From the
In the reaction between hydrogen peroxide and metmyoglobin, the
heme iron is oxidized to its ferryl-oxo form and the globin to protein
radicals, at least one of which reacts with dioxygen to form a peroxyl
radical. To identify the residue(s) that forms the oxygen-reactive
radical, we utilized electron spin resonance (ESR) spectroscopy and the
spin traps 2-methyl-2-nitrosopropane and
3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS). Metmyoglobin radical
adducts had spectra typical of immobilized nitroxides that provided
little structural information, but subsequent nonspecific protease
treatment resulted in the detection of isotropic three-line spectra,
indicative of a radical adduct centered on a tertiary carbon with no
bonds to nitrogen or hydrogen. Similar isotropic three-line ESR spectra
were obtained by spin trapping the oxidation product of tryptophan
reacting with catalytic metmyoglobin and hydrogen peroxide. High
resolution ESR spectra of DBNBS/trp and of the protease-treated
DBNBS/metMb were simulated using superhyperfine coupling to a nitrogen
and three non-equivalent hydrogens, consistent with a radical adduct
formed at C-3 of the indole ring. Oxidation of tryptophan by catalytic
metMb and hydrogen peroxide resulted in spin trap-inhibitable oxygen
consumption, consistent with formation of a peroxyl radical. The above
results support self-peroxidation of a tryptophan residue in the
reaction between metMb and hydrogen peroxide.
Oxidative damage to tissues has become a recurring theme as a
mechanism for the induction of a variety of medical conditions (1) including myocardial ischemia(2) , cancer(3) ,
and aging(4) . Myoglobin, a heme protein that is ubiquitous in
aerobic muscle tissues, has been shown to acquire peroxidative activity (5) that can result in the oxidative damage to a variety of
biological molecules, including proteins and membrane
lipids(6, 7, 8, 9) .
Since the
1950's, metMb has been known to react with hydrogen peroxide,
resulting in the one-electron oxidation of ferric heme to form ferryl
heme(10, 11) . The fate of the second oxidizing
equivalent available from the reduction of hydrogen peroxide to water
has been in question since the reaction was first reported(11) .
Free hydroxyl radical is one possibility, but this species has been
detected only under conditions of excess hydrogen peroxide where heme
damage has occurred(12, 13) . No evidence has been
obtained for formation of a porphyrin cation radical from metMb similar
to compound I of horseradish peroxidase, and recent results support the
formation of a protein-centered
radical(14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) .
However, the nature of the protein-centered radical is still under
debate. Many previous investigations have concluded that the free
radical is centered on a tyrosine
residue(17, 18, 19, 21, 23, 31) .
Recently, however, site-directed mutagenesis studies, in which all of
the tyrosine residues have been removed(26, 27) , and
ESR studies (24, 28) have demonstrated that another
amino acid residue in addition to tyrosine must also be involved. The
identity of the latter amino acid residue has not been determined.
In this investigation, we utilize the electron spin resonance (ESR)
spectroscopy spin-trapping technique using the nitroso-based spin traps
3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS)
The initial spin-trapping data obtained with
5,5-dimethyl-1-pyrroline N-oxide to discern the nature of the
electron-donating residue in metMb demonstrated that a free radical was
formed(20, 21, 24, 29) . The original
assignment to a peroxyl radical adduct (21, 22) was
subsequently demonstrated to be in error (24, 28), but no additional
structural information was obtained. Likewise, the whole-protein data
acquired in this study using the nitroso spin traps provided little
structural information ( Fig. 1and 3). The high degree of
rotational anisotropy observed in nitroso-spin trap/whole protein
radical adducts can be removed by proteolysis, which results in
fragmentation of the protein to form relatively freely rotating
peptides that give isotropic spectra(37) . Such was the case for
both DBNBS/metMb and MNP/metMb, resulting in a great enhancement of the
information content of the spectra (Figs. 1G and 2G).
