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INTRODUCTION |
Protein-centered free radicals are now recognized as intermediates
for an increasing number of enzymes (1). Examples include the thiyl (2)
and tyrosyl (3) radical centers of ribonucleotide reductase, the
tyrosyl radical of prostaglandin H synthase (4), the glycyl radical of
pyruvate formate-lyase (5), and the tryptophanyl radical of cytochrome
c peroxidase (6). Protein-centered radicals have also been
implicated in oxidative stress associated with pathology of
inflammatory disease(s) such as atherosclerosis and possibly in aging
(7, 8). At least some of the toxic effects of protein radicals, as
exemplified by protein-mediated initiation of lipid peroxidation
(9-12), involve translocation of radical centers from one amino acid
residue to another through both intramolecular (13) and intermolecular
(14) mechanisms. Also, it has been proposed that free radical damage in
proteins may well occur with a chain process similar to that for lipid
peroxidation (15). One example of radical translocation is provided by
the protein-centered radical(s) formed upon reaction of mammalian
myoglobin with H2O2 (16).
Myoglobin (Mb)1 shows a
limited peroxidase activity in the presence of
H2O2. Reaction of Mb and
H2O2 yields water with the concomitant
formation of ferryl (Fe(IV)=O) heme and a porphyrin radical cation
(16). At H2O2:protein ratios of
5, reaction of ferric or metmyoglobin (metMb) with H2O2
yields both ferryl (Fe(IV)=O) Mb and protein radicals (globin·)
(17). Although the ferryl Mb is stable for hours at room temperature and is characterized readily by electronic absorption spectroscopy (17), the identity of the Mb residue(s) that form globin· in the
presence of H2O2 has been the subject of some
controversy. The precise location of the radical on the protein likely
depends to some extent on both the mechanism by which it is formed and the stability of the radical species formed at a particular amino acid
residue (14). Globin· have been localized to tyrosine (18, 19)
and/or tryptophan residues (20) that effectively stabilize the odd
electron through extended delocalization over unsaturated bonds.
Additionally, globin· may undergo subsequent chemistry (20) and
is capable of oxidizing other biological molecules (21). Importantly,
hydroxyl radicals do not appear to be involved in the transfer of
oxidative damage to either protein or target molecules (22, 23).
Human Mb is similar in sequence to other mammalian myoglobins. One
significant difference, however, is the presence of
Cys110 (24). No other known mammalian Mb possesses a
reactive thiol group. Recently (25), we investigated the reaction of
human Mb and its C110A variant to evaluate the role of the reactive sulfhydryl group in the reaction of this protein with
H2O2. Similar to sperm whale and horse heart
Mb, reaction of human Mb and H2O2 yielded
radicals derived from Trp14 and from tyrosine residues
Tyr103 and Tyr146. Our data also indicated that
the reaction of human Mb and H2O2 differed from
the corresponding reaction of other myoglobin species by formation of
Cys110-derived thiyl radical and that bimolecular coupling
of thiyl radicals can lead to formation of a Mb homodimer through
intermolecular disulfide bond formation. The spin trapping agent DMPO
readily reacted with both Tyr103-phenoxyl and
Cys110-thiyl radicals where present in reactions of human
Mb and H2O2. However, the precise mechanism for
the formation of the Cys110-thiyl radical on wild type
human Mb was not elucidated. We now report further studies of the
mechanism of Cys110-thiyl radical formation in the reaction
of human Mb and H2O2 by the combined use of
site-directed mutagenesis, gel electrophoresis, and EPR spectroscopy.
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EXPERIMENTAL PROCEDURES |
Materials--
Horse heart Mb, iodoacetamide, trypsin (Type III,
10, 200 unit/mg protein), urea, EDTA,
2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO),
diethylenetriamine pentaacetic acid (DTPA),
ammonium-d-camphor-10-sulfonate, 2-methyl-nitrosopropane
(MNP), and DMPO were obtained from Sigma. DMPO was purified by stirring
solutions (1 M in 50 mM phosphate buffer, pH
7.4) with activated charcoal (100 mg/ml) in the dark. After 30 min, the
solution was filtered, and portions were stored at
80 °C prior to
use (26). MNP was used without further purification. Solutions of MNP
(500 mM in acetonitrile) were prepared immediately prior to
use. Tryptone and yeast extract were from Becton Dickinson (Sparks,
MD). Dithiothreitol and NaCl were obtained from Fisher Scientific (Fair
Lawn, NJ). H2O2 was from Bio-Rad. 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 the
ascorbate autoxidation analysis (27). Organic solvents and all other
chemicals employed were of the highest quality available.
Site-directed Mutagenesis--
Restriction enzymes were from New
England Biolabs or Life Technologies, Inc. DNA manipulations were
performed using procedures described in Ref. 28. Complementary
oligomers were synthesized, and DNA sequence analysis was performed at
the UBC Nucleic Acid and Protein Services Unit (University of British
Columbia, Canada). Site-directed mutagenesis was performed with the
pBluescript II KS (±) vector (Stratagene, La Jolla, CA). DNA was
amplified by the polymerase chain reaction with a high fidelity
PfuTurbo® DNA polymerase (Stratagene). Point mutations were confirmed
by DNA sequence analysis prior to protein expression in bacteria. Once the sequence was confirmed, the BamHI-HindIII
fragment from the amplified DNA that also contained the mutant Mb
coding was ligated to the BamHI-HindIII fragment
from the vector pMb3 (29) to yield the final expression
vector. The circular plasmid was then transformed to the appropriate
cell line for protein expression.
