ACCELERATED PUBLICATION
Oxidative Modification of Tryptophan 43 in the Heme Vicinity of
the F43W/H64L Myoglobin Mutant*
Isao
Hara
,
Takafumi
Ueno§¶,
Shin-ichi
Ozaki
,
Shinobu
Itoh**,
Keonil
Lee,
Norikazu
Ueyama, and
Yoshihito
Watanabe§¶§§
From the Department of Structural Molecular Science,
The Graduate University for Advanced Studies, Myodaiji, Okazaki
444-8585, Japan, the § Center for Integrative Bioscience,
Myodaiji, Okazaki 444-8585, Japan, the Faculty of Education,
Yamagata University, Kojirakawa, Yamagata 990-8560, Japan, the
** Department of Chemistry, Graduate School of Science, Osaka City
University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan, the
Department of Macromolecular Science,
Graduate School of Science, Osaka University, Machikaneyama 1-1, Toyonaka, Osaka 560-0043, Japan, and the ¶ Institute for
Molecular Science, Myodaiji, Okazaki 444-8585, Japan
Received for publication, July 2, 2001
 |
ABSTRACT |
The F43W/H64L myoglobin mutant
was previously constructed to investigate the effects of electron-rich
tryptophan residue in the heme vicinity on the catalysis, where we
found that Trp-43 in the mutant was oxidatively modified in the
reaction with m-chloroperbenzoic acid (mCPBA).
To identify the exact structure of the modified tryptophan in this
study, the mCPBA-treated F43W/H64L mutant has been digested
stepwise with Lys-C achromobacter and trypsin to isolate two oxidation
products by preparative fast protein liquid chromatography. The close
examinations of the 1H NMR spectra of peptide fragments
reveal that two forms of the modified tryptophan must have
2,6-disubstituted indole substructures. The 13C NMR
analysis suggests that one of the modified tryptophan bears a unique
hydroxyl group in stead of the NH2 group at the
amino-terminal. The results together with mass spectrometry (MS)/MS
analysis (30 Da increase in mass of Trp-43) indicate that oxidation
products of Trp-43 are 2,6-dihydro-2,6-dioxoindole and
2,6-dihydro-2-imino-6-oxoindole derivatives. Our finding is the first
example of the oxidation of aromatic carbons by the myoglobin mutant system.
| |
INTRODUCTION |
Myoglobin (Mb),1 a
carrier of molecular oxygen, can perform oxidation reactions in the
presence of hydrogen peroxide (H2O2), although
the activity is not as great as that of peroxidase (1-3). The
accumulated biochemical and biophysical data allow us to utilize Mb as
a heme enzyme model system, and various myoglobin mutants have been
constructed to elucidate structure-function relationship on the
activation of peroxides (3-5). For example, F43H/H64L Mb, one of the
distal histidine relocation mutants, exhibits the enhanced reactivity
with H2O2 and the longer lifetime of an active intermediate, a ferryl porphyrin radical cation
(O=FeIVporphyrin·+) (6). Therefore as
the results, the F43H/H64L mutant is able to catalyze the
sulfoxidation and epoxidation reaction at the rate comparable with the
values of peroxidases.
On the other hand, cytochromes P-450 (P-450) catalyze the hydroxylation
of a wide variety of substrates, including hydrocarbons and polycyclic
aromatic molecules (7, 8). The variance in reactivity of Mb and P-450
could arise from differences in the active site structure and the
arrangement of functional amino acid residues. The crystal structure of
P-450cam with d-camphor reveals that the substrate is
tightly bound in the hydrophobic heme pocket through hydrogen bonding
interaction with the hydroxyl group of Tyr-96 and the carbonyl oxygen
of d-camphor (Fig.
1A) (9). The distance between
the heme iron and C5 of d-camphor, the hydroxylation site,
is 4.2 Å. On the contrary, the active site of myoglobin is exposed to
the exterior and does not provide any specific interactions for
accommodating a foreign substrate with high affinity (10). Therefore,
it will be difficult for a ferryl porphyrin radical cation of Mb to
hydroxylate a substrate molecule, which is not bound in an appropriate
position nearby the heme. We hypothesize that a ferryl oxygen atom
transfer to aliphatic or aromatic molecules by Mb mutants might be
possible even without a proximal thiolate ligand if the substrates were fixed nearby the heme iron.
