Myoglobin catalyzes its own nitration

Full text of DOI:10.1021/ja015621m

5142
J. Am. Chem. Soc. 2001, 123, 5142-5143
Myoglobin Catalyzes Its Own Nitration
James L. Bourassa, Elizabeth P. Ives, Ann L. Marqueling,
Roman Shimanovich, and John T. Groves*
Department of Chemistry, Princeton UniVersity
Princeton, New Jersey 08544
ReceiVed February 1, 2001
ReVised Manuscript ReceiVed April 16, 2001
Myoglobin is thought to facilitate oxygen transport within
cells.1 However, alternative functions such as nitric oxide
scavenging have also been suggested.2 High myoglobin concen-
trations (10-4 M) are found in cardiac cells and striated muscle,1
both of which have been shown to use nitric oxide for intercellular
signaling.3 Likewise, a myoglobin-like neuroglobin has been found
recently in brain cells.3b In this light, the fast reaction of
oxymyoglobin with nitric oxide to afford metmyoglobin and
nitrate could be a physiologically important pathway.4 Enhanced
dissipation of NO could maintain the concentration gradients
needed for efficient NO signaling and mediate the respiratory
inhibition of cytochrome oxidase. These considerations have
prompted us to examine the reactions of metmyoglobin (metMb)
with peroxynitrite (PN), since the metMb-PN adduct, Fe(III)-
OO-NO, is the same species as is postulated for the reaction of
oxyMb with NO.4 We find that metMb catalyzes the decomposi-
tion of peroxynitrite to afford both nitrate and NO2, and further,
that metMb catalyzes its own nitration specifically at Tyr-103.
The decomposition of peroxynitrite, monitored at 302 nm at
pH 7.6 as we have previously described,5 was observed to
accelerate in the presence of 5-50 µM horse heart metMb. A
second-order rate constant k1 ) 1.03 × 104 M-1 s-1 was
determined for this process from the metMb concentration
dependence (Figure 1). Only metMb was observed during the
peroxynitrite decay, consistent with an earlier report.6 A slow
production of ferrylMb was observed after 10 s due to hydrogen
peroxide7 in the peroxynitrite solutions. No catalysis was observed
in the presence of 50 µM NaCN, indicating that cyanomyoglobin,8
which is formed under these conditions (λabs ) 422, 540 nm),
had no effect on peroxynitrite decay.
Figure 1. Pseudo-first-order rate constants for the decay of peroxynitrite
vs the concentration of metMb derived from stopped-flow kinetic data
monitoring peroxynitrite decay (λmon ) 302 nm) upon mixing equal
volumes of 1 mM OONO- in 2.5 mM NaOH and 20 mM Tris buffer,
pH 7.6. Second-order rate constant, k ) 1.03 × 104 M-1 s-1
.
Figure 2. Effect of myoglobin on the yield of nitrate and nitrite
determined directly by ion chromatography from the decomposition of
500 µM peroxynitrite in 20 mM Tris buffer, pH 7.6: (b) nitrate, (9)
nitrite, and (2) total of both. Values were corrected for nitrite levels in
the peroxynitrite stock solutions.
OO-NO intermediate (1) decomposes to afford ca. 80% nitrate
and 20% NO2 according to eq 1.