The similarity of the spectrum obtained by Pronase treatment of
DBNBS/metMb to that of DBNBS/trp strongly suggests that the
globin-centered radical resides on a tryptophan residue. The conclusion
that the radical is centered on the indole ring, and in particular on
C-3, is corroborated by the resolution of the superhyperfine coupling
from each of the lines of the primary triplet, which were very
convincingly simulated using coupling to a nitrogen, three
non-equivalent hydrogens, and the two equivalent meta-hydrogens from the DBNBS (Fig. 5A, ). Previous reports of similar spectra acquired from
tryptophan and DBNBS have assigned the radical to either C-3 of the
indole ring (34) or to the
The minor
difference between the highly resolved spectrum obtained from free
tryptophan and that obtained from the Pronase-treated DBNBS/metMb can
be accounted for by changes in the relative orientations of the
methylene protons induced by the steric effects of additional amino
acids on the amino and carboxyl groups of tryptophan that can increase
or decrease the magnitudes of hyperfine coupling constants(41) ,
as demonstrated with the spectra obtained using the trp-gly
The hydrogen peroxide-independent addition
of DBNBS to tryptophan, demonstrated in Fig. 3B,
suggested that this tryptophan adduct might be formed by direct
reaction of the spin trap with the tryptophan residues of the protein,
either before or after proteolysis. DBNBS has been shown to react with
biological compounds, including tryptophan, to give non-radical-derived
adducts(34) . The product of the ene reaction of DBNBS with an
unsaturated system is a hydroxylamine that must then be oxidized to
form an ESR-active nitroxide(34) . No radical adduct spectrum
was detected upon addition of hydrogen peroxide to the dialyzed control
incubation between metMb and DBNBS (Fig. 1E), showing
that the ene reaction using hydrogen peroxide to oxidize the proposed
hydroxylamine intermediate is not the pathway for DBNBS/metMb
formation. The inhibition of adduct formation by cyanide, which
coordinates heme iron and prevents access by the peroxide, thereby
eliminating peroxidase chemistry, demonstrated the requirement for heme
oxidation in the formation of the protein-centered radical adduct.
The results of the spin trapping experiments using MNP confirm the
hydrogen peroxide dependence of globin-centered radical formation and
also demonstrate that the radical adduct is centered on a tertiary
carbon. The observed difference between the a
Previous studies have suggested that a
tyrosine peroxyl radical is the spin-trapped radical in hydrogen
peroxide-treated metMb(21, 22) . A spin-trapping study
of radicals formed from tyrosine by pulsed radiolysis using MNP did not
report a spectrum at neutral pH, and spectra acquired in basic and
acidic solutions exhibited different hyperfine coupling constants from
those determined for the Pronase-treated metMb(42) .
The
present results provide strong evidence that the globin-centered
radical that is spin trapped following the reaction of metMb with
hydrogen peroxide is centered on a tryptophan residue. The formation of
tryptophan radicals in intact proteins has been
demonstrated(43, 44, 45) . The identification of
a tryptophan residue as an oxidized site is also consistent with the
studies performed by Ortiz de Montellano and
co-workers(26, 27) , in which a direct ESR signal
identical to that of the native protein is observed in the
site-directed mutant protein, which lacks all tyrosine residues.
However, the dityrosine cross-linking of sperm whale metMb (18) and the covalent binding of tyrosine to heme in horse
metMb(6) , as well as ESR studies(17, 23) , all
indicate that the oxidation of metMb by hydrogen peroxide forms one or
more tyrosine radicals. Transfer of the oxidative equivalent between
tryptophan and tyrosine residues is suggested by the similarity of
their oxidation potentials(46, 47) .
Previous
spin-trapping studies have demonstrated that oxygen competes with the
spin trap for the the primary radical, and direct ESR has demonstrated
that the radical detected in frozen solution is a peroxyl
radical(24, 28) . Tryptophan radical formed
photochemically has been shown to react with oxygen during its
decomposition(48, 49) . We found that tryptophan
oxidation by the catalytic peroxidase activity of metMb consumed oxygen
from the solution (Fig. 7), a reaction that was inhibited by the
inclusion of the spin traps DBNBS and MNP. That result is consistent
with the previously reported competition between the spin traps and
oxygen for the globin radical formed in the metMb/hydrogen peroxide
system(28) . Since free tryptophan radical reacts with oxygen,
it is likely that a tryptophan radical in a protein would also do so to
form a peroxyl radical. We examined a computer-generated space-filling
model calculated using theoretical van der Waals atomic radii from the
crystal structure of horse metMb(50) . Both tryptophan residues
of metMb were found to be in contact with the solvent, with tryptophan
7 having more surface contact than either of the tyrosine residues. The
generation of a protein-centered radical has been suggested to be
necessary for the oxidation of styrene by metMb and hydrogen peroxide
(51).