Preparation of Recombinant Proteins--
Transformed
Escherichia coli (strain AR68) containing plasmids for both
the wild type recombinant human myoglobin and the C110A variant (29)
were obtained from Prof. S. G. Boxer. The DNA bearing the Y103F
mutation was obtained by site-directed mutagenesis (above) and was used
to transform E. coli DHB10 cells. For transformed bacteria
(strain AR68), cells were grown at 28 °C in 10 × 2-liter flasks containing 2YT superbroth (1-liter flask: 16 g/liter tryptone, 10 g/liter yeast extract, and 5 g/liter NaCl) to an optical density (A600 nm) of 1.2-1.5. The expression of
recombinant Mbs was induced by immersing each flask into a water bath
(55 °C) for 5 min and then transferring the flask to an incubator
operating at 42 °C. However, for the case of transformed DHB10
cells, cultures were grown at 28 °C in 10 × 2-1iter flasks
containing 2YT superbroth to A600 = ~3-4, and
then the heat shock was applied in a similar fashion to that described
for transformed AR68 cells. All cultures were subsequently incubated
for a further 6-7 h, and the cells were harvested. Rapidly increasing
the culture temperature in this manner was essential as simply
increasing incubation temperature from 28 to 42 °C failed to induce
protein expression.
Myoglobin expressed by either cell line (isolated as a fusion product)
was purified by anion exchange chromatography (Whatman DE52 resin) as
described (29). The partially purified fusion protein(s) was
reconstituted with excess hemin (heme:protein = ~1.5) (Porphyrin
Products, Logan, UT), treated with trypsin (final concentration, ~0.1
µg/ml) to cleave the fusion segment and then further purified as
described (29). Under these conditions, recombinant myoglobin was
isolated as metMb. When required, proteins were concentrated by
centrifugal ultrafiltration (Centriprep-10 concentrators, Millipore).
The yield of recombinant protein obtained from E. coli DH10B
cells was significantly lower than for the original cell line used to
express human Mb; however, we were forced to use the former because of
unavailability of the Ar68 cells. As a result of the poor yields (
2
mg of protein/liter of culture medium), several protein samples were
pooled to obtain sufficient material for our various studies.
EPR Spectroscopy--
X-band EPR spectra (293 or 77 K) were
obtained with a Bruker ESP 300e spectrometer equipped with a Hewlett
Packard microwave frequency counter. Mb solutions (~0.5
mM in 50 mM sodium phosphate buffer, pH 7.4)
were treated with H2O2
(H2O2:protein = ~1.2-5 mol/mol) in both
the presence and absence of the appropriate spin trap (spin
trap:protein = 5-150 mol/mol). For spin trapping studies with
MNP, the reagent was added from concentrated stock solutions to
minimize effects of the organic solvent (final acetonitrile concentration in reaction mixtures, <5% v/v). Analyses of spin adducts were performed using samples (50 µl) of the reaction mixture transferred into capillary tubes with a glass pipette. The capillary was then placed into a quartz EPR tube, and the tube was transferred to
the cavity for EPR analysis at 293 K. The limit of detection of a
stable nitroxide (TEMPO) under identical conditions was determined to
be ~50 nM. Unless indicated otherwise, the time between
removal of the sample, transfer to the appropriate cell, and tuning the spectrometer was consistently <30 s. EPR spectra were obtained as an
average of 3-5 scans with modulation frequency 100 kHz and sweep time
of 84 s. Microwave power, modulation amplitude, and scan range
used for each analysis varied appropriately as indicated in the legends
to the figures. Hyperfine couplings were obtained by spectral
simulation using the simplex algorithm (30) provided in the WINSIM
program, which is available at the NIEHS, National Institutes of Health
web site. All hyperfine couplings are expressed in units of mT.
Simulations were considered acceptable if they produced correlation
factors of R > 0.85. Peak areas were determined by
integration with standard WINEPR software (Bruker). Yields of the
DMPO-Cys110 and DMPO-Tyr103 adducts were
determined by peak area comparison with 5 µM TEMPO prepared in 50 mM phosphate buffer, pH 7.4, and measured
under similar experimental conditions. DTPA (100 µM) was
included in all Mb solutions prior to the addition of
H2O2 to minimize the possibility of free
transition metal-mediated decomposition of peroxide by Fenton chemistry.
Circular Dichroism Spectroscopy--
CD spectroscopy was
performed using a JASCO model J-720 spectropolarimeter calibrated with
ammonium-d-camphor-10-sulfonate as described (31). Spectra
(190-250 nm) were recorded for samples placed in a water-jacketed,
cylindrical quartz cuvette (path length, 0.1 cm), and temperature was
controlled with a NESLAB model RT 100 circulating water bath operated
under computer control. The cuvette temperature was measured using a
NESLAB RS-2 remote sensor interfaced into a computerized data
acquisition system. Protein solutions (5-10 µm) were prepared in
sodium phosphate buffer (10 mM, pH 7 and 8).
Thermal denaturation curves were determined by monitoring ellipticity
at 222 nm as temperature was increased from 25 to 85 °C
(50 °C/h). Melting temperatures (Tm) are
reported as the means ± S.D. from three measurements. Melting
curves were transformed to ASCI text files for export to Scientist
software (MicroMath, Salt Lake City, UT), which was used to calculate
the first derivative of the curve for more accurate determination of
Tm. The irreversibility of myoglobin folding
under these conditions precluded determination of thermodynamic
parameters consistent with a previous study using horse heart Mb
(31).
