From the Department of Biochemistry and Molecular
Biology, Colorado State University, Fort Collins, Colorado 80523 and
the
Laboratory of Persistent Viral Diseases, Rocky Mountain
Laboratories, NIAID, National Institutes of Health,
Hamilton, Montana 59840
Received for publication, July 24, 2000, and in revised form, January 25, 2001
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ABSTRACT |
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Noncovalent bonding interactions of nitric oxide
(NO) with human serum albumin (HSA), human hemoglobin A, bovine
myoglobin, and bovine cytochrome c oxidase (CcO) have been
explored. The anesthetic nitrous oxide (NNO) occupies multiple sites
within each protein, but does not bind to heme iron. Infrared (IR)
spectra of NNO molecules sequestered within albumin, with NO present, support the binding of NO and NNO to the same sites with comparable affinities. Perturbations of IR spectra of the Cys34 thiol
of HSA indicate NO, NNO, halothane, and chloroform can induce similar
changes in protein structure. Experiments evaluating the relative
affinities of binding of NO and carbon monoxide (CO) to iron(II) sites
of the hemeproteins led to evidence of NO binding to noniron, nonsulfur
sites as well. With HbA, IR spectra of cysteine thiols and/or the
iron(II) N-O stretching region denote changes in protein structure due
to NO, NNO, or CO occupying noniron sites with an order of decreasing
affinities of NO > NNO > CO. Loss of NO from some, not all,
noniron sites in hemeproteins is very slow (t1/2 ~ hours). These findings provide examples in which NO and anesthetics
alter the structure and properties of protein similarly, and support
the hypothesis that some physiological effects of NO (and possibly CO)
result from anesthetic-like noncovalent bonding to sites within protein or other tissue components. Such bonding may be involved in mechanisms for control of oxygen transport, mitochondrial respiration, and activation of soluble guanylate cyclase by NO.
The mechanisms of anesthesia and the regulatory functions of
nitric oxide (NO)1 remain far
from clear. Potential roles of anesthetic-protein interactions in
anesthesia have become of increasing interest (1-5) as have the
interactions of NO with proteins (6-10). Volatile anesthetics occupy
hydrophobic sites within protein without formation of covalent bonds
with amino acid residues. In contrast, the reactions between NO and
receptor sites in proteins that have been studied extensively involve
covalent bond formation, as in metal nitrosyl and
S-nitrosothiol (-SNO) derivatives (7-10). Nitrosations
(i.e. attack of protein residues by NO+) on exposure of
proteins to NO with a suitable oxidant present are the only reactions
of NO that have been discussed other than the binding of NO to metal sites. Evidence of NO occupying sites within proteins as an uncharged diatomic free radical by means of anesthetic-like noncovalent bonding
has not been reported.
The binding of volatile anesthetics to serum albumins and the effects
of such binding on protein structure are considered in several recent
reports. Specific, saturable binding of halothane and other anesthetics
to bovine serum albumin and anesthetic-induced alterations in
the structure or carrier function of this protein have been shown
(11-16). The anesthetic nitrous oxide (NNO) was detected at sites
within human serum albumin (HSA) (5). Infrared (IR) spectra of bound
NNO molecules demonstrated sites of two types. The exposure of HSA to
NNO enhanced absorbance in the S-H stretching region of the IR spectrum
giving evidence of an NNO-induced change in protein conformation near
Cys34 (5). In 1992, NO was proposed to circulate in
mammalian plasma primarily in association with serum albumin, due to
formation of the S-nitrosothiol derivative (17). However,
the similarities in the structure and physical properties of NNO and NO
suggest the possibility that NO may also interact at some sites within albumin in the manner shown for NNO.
IR evidence of NNO molecules at multiple sites within hemoglobin,
myoglobin, and cytochrome c oxidase (CcO) has also been obtained (5, 18). NNO neither serves as a ligand to heme iron nor
reacts with thiols. The ability of NNO to alter hemeprotein structure
and function was shown by shifts in the IR spectra of cysteine thiols
of HbA (5) and by partial and reversible inhibitions of CcO (18). The
well established ability of NO to ligate to heme iron in HbA, Mb, and
CcO, and to copper B in CcO (19, 20), led to the assumption of metal
binding in proposed mechanisms for physiologically important reactions
of NO with hemeproteins. Reactions of NO with HbA that result in
S-nitrosothiol derivative formation have also received much
attention (8). However, alternative mechanisms whereby NO alters
hemeprotein structure and function by noncovalent anesthetic-like
bonding to sites that involve neither metal nor cysteine sulfur remain unexplored.