The present results provide strong evidence that the
globin-centered radical that is detected by ESR and spin trapping is
centered on a tryptophan residue. Spectral evidence strongly indicates
that the radical adduct is formed at the C-3 carbon of the indole ring,
which is also the most likely atom at which oxygen addition to form the
peroxyl radical occurs, as is shown in Fig. SI. Whether the
radical adduct is formed on only one or both of the tryptophan residues
in metMb, which a search of the metMb sequences in the PIR protein data
base revealed to be rigidly conserved among terrestrial vertebrates,
cannot be resolved at this time. The presence of the globin radical on
a tryptophan residue that is near the surface of the protein provides a
mechanism for the catalytic activity of metMb in the oxidation,
peroxidation, or epoxidation of large substrates that do not have ready
access to the heme iron.
Asterisk indicates a chiral carbon.
(
)and 2-methyl-2-nitrosopropane (MNP) to investigate
the nature of the amino acid residue modified in the reaction between
horse heart metMb and hydrogen peroxide. The spin trapping results
indicate that the globin radical that has been spin trapped is centered
on a tryptophan residue.
Materials
Horse heart metMb, proteinase
K, tryptophan, tyrosine, and diethylenetriaminepentaacetic acid were
acquired from Sigma. MNP dimer and sodium cyanide were obtained from
Aldrich. 3,5-dibromo-4-nitrosobenzenesulfonic acid sodium salt was
purchased from OMRF Spin Trap Source (Oklahoma City, OK). Pronase was
obtained from Boehringer Mannheim. Hydrogen peroxide was obtained from
Mallincrodt (St. Louis, MO). Chelex 100 was purchased from Bio-Rad.
Glycylglycyltryptophan (gly-gly-trp), glycyltryptophylglycine
(gly-trp-gly), and tryptophylglycylglycine (trp-gly-gly) were acquired
from Accurate Chemical and Scientific (Westbury, NY).
Tryptophan-indole-D was obtained from Cambridge Isotope
Laboratories (Cambridge, MA).
2,6-dideutero-3,5-dibromo-4-nitrosobenzenesulfonic acid was prepared by
Robert Sik (NIEHS), following the published procedure(32) .
Spin Trapping Experiments
All experiments
were done in 50 mM sodium phosphate buffer, pH 7.4, treated
with chelex by the batch method, and contained
diethylenetriaminepentaacetic acid at a final concentration of 50
µM. When MNP was used as the spin trap, metMb, tryptophan,
or peptides were dissolved into a solution of MNP (23 mM)
prepared by stirring overnight at room temperature. In all experiments,
hydrogen peroxide was added to the remaining reagents immediately
before transfer of the solution to a quartz ESR flat cell, which was
then positioned into the microwave cavity (TM) of a
Bruker ESP 300 spectrometer. Instrument parameters are reported in the
figure legends for each individual scan. Spectral simulations were
calculated and optimized to a minimum sum of squared residuals using a
program developed by David Duling (33) (available over the
Internet). When samples were subjected to dialysis, several milliliters
of sample were prepared, a small aliquot was scanned, and the remaining
sample was placed in dialysis tubing (molecular weight cutoff 3,500)
and dialyzed overnight against one 2-liter charge of 50 mM sodium phosphate buffer, pH 7.4. Following dialysis, a small
aliquot was removed and scanned, and the remaining sample was subjected
to protease treatment as described in the figure legends. Control
experiments were performed in parallel with complete incubations.
Oxygen Consumption
All experiments were
performed in chelex-treated 50 mM sodium phosphate buffer, pH
7.4, in the presence of 50 µM diethylenetriaminepentaacetic acid. When MNP was used, all further
reagents were dissolved in prepared MNP solutions. MetMb was added
after a base line had been established with no subsequent difference in
the base line being observed. Hydrogen peroxide (final concentration,
100 µM unless otherwise noted) was then added. Data were
collected using a YSI oxygraph equipped with a computer interface using
a Clark oxygen electrode.