Electronic Absorption Spectroscopy--
Electronic spectra were
obtained with a Cary 3E UV/Vis spectrophotometer. Mb solutions were
prepared in sodium phosphate buffer (50 mM, pH 7.4), and Mb
concentration was determined spectrophotometrically (
408 nm = 188,000 M
1
cm
1) (32). The pH-linked spectroscopic transitions of
metMb were monitored between 280 and 700 nm from pH 6.3 to 10.5, and
the corresponding pKa values were determined by
fitting absorbance data obtained at 580 nm to the Henderson-Hasselbach
equation with the program Scientist (MicroMath).
Protein Cross-linking--
Cross-linking experiments were
performed by incubating solutions of either wild type human Mb or
mixtures of the C110A and Y103F variants (protein concentration,
0.025-1 mM) with H2O2 (each in 50 mM sodium phosphate buffer, pH 7.4) at a
protein:H2O2 ratio of ~5 mol/mol and
37 °C. All samples contained the iron chelator DTPA (100 µM) to minimize the possibility of Fenton-type chemistry. After 30 min, the reaction mixture was diluted with 50 mM
sodium phosphate buffer, pH 7.4 (final protein concentration, 25 µM), and each of the carefully matched samples was
diluted further with nonreducing loading buffer (protein
solution:loading buffer = ~2 v/v). Nonreducing loading buffer
was prepared by mixing 7 ml of 0.5 M Tris-Cl
containing 0.4% SDS, 3.6 ml of glycerol, 1 g of SDS, and 1.2 mg
of bromphenol blue, and finally water was added to 10 ml of final
volume. Note that loading buffer did not contain either mercaptoethanol
or dithiothreitol because these reagents can reduce the dimeric product
(25). Dilute protein samples were then heated at 90-100 °C (5 min) and analyzed by SDS-PAGE (33) after staining with Coomassie Blue.
Statistical Analyses--
Statistics were performed with
Student's t test available in MS Excel (Microsoft), and
significant difference was accepted at p < 0.05.
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RESULTS |
Electronic Absorption Spectroscopy of the Y103F Variant--
To
investigate the relationship between Tyr103-phenoxyl
radicals and the formation of the Cys110-thiyl radical, we
sought to prepare the Y103F variant of human Mb. It has been reported
previously that the point mutation Y103F in recombinant sperm whale
myoglobin destabilizes the protein sufficiently that isolation of the
variant is precluded (13). For this species of Mb, the destabilizing
influence of the Y103F substitution can be offset by introduction of a
second point mutation, K102Q (13). In contrast, the expression of the
corresponding Y103F variant of human Mb afforded a stable protein
despite the presence of Lys102 (24) in the sequence for the
human protein. In view of this difference in stability between these
mammalian proteins to the alteration at Tyr103, we have
further characterized the Y103F variant Mb by electronic absorption and
CD spectroscopy and determined the pKa for titration
of the water molecule coordinated to the ferric iron of the variant and
wild type human proteins.
The electronic spectrum obtained from solutions of the Y103F variant of
human Mb exhibited Soret (409 nm) and visible bands (
max = 505 and 631 nm) similar to those of the wild type protein (Fig.
1A), consistent with
corresponding spectra reported for the wild type and Y103F/K102Q
variant of sperm whale Mb (see Fig. 4 in Ref. 13). The CD spectra for
wild type human Mb and the Y103F variant of human Mb (10 mM
phosphate buffer, pH 8) were also virtually identical (Fig.
1B) and indicated that the two proteins possess similar
helical content at pH 7 (not shown). The melting curves for each
protein afforded similar Tm values at pH 8:
Tm = 77.3 ± 0.5 and 76 ± 1 °C
(data represent means ± S.D., n = 3) for wild
type Mb and the Y103F variant, respectively (Fig. 1B,
inset). Melting curves determined for the two proteins at pH
7 (10 mM phosphate buffer) also afforded similar
Tm values: Tm = 80 ± 1 and 78.1 ± 0.2 °C for wild type human and Y103F variant proteins, respectively (not shown). These Tm
values were not significantly different independent of the pH of the
buffer employed as judged by t test analyses (p
0.05).

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Fig. 1.
Electronic absorbance and CD spectra of wild
type and Y103F variant Mb. A, electronic spectra were
obtained for wild type human Mb (solid line) and the
Y103F variant (broken line) (~5 µM; 50 mM phosphate buffer, pH 7.4). The inset shows
the Henderson-Hasselbach plots for wild type Mb (solid line)
and the Y103F variant (broken line) of human Mb (50 µM) over the pH range 6.3-10.5. B, CD spectra
were obtained with solutions of wild type (solid line) and
Y103F (broken line) Mb (10 µM; 10 mM phosphate buffer, pH 8) at 20 °C. The
inset shows corresponding first derivative melting curves
from the wild type (solid line) and Y103F (broken
line) Mb monitored over 25-85 °C. Results represent three or
more independent experiments.
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On the other hand, the pKa determined for the Y103F
variant was significantly lower (p < 0.05) than that
of the wild type protein (8.26 ± 0.09 and 8.85 ± 0.03, respectively, mean ± SD, n = 3) (Fig.
1A, inset). This difference in
pKa indicates that the Y103F point mutation probably
induces some subtle change in the heme binding site that alters the
binding of the water ligand. The crystal structure of a related
(K45R/C110A) variant of human Mb (24) indicates that Tyr103
resides near the surface of the protein and is the tyrosine residue nearest the heme group as is also the case for sperm whale Mb (34). For
the human protein, the point of closest approach is a phenyl carbon
that is ~3.44 Å from the edge of the heme group. Conceivably, a
minor reorganization of the heme pocket that is not detected by
electronic spectroscopy or by visible CD spectroscopy affords a lower
pKa for the acid-base transition in the Y103F
protein. Regardless of the basis for the difference in this pKa value, it is clear that the thermal stability
and the electronic absorbance characteristics of the Y103F variant are
comparable with that of the wild type protein.
