Manganese porphyrins as redox-coupled peroxynitrite reductases

Article abstract of DOI:10.1021/ja9801036

Superoxide (O₂^.-) and peroxynitrite (ONOO^-) have been implicated in many pathophysiological conditions. To develop novel catalysts that have both ONOO^- decomposition and O₂^.- dismutase activity, and to understand the mechanisms of these processes, we have explored the reactivity of 5,10,15,20-tetrakis- (N-methyl-4'-pyridyl)porphinatomanganese(III) [Mn(III)TMPyP] toward ONOO^- and 0₂^.-. The reaction of Mn(III)TMPyP with ONOO^- to generate an oxomanganese(IV) porphyrin species [(oxoMn(IV)] is fast, but Mn(III)TMPyP is not catalytic for ONOO^- decomposition because of the slow reduction of oxoMn(IV) back to the Mn(III) oxidation state. However, biological antioxidants such as ascorbate, glutathione, and Trolox rapidly turn over the catalytic cycle by reducing oxoMn(IV). Thus, Mn(III)TMPyP becomes an efficient peroxynitrite reductase when coupled with ascorbate, glutathione, and Trolox (k(c) ~2 x 10⁶ M^-1 s^-1), though the direct reactions of ONOO^- with these biological antioxidants are slow (88 M^-1 s^-1, 5.8 x 10² M^-1 s^-1 and 33 M^-1 s^-1, respectively). Mn(III)TMPyP is known to catalyze the dismutation of O₂^.-, and using stopped-flow spectrophotometry, the rate of Mn(III)TMPyP-catalyzed dismutation has been measured directly (k(c) = 1.1 x 10⁷ M^-1 s^-1). Further, O₂^.-, like the biological antioxidants, rapidly reduces oxoMn(IV) to the Mn(III) oxidation state (k ~10⁸ M^-1 s^-1), transforming Mn(III)TMPyP into a O₂^.--coupled ONOO^- reductase. Under conditions of oxidative stress and reduced antioxidant levels, Mn(III)TMPyP may deplete O₂^.- primarily as a function of its ONOO^- reductase activity, and not through its O₂^.- dismutase activity.

Full text of DOI:10.1021/ja9801036

J. Am. Chem. Soc. 1998, 120, 6053-6061
6053
Manganese Porphyrins as Redox-Coupled Peroxynitrite Reductases
Jinbo Lee, Julianne A. Hunt, and John T. Groves*
Contribution from the Department of Chemistry, Princeton UniVersity, Princeton, New Jersey 08544
ReceiVed January 9, 1998
Abstract: Superoxide (O2•-) and peroxynitrite (ONOO-) have been implicated in many pathophysiological
•-
conditions. To develop novel catalysts that have both ONOO- decomposition and O2 dismutase activity,
and to understand the mechanisms of these processes, we have explored the reactivity of 5,10,15,20-tetrakis-
(N-methyl-4′-pyridyl)porphinatomanganese(III) [Mn(III)TMPyP] toward ONOO- and O2•-. The reaction of
Mn(III)TMPyP with ONOO- to generate an oxomanganese(IV) porphyrin species [(oxoMn(IV)] is fast, but
Mn(III)TMPyP is not catalytic for ONOO- decomposition because of the slow reduction of oxoMn(IV) back
to the Mn(III) oxidation state. However, biological antioxidants such as ascorbate, glutathione, and Trolox
rapidly turn over the catalytic cycle by reducing oxoMn(IV). Thus, Mn(III)TMPyP becomes an efficient
peroxynitrite reductase when coupled with ascorbate, glutathione, and Trolox (kc ≈ 2 × 106 M-1 s-1), though
the direct reactions of ONOO- with these biological antioxidants are slow (88 M-1 s-1, 5.8 × 102 M-1 s-1
,
and 33 M-1 s-1, respectively). Mn(III)TMPyP is known to catalyze the dismutation of O2•-, and using stopped-
flow spectrophotometry, the rate of Mn(III)TMPyP-catalyzed dismutation has been measured directly (kc )
1.1 × 107 M-1 s-1). Further, O2•-, like the biological antioxidants, rapidly reduces oxoMn(IV) to the Mn(III)
oxidation state (k ≈ 108 M-1 s-1), transforming Mn(III)TMPyP into a O2•--coupled ONOO- reductase. Under
conditions of oxidative stress and reduced antioxidant levels, Mn(III)TMPyP may deplete O2•- primarily as a
function of its ONOO- reductase activity, and not through its O2 dismutase activity.
•-
Introduction
Administration of SOD enzymes has been shown to provide
protection from tissue damage associated with various types of
inflammatory diseases,1 but the limitations associated with using
enzymes for pharmaceutical intervention (e.g., bioavailability,
stability) have motivated a search for synthetic SOD mimics of
low molecular weight. For example, Riley et al. have developed
The reactive oxygen species superoxide (O2•-) and peroxy-
nitrite (ONOO-) have been implicated as important mediators
of tissue injury and cellular dysfunction under conditions of
toxic shock, inflammation, and oxidative stress.1,2 Although a
•-
large flux of O2 is produced by aerobic metabolism (1-5%
a series of manganese complexes that effectively catalyze the
of total oxygen consumption is estimated to be reduced to O2•-),3
•- 9,10
dismutation of O2
.
These agents inhibit neutrophil-
•-
the concentration of O2 is maintained at very low levels by
mediated cell injury in Vitro11 and neutrophil-mediated tissue
injury in vivo.12 Similarly, synthetic catalysts for fast ONOO-
decomposition may have important therapeutic applications, and
Stern et al. recently discovered that the water-soluble iron
porphyrins, 5,10,15,20-tetrakis(N-methyl-4′-pyridyl)porphina-
toiron(III) [Fe(III)TMPyP] and 5,10,15,20-tetrakis(2,4,6-tri-
methyl-3,5-sulfonatophenyl)porphinatoiron(III) [Fe(III)TMPS]
the superoxide dismutase (SOD) enzymes, which are found at
micromolar intracellular concentrations. These metalloenzymes
react with O2•- at rates in the range 108 M-1 s-1 for mitochon-
drial MnSOD4 and at the near-diffusion-limited rate of 2 × 109
M-1 s-1 for Cu,ZnSOD.5 The reaction rate for the formation
of ONOO- from O2 and nitric oxide (NO•) is even faster at
•-
6.7 × 109 M-1 s-1 6,7
.
Thus, when the level of NO• rises to
function as “peroxynitrite isomerases” to catalyze the rapid
micromolar concentrationssas it does, for example, during
- 13
isomerization of ONOO- to NO3
.