Spin Trapping a Free Radical Product of metMb and
H
Addition of
equimolar hydrogen peroxide to metMb in the presence of DBNBS resulted
in the detection of an ESR spectrum characteristic of a highly
immobilized nitroxide (Fig. 1A). Overnight dialysis of
the product resulted in only slight changes in the shape of the ESR
spectrum, indicating that the radical adduct has a molecular weight
greater than 3,500 (Fig. 1B). No spectra could be
detected in the absence of either metMb or hydrogen peroxide (Fig. 1, C, D, and F). Addition of
hydrogen peroxide to the hydrogen peroxide-free control after dialysis
did not result in detection of any ESR signal (Fig. 1E).
Inclusion of sodium cyanide (10 mM) prevented detection of the
immobilized nitroxide (data not shown). Treatment of the product with
Pronase resulted in the detection of a nearly isotropic three-line
spectrum with a hyperfine coupling constant of 13.6 G (Fig. 1G). Treatment of the product with proteinase K
resulted in detection of a spectrum similar to that observed when
Pronase was used (data not shown).
O
with DBNBS
Figure 1:
ESR
spectra obtained from a mixture of metMb and HO
in the presence of DBNBS. A, hydrogen peroxide (400
µM) was added to a solution of metMb (500 µM)
in the presence of DBNBS (10 mM). B, an aliquot of
the solution used in spectrumA after overnight
dialysis into 50 mM sodium phosphate buffer. C, MetMb
(500 µM) was added to a solution of DBNBS (10
mM). D, an aliquot of the solution used in spectrumC after overnight dialysis into 50 mM sodium phosphate buffer. E, hydrogen peroxide (400
µM) was added to an aliquot of the solution described in scanC after overnight dialysis. F, hydrogen
peroxide (400 µM) was added to DBNBS (10 mM). G, an aliquot of the solution used to obtain spectrumB 20 min after addition of Pronase (final concentration,
2 mg/ml). Instrument parameters were as follows for all scans:
modulation amplitude, 1 G; time constant, 0.33 s; scan time, 335 s;
gain, 1
10
; modulation frequency, 100 kHz;
microwave frequency, 9.80 GHz; microwave power, 20
mW.
Spin Trapping the Product of metMb and
H
When the
reaction between metMb and hydrogen peroxide was investigated with MNP,
the ESR spectrum of the resulting solution was similar to that observed
in the presence of DBNBS (Fig. 2A). Only moderate decay
of the immobilized nitroxide occurred upon dialysis (Fig. 2B). When hydrogen peroxide was excluded, a
three-line spectrum with a nitrogen hyperfine coupling constant of 17.2
G was detected, characteristic of di-t-butylnitroxide, a
decomposition product of MNP (Fig. 2D). The three-line
spectrum was removed by dialysis, confirming that it was not a
protein-centered radical adduct (Fig. 2E). Treatment of
the product with Pronase (Fig. 2F) resulted in the
detection of a three-line spectrum with aO
with MNP
=
15.5 G, which was dependent upon hydrogen peroxide (Fig. 2G); the ESR spectra obtained when proteinase K
was used in place of Pronase gave a similar spectrum, except that a
significant contribution from immobilized nitroxide was also observed
(data not shown).
Figure 2:
The ESR
spectra obtained after addition of HO
to metMb
in the presence of MNP. A, hydrogen peroxide (400
µM) was added to metMb (500 µM) in the
presence of MNP (23 mM). B, the ESR spectrum of an
aliquot of the solution used to obtain spectrumA after dialysis for 3 h in 50 mM sodium phosphate buffer,
pH 7.4. C, hydrogen peroxide (400 µM) was added
to a solution of MNP (23 mM). D, MetMb (500
µM) was added to a solution of MNP (23 mM). The
nitrogen hyperfine coupling constant (a = 17.2 G) is
characteristic of di-t-butyl nitroxide. E, an aliquot
of the solution used to obtain spectrumD was scanned
after overnight dialysis in 50 mM sodium phosphate buffer. F, Pronase (final concentration, 2 mg/ml) was added to an
aliquot of the solution used to obtain spectrumB.