Spin Trapping of Globin· in Wild Type Mb and the C110A and
Y103F Variant Proteins--
We next investigated the formation of
globin· in the reactions of wild type human Mb and both the
C110A and Y103F variants with H2O2 using the
spin traps DMPO and MNP. Addition of H2O2 to
solutions of wild type human Mb containing DMPO at DMPO:protein
10 mol/mol consistently produced a product with an EPR spectrum comprised of a broad four-line signal (Fig.
2A, solid line).
This broad signal was simulated well (Fig. 2A, broken
line) with hyperfine data previously reported for the trapping of
thiyl radicals on human Mb at Cys110
(DMPO-Cys110) (25). The treatment of the C110A variant of
human Mb with peroxide in the presence of DMPO under identical
conditions, however, consistently gave a more complex EPR spectrum
(Fig. 2B, solid line). This complex signal was
also detected in the corresponding reaction of horse heart Mb and
H2O2 (Fig. 2C) where the radical has
been assigned previously as resulting from the DMPO adduct of a
Tyr103-phenoxyl radical (DMPO-Tyr103) (20).
Thus, the DMPO adduct derived from the reaction of C110A variant of
human Mb and H2O2 was readily simulated (20)
with the reported coupling values (Fig. 2B, broken
line), and the globin· was assigned as a
DMPO-Tyr103 adduct. These data support the conclusion that
Cys110-thiyl radicals are thermodynamically stable end
products following reaction of the wild type protein with hydrogen
peroxide, whereas for Mb lacking Cys110, the
Tyr103-phenoxyl radical is the most stable globin·
produced.

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Fig. 2.
Trapping globin radicals on wild type human
Mb, C110A Mb and horse heart Mb with DMPO. Mb (0.5 mM
protein in 50 mM sodium phosphate buffer, pH 7.4) was mixed
with H2O2 (five equivalents) and DMPO (2.5 mM). After 2 min at 20 °C, EPR spectra were recorded for
wild type Mb (A, solid line); C110A variant Mb
(B, solid line); and horse heart Mb
(C). EPR simulations were performed with WINSIM software
(30) and afforded good fits to the data, r = 0.86 and
0.94 for the C110A and wild type proteins (A and
B, broken lines), respectively. EPR parameters:
microwave power, 5 mW; modulation amplitude, 0.1 mT; modulation
frequency, 100 kHz; sweep, 84 s. All protein solutions contained
DTPA (100 µM). Data represent three or more experiments
using different Mb preparations.
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In marked contrast to the analogous reaction with wild type human Mb,
no radical adducts were detected upon addition of
H2O2 to a solutions containing the Y103F
variant and DMPO (Y103F:H2O2:DMPO 1:5:
10
mol/mol/mol) (Fig. 3A).
Furthermore, at the highest trap efficiency investigated
(DMPO:H2O2:Y103F 1:5:~100 mol/mol/mol), only
a weak four-line EPR spectrum was obtained, and this spectrum exhibited
hyperfine coupling identical to that for the DMPO adduct derived from
trapping DMPO-Cys110 radicals (Fig. 3B). Under
these conditions, the corresponding reaction of wild type human Mb and
H2O2 gave both DMPO-Cys110 and
DMPO-Tyr103 adducts (e.g., see Fig. 2). Overall,
the concentrations of DMPO-Cys110 radicals measured from
the wild type and Y103F variant proteins varied by ~130-fold as
determined by double integration of the respective
DMPO-Cys110 EPR signals from the two proteins.
Collectively, these data independently confirm the alteration at
Tyr103 in the Y013F variant.

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Fig. 3.
Reaction of the Y103F variant Mb and
H2O2 in the presence of DMPO does not yield
significant concentrations of radical adducts. The Y103F variant
(0.5 mM protein in 50 mM sodium phosphate
buffer, pH 7.4) was mixed with H2O2 (five
equivalents) in the presence of DMPO. After 2 min at 20 °C, EPR
spectra were recorded for samples with DMPO:protein ratios of ~5
mol/mol (A) and ~100 mol/mol (B). EPR
parameters were as for Fig. 2. The EPR spectra are plotted with a
4-fold lower intensity range on the vertical axis than
spectra shown in Fig. 2. DTPA (100 µM) was present in all
reaction mixtures. Results represent three or more independent
experiments with different Mb preparations.
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Radical adducts of protein-derived tyrosine phenoxyl radicals have also
been reported to form with the nitroso spin trap MNP (20, 35, 36).
Therefore, to verify that replacement of Tyr103 resulted in
the loss of the Tyr103-phenoxyl radical, MNP was employed
in reactions of both the wild type and Y103F variant Mb with
H2O2 (Mb:H2O2:MNP
1:5:20 mol/mol/mol). For the case of wild type human Mb, reaction
mixtures treated with MNP afforded a radical with a three-line EPR
spectrum after 2 min at 20 °C (Fig.
4A). The signal was relatively
broad with the outer most line at lower g value broadened
almost to base line. The broad nature of the EPR signal is consistent
with a radical exhibiting restricted rotational motion on the EPR time scale and indicative of MNP trapping a radical on a large molecule (i.e. a protein). No EPR signal was detected in the absence
of protein, MNP, or H2O2 (Fig. 4,
B-D, respectively).

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Fig. 4.