These iron porphyrins
cerebral ischemia and near activated macrophagessNO• can
demonstrated profound activity in ONOO- related disease
•- 2
•-
effectively compete with SOD for O2
.
Accordingly, O2 ,
models.14,15
which may be toxic in its own right, is also efficiently converted
into ONOO-, an even more powerful oxidant, which is expected
to have free access to cell interiors by virtue of its ability to
cross phospholipid membranes.8 For these reasons, ONOO-
may play a major role in a wide variety of human diseases.
Our recent discovery that Mn(III)TMPyP shows high reactiv-
ity with ONOO- 8,16 has prompted us to explore the development
of novel catalysts with the potential for high ONOO- decom-
position and O2•- dismutation activity through an understanding
of the mechanisms and kinetics of these processes. The
* To whom correspondence should be addressed.
(1) Halliwell, B.; Gutteridge, J. M. C. Methods Enzymol. 1990, 186, 1.
(2) Beckman, J. S.; Koppenol, W. H. Am. J. Physiol. 1996, 271, C1424.
(3) Imlay, J. A.; Fridovich, I. J. Biol. Chem. 1991, 266, 6957.
(4) Gray, B.; Carmichael, A. J. Biochem. J. 1992, 281, 795.
(5) Fridovich, I. J. Biol. Chem. 1989, 264, 7761.
(6) Huie, R. E.; Padmaja, S. Free Radical. Res. Commun. 1993, 18, 195.
(7) Recently, it has been shown that the reaction rate for the formation
of ONOO- from O2•- and NO• is even faster, at 1.9 × 1010 M-1s-1 (Kissner,
R.; Nauser, T.; Bugnon, P.; Lye, P. G.; Koppenol, W. H. Chem. Res. Toxicol.
1997, 10, 1285).
(9) Riley, D. P.; Weiss, R. H. J. Am. Chem. Soc. 1994, 116, 387.
(10) Riley, D. P.; Lennon, P. J.; Neumann, W. L.; Weiss, R. H. J. Am.
Chem. Soc. 1997, 119, 6522.
(11) Hardy, M. M.; Flickinger, A. G.; Riley, D. P.; Weiss, R. H.; Ryan,
U. S. J. Biol. Chem. 1994, 269, 18535.
(12) Weiss, R. H.; Fretland, D. J.; Baron, D. A.; Ryan, U. S.; Riley, D.
P. J. Biol. Chem. 1996, 271, 26149.
(13) Stern, M. K.; Jensen, M. P.; Kramer, K. J. Am. Chem. Soc. 1996,
118, 8735.
(14) Stern, M. K.; Salvemini, D. PCT Int. Appl. WO95/31197, 1995.
(15) Salvemini, D.; Wang, Z. Q.; Stern, M. K.; Currie, M. G.; Misko,
T. P. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2659-2663.
(8) Marla, S. S.; Lee, J.; Groves, J. T. Proc. Natl. Acad. Sci. U.S.A. 1997,
94, 14243.
S0002-7863(98)00103-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/06/1998

6054 J. Am. Chem. Soc., Vol. 120, No. 24, 1998
Lee et al.
Scheme 1a
a
k1 ) 1.8 × 106 M-1 s-1; k2 ) 0.018 s-1; k3 ) 5.4 × 107, 1.3 × 105, and 7.0 × 106 M-1 s-1 for ascorbate, glutathione, and Trolox, respectively.
As described in the text and figure legends, all rates were determined at 23 °C in 50 mM phosphate buffer, pH 7.4.
manganese porphyrin Mn(III)TMPyP has been shown by
indirect assay to catalyze the dismutation of O2 at a rate of
results23 also reveal that Mn(III)TMPyP can demonstrate
profound changes in activity in the presence of biological
reductants.
•-
∼107 M-1 s-1 17
,
and both Mn(III)TMPyP and the structurally
related synthetic porphyrin 5,10,15,20-tetrakis(4-benzoic acid)-
porphinatomanganese(III) [Mn(III)TBAP] have proved effective
in protecting cells against oxidative stress.17-20
Here we report an elucidation of the mechanisms and kinetics
of the reactions of O2 and ONOO- with Mn(III)TMPyP.
•-
Specifically, we have found that Mn(III)TMPyP becomes a
highly effective peroxynitrite reductase24 when redox coupled
with the biological antioxidants ascorbate, glutathione, and
Trolox (a water-soluble analogue of R-tocopherol), which
rapidly reduce oxoMn(IV) back to the Mn(III) oxidation state
(see Scheme 1). We have also measured the rate of Mn(III)-
At a first glance, the protective ability of Mn(III)TMPyP17-20
is puzzling. Although the rate of O2 dismutation catalyzed
•-
by Mn(III)TMPyP (∼107 M-1 s-1) is rapid, that rate translates
into only ∼1% of the activity of native SOD on a molar basis.5
Similarly, the high reactivity of Mn(III)TMPyP with ONOO- 8,16
does not translate directly into high ONOO- decomposition
activity. As we have shown, ONOO- rapidly oxidizes Mn-
(III)TMPyP to an oxoMn(IV) species, but Mn(III)TMPyP alone
is not catalytic for ONOO- decomposition due to the slow
reduction of oxoMn(IV) back to the Mn(III) oxidation state.21
Fridovich et al. have suggested that Mn(III)TMPyP is reduced
to the Mn(II) state in vivo, at the expense of NADPH or
glutathione. Since the Mn(II) porphyrin can be reoxidized to
•-
TMPyP-catalyzed O2 dismutation directly for the first time,
using stopped-flow spectrophotometry, and our results are in
agreement with the published rates determined by indirect
assay17 and pulse radiolysis.22 Further, we have investigated
the interactions between Mn(III)TMPyP, ONOO-, and O2
,
•-
and we have discovered that O2•-, like the biological antioxi-
dants, can rapidly reduce oxoMn(IV)TMPyP to the Mn(III)
oxidation state, transforming Mn(III)TMPyP into a superoxide-
coupled peroxynitrite reductase. Significantly, the results
Mn(III)TMPyP by O2 with a rate constant of 4 × 109 M-1
•-
s-1 22
,
it was proposed that the protective effects of Mn(III)-
•-
described here indicate that Mn(III)TMPyP will deplete O2
•-
primarily as a function of a ONOO- coupled pathway at
biologically relevant ratios of O2•- and ONOO-. This surprising
result would explain the high biological activity of MnTMPyP.
TMPyP in vivo stem from its activity as a reductant: O2
•-
oxidoreductase rather than as a O2 dismutase.17 Our recent
(16) Groves, J. T.; Marla, S. S. J. Am. Chem. Soc. 1995, 117, 9578.
(17) Faulkner, K. M.; Liochev, S. I.; Fridovich, I. J. Biol. Chem. 1994,
269, 23471.