The F43W/H64L Mb mutant, which was previously constructed to
investigate effects of an electron-rich tryptophan residue on the
peroxygenase activity, appears to be a good model for examining the
hypothesis, because aromatic carbon atoms of tryptophan are fixed in
the heme vicinity (Fig. 1B) (12). Although the crystal structure of the F43W/H64L Mb is not available at the moment, the
calculated model structure suggests that the distances of Fe-C7 and
Fe-C6 are 4.9 and 5.5 Å, respectively. (A calculated structure
of F43W/H64L Mb was obtained by using the Insight II molecular modeling
program (Biosym MSI, San Diego, CA). The Trp-43 in the mutant is
generated by replacing His-43 of the F43H/H64L Mb (6) with a tryptophan
residue and minimizing the energy of the heme pocket.) The predicted
values are similar to the distance between C5 of d-camphor
and iron in P-450cam. Our earlier studies provided preliminary evidence
that Trp-43 in F43W/H64L Mb was oxidatively modified in the reaction
with m-chloroperbenzoic acid (mCPBA); however,
the exact structure of the modified product(s) remains to be elucidated
(12). The subject of this study is to identify the oxidized tryptophan
residue to determine whether or not the F43W/H64L Mb mutant is capable
of performing the oxidation of aromatic molecules.
| |
EXPERIMENTAL PROCEDURES |
The Oxidation of Trp-43 and the Isolation of Peptide Fragments
Bearing Trp-43--
F43W/H64L Mb (0.2 mM) in 50 mM potassium phosphate buffer at pH 7.4 was mixed with 4 equivalents of mCPBA at 4 °C. The modified protein (500 mg) was digested with Lys-C (1/100 w/w) in 100 mM Tris-HCl
buffer at pH 9.0 containing 2 M urea, and the mixture was
incubated at 25 °C for 24 h. The digestion was stopped by the
addition of trifluoroacetic acid (final concentration 1%). The
products were analyzed on an Äkta FPLC system (Amersham
Pharmacia Biotech) with a Vydac C-18 reverse phase column eluted
typically at a flow of 1.5 ml/min with a gradient of solvent A
(0.1% trifluoroacetic acid in water) into solvent B (20% acetonitrile
and 0.1% trifluoroacetic acid in water) over 200 min. The eluent was
monitored either at 280 nm for aromatic residues or 215 nm for amide
bonds in peptide fragments. The isolated fragments A and
B, which have been identified as modified Trp-43
linked with unmodified Asp-44-Arg-45-Phe-46-Lys-47 (W*DRFK in which the
modified tryptophan is designated as W*) (12), were further treated
with trypsin (~1/100 w/w) in 100 mM Tris-HCl buffer (pH
9.0) at 30 °C for 12 h and analyzed by the Äkta
FPLC system. Although A was not cleaved by trypsin,
B was digested to afford a peptide fragment B'
(W*DR). In a control experiment, the intact F43W/H64L mutant (250 mg)
was treated with Lys-C and trypsin, and the peptide fragment bearing
unmodified Trp-43 was isolated by preparative FPLC.
MS/MS Analysis of the Peptide Fragments--
The fragments
A and B isolated after Lys-C digestion were
directly analyzed on a Voyager DESTR (PerSeptive Biosystems) for
MALDI-TOF-MS. The spectrometer was calibrated with an angiotensin II
(molecular weight = 1046.2). 1 µl of the digested samples
containing ~10 pmol/µl were mixed with 1 µl of saturated
-cyano-4-hydroxycinnamic acid in water/acetonitrile (1:1) and
applied to the sample plate by the dried-droplet method.
NMR Spectroscopy--
1H and 13C NMR
spectra of A, B', and an intact peptide fragment
(Trp-43-Asp-44-Arg-45) were obtained either on a Unity Inova 600 MHz or on a Unity plus 600 MHz (Varian). 1H and
13C NMR measurements were undertaken in 20 mM
potassium phosphate buffer (pD 7.0) in D2O solution at
25 °C. 3-(Trimethylsilyl)propionic-2,2,3,3-d4 (TSP-d4) was used as an internal reference for
proton resonances. Complete proton resonance assignments were made
using DQF-COSY (13), TOCSY (14, 15), and ROESY (16) experiments. The ROE intensities of A and B' were obtained at 500 and 600 ms mixing time, respectively.
Amino Acid Sequence Analysis--
Amino acid sequences were
analyzed on a Protein Sequencer (Applied Biosystems, model Procise
494). The purified intact peptides (WDR) and the modified fragments
A and B' (~10 pmol) were analyzed on a Protein
Sequencer (Applied Biosystems, model Procise 494) by the Edman method.
