprotein of wild-type human
metmyoglobin was modified by exposure to NO in the presence of
dioxygen. Treatment of ferric wild-type human Mb and NO (method 3) at
mole ratios of NO:protein = 5 mol/mol resulted in an increase in
A330-350 nm as well as an increase in the
Soret band intensity (Fig. 2 and Table
I). As the NO-treated protein showed changes in absorbance over a relatively wide range of the absorbance envelope, we next investigated whether NO treatment altered the visible region (450-700 nm) of the Mb
solution spectrum. Importantly, no significant change was detected over
A450-700 nm as judged from the treatment of
relatively concentrated solutions of wild-type human holo-Mb with NO
under identical conditions (Fig. 2A, inset). In
contrast to the absorbance increase in the region 330-350 nm detected
in reaction mixtures of wild-type-human Mb and NO, solutions of the ferric C110A variant of human Mb treated with NO afforded no increased absorbance in this region (Fig. 2B), nor were there
observable changes between 450 and 700 nm (Fig. 2B,
inset). Thus, these data support the conclusion that the
increase in absorbance between 330 and 350 nm that results from the
aerobic treatment of wild-type human metmyoglobin with NO may be
derived from either direct modification of the protein or the overlap
of multiple absorbing species.

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Fig. 2.
Changes to the optical absorbance spectrum of
wild-type human Mb upon addition of NO. A solution of
either wild-type human metMb (panel A) or C110A variant of
human metMb (panel B) (each 5 µM protein in 2 mM phosphate buffer, pH 7.4) was analyzed before
(solid line) and after (dashed
line) exposure to NO (NO:protein = 5 mol/mol) generated
by method 3. Note the increase in intensity for both the Soret band
(A409 nm) and the region
A330-350 nm. Insets to panels
A and B show the respective visible regions
(A450-700 nm) of concentrated solutions of
either wild-type or C110A variant human Mb (each protein ~ 50 µM, NO:protein = 5 mol/mol) analyzed before
(solid line) and after (dashed
line) exposure to NO generated by method 3. The data
represent three independent analyses using different preparations of Mb
proteins.
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To evaluate the possibility of multiple species, the electronic
absorbance spectrum of the reaction mixture was measured before and
after purification by gel filtration (2x PD-10 column). Importantly, the A408 nm/A350 nm ratio was virtually
unchanged by this treatment, consistent with the notion that
low-molecular weight contaminants did not contribute to the detectable
changes in absorbance and that the protein was somehow modified by
reaction with NO in the presence of air. The yield of adduct increased significantly above that for the corresponding control
(i.e., addition of carefully matched volumes of 1 mM NaOH) with increasing concentration of NO
(NO:protein = 0-5 mol/mol), as judged by an NO-dependent increase in A350 nm (Fig.
3A). At NO:protein ratios >5 mol/mol, no
further increase in A350 nm was detected at least up to
NO:protein ratios
20 mol/mol (Fig. 3A). In contrast, addition
of chemically generated NO (Method 3) to the C110A variant of human Mb over the same range of NO:protein ratios resulted in no
significant change in A350 nm after removal of
low-molecular weight contaminants by gel filtration (Fig.
3A).

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Fig. 3.
Steady state measurements of changes to the
optical absorbance measured at 350 nm as a function of NO
concentration. Panel A, the change in optical
absorbance for solutions of metMb (each 5 µM protein in
50 mM phosphate buffer, pH 7.4) were measured at
A350 nm over a range of increasing NO:protein
(method 3) for wild-type human Mb (filled
squares), C110A variant of human Mb (filled
circles), and vehicle (1 mM NaOH) alone added to
wild-type human Mb (filled triangles) as a
control. Panel B shows the changes in
A350 nm for a solution of wild-type human Mb
treated with NO (NO:protein ~ 5 mol/mol) in 2 mM
(open squares), 20 mM
(open circles), and 50 mM phosphate
buffer (open triangles), respectively. Prior to
analyses, proteins were diluted into 50 mM phosphate
buffer, pH 7.4. Data represent mean ± S.D. of three independent
analyses using different Mb preparations. The asterisk
indicates significant difference from corresponding absorbance values
for both the control and C110A data (p < 0.05).