Note that the gain for this scan is 20-fold lower than in the other
scans. The value for a for the observed radical adduct is 15.5 G. G, Pronase (2 mg/ml) was added to an aliquot of the solution
used to obtain spectrumE. Instrument parameters for
scans A-C, E, and G were as follows:
modulation amplitude, 1 G; time constant, 0.33 s; scan time, 671 s;
receiver gain, 5
10
; all other parameters were as
reported for Fig. 1. For spectrumD, the time
constant was 0.66 s. For spectrumF, the receiver
gain was 2.5
10
.
Identification of the Amino Acid Residue
Modified
The three-line spectra obtained after treatment of
oxidized metMb with nonspecific proteases (Fig. 1G and
2F) are indicative of a tertiary carbon-centered radical
adduct. The lack of additional hyperfine structure in the spectrum
indicates that the tertiary carbon atom in the adduct has no bonds to
atoms with nuclear spin such as nitrogen. The most reasonably
oxidizable tertiary carbon atoms in proteins are C-3 of the indole ring
of tryptophan and C-4 of the phenol ring of tyrosine. The peroxidase
activity of metMb (5) was used to oxidize free amino acids to
identify the type of amino acid residue modified. Free tryptophan (20
mM), free tyrosine (saturated, approximately 3 mM),
or free histidine (20 mM) was added to a catalytic
concentration of metMb in the presence of a spin trap, and hydrogen
peroxide was added to the solution to initiate the reactions. No amino
acid-dependent radical adducts were detected when either tyrosine or
histidine was used with either spin trap. When tryptophan was studied
using DBNBS as the spin trap, a very strong three-line signal
(a = 13.6 G) nearly identical to that
observed from metMb and hydrogen peroxide after proteinase treatment
was detected (Fig. 3, A and B). The adduct was
also observed in the absence of metMb, and further study demonstrated
that the adduct formation was independent of hydrogen peroxide and
oxygen as well (Fig. 3B). The spontaneous ene reaction
between DBNBS and tryptophan to form a pseudo radical adduct has been
reported(34) . The broadening of the high field line in the
triplet in Fig. 3A relative to that in Fig. 3B is the result of slower molecular motion, consistent with a higher
molecular weight of the radical adduct(s) from protease-treated metMb.
Figure 3:
The ESR spectra obtained from the
protease-treated DBNBS/metMb radical and from free tryptophan and
DBNBS. A, a 50-G scan of the proteinase K-treated dialyzed
sample obtained from the reaction of metMb and hydrogen peroxide in the
presence of DBNBS. B, DBNBS (10 mM) was added to
tryptophan (20 mM) in N-saturated solution under
an atmosphere of N
, and the solution was aspirated into a
quartz ESR flat cell prepositioned in the microwave cavity of a Bruker
ER300 ESR spectrometer. C, the optimized computer simulation
of spectrumB with hyperfine coupling constant a
= 13.6 G. The instrument conditions used for the experimental
scans were the same as reported for Fig. 1.
When tryptophan was incubated with MNP, hydrogen peroxide, and
catalytic concentrations of metMb, a three-line ESR spectrum was
detected with hyperfine coupling constant a = 16.1 G (Fig. 4B). The detected
spectrum was different from the spectrum obtained from Pronase-treated
metMb, which had a nitrogen hyperfine coupling constant a
= 15.5 G (Fig. 4A). When either
hydrogen peroxide or metMb was removed from the incubation, a
three-line spectrum with a nitrogen hyperfine coupling constant
characteristic of di-t-butylnitroxide was detected (data not
shown). A radical adduct of N-acetyl-tryptophan with MNP has
been reported with a nitrogen hyperfine coupling constant identical to
that which we detected from the protease-treated MNP/metMb (35). The
presence of amide bonds at the amino terminus of the amino acid residue
is expected to have similar effects on the nitrogen hyperfine coupling
constant of the radical adduct(35) . To investigate that
possibility, we used metMb as a peroxidase to oxidize the tryptophan
residues in the tripeptides trp-gly-gly, gly-trp-gly, and gly-gly-trp
using MNP as a spin trap to detect the resulting radicals. The
hyperfine coupling constants for the observed spectra range from 16.1 G
for trp-gly-gly to 15.75 G for gly-gly-trp, indicating that the
difference between free tryptophan and the protein-derived adduct could
be due to the amino acid residues that retain amide bonds to the
tryptophan residue during nonspecific protease treatment of oxidized
metMb, particularly on its
-amine.