Much interest in possible physiological roles of carbon monoxide (CO),
including a messenger role in the nervous system and the activation of
soluble guanylate cyclase, has developed recently (21-23). The
biochemical mechanisms related to these effects of CO have not been
elucidated, but those proposed thus far have considered the ability of
CO to bind to iron(II) of hemeproteins. The occupancy of nonmetal
protein sites by CO, as with NO, remains unstudied.
We report here experimental results that indicate NO can reversibly
occupy sites within HSA and hemeproteins in the manner of NNO (and
other anesthetics) and, in so doing, can induce changes in the
properties and structures of the proteins. These findings provide
support for considering NO-protein interactions of this type as
potentially important in at least some of the physiological roles of
NO. Limited evidence of the ability of CO to occupy noniron as well as
iron(II) sites in hemoglobin is also presented.
Materials--
Fatty acid- and globulin-free human serum albumin
(99% from Sigma) was used as received. Recrystallized heart cytochrome
c oxidase and myoglobin were isolated and purified from
fresh bovine heart as was hemoglobin A from human blood via methods
described earlier (24-26). Nitric oxide was obtained as
15N16O (99% from Cambridge Isotope
Laboratories) and as 5% 14N16O, 95%
N2 (technical grade, Air Products and Chemicals). Both NO
gases were treated with 1 M aqueous KOH to remove
NO2 (27). Other materials included NNO (99% from General
Air Services and Supply), CO (99.5% from General Air Services and
Supply), N2 (99.9% from General Air Services and Supply),
halothane (99.99% from Halocarbon Laboratories), and chloroform (99%
from Fisher).
Albumin Studies--
Solutions of HSA in 200 mM
Tris-Cl buffer, pH 7.2, were made anaerobic by evacuation and flushing
with nitrogen. HSA solutions were exposed to varying amounts of
15N16O, NNO, and/or nitrogen to give a total
pressure of 1 atm. Halothane and chloroform were added to HSA solutions
as liquids. In each case the treated solution was allowed to stand for
at least 1 h prior to recording IR spectra. All operations were
carried out at 20 °C in both albumin and hemeprotein studies.
Hemeprotein Studies--
Hemeprotein solutions were prepared in
200 mM sodium phosphate buffer, pH 7.2, under strictly
anaerobic conditions as described earlier (19, 28). Several methods
were used to detect the relative affinities of the proteins for NO and
CO and the slow release of NO. In one method a solution (2.5 ml) of
deoxy-Mb (or deoxy-Hb), 5 µM in heme and CcO (unliganded
and fully-reduced) at 10 µM in heme A was prepared in a
sealed 1-cm path length spectrophotometer cell. A NO/N2 gas
mixture or CO was gradually added incrementally via a gas-tight syringe
under atmospheric pressure. The visible/Soret spectrum was measured
after each addition and second-derivative analysis of the Soret
spectrum carried out. In a second procedure, CcONO was prepared
initially from a solution (2.5 ml) of CcO (10 µM in heme
A) by slowly adding, at atmospheric pressure, an amount of
NO/N2 (~0.3 ml) that was just sufficient for the complete
ligation of heme a3 iron(II) with NO as observed in
visible/Soret spectra. N2 was then passed over the solution
for 15 min to remove the dissolved free NO, as well as the NO present
above the solution, without decreasing metal-bound NO. A solution of
deoxy-Mb or deoxy-Hb at 8-10 mM in heme was introduced
into the CcONO solution with a gas-tight syringe to give a solution
containing 5 µM Mb or Hb. Changes in visible/Soret
spectra were monitored over 24 h. The Mb or Hb level was then
increased to 15 µM and the visible/Soret spectra followed
for another 24 h. An analogous procedure was used to measure the
relative affinities of these hemeproteins for CO. In a third method the
order of protein addition was reversed. A solution (2.5 ml) of 5 µM deoxy-Mb or deoxy-Hb was exposed to 0.3 ml of
NO/N2, which was just sufficient to saturate the heme irons
with NO as shown in visible/Soret spectra, followed by exposure to
N2 for 15 min. Changes in visible/Soret spectra were
monitored for 48 h after the addition of 10 µM CcO.
Spectra were observed for an additional 24 h after raising the Mb
or Hb levels to 15 µM.
To evaluate the number of nonmetal sites for NO in Hb, a solution (2.5 ml) of 5 µM deoxy-Hb was exposed to 0.3 ml of
NO/N2. This amount of NO was sufficient to saturate
essentially all the heme iron sites with NO, but not necessarily all
the noniron sites. After flushing with N2 for 15 min, an
additional 5 µM deoxy-Hb was added. Three additional 5 µM increases in Hb concentration were made after 4-, 5-, and 12-h intervals, respectively. Visible/Soret spectra were monitored throughout.