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The increase in A330-350 nm upon addition of
NO to wild-type human metmyoglobin is similar to that observed for S-nitrosoglutathione (GS-NO) obtained from
reaction of reduced GSH and NO generated from either method 1 or method
3 (Table I), or by comparison with data previously reported (36, 45).
The relatively weak absorbance at ~544 nm (
< 50 M
1 cm
1)
also associated with GS-NO formation (36, 45) was not
observed in the corresponding reactions of NO and wild-type human Mb
owing to the complication of overlap with the visible bands of the
protein. Despite the inability to detect the weak absorption at
540-550 nm, we tentatively concluded that the aerobic reaction of
human metmyoglobin with NO results in S-nitrosation of the
human protein at Cys110. Similar to the case for
GS-NO (45), the yield of S-nitrosated human Mb
was dependent on the concentration of phosphate employed in the
reaction mixture, as reflected by the decrease in
A350 nm with increasing phosphate over the
range 2-50 mM (Fig. 3B). Under physiological
conditions (50 mM phosphate, pH 7.4), the extent of
S-nitrosation was ~40-45% of that obtained at the lowest
phosphate concentration employed here (i.e., 2 mM phosphate). Importantly, DeaNO solutions (
1
mM) did not exhibit any absorbance over the range 300-700
nm when added to the buffer (2, 20, or 50 mM phosphate, pH
7.4) alone to act as control (data not shown).
To eliminate interference from the delta, Soret, and visible bands of
the holoprotein, we next investigated the electronic spectrum of
apoprotein prepared from wild-type human Mb and the C110A variant
before and after treatment with NO (method 1). Upon treatment of the
wild-type human apo-Mb with NO (NO:protein = 2 mol/mol), the
electronic absorbance spectrum of the reaction mixture exhibited new,
broad maxima at 333 and 545 nm (see filled lines
in Figs. 4, A and
B, and respective insets). The broadened maxima
at 333 nm in reaction mixtures of apo-Mb and NO may explain the wide
range increase in absorbance detected at 333-350 and 409 nm for
corresponding reactions of NO and holo-Mb (see above). These maxima
were not observed prior to NO-treatment (data not shown) and are
consistent with S-nitrosation of the wild-type human apo-Mb.
By contrast, treatment of either apo-Mb from the C110A variant or
wild-type human apo-Mb, previously modified with iodoacetamide,
resulted in a significant decrease at 333 nm and near elimination of
the broad band at 545 nm (dashed lines in Fig. 4
(A and B) and respective insets).
Together, these data strongly support the argument that
S-nitrosation occurs at Cys110 of wild-type
human Mb. In the absence of the overlapping Soret band, the
A333 nm/A545 nm ratio
obtained for the S-nitrosated apo-Mb was 81 ± 5 (mean ± S.D., n = 3). The value of this ratio is
similar to that (
335 nm (3869 M
1
cm
1)/
545 nm (47 M
1
cm
1) ~ 82) reported (36) for
S-nitrosated bovine serum albumin, another protein with a
single reactive sulfhydryl (29, 46, 47) that is readily
S-nitrosated by aerobic addition of NO (36). Molar
absorptivity values of
333 nm = 3667 ± 90 and
545 nm = 44 ± 3 M
1 cm
1
(mean ± S.D.) were obtained by plotting the apomyoglobin protein dependent change in A333 nm (e.g.
Fig. 4C) and A545 nm (data not
shown), respectively. Notably, employing
333 nm to the
relative increase at A330-350 nm observed for
solutions of untreated versus NO-treated holo-Mb (data taken from Fig. 2) yields ~1-1.2 NO/human Mb mol/mol, consistent with stoichiometric conversion to the NO adduct.

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Fig. 4.