Figure 4:
Comparison of the ESR spectra obtained
from Pronase-treated MNP/metMb radical adduct and from the oxidation of
tryptophan in the presence of MNP. A, a 50-G scan of the
solution scanned in Fig. 2F. B, hydrogen peroxide
(100 µM) was added to tryptophan (20 mM) in the
presence of metMb (50 µM) and MNP (23 mM). C, the optimized computer simulation of the spectrum shown in B. Instrument settings: modulation amplitude, 1 G; time
constant, 0.67 s; scan time, 671 s; receiver gain for spectrumB, 1 10
; receiver gain for spectrumA, 2.5
10
. All other
spectrometer settings were the same as reported for Fig.
1.
Resolution of the Superhyperfine Coupling of
DBNBS/Tryptophan Radical Adducts
The ene reaction between
DBNBS and tryptophan results in the formation of a pseudo radical
adduct that has an identical structure to the adduct formed through a
radical pathway(34) . The adduct is produced in sufficiently
high concentration to resolve small hyperfine coupling constants from
magnetic nuclei to the carbon at which the radical adduct is
formed (See for the structure of the adduct). With the use
of a smaller modulation amplitude (0.1 G), each broad line of the
observed three-line spectrum was determined to be composed of a number
of overlapping lines (Fig. 5A). The middle line of the
high resolution spectrum of DBNBS/trp was simulated using contributions
from a nitrogen atom, three non-equivalent protons, and the expected
two meta-protons of the spin trap, and the simulation was
optimized using an iterative process in which each parameter is varied
until the minimum residual is achieved. Details of the method and the
program utilized have been published(33) . A 0.1-G modulation
amplitude scan of the middle line of the radical adduct produced by
Pronase treatment of DBNBS/metMb gave rise to a similar, but not
identical, superhyperfine structure (Fig. 5E). To
account for effects of amide bonds to the tryptophan in the
protease-treated metMb sample, the tripeptides trp-gly-gly,
gly-trp-gly, and gly-gly-trp were studied in a similar manner (Fig. 5, B-D). The hyperfine coupling constants
from the optimized simulation for each experimental spectrum are
reported in . To identify the atoms contributing hyperfine
coupling in the various DBNBS adducts, indole-deuterated tryptophan and
DBNBS deuterated on the meta positions were utilized.
Deuteration of DBNBS removed the hyperfine interaction of two
equivalent protons, which had contributed a 1:2:1 triplet to the
superhyperfine structure, most notably in the outermost shoulders in
the spectrum (Fig. 6, B compared to A),
demonstrating the contribution of the meta-protons of the spin
trap to the spectrum. When indole-deuterated tryptophan was used, a
proton hyperfine coupling was replaced by a much smaller deuterium
coupling, demonstrating hyperfine interaction with an indole ring
hydrogen (Fig. 6C). The use of both deuterated
tryptophan and deuterated spin trap greatly simplified the spectrum in
the expected manner (Fig. 6D).
Figure 5:
High
resolution scans and computer simulations of the middle line of the
spectra from the reaction of tryptophan derivatives with DBNBS.
Computer simulations were calculated from the hyperfine coupling
constants reported in Table I and were the result of differential
minimization optimization (30). Simulations (dashedlines) are shown superimposed on the experimental spectra (solidlines). A, hydrogen peroxide (100
µM) was added to a solution containing metMb (25
µM), tryptophan (20 mM), and DBNBS (10
mM). B, hydrogen peroxide (100 µM) was
added to the tripeptide gly-trp-gly (saturated solution) in the
presence of metMb (25 µM) and DBNBS (10 mM). C, hydrogen peroxide (100 µM) was added to the
tripeptide gly-gly-trp (20 mM) in the presence of metMb (25
µM) and DBNBS (10 mM). D, hydrogen
peroxide (100 µM) was added to the tripeptide trp-gly-gly
(20 mM) in the presence of metMb (25 µM) and
DBNBS (10 mM). E, Pronase (2 mg/ml, final
concentration) was added to a fraction of a dialyzed solution prepared
by addition of hydrogen peroxide (400 µM) to metMb (500
µM) in the presence of DBNBS (10 mM), and the
spectrum was recorded after a 40-min incubation period to ensure
complete proteolysis of the metMb. Instrument settings for scans A-D were as follows: modulation amplitude, 0.1 G; time
constant, 0.65 s; scan range, 15 G; scan time, 5368 s; receiver gain, 5
10
; microwave power, 0.2 mW. Instrument settings
for scanE were: modulation amplitude, 0.1 G; time
constant, 0.33 s; scan range, 15 G; scan time, 2684 s; receiver gain, 5
10
; microwave power, 20
mW.