The stoichiometry of this myoglobin-induced decomposition
of peroxynitrite was examined by directly monitoring changes in
the nitrite and nitrate yields with varying [metMb](Figure 2). The
nitrite yield decreased nearly 4-fold with increasing [metMb],
leveling off at 11% above 50 µM Mb. That nitrite was produced
even at high [metMb] suggested that NO2 was produced during
catalysis which afforded nitrite and nitrate upon hydrolysis. We
can estimate from this nitrite yield that the proposed MbFe(III)-
Electrospray mass spectroscopy of metMb samples after
peroxynitrite treatment showed significant nitration of myoglobin,
consistent with the production of NO2. After incubation of metMb
with 3 mM peroxynitrite at pH 7.6, the ES/MS spectrum showed
an envelope of peaks that analyzed for a species with m/e 16998
(80%) accompanied by weaker peaks corresponding to ∼20%
metMb (m/e 16953) (see Supporting Information). This mass
change is consistent with mono-nitration of the protein.9 Con-
centrations of peroxynitrite of 0.5, 1, and 2 mM reacted with 100
µM metmyoglobin led to ∼20, 36, and 50% nitration, as estimated
from ES/MS data. Likewise, repeated exposure of 100 µM oxyMb
to aqueous NO in the presence of air and 5 mM ascorbate gave
MS evidence for ca. 10% Mb nitration.10
(1) (a) Suzuki, T.; Imai, K. Cell. Mol. Life Sci. 1998, 54, 979. (b)
Wittenberg, B. A.; Wittenberg, J. B. Annu. ReV. Physiol. 1989, 51, 857. (c)
Takahashi, E.; Endoh H.; Doi, K. Biophys. J. 2000, 78, 3252.
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Chu, K.; Groves, J. T. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 2370-2374.
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Lee, J.; Hunt, J. A.; Groves, J. T. J. Am. Chem. Soc. 1998, 120, 6053. (c)
Lee, J.; Hunt, J. A.; Groves, J. T. J. Am. Chem. Soc. 1998, 120, 7493.
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10.1021/ja015621m CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/03/2001

Communications to the Editor
J. Am. Chem. Soc., Vol. 123, No. 21, 2001 5143
Scheme 1
Figure 3. Nitration of fluorescein during stopped-flow spectroscopy
monitored at λmon ) 520 nm. Nitrofluorescein yields were 24% as
determined from ∆OD. Solid line: Kinetic trace for 30 µM fluorescein
in 20 mM Tris buffer (pH 7.6) + 1 mM OONO- in 2.5 mM NaOH,
kobs ) 0.058 s-1. Dashed line: Same as above with 50 µM metMb (horse
A mechanism consistent with these observations is shown in
Scheme 1. The rate constants shown were determined from
stopped-flow kinetic data as we have described.5 Initial reaction
of PN with metMb is proposed to afford an intermediate adduct,
metMb-PN, that decomposes via O-O bond homolysis yielding
ferrylMb and NO2.5,15 Rebound of NO2 with ferrylMb within the
heme cavity would give metMb and nitrate as the major product.16
Cage escape of NO2 must also occur with ca. 20% efficiency to
account for the observed nitrite and metMb nitration.
The reduction of ferrylMb by peroxynitrite was observed
directly. FerrylMb was produced by incubation of metMb (100
uM) for 10 min with 2 equiv of H2O2.7 Stoichiometric addition
of PN, as monitored by stopped-flow techniques,5 caused the
reduction of ferrylMb to metMb (k2 ) 7.7 × 103 M-1 s-1) and a
complete suppression of fluorescein nitration. This explains why
ferrylMb is not seen during PN decomposition, as suggested by
Herold,6 and it shows that the reaction between ferrylMb and PN
does not produce a nitrating species.17
In summary, metMb catalyzes the decomposition of peroxy-
nitrite at biologically relevant concentrations. The products are
nitrate (80%) and NO2 (20%). At high peroxynitrite concentra-
tions, this process leads to nitration of the surface tyrosine-103.
Small but significant amounts of myoglobion nitration were also
observed upon addition of NO to oxyMb. More detailed studies
of myoglobin interactions with peroxynitrite and NO are under
way.18
heart) added to fluorescein, kobs ) 0.41 s-1
.
The nitration of myoglobin was also observed after the addition
to metMb of H2O2 and NO2- in concentrations similar to that of
PN (1-3 mM).11 However, the addition of catalase (1 µM) to
the metMb solutions proved adequate to completely eliminate this
process while for the 3 mM PN case, catalase reduced the amount
of myoglobin nitration observed by only 20%. Cyano-Mb showed
reduced (<50%) nitration levels. Thus, the majority of the
myoglobin nitration must have derived from the peroxynitrite
reactivity with the heme center. Nitrated Mb catalyzed PN
decomposition at an undiminished rate.