Optical spectra from solutions of both
wild-type and C110A variant of human apo-myoglobin after NO
treatment. The change in A250-700 nm for
solutions of apo-Mb (2 µM protein, 50 mM
phosphate buffer, pH 7.4) after exposure to ~2 mol eq of NO (method
1). Panel A, spectra correspond to NO-treated wild-type
human apo-Mb (solid line) and NO-treated
apoprotein from the C110A variant (dashed line).
Panel B shows NO-treated wild-type apo-Mb before
(solid line) and after treatment with
iodoacetamide (dashed line). Insets to
panels A and B show visible electronic spectra
(500-600 nm) from solutions of corresponding Mb at 50 µM
protein concentration. Panel C shows the effect of
increasing apo-Mb concentration (~0-80 µM) on
formation of S-nitroso apo-Mb (NO:apo-Mb~2 mol/mol).
Inset to panel C shows linear relationship
between [apo-Mb] and
A333 nm: 333 nm is determined as
the slope of the line. The data represent three independent analyses
using different Mb preparations.
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FTIR Analyses of the Reaction Mixture from Wild-type Human
Holo-Mb and NO--
As the evidence from electronic absorbance spectra
from reaction mixtures of wild-type human Mb and NO supported the
formation of S-nitrosated wild-type human Mb, we sought to
confirm that NO reacted specifically with the sulfhydryl group at
Cys110. The S-H stretching vibration of the sulfhydryl(s)
in human hemoglobin is readily detected by IR spectroscopy in
concentrated solutions of the protein,
~ 2550-2580
cm
1 (48, 49). Thus, to confirm that the
reaction of aerated solutions of ferric wild-type human Mb and NO
(method 3) resulted in the modification of the protein at
Cys110, solutions of wild-type and C110A variant of Mb were
analyzed by FTIR spectroscopy before and after NO treatment (Fig.
5). In the absence of NO, aqueous
solutions of wild-type human holo-Mb exhibited a weak and broad
vibration at
~ 2552 cm
1 with
half-height peak width 
1/2 ~ 10-12
cm
1 (Fig. 5A). Concentrated
solutions of human apo-Mb also exhibited a weak vibration at
~ 2551 cm
1 with

1/2 ~ 8-10 cm
1
(Fig. 5B), while the C110A variant of human Mb did not (Fig. 5E). Solutions of reduced-GSH (100 mM) measured
under identical conditions as a control also exhibited a peak at
~ 2550 cm
1 (data not shown). The
band intensity for the wild-type human holo-Mb was severely reduced at
pH 10, above the pKa for a reduced thiol (49) (data
not shown). Together, these data support the assignment of an S-H
stretching band for Cys110 in human Mb at 2551-2552
cm
1. After treatment of either wild-type
human holo- or apo-Mb with NO (NO:protein = 5 mol/mol), the band
intensity for the S-H stretch was significantly diminished
(cf. Fig. 5, panels A and C
and panels B and D, for holo- and
apo-Mb, respectively). Overall, these data strongly support the
conclusion that the sulfhydryl at Cys110 of human Mb can be
detected directly by FTIR spectroscopy and that this residue is
specifically modified by aerobic exposure to NO independent of the
presence or absence of the heme prosthetic group.

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Fig. 5.
Infrared stretching vibration of the human Mb
sulfhydryl group before and after treatment with NO. Aerobic
solutions of pooled wild-type human holo-metMb or apo-Mb (each 5-6
mM protein in 2 mM phosphate buffer, pH 7.4)
were analyzed by FTIR before (panels A and B) and
after (panels C and D) exposure to NO
(NO:protein = 5 mol/mol) generated by method 3,
respectively. Panel E shows the corresponding IR region for
a concentrated solution (6 mM) of the C110A variant of
human Mb in the absence of NO treatment.
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Mass Analyses of Reactions of Wild-type Human Holo- and
Apo-Mb and NO--
To establish unambiguously that wild-type human Mb
was S-nitrosated by reaction with NO in a 1:1 stoichiometric
reaction, mass analyses were performed on holo- and apo-Mb in both the
presence and absence of NO. The mass of wild-type human Mb was
determined first by ESI-MS under ionization conditions that produce
mass-to-charge (m/z) distributions for holo-Mb (Fig.