To evaluate the effectiveness of N2 flushing for removal of
unbound NO, 0.3 ml of NO/N2 was introduced into 2.5 ml of
anaerobic buffer. After passing N2 above the solution for
15 min, deoxy-Mb or deoxy-Hb was added to a level of 5 µM. Visible/Soret spectra of the solution were monitored
for 48 h to ascertain if any MbNO or HbNO formed in the solution.
To evaluate the effects of NNO, CO, N2, and excess NO on
the IR spectra of solutions of hemeprotein nitrosyls, concentrated protein solutions were required. Solutions of oxy-Hb (9 mM
in heme) and oxy-Mb (9 mM) were made anaerobic and fully
reduced by repeated evacuations and exposures to N2 for 30 min, followed by treatment with sodium dithionite in slight excess.
These solutions were exposed to increasing levels of NO/N2
in small increments. After each treatment with NO, a small amount of
solution was placed in an N2-flushed IR cell in which both
the IR spectrum from 4000 to 1000 cm Recording of Spectra--
The methods followed to record the IR
spectra were as described earlier (5, 10). IR spectra were recorded at
20 °C in cells with CaF2 windows with a PerkinElmer
Model 1800 FTIR spectrophotometer equipped with a
mercury/cadmium/tellurium detector and interfaced with a PerkinElmer
7700 data station. Path lengths were 100 µm for S-H spectra and 6 µm for NNO and NO spectra. For each spectrum a 1000-scan
interferogram was collected in single beam mode with a
2-cm Albumin Experiments
Perturbations of the SH-IR Spectrum of Cys34 by NO,
NNO, Halothane, and Chloroform--
The increases in absorbance near
2560 cm
Saturation of a solution of HSA with either halothane or chloroform
resulted in a S-H band (Fig. 2) that was
broader and at higher wavenumber (~2576 cm Perturbation of IR Spectra of NNO Molecules within HSA--
The
NNO-IR spectrum obtained when HSA was exposed only to NNO is shown in
Fig. 3A. As reported earlier
(5), curve-fitting reveals two major bands at 2220 cm
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and the
visible/Soret spectrum from 700 to 350 nm were recorded. The degree of
saturation of the heme iron(II) sites by NO was calculated from Soret
spectra. Exposure to NO was continued until all iron sites had bound
NO. Finally more NO, that represented 50% of the total NO already
added, was administered. IR spectra were also recorded following
exposures of the solutions to N2, NNO, and CO.
1 resolution and a 1-cm
1 interval.
Reference spectra were recorded under identical scan conditions. SH,
NNO, and NO IR spectra were obtained by subtracting a reference
solution spectrum from the sample spectrum followed by baseline
corrections in the regions from 2630 cm
1 to 2500 cm
1, 2260 cm
1 to 2180 cm
1,
and 1700 cm
1 to 1400 cm
1, respectively. To
obtain the spectra for the NNO molecules within protein the spectra for
NNO in buffer alone was subtracted from the NNO spectrum for the
protein solution (5). Curve fitting analysis was carried out using the
CURVEFIT function of Spectra Calc software (Galactic Industries Corp.).
Visible/Soret spectra were recorded with a Cary 2200 spectrophotometer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 in the IR spectrum of an anaerobic solution of
HSA at pH 7.2 that resulted from exposure of the solution to NNO or NO
are shown in Fig. 1. Curve-fitting of the
absorbance induced by NNO with 100% Gaussian profiles, as in spectrum
C, gives two bands: a major band at 2563 cm
1 with about
85% of total intensity and a minor band at 2579 cm
1.
Assignment of these bands to S-H stretch vibrations was discussed earlier (5, 19, 29-31). With NO, as in spectrum F, curve-fitting yields one band at 2563 cm
1, the same wavenumber as found
for the major band with NNO, and a second less intense band at 2548 cm
1. The insets of Fig. 1 show the dependence
of band intensities on partial pressures of NNO and NO. Raising the
partial pressure beyond 0.6 atm did not increase S-H band intensity
with either gas. One-half maximum absorbance was achieved at 0.35 atm
for NNO and 0.47 atm for NO.
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Fig. 1.
S-H infrared bands for HSA generated by NNO
and NO. An anaerobic solution of 2 mM HSA was exposed
to NNO or NO at the partial pressures indicated, and allowed to stand
for 1 h prior to recording the spectrum. Total pressure was
brought to 1 atm with N2. The reference solution used was 2 mM HSA in buffer. Dashed lines of C
and F represent bands of 100% Gaussian profile from
curve-fitting into two bands with maxima at 2563 cm 1 and
2579 cm
1 and widths at one-half maximum absorbance of 22 cm
1 and 13 cm
1, respectively, for
C, and maxima at 2563 cm
1 and 2548 cm
1 and widths at one-half maximum absorbance of 25 cm
1 and 22 cm
1, respectively, for F. Insets show plots of maximum absorbance versus
variations in NNO or NO partial pressure. One-half maximum absorbance
was achieved at 0.35 atm with NNO and 0.47 atm with NO.