Figure 6:
Effects of
deuteration on the high resolution ESR scans of the reaction between
tryptophan and DBNBS in the presence of catalytic metMb. Computer
simulations (dashedlines) were obtained as reported
in Fig. 5. ESR parameters are the same as reported in Fig. 5. All
reactions were initiated by the addition of hydrogen peroxide (100
µM) to solutions containing tryptophan (20 mM),
metMb (25 µM), and DBNBS (10 mM). A,
non-deuterated tryptophan and DBNBS. This spectrum is repeated from
Fig. 5 to allow comparison. B, non-deuterated tryptophan and
2,6-D-DBNBS. C, indole-D
-tryptophan
and non-deuterated DBNBS. D, indole-D
-tryptophan
and 2,6-D
-DBNBS.
Oxygen Consumption by
Tryptophan
Incubation of tryptophan (20 mM) with
metMb (40 µM) and hydrogen peroxide (100 µM)
resulted in the rapid consumption of oxygen (Fig. 7, curveA), which was inhibited by inclusion of 23 mM MNP (Fig. 7, curveB) and by 10 mM DBNBS (Fig. 7, curveC). The rate of
oxygen consumption was dependent upon the concentration of metMb (data
not shown). Slight oxygen evolution was detected when hydrogen peroxide
was added to metMb in the absence of tryptophan, consistent with the
known catalase activity of metMb (Fig. 7D)(5) .
No oxygen consumption was observed when tryptophan was replaced by
either tyrosine or histidine (data not shown).
Figure 7:
Oxygen consumption by solutions of
tryptophan in the presence and absence of spin traps. In each reaction,
hydrogen peroxide (100 µM) was added to a solution that
already contained metMb (50 µM) at the time indicated by
the solidarrow. A, tryptophan (20
mM). B, tryptophan (20 mM) + MNP (23
mM). C, tryptophan (20 mM) + DBNBS (10
mM). D, the no tryptophan control. Additional
hydrogen peroxide was added to demonstrate the catalase-like activity
of metMb at the times indicated with the brokenarrows.
-carbon of the amino
acid(37) . The latter assignment seems unlikely due to the lack
of the expected hyperfine coupling to the
-amine nitrogen.
Previous studies using different techniques have demonstrated that
tryptophan radicals centered on the indole ring are formed with
significant free electron density residing on
C-3(35, 38, 39) . Calculated electron densities
of the indole ring-centered tryptophan radical have shown that the
greatest electron density resides on C-3(40) .
tripeptides. In addition, the molecular inhomogeneity, resulting
from nonspecific protease treatment of the intact DBNBS/metMb and
reflected in the increased linewidth used in the optimized simulation
of the metMb-derived adduct (), contributes to the
deviation between the protein-derived and tryptophan spectra. Finally,
the formation of the radical adduct at C-3 of the indole ring creates a
chiral center that results in the possibility of the formation of a
pair of diastereomers, which could themselves have non-identical
spectra, thereby also contributing significant line
broadening(41) .
for
the Pronase-treated MNP/metMb and for MNP/trp can be accounted for by
the presence of amide(s) on the primary amine, the primary carboxylic
acid, or both groups of tryptophan in the hydrolysate, as demonstrated
by the observed a
values for the tryptophan
tripeptides. Effects of amidation, ionization, and acetylation of the
functional groups of tryptophan similar to those observed here have
been reported(37) .
Figure SI:
Scheme I.
Table: Hyperfine coupling constants in Gauss
used to simulate the ESR spectra from solutions of tryptophan and DBNBS
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.