6). Consistent with our previous study
(29), the mass determined for human holo-Mb was 17,670 ± 2 atomic
mass units, which compares well with a mass of 17,669 atomic mass units
predicted from the amino acid sequence (Fig. 6A,
inset). The small shoulder on each peak in the
m/z distribution of each reaction mixture results from
residual phosphate contamination in the samples. Importantly, the peaks
in the m/z distribution for the NO-treated sample increased
by 2-3 units (cf. Fig. 6, A and B) to
yield a mass for the nitrosated human Mb of 17,698 ± 5 atomic
mass units, i.e. an increase of 28 ± 4 atomic mass units (mean ± S.D., n = 5) (cf.
insets from Fig. 6, A and B). This
increase in mass of ~30 units corresponds to the addition of one
molecule of NO to each molecule of protein. No other significant adduct(s) was detected in the reaction mixture under conditions employed to optimize S-nitrosation of the wild-type human Mb
(i.e. nitrosation reactions performed in low phosphate, 2 mM phosphate, pH 7.4), indicating near complete conversion
to the S-nitrosated holoprotein.

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Fig. 6.
Mass analyses of the wild-type human holo-Mb
before and after treatment with NO. Solutions of wild-type human
holo-Mb (10 µM protein in 2 mM phosphate
buffer, pH 7.4) were analyzed by ESI-MS and the mass-to-charge
(m/z) ratios shown for the protein before (panel
A) and after (panel B) exposure to NO (NO:protein = 5 mol/mol) generated by method 3, respectively. The respective
insets show the deconvoluted mass value for the
corresponding m/z distribution. The data represent five
analyses using at least two different Mb preparations. amu,
atomic mass units.
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To determine whether the NO adduct was derived from sulfhydryl
S-NO rather than a heme-NO complex, mass spectrometry was
performed on preparations of wild-type human apo-Mb and
S-nitrosated wild-type human apo-Mb (Fig.
7) under conditions that favor the
detection of apo-Mb (39). The m/z distribution of authentic
human apo-Mb, accumulated with an expanded scale (1400
m/z
2200), exhibited relatively sharp peaks (Fig.
7A). The m/z distribution (m/z (charge in parentheses) = 1421 (12+), 1550 (11+), 1705 (10+), 1894 (9+), and 2131 (8+)) yielded a mass of 17,053 ± 1 atomic mass units (Fig. 7A, inset). This mass compares well with
the predicted mass of 17,053 atomic mass units for wild-type human
apo-Mb. The m/z distribution of dilute samples from the
reaction of authentic apo-Mb and NO (method 1 or 3) were shifted 2-5
units compared with the untreated apo-Mb (m/z (charge in
parentheses) = 1424 (12+), 1553 (11+), 1708 (10+), 1899 (9+), and
2136 (8+)) and yielded a mass of 17,082 ± 3 atomic mass units.
Thus, the mass of the NO-modified apoprotein also corresponded to an
increase in protein mass of ~29 ± 4 (mean ± S.D.,
n = 5) relative to wild-type human apo-Mb
(cf. Fig. 7, A and B, and respective
insets), indicating that the apoprotein was readily
nitrosated in the absence of the heme prosthetic group. Finally, to
show unambiguously that the NO adduct results from sulfhydryl
(S-) rather than heme-nitrosation in the holoprotein,
samples of reaction mixture from holo-Mb and NO (method 3) at mole
ratios of NO:protein = 5 mol/mol, which showed a mass increase of
~30 atomic mass units, were adjusted to pH 2.5 with HCl so that heme
could be extracted from the protein (32). Analysis of the resulting
apoprotein fraction by ESI-MS yielded an m/z distribution
and corresponding mass value identical to that for authentic wild-type
human apo-Mb treated aerobically with NO (cf. Fig. 7,
B and C, and respective insets).
Together, these data strongly support the conclusion that the reaction
of ferric wild-type human Mb and NO in the presence of air yields S-nitrosated Mb.

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Fig. 7.