1) than
occurred with either NNO or NO (Fig. 2). The spectra obtained with
NNO/halothane and NNO/chloroform mixtures reflected a combination of
the spectra exhibited by the individual anesthetics. Mixtures of NO and
NNO resulted in NO-type and NNO-type S-H bands with their relative
contributions varying as the ratio of their partial pressures are
changed (Fig. 2).
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Fig. 2.
S-H infrared bands for HSA generated by
mixtures of NNO with halothane, chloroform, and NO. Conditions as
in Fig. 1. A solution of HSA was treated as indicated. A,
exposure to 0.74 atm NNO, 0.26 atm N2. B,
saturated with chloroform (~62 mM). C,
solution B exposed to 0.74 atm NNO, 0.26 atm N2.
D, saturated with halothane (~17 mM).
E, solution D exposed to 0.74 atm NNO, 0.26 atm
N2. F, 1 atm NO. G, 0.8 atm NO, 0.2 atm NNO. H, 0.5 atm NO, 0.5 atm NNO. I, 0.2 atm
NO, 0.8 atm NNO. K, 1 atm NNO.
1
and 2225 cm
1 and minor "hot bands" at lower
wavenumbers. Spectra B, C, and D of Fig. 3 exhibit a reduction in the
intensity of the 2225 cm
1 band relative to the 2220 cm
1 band as the partial pressure of NNO relative to NO
decreases. Spectra A and B are nearly identical indicating that the NNO
sites are essentially as fully occupied by NNO at 0.8 atm NNO and 0.2 atm NO as when NNO is 1 atm with NO absent. However, spectra C and D
show that at lower NNO/NO pressure ratios, the intensity of the 2225 cm
1 band relative to the 2220 cm
1 band
decreases, as does the combined band intensity. Thus, at NO partial
pressures greater than 0.2 atm, occupancy by NNO at both NNO sites
decreases, but more so at the more polar 2225 cm
1 site.
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Fig. 3.
Perturbations of IR spectra of NNO molecules
within HSA by NO. Conditions as in Fig. 1. A solution of HSA was
exposed to gases as indicated. A, 1 atm NNO. B,
0.8 atm NNO, 0.2 atm NO. C, 0.5 atm NNO, 0.5 atm NO.
D, 0.2 atm NNO, 0.8 atm NO. Curve-fitting used 100%
Gaussian profiles.
Hemeprotein Experiments
Relative Affinities of Iron(II) Sites in Hb, Mb, and CcO for NO and
CO--
The visible/Soret spectra for unliganded fully reduced Hb, Mb,
and CcO and their iron(II) nitrosyl complexes (Fig.
4) served as reference spectra for
determining the binding of NO to heme iron(II) in these proteins. For
example, spectra of a solution of deoxy-Mb and unliganded fully reduced
CcO were recorded as the solution was exposed to increasing volumes of
a 5% NO, 95% N2 gas mixture (Fig.
5). With NO absent, the second-derivative minimum at 441 nm represents the combination of unresolved bands for
deoxy-Mb at 435 nm and unliganded CcO at 444 nm. Exposure to 0.2 ml of
gas mixture gave spectra consistent with only partial conversion of
deoxy-Mb to MbNO. Exposure to 0.4 ml resulted in second-derivative
minima at 421 and 444 nm, as expected for Mb being present as mainly
MbNO and CcO remaining mostly unliganded. Increasing the volume of gas
to 0.6 ml resulted in bands due to both MbNO and CcONO. The addition of
deoxy-Mb to a solution of CcONO resulted in the rapid transfer of
NO from heme a3 of CcONO to Mb heme iron(II) (Fig.
6B). Similar experiments with
CO demonstrated the greater affinity of Mb for CO (data not shown).
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Visible/Soret spectra of a solution of deoxy-Hb and unliganded CcO,
upon exposure to increasing volumes of NO, demonstrated that HbNO
formed first leaving CcO unliganded (data not shown). Similar
experiments with CO indicated HbCO formed first. Following the addition
of deoxy-Hb to a solution containing CcONO a slow transfer of NO from
heme a3 of CcONO to Hb occurred (Fig.
7, A-D). Experiments with
CcOCO and deoxy-Hb revealed a fast transfer of CO from CcOCO to Hb,
which was complete within 5 min.