Trapping globin radicals on wild type and
Y103F human Mb proteins with MNP. Wild type Mb (0.5 mM
protein in 50 mM sodium phosphate buffer, pH 7.4) was mixed
with H2O2 (five equivalents) and MNP (20 mM). After 2 min at 20 °C, EPR spectra were recorded for
the complete system (A); same as in A but in the
absence of protein (B); same as in A but in the
absence of MNP (C); same as in A but in the
absence of H2O2 (D); and same as in
A after incubation with "Pronase" (1000 units/ml,
E). F, treatment of the Y103F Mb under identical
conditions as for A did not yield a detectable MNP radical
adduct. EPR parameters were as for Fig. 2. Results represent three or
more independent experiments using pooled protein.
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Addition of the nonspecific protease "Pronase" (1000 units/ml) to
reaction mixtures containing the MNP adduct resulted in the digestion
of the protein into small peptide fragments. Subsequent analyses of the
digest by EPR yielded a spectrum with a sharp triplet signal (Fig.
4E). Hyperfine couplings were obtained by spectral
simulation to high correlations (r = 0.97, not shown) using aN = 14.3 ± 0.1 mT,
a
H = 10.6 ± 0.2 mT, and
a
H = 1.2 ± 0.2 mT. These
hyperfine couplings are identical to that determined previously (20,
35) for MNP adducts of Tyr103-phenoxyl radicals on horse
heart Mb. On this basis, the radical detected here with wild type human
Mb was also assigned as the MNP adduct of the
Tyr103-phenoxyl radical. In contrast, the corresponding
reaction of the Y103F variant of Mb and H2O2
failed to produce a detectable MNP adduct under identical reaction
conditions (Fig. 4F). Higher mol ratios of MNP:protein
(
100 mol/mol) also failed to yield a detectable EPR signal (not
shown). These latter results independently verify that no tyrosyl
radical that is accessible to water-soluble spin traps forms upon
reaction of the Y103F Mb and peroxide.
DMPO Concentration-dependent Changes in the
Distribution of DMPO-globin· Adducts--
At higher DMPO
trapping efficiencies, both DMPO-Cys110 and
DMPO-Tyr103 radical adducts could be detected in the
reaction of wild type human Mb and peroxide (25). These results are
consistent with the idea that these different globin· may be
related by an electron transfer reaction. To investigate the putative
electron transfer between Cys110 and Tyr103, we
tested the effect of systematically increasing DMPO:protein ratios on
the distribution of globin· adducts derived from the
reaction of wild type human Mb and H2O2.
In agreement with our previous study (25), treatment of the
globin· formed by reaction of wild type Mb and
H2O2 with DMPO (over DMPO:protein ranging
25-150 mol/mol) resulted in distinct changes in both the type and the
yield of DMPO adduct detected by EPR spectroscopy (Figs
5). These changes are attributed to the
increasingly efficient trapping of the Tyr103-phenoxyl
radical (25). Computer simulation of the data obtained at various mol
ratios of DMPO: Mb (e.g., see Fig. 2B,
broken lines) coupled with double integration (37, 38)
afforded the relative mol fraction of DMPO-Cys110 or
DMPO-Tyr103 adducts in the composite EPR spectrum for a
given DMPO concentration (Fig. 6).
Maximum concentrations of DMPO-Cys110 and
DMPO-Tyr103 were determined under experimental conditions
that provided the EPR signal of each in the absence of the other to
define a ratio for
(DMPO-Tyr103)max/(DMPO-Cys110)max
of 1.18 ± 0.25 (means ± S.D., n = 3).
Therefore, the DMPO-dependent change in product
distribution is consistent with a near 1:1 stoichiometric relationship
between DMPO-Tyr103 and DMPO-Cys110 radicals
and is further support for the idea that the thiyl radical is formed by
transfer of an electron from Cys110 to the
Tyr103-phenoxyl radical.

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Fig. 5.
Trapping of DMPO-Tyr103 and
DMPO-Cys110 adducts from reactions of wild type human Mb
and H2O2. Wild type Mb (0.5 mM
protein in 50 mM sodium phosphate buffer, pH 7.4) was mixed
with H2O2 (five equivalents) and increasing
concentration of DMPO. After 2 min of reaction at 20 °C, EPR spectra
were recorded for samples with DMPO:protein ratios of 5 mol/mol
(A); 20 mol/mol (B); 50 mol/mol (C);
and 100 mol/mol (D). EPR parameters were as for Fig. 2. DTPA
(100 µm) was present in all reaction mixtures. Results represent
three or more experiments with different preparations of human
Mb.
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Fig. 6.
DMPO-dependent changes to the
relative mol fraction of DMPO-Cys110 and
DMPO-Tyr103 in composite EPR spectra from the reaction of
wild type Mb and H2O2. Relative
contributions of the DMPO-Cys110 (squares) and
DMPO-Tyr103 (circles) adducts were determined
from composite spectra by simulation (30) and double integration and
are plotted as the relative mol fraction as a function of the
DMPO:protein ratio. Peroxide was maintained at
H2O2:protein ~5 mol/mol, and wild type Mb was
0.5 mM for all DMPO concentrations investigated. The
absolute concentrations of DMPO adduct were determined to be 0.26 ± 0.02 (mean ± S.D.; three independent measurements) and
0.22 ± 0.03 spins/mol Mb protein for the DMPO-Tyr103
and DMPO-Cys110 adducts, respectively.