Mass analyses of authentic wild-type human
apo-Mb before and after treatment with NO. Solutions of authentic
wild-type human apo-Mb (~10 µM protein in 2 mM phosphate buffer) were analyzed by electrospray mass
spectrometry and the m/z ratios shown for the apoprotein
(panel A) before and (panel B) after exposure to
NO (NO:protein = 5 mol/mol) generated by method 1, respectively.
Panel C shows the mass analyses performed on apoprotein
derived from samples of holo-Mb after treatment with NO and removal of
heme. Respective insets show the deconvoluted mass values
for corresponding m/z distributions. The data represent five
analyses using at least two different Mb preparations. amu,
atomic mass units.
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Other minor peaks were detected in the mass analyses of the reaction
mixtures (Fig. 7, B and C, diamonds).
The minor peaks are attributed to the incomplete conversion of holo-Mb
to apo-Mb. As these peaks were resolved with low signal to noise
ratios, they were not used to characterize the NO adduct. Mass peaks
obtained from aerobic solutions of wild-type apo-Mb treated with NO
were broader than those for the corresponding untreated apoprotein (Fig. 7, compare A with B and C). This
peak broadening did not likely result from sample inhomogeneity but
rather from accumulation of mass data with a lower resolution setting
for NO-treated versus untreated apo-Mb, respectively. This
change in resolution parameter was necessitated by the consistently
lower peak intensities detected in the dilute samples of reaction
mixture used for mass analyses of S-nitrosated apo-Mb.
Solutions of S-nitrosated apo-Mb were not concentrated prior
to mass analyses to minimize the potential for decomposition of any
S-nitrosothiol protein subsequent to gel filtration.
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DISCUSSION |
The reactions of NO with heme iron, non-heme iron, and sulfhydryl
groups are thought to be important mechanisms for regulating NO
in vivo (see Introduction). Human Mb contains both a heme
prosthetic group and a protein sulfhydryl at Cys110.
Interestingly, human Mb differs from all other known Mbs by the
presence of the sulfhydryl at Cys110. The sulfhydryl group
in human Mb significantly modifies its reaction with hydrogen peroxide
(29) and can be reasonably expected to modify the reaction of the
protein with other reactive biological agents such as NO. In addition
to the reaction of NO and ferrous Mb to yield heme-NO complexes (18,
19), and oxy-Mb induced oxidation of NO to
NO3
(26), wild-type human Mb is now
shown for the first time to react aerobically with NO in
vitro to yield a derivative that is S-nitrosated at
Cys110. The evidence we have obtained by combining FTIR,
mass spectrometry, and optical absorbance spectroscopy together with
EPR analyses of aerobic reaction mixtures of wild-type human Mb and NO
strongly support this conclusion. Thus, it is conceivable that
wild-type human Mb interacts with NO by a variety of mechanisms (and
under a variety of conditions) to yield both heme iron and
S-nitrosated protein and possibly modulate the biological
activity of NO and/or the lifetime of NO under physiological conditions.
Glutathione is a widely distributed low molecular weight thiol that is
present in high concentration (0.5-10 mM) inside cells (50). Kinetic analyses (45, 51, 52) of the aerobic nitrosation reaction
of reduced GSH and NO have proven that the nitrosating reagent is not
NO itself but rather a higher oxidation state of the gas. That
GS-NO formation requires the presence of NO and oxygen
supports this conclusion (45, 51). Furthermore, evidence that the
rate-limiting process in the formation of GS-NO is actually the reaction of NO and dioxygen to yield N2O3
supports the conclusion that the likely nitrosylating reagent is
N2O3 rather than NO (45). That anaerobic
treatment of ferrous Mb with NO gave only the nitrosyl-Mb, and that
S-nitrosation of the ferric- or apo-Mb required the presence of both NO and dioxygen, is completely consistent with the proposal that higher oxides of NO are responsible for the
S-nitrosation of the human protein. The dependence of the
overall yield of S-nitrosated Mb on the concentration of
phosphate in the reaction buffer is also consistent with that
previously reported for S-nitrosothiols (45).