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Discrimination between Hb and Mb as iron(II) nitrosyl or carbonyl
species was more readily achieved by use of ligand IR spectra than
visible/Soret spectra (19, 20). Both NO and CO were shown by N-O or C-O
stretching bands to bind preferentially to the iron(II) of Mb in an
anaerobic solution of deoxy-Mb and deoxy-Hb, equimolar in heme (data
not shown). However, based on C-O stretching band parameters (32), when
CO was added to an aerobic solution of oxy-Mb and oxy-Hb, equimolar in
heme, the order of affinities for CO was shown to be Hb subunits > Hb
subunits > Mb.
The visible/Soret spectrum of a solution of CcCCO or HbCO upon exposure to NO underwent immediate changes that were consistent with the conversion of the carbonyl species to the nitrosyl species. NO was less effective in displacing CO from MbCO. If a solution of MbCO had been prepared by exposing deoxy-Mb only to the amount of CO needed to completely saturate iron(II) sites, the displacement of CO by NO was much slower than in a similar experiment with HbCO. Furthermore, CO at high levels slowly displaced the heme-bound NO of MbNO, if the MbNO solution had been prepared with the minimum amount of NO needed to saturate all iron(II) sites with NO. Thus, the order of affinities of NO > CO is more pronounced for CcO and Hb than for Mb.
Evidence of Slow Dissociation of NO from Noniron Sites in CcO and Hb-- The visible/Soret spectrum 35 min after the addition of deoxy-Mb to a solution of CcONO (Fig. 6C), which indicated only MbNO and ligand-free CcO were present, changed upon standing. The spectrum recorded after 120 min (Fig. 6D) revealed that now all the heme a3 of CcO, as well as all Mb heme, contained iron(II)-bound NO. Analogous experiments, except for the substitution of Hb for Mb, resulted in similar, but slower, spectral changes (Fig. 7).
A solution (2.5 ml) of 5 µM deoxy-Hb was exposed to 0.3 ml of NO/N2 to saturate all heme iron(II) sites with NO and then surface-flushed with N2 for 15 min to completely remove all the NO in buffer. Following the addition of another 5 µM deoxy-Hb, the second-derivative Soret minimum at 433 nm due to deoxy-Hb decreased slowly as a minimum at 418 nm (HbNO) increased. After 3 h, only HbNO was present. Similar changes in spectra occurred over 4 h following the addition of a second 5 µM deoxy-Hb, and, over 5 h, after a third 5 µM addition of deoxy-Hb. However, no further spectral changes occurred over 24 h following a fourth addition of 5 µM deoxy-Hb. These findings support the slow release of NO from noniron sites in both CcO and Hb with the released NO subsequently binding to heme iron(II) of added Mb or Hb.
Perturbations of IR Spectra of MbNO and HbNO Solutions in the N-O
Stretching Region by NO, NNO, CO, and N2--
Fig.
8 shows IR spectra of a NO-saturated MbNO
solution with a CO-saturated solution of MbCO used as reference.
Curve-fitting of the asymmetric absorbance in Fig. 8A
yielded three bands with maxima at 1611.5 ± 0.1, 1603.5 ± 0.5, and 1595.3 ± 0.2 cm1, widths at one-half
maximum absorbance of 9.2 ± 0.4, 8.4 ± 0.4, and 9.0 ± 0.2 cm
1, and relative integrated areas of 15:4:1,
respectively. After N2-flushing, the spectrum obtained
(Fig. 8B) can be curve-fitted as only two bands with maxima
at 1611.8 ± 0.1 and 1604.8 ± 0.1 cm
1, widths
at one-half maximum absorbance of 8.4 ± 0.6 and 9.1 ± 0.3 cm
1, and relative integrated areas of 3:1, respectively.
Flushing of NO-saturated solutions of HbNO with N2 also
perturbs the IR spectra (Fig. 8, C and D).
Curve-fitting yielded two bands for the NO-saturated solution and three
bands for the N2-flushed solution (data not shown).
Asymmetry decreased on N2-flushing with Mb but increased
with Hb. Flushing with CO or NNO also affected the degree of asymmetry
of HbNO solutions (Fig. 8, D and E). These
asymmetry differences are reflected in the widths of absorbances
recorded at one-tenth maximum absorbance. Thus, flushing the solutions with NNO, CO, or N2 altered the recorded spectrum without
reducing the amount of NO bound to iron(II).
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Perturbations of Thiol IR Spectra of Hb by NO, NNO, CO, and
O2--
The S-H stretching bands due to 93,
112, and
104 cysteines were perturbed very little when a CO-saturated
solution of HbCO or a NO-saturated solution of HbNO was flushed with
N2 (Fig. 9, A-D).