The site of metMb nitration was determined by LC/MS analysis
of tryptic digests of the reaction mixture that showed all of the
typical myoglobin fragments.12 A single new peak in the digest
was observed at 42 min with m/e 1931 that displayed a strong
UV absorbance at λmon ) 420 nm characteristic of nitrotyrosine.9
There was correspondingly diminished intensity of a peak at 41
min (m/e 1886), the fragment corresponding to residues 103-
118. This unambiguously identifies Tyr103 as the site of nitration.
This tyrosine is located at the protein surface about 6 Å (edge-
to-edge) from the heme. The absence of a peak in the HPLC trace
in the region of authentic O2N-Tyr-Lys (2.6 min) indicated
negligible nitration at Tyr146.13
MetMb nitration was suppressed in a concentration-dependent
manner upon addition of 10-300 µM fluorescein, a detector, and
scavenger of NO2.14 At the higher concentrations of fluorescein,
no significant metMb nitration was observed. Fluorescein was
nitrated under these conditions. Figure 3 shows the time course
for the appearance of nitrofluorescein observed at 520 nm upon
the addition of PN. The same spectral changes, including
isosbestic points at 512 nm, were observed for fluorescein nitration
during PN decay in the presence of 100 µM metMb. The rate of
nitration increased proportionately with the rate of peroxynitrite
decomposition. Since the majority of the peroxynitrite flux passes
through the metMb-catalyzed pathway under these conditions,
these results indicate that the metMb-catalyzed decay of peroxy-
nitrite produces a freely diffusing nitrating species, also consistent
with nitrogen dioxide production.
Acknowledgment. Support of this research by the National Institutes
of Health (GM36928 to J.T.G. and GM20393 (NRSA to J.L.B.) is
gratefully acknowledged. We also thank Drs. John Eng, Saw Kyin, and
Dorothy Little for help with the protein digests and mass spectroscopy
and Prof. Robert Austin for stimulating discussions.
Supporting Information Available: LC/electrospray MS data for
nitrated myoglobin (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
JA015621M
(15) (a) Daiber, A.; Herold, S.; Schoneich, C.; Namgaladze, D.; Peterson,
J. A.; Ullrich, V. Eur. J. Biochem. 2000, 267, 6729-6739. (b) Rao, S. I.;
Wilks, A.; Ortiz de Montellano, R. R. J. Biol. Chem. 1993, 268, 803.
(16) (a) Stern, M. K.; Jensen, M. P.; Kramer, K. J. Am. Chem. Soc. 1996,
118, 8735-8736. (b) Shimanovich, R.; Groves, J. T. Arch. Biochem. Biophys.
2001, 387, 307-317.
(17) Clark electrode measurements detected the production of 87 µM (87%
based on ferrylMb) oxygen upon addition of peoxynitrite, cf.: McKee, M. L.
J. Am. Chem. Soc. 1995, 117, 1629.
(11) Eiserich, J. P.; Cross, C. E.; Jones, A. D.; Halliwell, B.; van der Vliet,
A. J. Biol. Chem. 1996, 271, 19199.
(12) Mann, M.; Meng, C. K.; Fenn, J. B. Anal. Chem. 1989, 61, 1702.
(13) These analyses were performed on a Waters 717 HPLC using a Delta
Pak C18 column with 10-50% CH3CN in water.
(18) We have recently shown with Y. Watanabe that double mutant
metmyoglobins (H64D/V68L and F43H/H64L) decomposed peroxynitrite at
accelerated rates (kcat ) 7.7 × 105 and 2.3 × 105 M-1 s-1, respectively) also
with extensive protein nitration. Details will be presented elsewhere.
(14) Jiang, Q.; Hurst, J. K. J. Biol. Chem. 1997, 272, 32767.