Results and Discussion
(18) Gardner, P. R. Arch. Biochem. Biophys. 1996, 333, 267.
(19) Gardner, P. R.; Nguyen, D. H.; White, C. W. Arch. Biochem.
Biophys. 1996, 325, 20.
(20) Szabo, C.; Day, B. J.; Salzman, A. L. FEBS Lett. 1996, 381, 82.
(21) Groves, J. T.; Lee, J.; Marla, S. S. J. Am. Chem. Soc. 1997, 119,
6269.
We have shown that the water-soluble manganese porphyrin,
Mn(III)TMPyP, is an excellent marker for ONOO- due to the
(23) Lee, J.; Hunt, J. A.; Groves, J. T. Bioorg. Med. Chem. Lett. 1997,
7, 2913.
(22) Faraggi, M. In Oxygen Radicals in Chemistry and Biology; Bors,
W., Saran, M., Tait, D., Eds.; Walter de Gruyter: Berlin, Germany, 1984;
pp 419-430.
(24) While the term peroxynitrite reductase has been used here, it should
be noted that the general nature of the reaction is analogous to peroxidase
activity.

Mn Porphyrins as Redox-Coupled ONOO- Reductases
J. Am. Chem. Soc., Vol. 120, No. 24, 1998 6055
Figure 1. Decomposition of 100 µM ONOO- monitored at 302 nm:
(a) in 25 mM pH 7.4 phosphate buffer at 25 °C, with a first-order rate
of 0.35 s-1; (b) in the presence of 10 µM Mn(III)TMPyP, with a rate
of 0.35 s-1; (c) in the presence of 100 µM ascorbate, with a first-order
rate of 0.29 s-1; (d) in the presence of 10 µM Mn(III)TMPyP and 150
Figure 2. (a) Plot of the apparent first-order rate (kobs) of the reaction
between ONOO- and antioxidants such as ascorbate (O), glutathione
(∆), and Trolox (]) under pseudo-first-order conditions vs the
concentrations of these antioxidants. Linear least-squares fitting of the
experimental data yielded a second-order rate constant of 88 (R )
0.997), 5.8 × 102 (R ) 0.997), and 33 M-1 s-1 (R ) 0.994) for
ascorbate, glutathione, and Trolox, respectively, (b) ONOO- decom-
position catalyzed by various concentrations of Mn(III)TMPyP in the
presence of biologically significant concentrations of antioxidants [150
µM ascorbate (O), 2 mM glutathione (∆), or 150 µM Trolox (])].
Antioxidants were added to the Mn(III)TMPyP solutions immediately
before the stopped-flow experiments to minimize the slow autoxidation
of antioxidants catalyzed by Mn(III)TMPyP under air. The apparent
first-order rates correlated linearly with the catalyst concentrations with
kc of 2.2 × 106 (R ) 0.994), 3.2 × 106 (R ) 0.992), and 1.1 × 106
M-1 s-1 (R ) 0.999) for ascorbate, glutathione, and Trolox, respectively.
µM ascorbate, with a rate of 22.4 s-1
.
diagnostic formation of an oxoMn(IV) intermediate.8,16 The
reaction of Mn(III)TMPyP with ONOO- to generate oxoMn-
(IV) is among the fastest reactions known for ONOO-,25 with
a second-order rate constant of 1.8 × 106 M-1 s-1 8
. Neverthe-
less, Mn(III)TMPyP is not an efficient catalyst for ONOO-
decomposition because of the relative stability of oxoMn(IV).
In typical experiments, rates of ONOO- decomposition were
measured with stopped-flow spectrophotometry by monitoring
the ONOO- absorbance at 302 nm. As shown in Figure 1, the
presence of 10 µM Mn(III)TMPyP did not significantly ac-
celerate the spontaneous decay of ONOO- at pH 7.4 (Figure
1b). Similarly, ascorbate alone did not significantly affect this
spontaneous reaction (Figure 1c). However, the incorporation
of near stoichiometric amounts of ascorbate with the manganese
porphyrin dramatically reduced the half-life (t1/2) of ONOO-
∼70-fold, from 2 s to 0.03 s (Figure 1d).
In marked contrast, Mn(III)TMPyP efficiently utilized ascor-
bate as the electron source for the reduction of ONOO-,
displaying peroxynitrite reductase activity (Figure 2b). The
turnover rate of the Mn(III)TMPyP-catalyzed decomposition of
ONOO- in the presence of 1.5 equiv of ascorbate was dependent
on the concentration of the catalyst (Figure 2b). The catalytic
rate constant (kc) was determined to be 2.2 × 106 M-1 s-1, which
is nearly equivalent to the formation rate of oxoMn(IV) by
ONOO-.8 This suggests that oxoMn(IV) formation becomes
the rate-limiting step in the catalytic cycle of ONOO- decom-
position when ascorbate is present (Scheme 1).
Peroxynitrite Decomposition by the Mn(III)TMPyP-
Ascorbate Redox Couple. The direct uncatalyzed reaction of
ONOO- with ascorbate was found to be slow, first order in
ONOO-, and first order in ascorbate. The apparent first-order
rate constant for ONOO- depletion, as determined by stopped-
flow spectrophotometry, correlated linearly with the concentra-
tion of ascorbate under pseudo-first-order conditions (excess
ascorbate), yielding a second-order rate constant of 88 M-1 s-1
at 25 °C in 25 mM pH 7.4 phosphate buffer (Figure 2a). This
is consistent with the results of Bartlett et al. and further supports
the notion that ascorbate alone cannot play a significant direct
role in the defense against ONOO-.26
Reduction of OxoMn(IV) by Ascorbate. Mn(III)TMPyP
does not catalyze ONOO- decomposition in the absence of
ascorbate because the conversion of oxoMn(IV) back to Mn-
(III) to complete the catalytic cycle has a first-order rate constant
of ∼0.018 s-1, much slower than the spontaneous decay of
ONOO- (0.35 s-1) under these conditions (Figure 3a).21 As
we have recently shown,21 the oxidation of Mn(III)TMPyP by
(25) Floris, R.; Piersma, S. R.; Yang, G.; Jones, P.; Wever, R. Eur. J.
Biochem. 1993, 215, 767.
(26) Bartlett, D.; Church, D. F.; Bounds, P. L.; Koppenol, W. H. Free
Radical. Biol. Med. 1995, 18, 85.
-
HSO5 rapidly and stoichiometrically forms an oxoMn(IV)
intermediate via an oxoMn(V) precursor. We have measured
the rate of reduction of oxoMn(IV) to Mn(III) by ascorbate in

6056 J. Am. Chem. Soc., Vol. 120, No. 24, 1998
Lee et al.