Incubations of solutions of HbCO, HbNO, and HbO2 saturated with CO, NO, and O2, respectively, under a small volume of
NNO also had only small effects on the S-H bands (Fig. 9, E,
G, and I). However, incubations under a larger volume
of NNO resulted in substantial shifts in spectra for the
93 and
112 cysteines (Fig. 9, F, H, and K). NNO-IR
spectra recorded for solutions E to K of Fig. 8 (data not shown)
demonstrated that exposure to the larger volume of NNO resulted in a
5-fold larger concentration of NNO molecules at sites within protein
than when the smaller volume of NNO was used. Furthermore, the
solutions exposed to the larger volume of NNO exhibited a band near
2226 cm
1 (the band for NNO molecules at the most polar
sites) that was more intense than the bands for NNO at less polar
sites. The solutions of HbCO, HbNO, and HbO2 that had been
exposed to the larger volume of NNO, were each subsequently incubated
with excess CO, NO, or O2, respectively, for 30 min. In
each case, the NNO-IR spectrum demonstrated that the incubation reduced
the overall NNO band intensity by about 80%, with the greater loss at
the more polar sites. The preferential loss of NNO at the more polar
sites was greatest for incubation with NO, and least with
O2. Similar incubations with N2 reduced NNO
band intensities to the same extent at each site. The IR spectra of the
cysteine thiols of HbCO, HbNO, and HbO2 were perturbed by
exposures to NO, NNO, CO, and/or O2 without altering the
extent of ligand binding to heme iron.
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DISCUSSION |
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Albumin Studies--
The IR spectra reported here provide a means
for detecting alterations in HSA conformation in solution in areas of
the Cys34 thiol and NNO binding. HSA is a single
polypeptide with 585 residues containing 34 cysteines paired in
disulfide bridges and one free cysteine (Cys34) (33).
Crystal structure determinations reveal a heart-shaped molecule
containing three domains: I (residues 1-195), II (196), and III
(384), with similar three-dimensional structures (34, 35). Each
domain can be divided into two subdomains A and B that contain six and
four -helices, respectively. The subdomains are linked by an
extended loop. Cys34 is located in a short loop linking
helices h2 and h3 in subdomain IA. The recent structure at 2.5-Å
resolution places Cys34 on the surface of the protein (35).
The thiol side chain is positioned toward the interior of the protein
surrounded by side chains of Pro35, His39,
Val77, and Tyr84 that appear to isolate the
thiol from external reactants. However, the difference electron density
map did not provide detailed information on the immediate environment
of the thiol. Furthermore, the structure in solution may differ from
the crystal structure in the steric relationships among the amino acid
side chains near Cys34.
The direct measurement of the stretching vibration of the S-H bond in IR spectra provides S-H band parameters that characterize the S-H bond and reflect interactions of the thiol with its environs. At the neutral pH used here, the S-H stretching band is expected to be very weak or undetectable as a result of extensive ionization of this unusually acidic thiol (36). Therefore, the detection of S-H IR bands reflected a change in protein conformation that reduced the acidity of the thiol.
The frequency of an S-H stretching band increases with increasing bond
strength and the intensity increases as the strength of the dipole
associated with the S-H bond increases (5, 30). The Cys34
S-H stretching band at 2563 cm1, as induced by both NNO
and NO, suggests a nonpolar environment and hydrogen bonding between
the thiol and an adjacent proton acceptor such as His39 or
Tyr84. The slight asymmetry of the NNO-induced band of Fig.
1C is consistent with one major protein conformation and one
minor conformation that exhibit maxima at 2563 cm
1 and
2579 cm
1, respectively. The wavenumber difference
indicates that the S-H bond strength is greater in the latter than the
former. The absorbance induced by NO also supports the generation of
two significantly different thiol environments, one of which, with a
band at 2563 cm
1, is identical to the major environment
induced by NNO. The second environment, in which the S-H bond is
weaker, since the wavenumber (2548 cm
1) is lower, is not
generated by NNO. The affinities of NNO and NO for the sites that give
rise to the S-H bands are nearly identical, although bands are first
detected at lower pressures with NNO than NO (Fig. 1,
insets). Spectra for mixtures of NNO and NO (Fig. 2) are
consistent with mixtures of NNO-type and NO-type protein conformations.
The higher wavenumbers for the S-H bands generated by halothane and
chloroform support stronger S-H bonds and weaker hydrogen bonding than
are found with either NNO or NO. Our findings indicate that NO and
anesthetics may also affect physiological roles of the albumin-free
thiol, including its reactions with other thiols, metal ions, and
oxidants (17, 33, 37-39).