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SDS-PAGE Analyses of Reactions of Wild type and Y103F Variant
Proteins and H2O2--
The evidence obtained
from spin trapping studies (above) were consistent with the transfer of
an electron from Cys110 to a Tyr103-phenoxyl
radical that is formed as a result of the reaction of wild type human
Mb and H2O2. This electron transfer reaction
may be intramolecular or intermolecular or both. To evaluate this issue, we studied the dependence of intermolecular disulfide bond formation that results from the reaction of wild type Mb with peroxide
on the concentration of protein. In this experiment, wild type human Mb
(25-1000 µM) was incubated at 37 °C in either the
absence or presence of H2O2 (at a constant
ratio of H2O2:protein ~5:1 mol/mol). After 30 min (a time previously determined to be optimal for formation of the
disulfide dimer (25)), reaction mixtures were diluted with 50 mM phosphate buffer, pH 7.4, so that the final total
protein concentrations of all the samples were carefully matched prior
to gel electrophoresis. Subsequent SDS-PAGE analyses of the various
reaction mixtures showed that two protein bands were present in samples
treated with H2O2
for each of the protein concentrations investigated (Fig. 7).
Comparison with molecular mass markers indicated that these
species have masses of ~34 and 17 kDa, so these bands were assigned
as the dimeric product and monomeric wild type proteins, respectively (25). Overall, the intensity of the band corresponding to the dimeric
product increased in samples that initially contained greater protein
concentration (Fig. 7, lanes 2-6). No dimer product was
detected in the absence of H2O2 (Fig. 7,
lane 7).

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Fig. 7.
Reaction of wild type human Mb and
H2O2 affords a cross-linked dimer that
increases in yield with increasing protein concentration. Human Mb
(25-1000 µM) was treated with
H2O2 (at H2O2:protein 5 mol/mol for all protein concentrations investigated). After 30 min at
37 °C all reaction mixtures were diluted to 25 µM in
protein using 50 mM phosphate buffer, pH 7.4, and the
carefully matched samples were analyzed by SDS-PAGE. Lane 1,
protein standard; lanes 2-6, human Mb initially at 25 (lane 2), 50 (lane 3), 250 (lane 4),
500 (lane 5), and 1000 µM (lane 6),
respectively. Lane 7 shows control human Mb incubated in the
absence of H2O2.
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In contrast to the reaction of wild type human Mb and
H2O2, reaction of the Y103F variant and
H2O2 failed to yield a dimeric product even
after incubation with peroxide for up to 60 min at 37 °C (Fig.
8A). These data are consistent
with a critical role for Tyr103 in the formation of
Cys110-thiyl radicals (i.e. one of the
intermediate radicals that is formed prior to bimolecular disulfide
bond formation). If the reactions leading to formation of the
Cys110-thiyl radical were solely intermolecular processes,
then such a mechanism would be expected to result in dependence on the
concentration of target protein. Therefore, we considered that the
protein-dependent increase in dimeric product (Fig. 7) was
indicative of an intermolecular electron transfer event that yielded
the Cys110-thiyl radical. To test whether
Cys110-thiyl radical formation involved intermolecular
electron transfer to reduce the phenoxyl radical at Tyr103,
we investigated the reaction of a mixture of Y103F (lacking Tyr103 but with Cys110) and C110A (lacking
Cys110 but containing Tyr103) variant proteins
with H2O2 (at
C110A:Y103F:H2O2 ~2:1:5 mol/mol/mol). Despite
the inability of either protein to form an intermolecular disulfide
bond upon treatment with H2O2 individually (see
Fig. 8A and also Fig. 7B in Ref. 25), treatment
of the mixture of variants with H2O2 afforded
the dimeric product (Fig. 8B). Overall, the yield of dimer
obtained from the mixture of C110A/Y103F Mb was similar to that from
the wild type protein treated with peroxide under identical conditions.
Interestingly, the yield of dimeric product could be increased in
independent studies by changing the ratio of C110A:Y103F. For example,
increasing the content of C110A from 0.5 to 3 mol equivalents resulted
in an increased intensity of the protein bands assigned as the dimeric
product. These latter data indicate that the concentration of
Tyr103-phenoxyl radical in the C110A variant was important
in generating the Cys110-thiyl radical in the Y103F variant
and, in turn, in determining the overall yield of disulfide homodimer
product (not shown).

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Fig. 8.
Treatment of Y103F Mb with
H2O2 does not produce a disulfide cross-linked
product unless C110A Mb is also present in the reaction mixture.
Y103F Mb was incubated with H2O2
(H2O2:total protein = ~5 mol/mol) in
either the absence (A) or presence (B) of C110A
Mb at C110A:Y103F ~2 mol/mol. A, lane 1, mass
standard; lanes 2 and 3, authentic wild type
protein before and after incubation with H2O2,
respectively; lanes 4-8, Y103F variant and
H2O2 after incubation at 37 °C for periods
of 0 (lane 4), 5 (lane 5), 15 (lane
6), 30 (lane 7) and 60 min (lane 8),
respectively. B, lane 1, mass standard;
lane 2, protein before H2O2
addition; lanes 3-6, reaction mixture after incubation at
37 °C for 5 (lane 3), 15 (lane 4), 30 (lane 5), and 60 min (lane 6),
respectively. Lane 7 shows authentic disulfide
product from reaction of wild type human Mb and peroxide. Note the
protein standard used in B differs from that in
A.
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DISCUSSION |
Formation of ferryl-heme and a protein-centered radical
(globin·) upon reaction of metMb with
H2O2 is well established (see the Introduction). Recently (25), we investigated the reaction of human Mb
and its C110A variant to evaluate the role of the unique sulfhydryl
group in the reaction of this protein with H2O2
and demonstrated by low temperature EPR spectroscopy that the human protein undergoes radical formation at Trp14,
Tyr103, and Cys110 in this reaction. The spin
trapping studies that are reported here using both the C110A and the
Y103F variants of human Mb independently confirm the assignment of the
Tyr103-phenoxyl and Cys110-thiyl radicals on
the wild type human protein. Additionally, comparison of the reactions
of C110A and Y103F Mb with H2O2 afforded a
useful means of evaluating the precise mechanism of
Cys110-thiyl radical generation in the reaction of peroxide
with wild type human Mb.