S-Nitrosation of proteins, however, typically occurs
with a rate constant ~10-fold lower than that for low molecular
weight reduced thiols (51). Therefore, to compete with the
S-nitrosation of reduced GSH and other low molecular weight
reduced thiols, intracellular concentrations of protein sulfhydryl must
be at least an order of magnitude greater than the corresponding GSH concentration. Since human Mb possesses only a single sulfhydryl per
molecule of protein, the role of S-nitrosated human Mb may be limited to human cells where the concentration of Mb is sufficiently high that the reaction of NO and the protein sulfhydryl is favorable. Interestingly, Mb is reportedly present in relatively high
concentration in human heart and skeletal tissue (2.1-6 mg/g of wet
tissue (Refs. 20-22)). Although Mb is known to be present in human
smooth muscle (23), the concentration of the protein in these cells is
not known.
Both NO and N2O3 are reported (26) to be
capable of oxidizing horse heart oxy-Mb to produce metmyoglobin and
nitrate in both the presence and absence of dioxygen. Our observation
that low molar excess of NO (NO:protein
5 mol/mol) readily
oxidized wild-type human oxy-Mb to metmyoglobin in the presence of
dioxygen is consistent with this report. Under physiological
conditions, a majority of Mb is both reduced and oxygenated. Thus, it
may be argued that the oxidation of human oxy-Mb to metmyoglobin by NO,
NO2, or N2O3 may occur in
preference to S-nitrosation of the protein. Despite the
rapid rate of reaction of NO with oxy-Mb (k ~ 4 × 107 M
1
s
1 (Ref. 26)),
N2O3-mediated oxidation of oxy-Mb is known to
be relatively slow (k ~ 4 × 102
M
1 s
1
(Ref. 26)). Furthermore, N2O3 (generated from
the relatively rapid reaction of NO and dioxygen (k ~ 2 × 106 (Ref. 45)) reacts with reduced thiols with
rate constant k ~ 2 × 105-2 × 106 M
1
s
1 (45, 51). Together, these data suggest
that formation of an S-nitrosothiol on human Mb may compete
with the oxidation of oxy-Mb under conditions that favor the oxidation
of NO to N2O3. Importantly, a similar argument
can be made for the reaction of human oxy-hemoglobin with NO; however,
we note that at present in vivo S-nitrosation is regarded by
some (11, 52-54) but not others (55) to be a physiologically relevant
reaction of hemoglobin. That wild-type human metmyoglobin may also be
S-nitrosated in the presence of low molar excess of NO
affords the intriguing possibility that this protein might regulate the
availability of NO by several modes. Thus, human Mb may oxidize excess
NO to nitrate while also preserving NO bio-activity through formation of the S-nitrosated protein.
As discussed in the Introduction, NO is responsible for a variety of
physiological processes. Although S-nitrosothiols are stable
under physiological conditions, they can decompose to yield the
corresponding disulfide and NO (12) and thereby participate in the
regulation of NO release and activity. Furthermore, the decomposition
of S-nitrosothiols can be enhanced by increased temperature
(12), photolysis (37, 56, 57), or reaction with transition metals (37).
With our demonstration that human Mb can also yield an
S-nitrosated protein in the presence of NO and dioxygen,
this protein joins the S-nitrosoproteins,
S-nitrosohemoglobin and S-nitrosoalbumin as a
well characterized sulfhydryl-containing protein that may exhibit
potent NO-mediated biological activity in vivo (36).
Overall, combined data from FTIR spectroscopy, EPR spectroscopy, mass
spectrometry, and electronic absorption spectroscopy strongly support
the conclusion that both heme or sulfhydryl complexes of NO can form in
human Mb. Whether S-nitrosated wild-type human Mb is capable
of releasing biologically active NO is yet to be determined. However,
if NO release from S-nitrosated human Mb can occur, then
this form of the protein could represent a previously unrecognized pool
of biologically active NO that may participate in the regulation of
intracellular function of human tissues that contain Mb in abundance,
e.g. human smooth muscle.