Available evidence does not permit identification of the precise sites
within HSA that, when occupied by NNO, NO, halothane, or chloroform,
result in an altered Cys34 thiol environment. However,
NNO-IR spectra do give insight into the polar nature of NNO sites. The
wavenumber values of 2225 cm1 and 2220 cm
1
for NNO-IR bands reflect environments with polarities between those in
highly polar water and in a nonpolar alkane, wherein bands are found at
2230 cm
1 and 2215 cm
1, respectively (5,
40-42). Based on known effects of solvents on NNO-IR spectra, we
conclude that the more polar 2225 cm
1 site may involve a
peptide bond carbonyl, a carbonyl plus an aromatic ring, or two
aromatic rings. One aromatic ring from phenylalanine, tyrosine, or
tryptophan may account for the limited polarity of the 2220 cm
1 site (5). The aromatic structures of these amino
acids are polar, yet hydrophobic (43, 44). The finding that NO competes with NNO more effectively at the more polar (2225 cm
1)
site is consistent with the expectation that NO bonding may have
stronger contributions from dipole-dipole and/or dipole-induced dipole
interactions (10).
Recent studies provide support for interactions of both halothane and chloroform with tryptophans of HSA and bovine serum albumin (13, 14). Considerations of the bonding interactions involved and the frequencies of NNO-IR bands make tryptophan an attractive binding site for both NNO and NO. The single tryptophan of HSA (Trp214), conserved in mammals, is found between helices h2 and h3 of domain IIA and has a key structural role in formation of the subdomain IIA-binding site (33, 35). A potentially important factor in the ability of a compound, by binding at this site, to alter the environment of Cys34 thiol located in subdomain IA, is the attachment of the tail of subdomain IIA to the interface region between subdomains IA and IB by hydrophobic interactions and hydrogen bonds. Future studies of effects, if any, of NO and NNO on Trp214 fluoresence may clarify the nature of the interactions involved.
The findings reported here demonstrate the ability of each of the anesthetics, NNO, halothane, and chloroform, to alter HSA structure in the environment of the free thiol of Cys34. NO closely mimics NNO in its effects on protein structure, and in binding to protein sites. It is noteworthy that NO can modify HSA structure without formation of the S-nitrosothiol derivative. However, consideration of the possible in vivo significance of such reactions of NO with HSA must be made with an appreciation of the much higher levels of NO used here than are currently expected to occur in vivo.
Hemeprotein Studies-- The possibility that NO can bind to nonheme iron(II) sites in hemeproteins that are similar to the multiple sites shown earlier to be occupied by NNO (5) was explored. The binding of NO and CO to iron(II) sites of the hemeproteins was established by the visible/Soret spectra of the heme and IR spectra of NO and CO bound as ligands to iron(II). Under anaerobic conditions, the relative affinities of both NO and CO for heme iron(II) sites decreased in the order Mb > Hb > CcO. However, when oxygen as well as CO was present, Hb had a greater affinity for CO than did Mb. Although each protein has a greater affinity for NO than CO, the difference in the affinities is much less with Mb than with Hb or CcO.
The spectra of Figs. 6 and 7 demonstrate that CcO sequestered NO reversibly at sites other than iron(II). The noniron sites became occupied by NO rapidly, and retained NO when the solution was flushed with sufficient N2 to remove all NO from protein-free buffer. However, several hours were required for the loss of NO from all these sites as shown by the binding of the released NO to iron(II) sites of Mb or Hb. NO was also retained at noniron sites within Hb from which dissociation of NO was very slow. The number of "slow release" noniron sites approximates the number of NNO sites estimated earlier (5), a finding consistent with, but not proof of, NO and NNO binding at the same sites.
The IR spectra in the N-O stretching regions for solutions of MbNO and
HbNO are altered by exposure to NNO, CO, or N2 (Fig. 8).
The changes can be attributed to shifts in the Amide I spectrum of the
protein rather than to perturbations of the true N-O stretching vibration. The absorbance recorded represents the sum of the true N-O
stretching vibration plus the difference between the Amide I spectrum
of the sample solution and the Amide I spectrum of the reference
solution when a CO-saturated solution of the respective protein
carbonyl was used as reference. In each case, curve-fitting revealed
one fitted band that was consistent with a N-O stretching band with
parameters identical to those found earlier when the reference solution
used contained 15N16O (1611 cm1
for MbNO and 1617 cm
1 for HbNO) (19, 28). The problems
associated with isolating the true N-O stretching band from the protein
Amide I bands that appear in the same region of the IR spectrum have
been discussed (10, 19, 28). Effects of N2-flushing on the
spectrum can be attributed to changes in protein secondary structure as
the result of the removal of NO from noniron sites from which NO
dissociates more rapidly than from the "slow release" sites
discussed above. The smaller effects of flushing with NNO, than with
N2, suggests that NNO replaces NO at sites, and thereby
causes only a small change in protein structure. A shift in the protein
Amide I spectrum without displacing NO at iron(II), that occurs upon
flushing with CO, can also result from loss of NO from noniron sites
with partial or complete replacement with CO. The spectral shifts of
Fig. 8 are consistent with NO occupying noniron protein sites from
which NO can be dissociated, or replaced.