Identifying Key Residues Involved in globin· Formation on
Wild type Human Mb--
Spin trapping studies with the spin trap DMPO
showed that the Cys110-thiyl radical is trapped exclusively
using relatively low DMPO:Mb ratios (<10 mol/mol), indicating that
this radical is thermodynamically favored under these conditions
(i.e. Cys110 acts as a radical sink).
Conversely, our observations that the Tyr103-phenoxyl
radical is detected in the absence of all other DMPO-globin adducts at
high DMPO trapping efficiency (DMPO:protein >100 mol/mol), where the
rates of secondary reactions are overcome by the kinetics of the
trapping reaction, supports the argument that a
Tyr103-derived globin· is formed before a radical
center is generated at position Cys110. The observation of
a near 1:1 stoichiometry for the yield of DMPO-Tyr103 and
DMPO-Cys110 radical adducts with increasing mol ratios of
DMPO: Mb indicates that electron transfer occurs from
Cys110 to Tyr103.
Mechanism of Electron Transfer Between Tyr103 and
Cys110 Residues in Human Mb--
Having established that
tyrosyl phenoxyl and thiyl radicals form sequentially on the globin
portion of wild type Mb and that these radicals are generated through
an electron transfer process, we next determined whether this process
occurs through an intra- or intermolecular mechanism. Because the
three-dimensional structure of wild type human Mb has not been
reported, the best alternative structure that is available for
estimating distances between structural elements of this protein is the
structure of the K45R/C110A variant of this protein (24). In the
structure of this variant determined at 2.8 Å resolution,
Tyr103 is the tyrosine residue nearest the heme group, and
the shortest distance separating these two group is ~3.4 Å. This
distance is similar to the corresponding distance in both sperm whale
(13, 34) and horse heart (39) Mb. In the human protein, however, the
sulfhydryl group of Cys110 is expected to be ~9-10 Å from the heme edge, and the distance between the Cys110 and
Tyr103 residues is ~12 Å (24).
Formation of Tyr103-phenoxyl radicals prior to formation of
Cys110-thiyl radicals is consistent with the proximity of
Tyr103 to the heme group. The relatively large distance
separating the Tyr103 and Cys110 residues
decreases the efficiency of intramolecular electron transfer between
these residues, although long range intramolecular through-bond
processes are known to occur in heme-proteins and therefore cannot be
excluded (40). Interestingly, tyrosyl phenoxyl radicals (generated from
reaction of horseradish peroxidase, H2O2, and
free tyrosine) have been demonstrated to oxidize thiols to thiyl
radicals by an intermolecular mechanism (41). Therefore, it is not
inconceivable that relatively rapid intermolecular electron transfer
from Cys110 (on one protein molecule) to
Tyr103-phenoxyl radical (on a second protein molecule)
results in the formation of the thiyl radical at Cys110 of
human Mb. To evaluate this possibility, we studied formation of the
dimeric product from reactions of human Mb and peroxide using
SDS-PAGE.
The accumulation of the disulfide homodimer in reactions of human metMb
and H2O2 follows the simplified mechanism
outlined in Reactions 1-R4.
Human metMb reacts with hydrogen peroxide to yield a ferryl heme
(Fe(IV)-oxo) porphyrin radical cation
((Globin-Fe(IV)=O)·+) that is related to the
compound I formed by peroxidases (Reaction 1). Formation of
Tyr103-phenoxyl radicals may then result from an
intramolecular electron transfer reaction (rate constant
ket1) in which the porphyrin radical cation is
reduced by Tyr103 as shown in Reaction 2 (13). The
resulting Tyr103-phenoxyl radical is the initial radical
trapped by DMPO on wild type human Mb at high DMPO:protein ratios.
Subsequently, a second electron transfer reaction (rate constant
ket2) affords the Cys110-thiyl
radical and regenerates the Mb protein (Reaction 3). Finally, Cys110-thiyl radicals react in a bimolecular reaction to
generate the disulfide homodimer (Reaction 4).
Varying the protein concentration may affect the yield of the homodimer
in two ways. First, if formation of the Cys110-thiyl
radical involves an intermolecular process(es), then the yield of this
radical and the disulfide-linked homodimer should be directly dependent
on protein concentration. That is, the yield of the homodimer is
related to the efficiency of the intermolecular electron transfer from
the sulfhydryl group at Cys110 to the tyrosyl phenoxyl
radical at Tyr103 (Reaction 4). At higher protein
concentration, this intermolecular process would occur with greater
efficiency to generate a higher concentration of
Cys110-thiyl radicals that in turn generates greater
concentrations of homodimer. Overall, the rate of accumulation of the
homodimer is proportional to both the concentration of
Cys110-thiyl radical and the absolute concentration of
protein as described by the process shown (Reaction 4). Alternatively,
if the mechanism for Cys110-thiyl radical formation
involves intramolecular reduction of the Tyr103-phenoxyl
radical by Cys110, then the yield of
Cys110-thiyl radical and the overall protein-standardized
yield of homodimer would be independent of protein concentration.