Amino Acid Composition Analysis--
Amino acid composition was
analyzed on a High Speed Amino Acid Analyzer (Hitachi, model
L-8500A) to confirm the modification site of A. The fragment
A (2 nmol) was hydrolyzed by heating in 25 µl of 4 M methanesulfonic acid at 110 °C for 24 h. The
reaction solution was neutralized with 25 µl of 3.5 M
NaOH. The peptide sample was loaded on a High Speed Amino Acid Analyzer (Hitachi, model L-8500A) with a post-column method.
| |
RESULTS AND DISCUSSION |
The oxidative modification of Trp-43 in F43W/H64L Mb has been
performed by adding mCPBA in 50 mM potassium
phosphate buffer at pH 7.4. The transition from 5- to 6-coordinated
ferric high spin state is a good indication of the protein modification
(Scheme 1) (17-21). The
mCPBA-treated F43W/H64L mutant is digested with Lys-C
achromobacter, and two peptide fragments A and B, which consist of Trp-43(modified)-Asp-44-Arg-45-Phe-46-Lys-47 (W*DRFK),
are purified by FPLC. Fig. 2 shows the
comparison of the MS/MS spectra of unmodified WDRFK fragment and the
modified fragment A. The difference in the spectra provides
the direct evidence for the modified site to be Trp-43, and the
increased mass number of 30 Da could correspond to the addition of two
oxygen atoms and loss of two protons. The MS/MS spectrum of the
fragment B is identical to that of the fragment
A. Prior to the NMR analysis, A and B
have been further treated with trypsin to simplify the NMR spectra.
While the fragment B affords a shorter peptide defined as
B' (W*DR), trypsin does not cleave the carboxyl side
of arginine in the fragment A. Therefore, we have performed
NMR analysis of the intact WDR fragment and modified fragments
A (W*DRFK) and B' (W*DR).
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Fig. 2.
MS/MS analysis of the intact WDRFK fragment
(A) and product A (W*DRFK) performed on MALDI-TOF
(B) (PerSeptive Biosystems, Voyager DESTR). The
total masses for the intact and modified fragments are 751.5 and 781.5, respectively. Mass units of the major b and y fragment ions are shown.
Essentially the same mass pattern is observed for product
B.
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|
Although the 1H NMR spectrum of the intact WDR fragment
exhibits signals derived from five tryptophan protons, only three
proton signals of the tryptophan appear in the aromatic region for the modified product A (W*DRFK) (Fig.
3, Table
I). The close examination of the
1H NMR spectrum of A reveals that one proton
signal at 6.60 ppm couples with two different proton signals with
coupling constants of J = 8.2, 2.3 Hz. Two other proton
signals appear at 6.58 and 7.19 ppm with coupling constants of
J = 2.3 and 8.2 Hz, respectively. The only structure
that could afford such a coupling pattern is a 2,6-disubstituted indole
substructure as shown in Fig. 4;
i.e. the resonance at 6.58 ppm, 6.60 ppm, and 7.19 ppm are
unambiguously assigned to the protons at C7, C5, and C4,
respectively.
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Table I
1H NMR chemical shifts ( in ppm) of Trp-43 of the intact WDR
fragment, product A (W*DRFK), and product B' (W*DR)
The NMR spectra were obtained in 20 mM potassium phosphate
buffer (pD 7.0) in D2O at 25 °C on a Unity Inova 600 MHz NMR
and a Unity plus 600 MHz spectrometer (Varian). The chemical shifts are
referred to TSP-d4.
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|
The coupling between proton signals at C4 and C5 observed in the COSY
spectrum also support the assignment. Furthermore, the correlation of
the C4-H with C
-H (4.14 ppm) and C
-H
(2.65 ppm) is observed by the ROESY experiments. The combination of the
COSY, ROESY, and TOCSY methods allows us the complete assignment for
the fragment A as summarized in Table
II. Essentially the same assignment can
be applied to the interpretation for the NMR spectra of B'.
Two doublet signals at 6.36 ppm (J = 2.5 Hz) and 6.91 ppm (J = 8.5 Hz) are assigned to the C7 and C4 protons
of the modified Trp-43 in B', respectively, and the
resonance at 6.26 ppm (J = 8.5, 2.5 Hz) is derived from
C5-H (Table I). Therefore, the tryptophan residue in the fragment B' (W*DR) also must have the 2,6-disubstituted indole
substructure (Fig. 4). The coupling pattern in the 1H NMR
spectrum of the fragment B is essentially the same as those
observed in A and B' spectra.
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Table II
1H NMR chemical shifts ( in ppm) for the residues in the
intact WDR fragment, product A (W*DRFK), and product
B' (W*DR)
The NMR spectra were obtained in 20 mM potassium phosphate
buffer (pD 7.0) in D2O at 25 °C on a Unity Inova 600 MHz NMR
and a Unity plus 600 MHz spectrometer (Varian). The chemical shifts are
referred to TSP-d4.