Table 1. Kinetic Evaluation of Mn(III)TMPyP-antioxidant
Redox Couples for the Decomposition of ONOO-
-
kONOO
kMn(IV)fMn(III)
(M-1 s-1
kc
antioxidant
(M-+1asn-tio1xidant
)
)
(M-1 s-1
)
none
0.018 s-1
ascorbate
glutathione
Trolox
88
5.4 × 107
1.3 × 105 b
7.0 × 106 c
2.2 × 106
3.2 × 106
1.1 × 106
5.8 × 102
33a
a In all the Trolox reactions, ONOO- decay was monitored at 289
nm to avoid interference from the Trolox phenoxyl radical and quinone
interconversion (289 nm is the isosbestic point). b The rate of oxoMn-
(IV) reduction by glutathionewas determined by a double-mixing
stopped-flow experiment similar to that described for ascorbate (see
text). The second-order rate constant was obtained by a linear plot of
kobs (s-1) vs glutathione concentration (R ) 0.999). c The rate of
oxoMn(IV) reduction by Trolox was obtained by a second-order kinetic
analysis of the experimental data from a stoichiometric reaction between
oxoMn(IV) and Trolox in a double-mixing stopped-flow experiment
analogous to that described for ascorbate (plot of (A0 - A∞)/(A - A∞) vs
time, R ) 0.996).
(Figure 2b). Curiously, though the rate-limiting step in the
catalytic decomposition of ONOO- is the oxoMn(IV) formation
with a second-order rate constant of 1.8 × 106 M-1 s-1, kc for
the Mn(III)TMPyP-glutathione redox couple is slightly higher
than expected. This may be due to the acceleration of oxoMn-
(IV) formation by the trans-effect if the axial ligand of the
manganese porphyrin is the thiolate of glutathione instead of a
water molecule. The estimated kc for the Mn(III)TMPyP-
Trolox redox couple is slower than expected because of residual
interference from the Trolox phenoxyl radical and quinone
interconversion.
Protection Against Oxidation and Nitration by Peroxyni-
trite. Significantly, these Mn(III)TMPyP-antioxidant redox-
coupled systems completely protected all-trans-retinoic acid
(RA) in phospholipid vesicles from ONOO- oxidation. Addi-
tion of 250 µM ONOO- to 40 µM RA in small unilamellar
vesicles resulted in a complete loss of the RA chromophore,
indicating oxidation (Figure 4a). At concentrations as low as
2 µM, Mn(III)TMPyP redox coupled with 300 µM ascorbate
protected 99% of the RA chromophore from ONOO- oxidation.
Further, the Mn(III)TMPyP-antioxidant redox couples also
prevented metal-catalyzed nitration of phenols by ONOO-
(Figure 4b). Reaction of 1 mM ONOO- with 1 mM 4-hydroxy-
phenylacetic acid (HPA) in the presence of 5 µM Mn(III)TMPyP
yielded 37% of 4-hydroxy-3-nitrophenylacetic acid (nitro-HPA).
Addition of ascorbate, Trolox, or glutathione offered dose-
dependent protection of HPA. The presence of 2 equiv (2 mM)
of the antioxidants completely prevented the nitration of HPA
by ONOO-. Interestingly, the efficacy of these biological
antioxidants in preventing phenolic nitration followed a trend
similar to that of their reduction of oxoMn(IV).
Figure 3. Double-mixing stopped-flow experiments to determine the
rate of reduction of the oxoMn(IV) intermediate by ascorbate, in which
Mn(III)TMPyP was first mixed with HSO5- to generate 10 µM oxoMn-
(IV) and (a) pH 7.4 phosphate buffer or (b) 10 µM ascorbate was then
added in the second mixing step after a 5 s age time, to provide final
concentrations of 5 µM MnTMPyP and 5 µM ascorbate. The disap-
pearance of oxoMn(IV) was monitored at 428 nm, and the appearance
of Mn(III) was monitored at 462 nm; for clarity, only the traces at 428
nm are shown. Inset: A second-order analysis of the reaction of 10
µM oxoMn(IV) and 10 µM ascorbate, monitored at 462 nm, in which
(A0 - A∞)/(A - A∞) was plotted against time. Linear least-squares fitting
of the plot gave a second-order rate constant of 5.4 × 107 M-1 s-1 (R
) 0.998).
a double-mixing experiment. When the oxoMn(IV) intermediate
was generated by mixing Mn(III)TMPyP with 1 equiv of
HSO5-, subsequent addition of 1 equiv of ascorbate greatly
enhanced the reduction of oxoMn(IV) to Mn(III) (Figure 3b).
The kinetic trace monitored at 428 nm shows the reduction of
oxoMn(IV). Both nonlinear regression fitting of the trace at
462 nm and second-order kinetic analysis of the trace at 428
nm gave a second-order rate constant of 5.4 × 107 M-1 s-1 for
the reduction of oxoMn(IV) by ascorbate (Figure 3b inset). This
rapid reduction results in the peroxynitrite reductase activity of
Mn(III)TMPyP, which is only limited by the rate of oxoMn-
(IV) formation in the catalytic cycle (k1 in Scheme 1).
Glutathione and Trolox as Mn(III)TMPyP-Coupled Anti-
oxidants. Glutathione and Trolox were examined as candidates
for redox coupling with Mn(III)TMPyP for the catalytic
decompostion of ONOO-. The direct reactions of ONOO- with
glutathione and Trolox are relatively slow, as in the case of
ascorbate (Figure 2a). The second-order rate constants are 5.8
× 102 and 33 M-1 s-1 for glutathione and Trolox, respectively.
Both antioxidants rapidly reduced oxoMn(IV) to Mn(III)TMPyP,
with second-order rate constants of 1.3 × 105 M-1 s-1 for
glutathione and 7.0 × 106 M-1 s-1 for Trolox (Table 1); thus,
both of these reducing agents effectively complete the catalytic
cycle of ONOO- decomposition shown in Scheme 1. Interest-
ingly, the rates of oxoMn(IV) reduction by ascorbate, Trolox,
and glutathione follow the order of the reduction potentials of
these biological antioxidants.27
Mn(III)TMPyP-Catalyzed Superoxide Dismutation. The
SOD activity of Mn(III)TMPyP was evaluated by directly
observing the decay of O2•- at 245 nm28 by using a specialized
four-syringe setup of the stopped-flowed spectrophotometer, as
described in the Experimental Section. In the absence of Mn-
•-
(III)TMPyP, self-dismutation of O2 at pH 7.4 in 50 mM
phosphate buffer was second order in O2•- with a rate constant
(kself) of 7.34 × 105 M-1 s-1 (Figure 5a), which is typical for
•-
H+-catalyzed dismutation.5,22,28,29 The decay of O2 was
significantly accelerated by the introduction of Mn(III)TMPyP
at low concentrations (Figure 5b). Taking into consideration
both the self-dismutation and the Mn(III)TMPyP-catalyzed
Mn(III)TMPyP catalyzed the rapid decomposition of ONOO-
in the presence of physiologically significant concentrations of
glutathione (2 mM) and Trolox (150 µM), with catalytic rate
constants (kc) of 3.2 × 106 and 1.1 × 106 M-1 s-1, respectively
(28) Riley, D. P.; Rivers, W. J.; Weiss, R. H. Anal. Biochem. 1991, 196,
344.