Therefore, the increased yield of disulfide dimer observed with
increasing concentration of human Mb strongly supports the argument
that Cys110-thiyl radical formation (Reaction 3) involves
an intermolecular electron transfer reaction. That the disulfide dimer
is detected in a reaction mixture of the Y103F and C110A variants and
H2O2 confirms that Cys110-thiyl
radicals are generated on the Y103F variant by reaction with
Tyr103-phenoxyl radicals on a molecule of the C110A variant
and is further evidence to support the occurrence of an intermolecular
electron transfer reaction. Furthermore, the dependence of homodimer
yield on the initial concentration of C110A in the mixed variant
reactions with peroxide (data not shown) emphasizes the role of the
Tyr103-phenoxyl radical in generating
Cys110-thiyl radicals by an intermolecular reaction. That
is, the concentration of Tyr103-phenoxyl radical in the
mixture is dependent on the starting concentration of C110A variant,
and increasing this concentration results in more efficient generation
of the Cys110-thiyl radical on the Y103F variant present in
the reaction mixture that in turn yields higher concentrations of
disulfide dimer.
Alternate Mechanisms for Cys110-thiyl Radical Formation
in Human Mb--
Direct oxidation of the sulfhydryl group at
Cys110 in the wild type protein by peroxide might produce
Cys110-thiyl radicals (i.e. a one-electron
oxidation product), but thiol oxidation in this manner conflicts with
the expectation that H2O2 acts as a potent
two-electron oxidant. Furthermore, as evidence against this potential
mechanism of direct Cys110-thiyl radical generation,
incubation of Y103F (a protein that contains Cys110) with
H2O2 gave only trace concentrations of
DMPO-Cys110 and no detectable disulfide product (Figs. 3
and 8A, respectively). Interestingly, cysteine residues
within a protein may be oxidized to the sulfenic acid
(RS-OH) by added peroxide, and this reaction may be reversed
in the presence of suitable reductants (42, 43). Conversion of cysteine
to the corresponding sulfenic acid in this manner does not involve
thiyl radical formation. In addition to Tyr103-phenoxyl
radicals, Trp14-derived peroxyl radicals are also formed on
wild type human Mb (25) and other mammalian Mbs (20).
Trp14-peroxyl radicals are reportedly (20, 44) capable of
reacting with a wide range of substrates (including other free amino
acids). Therefore, another possible mechanism that may result in
Cys110-thiyl radical generation on wild type human Mb is
through reduction of Trp14-derived radicals by the
sulfhydryl at Cys110 (again via either an intra- or
intermolecular pathway). However, any potential electron transfer
between Trp14 and Cys110 can be ruled out in
reactions of wild type human Mb and H2O2 by way
of a similar argument against direct
H2O2-mediated oxidation of Cys110.
That is, despite the presence of Trp14 and
Cys110 in the Y103F variant, reaction of this protein with
H2O2 failed to yield either a significant
concentration of DMPO-Cys110 or detectable quantities of
disulfide-coupled Mb homodimer. Thus, it is unlikely that
Cys110 can reduce a Trp14-centered radical.
Another means by which protein thiol groups may be oxidized is through
direct oxidation of the sulfhydryl by compound I in a manner similar to
peroxidase-catalyzed mono-oxygenase reactions of the P450 class of
heme-proteins (45). However, by analogy to the reaction of compound I
of Mb and organic sulfides to yield sulfoxides (46, 47), oxygenation
reactions involve direct oxygen transfer that does not generate thiyl
radicals. Yet another oxidant remaining in reaction mixtures of Mb and
H2O2 that is also capable of oxidizing a
variety substrates (48, 49) is the ferryl (Fe(IV)=O) heme center. For
example reaction mixtures of horse heart Mb and
H2O2 reportedly show a lipoxygenase-like activity that leads to peroxidation of low molecular mass
polyunsaturated lipids (49). However, a substrate that is oxidized
directly by ferryl (Fe(IV)=O) heme is limited by the requirement that
it must gain sufficient proximity to the heme binding site to permit reaction with the activated oxygen and then leave the heme cavity as an
oxygenated product (49). Although a thiyl radical would not be expected
to form in this manner, it it conceivable that a sulfoxide group could
be produced in this two-electron oxidation process. Additionally, if
electron transfer occurred between the edge of ferryl heme and
Cys110, then treatment of the Y103F variant with
H2O2 would be expected to yield significant
amounts of DMPO-Cys110 and detectable homodimer, which is
not the case.
One final alternate explanation for our data is that the rate of the
reaction leading to the formation of the homodimer is dependent on the
half-life of the Cys110-thiyl radical. At lower protein
concentrations, Cys110-thiyl radicals may decompose before
taking part in the bimolecular termination reaction described by
reaction (4), whereas at higher protein concentration this may not be
the case. This dependence is difficult to substantiate, and pathways
for the decay of Cys110-thiyl radicals (other than Reaction
4) have not been identified. Therefore, the dependence of homodimer
formation on the half-life of Cys110-thiyl radicals remains
a plausible explanation for our data.
Importantly, we reiterate that hydroxyl radicals do not appear to be
involved in the transfer of oxidative damage to the protein (22, 23) at
least under the experimental conditions employed here. In any case, the
risk of hydroxyl radical formation in the current study has been
prevented by the use of the iron chelator DTPA throughout.
In summary, we have investigated the reactions of wild type human Mb
and both the C110A and Y103F variants to evaluate the mechanism of
Cys110-thiyl radical formation. Our studies outlined in
this work indicate that the mechanism leading to
Cys110-thiyl radical formation involves intermolecular
electron transfer. In contrast, alternate mechanisms of thiol oxidation
including direct oxygenation of the protein-thiol do not lead to thiyl
radical production. Overall, we have combined use of site-directed
mutagenesis, SDS-PAGE, and EPR spectroscopy to demonstrate that the
Cys110-thiyl radical can result from an intermolecular
electron transfer reaction in which the Tyr103-phenoxyl
radical is reduced by the sulfhydryl group at Cys110.