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|
Amino acid sequence and composition analyses provided us with a
clue to identify the structural differences in the modified tryptophan of the fragment A and B'. First of
all, A does not react with phenylisothiocyanate, and the
Edman degradation does not give us any sequence information, while the sequence analysis for B' shows Trp*-43(not
detected)-Asp-44-Arg-45 sequence. Second, amino acid composition
analysis of A reveals that the fragment consists of Asp,
Arg, Phe, and Lys; however, the tryptophan residue is not identified
due to its modification. The results imply that the amino terminus of
the fragment A is protected, but the residues except for
Trp-43 are intact. To clarify the amino-terminal structure, we have
measured 13C NMR of A. The spectrum exhibits a
unique signal at 71.1 ppm, which could be assigned as a signal from the
hydroxylated carbon atom. Since the fragment A does not
contain a serine nor a threonine residue, the comparison of chemical
shift for the carbon in tryptophan (56.1 ppm), indole-3-lactic acid
(70.6 ppm), and A (71.1 ppm) suggest that the terminal amino group in A is replaced with the hydroxyl moiety. Thus, we
conclude at the moment that the indole substructure in the modified
tryptophan is 2,6-dihydro-2-imino-6-oxoindole in A (X=O, Y=NH in Fig. 4) and
2,6-dihydro-2,6-dioxoindole in B terminal (X=Y=O). The
proposed structures are consistent with the increase in the
30-Da mass unit with respect to the intact tryptophan. Although
further studies are required to clarify the mechanism, a cyclic
intermediate generated through the reaction of the terminal amine with
the carbonyl carbon atom at the C2 position followed by hydrolysis
might be involved to afford 2,6-dihydro-2-imino-6-oxoindole substructure without changing the total mass of the peptide (Scheme 2).
It should be noticed that trypsin somehow recognizes the structural
difference in the amino-terminal of pentapeptide A (W*DRFK)
and prevents hydrolysis of the carboxyl side of arginine to yield
A' (W*DR). More interestingly, the fragment A is
found to be produced from B during the incubation with trypsin or Lys-C, while B is stable in aqueous solution in
the absence of peptidases.
Since the mixing of mCPBA and tryptophan does not produce
the modified tryptophan, a ferryl porphyrin radical cation generated in
the reaction of F43W/H64L Mb with mCPBA is presumably a
catalytic species to form 6-hydroxytryptophan either directly or via
the epoxidation followed by hydrolysis (Scheme
3). Previous studies using Fremy's salt
as the oxidant indicate that 6-hydroxy-3-methylindole is readily
oxidized to yield 3-methylindole 2,6- and 6,7-dione, and 6,7-dione is
reported to be a minor product due to its instability (yield of
3-methylindole 2,6-dione = 63%, 6,7-dione = 7%) (22). We
expect that 6-hydroxytryptophan could further be oxidized with mCPBA (Scheme 3A) or compound I (Scheme 3B) by overall four
electron oxidation to produce the 2,6-dihydro-2,6-dioxoindole
substructure.
In summary, the indole substructure of Trp-43 in F43W/H64L
Mb is oxidatively transformed into 2,6-dihydro-2,6-dioxoindole in the
presence of mCPBA. Our finding is the first example of the
oxidation of aromatic carbons by the myoglobin mutant system as far as
we know. The results implicate that a ferryl oxygen atom transfer to
aromatic molecules would be possible by a heme enzyme with a
non-thiolate ligand if the substrates were fixed nearby the heme iron.
Our results would coincide with recent genetic analysis, suggesting
that a hemoenzyme similar to cytochrome c peroxidase with an
imidazole as the proximal ligand is involved in biosynthesis of
tryptophan tryptophylquinone, a novel cofactor bearing indole 6,7-dione
moiety, in methylamine dehydrogenase (11, 23-24). In addition,
we have reported herein that a unique amino-oxo exchange reaction of
the amino-termnal 2,6-dihydro-2,6-dioxoindole was performed by
Lys-C and trypsin.
| |
FOOTNOTES |
*
This work was supported by Grants-in-aid for Scientific
Research 11490036 and 11228208 (to Y. W.), 13740384 (to T. U.), and 10680575 and 13780496 (to S. O.).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.
§§
To whom correspondence should be addressed: Institute for
Molecular Science, Myodaiji, Okazaki, 444-8585, Japan. Tel.:
81-564-55-7430; Fax: 81-564-54-2254; E-mail:
yoshi@ims.ac.jp.
Published, JBC Papers in Press, July 31, 2001, DOI 10.1074/jbc.C100371200
| |
ABBREVIATIONS |
The abbreviations used are:
Mb, myoglobin;
P-450, cytochromes P-450;
mCPBA, m-chloroperbenzoic acid;
FPLC, fast protein liquid
chromatography;
MS, mass spectrometry;
MALDI-TOF, matrix-assisted laser
desorption ionization time-of-flight;
TSP-d4, (trimethyl-silyl)propionic-2,2,3,3-d4.
| |
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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ABSTRACT
INTRODUCTION