(27) Buettner, G. R. Arch. Biochem. Biophys. 1993, 300, 535.
(29) Bielski, B. H. J. Photochem. Photobiol. 1978, 28, 645.

Mn Porphyrins as Redox-Coupled ONOO- Reductases
J. Am. Chem. Soc., Vol. 120, No. 24, 1998 6057
Figure 5. Decay of O2•- in 50 mM, pH 7.4 phosphate buffer at 25 oC
(a) in the absence of porphyrin and (b) in the presence of 3 µM Mn-
(III)TMPyP. The solid lines in (a) and (b) are the nonlinear least-squares
fitting of the experimental data into second-order kinetics (R ) 0.999)
and eq 3 (R ) 0.997), respectively. Inset: Linear least-squares fit of
the pseudo-first-order rate constants vs Mn(III)TMPyP concentrations
(R ) 0.999).
Figure 4. Protection against oxidation and nitration by peroxynitrite.
(a) Difference UV-vis spectra: the solid trace shows the reaction of
250 µM ONOO- with 40 µM all-trans-retinoic acid in small unilamellar
vesicles, while the dotted trace shows the same reaction but in the
presence of 2 µM Mn(III)TMPyP and 300 µM ascorbate. Each
difference spectrum is obtained by subtraction of a spectrum measured
after addition of ONOO- (and ascorbate) from a spectrum measured
before addition of ONOO-. (b) HPLC analysis of nitro-HPA produced
in the reactions of 1 mM HPA and 1 mM ONOO- in the presence of
5 µM Mn(III)TMPyP and various concentrations (0, 0.25, 0.5, 1, 2, 5
mM) of ascorbate, glutathione*, or Trolox.
3. A typical trace is shown in Figure 5b. The catalytic rate
constant of Mn(III)TMPyP-catalyzed dismutation of O2•- is 1.1
× 107 M-1 s-1, determined from the slope of a linear plot of
kobs vs Mn(III)TMPyP concentrations (Figure 5b inset). This
rate is in agreement with the results obtained by indirect assay17
and pulse radiolysis.22 The presence of ascorbate did not affect
the observed rate of Mn(III)TMPyP-catalyzed dismutation of
O2•-. The SOD activity of an anionic manganese porphyrin,
MnTBAP, was also examined. Mn(III)TBAP was inefficient
in decomposing O2•-, with a catalytic rate slower than the rate
•-
dismutation, the rate of O2 decomposition can be expressed
as eq 1
•-
of O2 self-dismutation, which represents the lower limit of
the stopped-flow method.
Mn(III)TMPyP effects the dismutation of O2 via a rate-
limiting reduction (k1) of Mn(III) to Mn(II) and a fast oxidation
(k2) of Mn(II) back to Mn(III),22 as shown in Scheme 2.
Accordingly, Mn(III)TMPyP remained in the Mn(III) oxida-
•-
-d[O2•-]/dt ) kself[O2•-] + kobs[O2
kobs ) kc[Mn(III)TMPyP]
]
(1)
•-
in which
(2)
•-
tion state during the entire course of O2 dismutation, as
determined by monitoring the porphyrin absorbance at 462 nm.
The Mn(II) species can be considered a transient reactive
intermediate, since the oxidation of Mn(II) by O2•-, with an
Integration of eq 1 yields the following expression for the
concentration of O2 with time.
•-
estimated rate constant of ∼4 × 109 M-1 s-1 22
,
is almost 3
[O2•-]0 kobs exp(-kobst)
kobs + kself[O2•-]0(1 - exp(-kobst))
•-
orders of magnitude faster than the reduction of Mn(III) by O2
.
[O2•-] )
(3)
Applying a steady-state approximation to the intermediate Mn-
(II) species, kobs can be expressed as eq 4.
The set of first-order rate constants, kobs, was extracted by
nonlinear least-squares fitting of the experimental data into eq
kobs ) 2k1[Mn(III)TMPyP]
(4)

6058 J. Am. Chem. Soc., Vol. 120, No. 24, 1998
Lee et al.
Scheme 2a
a k1 ) 5.5 × 106 M-1 s-1; k2 ) 4 × 109 M-1 s-1; k3 ) 1.16 × 106 M-1 s-1; k4 ) 1 × 108 M-1 s-1; k5 ) 4.5 × 109 M-1 s-1; k6 ) 7.34 × 105
M-1 s-1; k7 ) 1.17 × 103 M-1 s-1; k8 ) 9 × 108 M-1 s-1. As described in the text and figure legends, the rates k1, k3, k4, and k6 were determined
at 23 °C in ∼8% DMSO/50 mM phosphate buffer, pH 7.4, while k7 was determined at 23 °C in 50 mM phosphate buffer, pH 7.4. The values for
the remaining rates (k2,,22 k5,22 and k853) are taken from the literature.
Thus,
To demonstrate O2•--coupled Mn(III)TMPyP reduction of
ONOO-, we again employed the extended-plate, four-syringe
setup of the stopped-flow spectrophotometer (described in the
Experimental Section). However, we departed from the usual
procedure for monitoring the dismutation of O2•-, which
involves loading KO2/DMSO in the first syringe so that it mixes
with buffer from the second syringe in the aging loop before
reaching the detector cell. Instead, we loaded ONOO- in the
first syringe, Mn(III)TMPyP in the second syringe, and KO2/
DMSO in the third syringe. As a result, ONOO- and Mn(III)-
TMPyP mixed in the aging loop, producing a mixture of
oxoMn(IV) and the excess ONOO-, which then reacted with
k1 ) kc/2 ) 5.5 × 106 M-1 s-1
(5)
Superoxide-Coupled Mn(III)TMPyP Reduction of Per-
•-
oxynitrite. The fast reduction of Mn(III) to Mn(II) by O2
suggested that O2•-, like the biological antioxidants, would also
rapidly reduce oxoMn(IV) to Mn(III). Accordingly, we de-
•-
signed an experiment to determine whether O2 could drive
the reduction of ONOO- in the presence of Mn(III)TMPyP.