The IR spectra of thiols and sequestered NNO molecules of Fig. 9 provide evidence for the occupancy of noniron protein sites in Hb by NO, NNO, CO, and O2. The Hb cysteine S-H stretching bands, although relatively weak, have the advantage of being in a region of the spectrum where other protein bands are absent (5, 30). NNO-IR spectra provide direct evidence that when solutions of HbCO, HbNO, and HbO2, which had been saturated with NNO, were subsequently exposed to CO, NO, or O2, respectively, the loss of NNO from the more polar sites was greater than from the less polar sites. The effectiveness in displacing NNO decreased in the order NO > CO > O2. These findings suggest that each of these gases may be able to influence Hb function by occupying noniron sites.
Conclusions-- The broader implications of these findings include the expectation that NO, volatile anesthetics, and probably CO may occupy similar sites within many proteins and, thereby, alter the structure and function of the protein. Such sites may also provide safe havens for NO by limiting its accessibility to external reactants. The precise nature of such sites remains elusive, but aromatic residues are attractive possible components. The ability of aromatic ring structures to bind NO, tightly and reversibly, is shown unequivocally by the NO binding between the cofacial aromatic groups of so-called "Venus fly trap" organic compounds recently reported by Kochi and co-workers (45, 46). The ability to detect NNO at different sites within protein by IR spectroscopy, coupled with the evidence found in support of NO and NNO binding at the same sites, illustrate the utility of NNO-IR spectroscopy for detecting potential nonmetal sites for NO binding via noncovalent bonding.
Physiologically important areas in which control by NO via noncovalent interactions of NO with protein may occur include oxygen transport, mitochondrial respiration, and the activation of soluble guanylate cyclase. Failure to invoke such interactions with hemoglobin may be the reason why considerations of only the reactions of NO with the thiols and irons of hemoglobin have failed to provide a satisfactory mechanism for control of oxygen transport by NO (47). NO has been suggested to regulate CcO activity by forming metal nitrosyls at heme iron and/or copper (20, 48). Both NO and NNO inhibit CcO reversibly (18, 48). Since NNO does not serve as a ligand to these metals, the partial, reversible inhibition by NNO appears due to the noncovalent bonding of NNO molecules to one or more of the nonmetal sites that are detected in IR spectra (5, 18, 28). Noncovalent bonding of NO to NNO sites, and possibly other sites, provides new mechanisms for the control of CcO activity by NO.
NO binding to protein sites via noncovalent bonding, as well as to heme
iron(II), may be particularly important in the activation of guanylate
cyclase by NO and provides an explanation for findings recently
reported by Marletta and co-workers (49). Activation was shown not to
occur upon NO binding to heme iron, as had been widely assumed (49).
Activation requires a subsequent step, the rate of which is dependent
on NO concentration. The second step involves conversion of
six-coordinate heme iron to a five-coordinate species, a conversion
apparently induced by NO binding to an unknown site that is not heme
iron. This loss of the ligand trans to NO is a process analogous to the
change in heme coordination in HbA nitrosyl that is induced by inositol
hexaphosphate as discovered in early studies of protein allosteric
effects (50, 51). The porphyrin -electron system of the heme and the
aromatic amino acids widely found adjacent to the heme in hemeprotein
structures (52) provide potential sites for noncovalent bonding of NO
in hemeproteins. The availability of such sites, and the slow
dissociation of NO from them, suggests that hemeproteins are likely to
bind NO via noncovalent bonding much more avidly than occurs with HSA. Furthermore, the activation of soluble guanylate cyclase and some of
the other effects of CO (21-23) may also involve noncovalent interactions of CO with protein.
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ACKNOWLEDGEMENTS |
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We thank Dr. Byron Caughey for helpful discussions and Bob Evans, Gary Hettrick, and Anita Mora for graphics assistance.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service Grant HL-15890 and a gift from Apex Bioscience, Inc.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.
§ Present address: Dept. of Chemistry, Colorado State University, Fort Collins, CO 80523.
¶ Present address: Amersham Pharmacia Biotech, Inc., 800 Central Ave., Piscataway, NJ 08954.
** To whom correspondence should be addressed: Rocky Mountain Laboratories, 903 4th St., Hamilton, MT 59840. Tel.: 406-363-9440; Fax: 406-363-9286; E-mail: wcaughey@nih.gov.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M006588200
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ABBREVIATIONS |
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The abbreviations used are: NO, nitric oxide; HSA, human serum albumin; CcO, cytochrome c oxidase; HbA, adult human hemoglobin; Mb, myoglobin; IR, infrared; NO/N2, 5% NO, 95% N2; atm, atmosphere.
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