•-
First, the direct reaction of oxoMn(IV) with O2 was
monitored by stopped-flow spectrophotometry (Figure 6a) to
confirm our expectation that O2•- would rapidly reduce oxoMn-
(IV) to Mn(III). Indeed, this reduction rate was at the upper
detection limit of the stopped-flow spectrophotometer, even for
the near stoichiometric reaction of 5 µM oxoMn(IV) with ∼5-
10 µM KO2. However, from the data in Figure 6a, the second-
order rate constant could be estimated to be on the order of 1
× 108 M-1 s-1. Therefore, this fast reduction of oxoMn(IV)
by O2•- could turn over the cycle of Mn(III)TMPyP-catalyzed
decomposition of ONOO-, which is again limited by the rate
of generation of oxoMn(IV) by ONOO- (Scheme 2). In the
less polar medium in which these experiments were conducted
(∼8% DMSO/pH 7.4, 50 mM phosphate buffer), oxidation of
Mn(III) to oxoMn(IV) by ONOO- has a rate constant of 1.16
× 106 M-1 s-1 (Figure 6b), which is slightly slower than that
in pure phosphate buffer.30
•-
O2 in the detector cell.
With the use of this special setup, we monitored the reaction
of 5 µM Mn(III)TMPyP with 100 µM ONOO- and ∼160 µM
O2•-; the data are shown in Figure 7, trace a. As can be seen,
the manganese porphyrin, monitored at 462 nm, was maintained
•-
as Mn(III) by rapid O2 reduction of oxoMn(IV) during the
•-
initial phase of the reaction (ca. 25 ms). Only after O2 had
been fully consumed was Mn(III) oxidized by excess ONOO-
to form oxoMn(IV). A control experiment in the absence of
•-
O2 showed very different behavior (Figure 7, trace c): only
oxidation of Mn(III) by ONOO- was observed under these
conditions. Taken together, these experiments show that Mn-
(III)TMPyP can act as a O2•--coupled ONOO- reductase,
introducing a new pathway for O2•- removal by Mn(III)TMPyP.
We present in Scheme 2 a mechanism for Mn(III)TMPyP-
catalyzed O2•- removal. In this bifurcated process, the left fork
describes the usual Mn(III)-Mn(II) dismutation cycle, while
the right fork describes the oxoMn(IV)-Mn(III) ONOO-
The reduction rate of oxoMn(IV) by H2O2, the dismutation
product of O2•-, was also measured by double-mixing stopped-
flow experiments, which gave a second-order rate constant of
1.17 × 103 M-1 s-1. This result served to limit the significance
of a peroxide-dependent cycle, and was an important control
since H2O2 is the major impurity in the ONOO- stock solutions.
•-
reductase cycle. Furthermore, O2 can react with nitrogen
dioxide (NO2) at the near diffusion-controlled rate of 4.5 × 109
M-1 s-1 to form peroxynitrate (O2NOO-)31 in the right-hand
fork of Scheme 2. A reassuring confirmation of this reaction
(30) This is consistent with our proposition that heterolytic cleavage of
the ONOO- O-O bond to generate oxoMn(V) is followed by fast one-
-
electron NO2 reduction to form oxoMn(IV) (see refs 8 and 16).
(31) Løgager, T.; Sehested, K. J. Phys. Chem. 1993, 97, 10047.

Mn Porphyrins as Redox-Coupled ONOO- Reductases
J. Am. Chem. Soc., Vol. 120, No. 24, 1998 6059
Figure 7. Reaction of 100 µM ONOO-, 5 µM Mn(III)TMPyP, and
∼160 µM O2 (0.8 equiv based on ONOO- concentration). (a) The
•-
solid trace is the experimental data monitoring Mn(III) concentration.
(b) The dotted trace is a kinetic simulation based on the eight possible
reactions shown in Scheme 2, with no adjustable parameters. (c) The
bottom trace is the same reaction as in (a), except in the absence of
•-
O2
.
consequence of imbalance in the cellular levels of prooxidants
and antioxidants.34,35 Excessive production of reactive oxygen
•-
species, including O2 and ONOO-, results in much lower
concentrations and even depletion of antioxidants. Nevertheless,
we have shown here that Mn(III)TMPyP has the potential to
•-
catalyze the concurrent removal of O2 and ONOO- under
these pathological conditions.
In the new bifurcated mechanism for Mn(III)TMPyP-
catalyzed elimination of O2 (Scheme 2), Mn(III)TMPyP
•-
•-
Figure 6. (a) The reduction of 5 µM oxoMn(IV) by ∼5-10 µM O2
.
performs both as a SOD mimic (left fork) and as a O2•- coupled
ONOO- reductase (right fork). It is instructive to consider the
ramifications of these results in biological situations. The
steady-state concentrations of NO•, O2•-, and ONOO- under
physiological conditions are highly tissue dependent. Physi-
ological NO• concentrations span 3 orders of magnitude, from
the 5 nM required for guanylate cyclase activation to the ∼4
µM measured under conditions of cerebral ischemia.2,36 Cellular
The oxoMn(IV) intermediate was prepared by the reaction of 5 µM
Mn(III)TMPyP with 5.5 µM HSO5-. The disappearance of oxoMn-
(IV) was monitored at 428 nm, and the reappearance of Mn(III) was
monitored at 462 nm. (b) Linear plot of the first-order rate constants
(kobs) of oxoMn(IV) formation vs ONOO- concentrations (R ) 0.996).
scheme derives from a simulation of the data. Taking into
consideration all eight possible reactions shown in Scheme 2,
with all eight rate constants individually determined, the
simulated plot reproduced the experimental data with no
adjustable parameters (Figure 7, trace b).
•-
SOD activity keeps the concentrations of O2 very low, with
typical steady-state concentrations of ∼10-10 M.3,37 As for
ONOO-, nanomolar concentrations were detected in agonist-
induced endothelial cells.38 Assuming the steady-state concen-
trations of O2•- and ONOO- to be ∼10-10 and ∼4 × 10-9 M,
respectively, the manganese porphyrin catalyst inventory would
be 60% Mn(III) and 40% oxoMn(IV) if the oxoMn(IV)
concentration is treated as a steady state. Thus, the partition
Biological Implications. Cells exist in a reducing environ-
ment rich in antioxidants. However, as we23 and others26,32 have
shown, the reactions of ONOO- with these biological reductants
are quite slow, indicating that antioxidants such as ascorbate,
glutathione, and tocopherol alone cannot play a direct role in
the defense against ONOO-. In fact, ONOO- readily oxidizes
and nitrates proteins long before the endogeneous antioxidants
have been depleted in human plasma.33 By contrast, we have
shown that Mn(III)TMPyP can efficiently utilize ascorbate,
glutathione, and Trolox as the electron source for the reduction
of ONOO-. Thus, under normal conditions, the ONOO-
reductase activity of Mn(III)TMPyP may protect cells from
oxidative damage.
•-
of O2 down the two forks of the bifurcated pathway shown
in Scheme 2 can be evaluated as in eq 6, where [O2•-]L and
•-
•-
[O2
]
R
represent the concentrations of O2 decomposed
through the left-hand and the right-hand forks, respectively.
•-
[O2
[O2
]
]
k1‚[Mn(III)]
k4‚[Mn(IV)]
L
)
) 0.08
(6)
•-
R
Conversely, under conditions of oxidative stress and depleted
antioxidant levels, Mn(III)TMPyP can protect cells from both
(34) Scandalios, J. G. OxidatiVe Stress and the Molecular Biology of
Antioxidant Defenses; Cold Spring Harbor Laboratory Press: New York,
1997.
ONOO- and O2 mediated injury. Oxidative stress is the
•-
(32) Koppenol, W. H.; Moreno, J. J.; Pryor, W. A.; Ischiropoulos, H.;
Beckman, J. S. Chem. Res. Toxicol. 1992, 5, 834.
(33) Van der Vliet, A.; Smith, D.; O’Neill, C. A.; Kaur, H.; Darley
Usmar, V.; Cross, C. E.; Halliwell, B. Biochem. J. 1994, 303, 295.
(35) Sies, H. OxidatiVe Stress; Academic: Orlando, 1985.
(36) Malinski, T.; Bailey, F.; Zhang, Z. G.; Chopp, M. J. Cereb. Blood
Flow Metab. 1993, 13, 355.
(37) Gardner, P. R.; Fridovich, I. J. Biol. Chem. 1992, 267, 8757.

6060 J. Am. Chem. Soc., Vol. 120, No. 24, 1998
Lee et al.
•-
disodium phosphate were purchased from Sigma. Sodium l-ascorbate,
Trolox, glutathione, all-trans-retionoic acid, 4-hydroxyphenylacetic acid,
potassium superoxide (KO2), potassium peroxymonosulfate (HSO5-),
and 4,4′,4′′,4′′′-(21H,23H-porphine-5,10,15,20-tetraaryl)tetrakis(benzoic
acid) (H2TBAP) were obtained from Aldrich. Mn(III)TBAP(Cl) was
prepared by metalation of H2TBAP under standard conditions.50
Peroxynitrite was prepared from the reaction of acidic H2O2 with sodium
nitrite following the published procedure.16 All the solvents were
analytical grade. Dry DMSO was distilled under reduced pressure from
calcium hydride. Water used in all the experiments was distilled and
deionized (Millipore, Milli-Q).
Significantly, more than 90% of the O2 will be depleted
via the ONOO--coupled pathway, given the rate constants we
have measured herein. Therefore, though Mn(III)TMPyP has
only modest SOD activity, it could be expected to effectively
remove O2•- even in the absence of antioxidants in tissues under
oxidative stress.
Conclusions
We have shown that a water-soluble manganese porphyrin,
Mn(III)TMPyP, can become an efficient peroxynitrite reductase
when redox coupled with biological antioxidants, though the
direct reactions of ONOO- with these antioxidants are slow.
Further, these Mn(III)TMPyP-antioxidant redox couples pro-
tected membrane components from oxidation and protected
phenols from nitration. Because cells exist in an environment
replete with antioxidants, including vitamin C (ascorbate),39,40
glutathione,41 and vitamin E (tocopherol),42,43 the ONOO-
reductase reactivity of manganese porphyrins may play an
important role in the protection of cells from oxidative stress
Kinetics of Peroxynitrite Decomposition. The kinetic profiles of
Mn(III)TMPyP-catalyzed decomposition of ONOO- in 25 mM phos-
phate pH 7.4 buffer at 25 °C were recorded on a HI-TECH SF-61 DX2
rapid-mixing stopped-flow spectrophotometer. In the presence of fixed
concentrations of ascorbate (150 µM), glutathione (2 mM), or Trolox
(150 µM), the porphyrin concentration was varied from 2 to 15 µM.
The decay of ONOO- (100 µM) was monitored at 302 nm. In a similar
experiment, the rate of reaction of Mn(III)TBAP and ascorbate (150
µM) with ONOO- (100 µM) was found to be about 40 times slower
than that of Mn(III)TMPyP/ascorbate: kc ) 5.0 × 104 M-1 s-1, obtained
from the slope of a linear plot of kobs vs Mn(III)TBAP concentrations
(R ) 0.999). The reduction rates of oxoMn(IV) intermediates by
antioxidants (ascorbate, glutathione, or Trolox) were measured directly
by performing double-mixing experiments: the oxoMn(IV) intermedi-
in O2 and ONOO- related diseases.
•-
We have measured the rate of Mn(III)TMPyP-catalyzed
dismutation directly for the first time, using stopped-flow
spectrophotometry. The catalytic rate constant we determined
(1.1 × 107 M-1 s-1) agrees well with the results obtained by
indirect assay17 and pulse radiolysis.22 Further, we have
determined that O2•-, like the above-mentioned biological
antioxidants, can rapidly reduce oxoMn(IV) to the Mn(III)
oxidation state, transforming Mn(III)TMPyP into a O2•--coupled
ONOO- reductase. Under the extreme conditions of oxidative
stress and antioxidant depletion within a cell, the concomitant
-
ates were fully generated by HSO5 during the first mixing step, and
then the antioxidant was added in the second mixing step. The recovery
of Mn(III)TMPyP and the disappearance of oxoMn(IV) complexes were
monitored at 462 and 428 nm, respectively.
Protection Against Oxidation and Nitration. Small unilamellar
vesicles (SUV) containing 40 µM all-trans-retinoic acid (RA) were
prepared following the literature procedure.51 The membrane-bound
substrate was mixed with 250 µM ONOOj, and the extent of oxidation
was evaluated as the decrease in absorbance at 340 nm of the RA
chromophore. Protection of RA oxidation was attempted by adding
Mn(III)TMPyP (2, 5, and 10 µM) in conjunction with ascorbate (300
µM). Reaction of 1 mM ONOO- with 1 mM HPA in the present of
5 µM Mn(III)TMPyP produced nitro-HPA, which was quantitated by
reverse-phase HPLC analysis (Waters Delta PAK 5 µ C18 300 Å
column; gradient of methanol and 5 mM pH 7.4 phosphate buffer (v/
v): 10:95 at 0 min, 40:60 at 10 min). Ascorbate, Trolox, or glutathione
(0.25, 0.5, 1, 2, 5 mM) was added to the reaction mixtures to prevent
the nitration of HPA.
removal of both O2 and ONOO- mediated by the ONOO-
reductase activity of Mn(III)TMPyP may become significant
in the cell-protective mechanism of this and related metallopor-
phyrins.44
•-
Finally, the dissection described herein of the mechanisms
•-
of the reactions of Mn(III)TMPyP with O2 and ONOO-, in
the presence and absence of antioxidants, may have significant
biological implications beyond elucidation of the cell-protective
effects of the metalloporphyrins. ONOO- is known to react
rapidly with a host of metalloenzymes, such as myeloperoxidase
(k > 2 × 106 M-1 s-1),25 Cu,ZnSOD (k ) 103-105 M-1 s-1),45
MnSOD,46 and P450-like enzymes such as nitric oxide syn-
thase47 and prostacyclin synthase,48 and cytochrome c.49 Thus,
Superoxide Dismutation. A DMSO solution of KO2 (∼2 mM) was
prepared following the published procedure.28 The dismutation of O2
•-
was monitored directly at the O2•- absorbance (245 nm), using a special
setup of the four-syringe, double-mixing sample-handling unit to reduce
changes in the refractive index due to the mixing of DMSO and
buffers.52 In this stopped-flow setup, the KO2/DMSO solution was
loaded in syringe A (0.5 mL) and 50 mM pH 7.4 phosphate buffer
was loaded in syringes B (2.5 mL), C (0.5 mL), and D (2.5 mL)
(syringes A, B, C, and D were labeled from left to right). All four
syringes were pushed up simultaneously by the extended plate; the
contents of syringes A and B were first mixed in an aging loop, and
then mixed with the contents of syringes C and D. A 12-fold dilution
of the KO2/DMSO solution was achieved by using this two-stage
mixing, thus minimizing the interference due to DMSO/water interac-
tion. In the SOD activity assays, manganese porphyrin solutions (either
Mn(III)TMPyP or Mn(III)TBAP) were loaded into syringe C.53
•-
the reactions of O2 and ONOO- with MnTMPyP and other
metalloporphyrins may provide an instructive model for under-
standing reactions of oxidants with these important biological
targets.
Experimental Section
Materials. 5,10,15,20-Tetrakis(N-methyl-4′-pyridyl)porphinatoman-
ganese(III) chloride [Mn(III)TMPyP(Cl)] was purchased from Mid-
Century Chemical. Anhydrous monosodium phosphate and anhydrous
(38) Kooy, N. W.; Royall, J. A. Arch. Biochem. Biophys. 1994, 310,
352.
For the O2•--coupled ONOOj reduction experiment, ONOOj, Mn-
(III)TMPyP, KO2/DMSO, and 50 mM pH 7.4 phosphate buffer were
placed in syringes A, B, C, and D, respectively. Though the four
syringes were pushed up simultaneously by an extended plate, ONOO-
(39) Margolis, S. A.; Davis, T. Clin. Chem. 1988, 34, 2217.
(40) Behrens, W. A.; Made`re, R. Anal. Biochem. 1987, 165, 102.
(41) Stryer, L. Biochemistry, 4th ed.; W. H. Freeman and Company: New
York, 1995; p 731.
(42) Sies, H.; Murphy, M. E. J. Photochem. Photobiol. 1991, B 8, 211.
(43) Burton, G. W.; Traber, M. G. Annu. ReV. Nutr. 1990, 10, 357.
(44) Hunt, J. A.; Lee, J.; Groves, J. T. Chem. Biol. 1997, 4, 845.
(45) Gryglewski, R. J.; Palmer, R. M. J.; Moncada, S. Nature 1986, 320,
454.
(46) MacMillan-Crow, L. A.; Crow, J. P.; Kerby, J. D.; Beckman, J. S.;
Thompson, J. A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 11853.
(47) Pasquet, J. P. E. E.; Zou, M. H.; Ullrich, V. Biochimie 1996, 78,
785.
(49) Thomson, L.; Trujillo, M.; Telleri, R.; Radi, R. Arch. Biochem.
Biophys. 1995, 319, 491.
(50) Groves, J. T.; Neumann, R. J. Am. Chem. Soc. 1989, 111, 2900.
(51) Huang, C. Biochemistry 1969, 8, 344.
(52) See Hi-Tech Scientific Application Note AN.010.S60 for more
detail.
(53) Forni, L. G.; Mora-Arellano, V. O.; Packer, J. E.; Willson, R. L. J.
Chem. Soc., Perkin Trans. 2 1986, 1.
(48) Zou, M. H.; Ullrich, V. FEBS Lett. 1996, 382, 101.

Mn Porphyrins as Redox-Coupled ONOO- Reductases
J. Am. Chem. Soc., Vol. 120, No. 24, 1998 6061
was first mixed with Mn(III)TMPyP in an aging loop to form oxoMn-
(IV)TMPyP, and then mixed with KO2/DMSO. Significant mixing
artifacts at the beginning of the kinetic traces (∼5 ms) were observed
because the KO2/DMSO solution did not go through the aging loop to
mix with buffer first. As a control experiment, the reaction of 100
µM ONOO- and ∼160 µM O2•- was monitored. The presence of the
O2•- did not significantly affect the rate of decomposition of ONOO-,
of the first-order rate constants, kobs (s-1), vs H2O2 concentrations (R
) 0.999).
Reaction kinetics were simulated by using the Chemical Kinetics
Simulator developed by IBM Almaden Research Center. The general
settings for the simulation shown in Figure 7 are as follows: total
number of molecules ) 108; record state at interval ) 75; random
number seed ) 12947.
•-
suggesting that this reaction is slower than the self-dismutation of O2
Acknowledgment. Support of this research by the National
Institutes of Health (GM36928), the National Science Founda-
tion for the purchase of 500 and 600 MHz NMR spectrometers,
and Princeton University for the purchase of a stopped-flow
spectrophotometer are gratefully acknowledged. We acknowl-
edge the excellent technical assistance of Steven C. Tizio.
J.A.H. is the recipient of an NIH NRSA fellowship (GM18490).
under the experimental conditions.
To determine the significance of H2O2-dependent ONOOj reduction,
the reduction rate of the oxoMn(IV) intermediate by H2O2 was measured
in a double-mixing experiment analogous to those described above for
the antioxidants. The oxoMn(IV) intermediate was generated by mixing
5 µM Mn(III)TMPyP with 5 µM HSO5- in the first mixing step. After
a 5 s age time, addition of H2O2 under pseudo-first-order conditions in
the second mixing step resulted in the reduction of oxoMn(IV) to Mn-
(III). The second-order rate constant was obtained from the linear plot
JA9801036