Mechanism of iron porphyrin reactions with peroxynitrite

Article abstract of DOI:10.1021/ja980507y

Peroxynitrite (ONOO^-) is a major cytotoxic agent that has been implicated in a host of pathophysiological conditions; it is therefore important to develop therapeutic agents to detoxify this potent biological oxidant, and to understand the modes of action of these agents. Water-soluble iron porphyrins, such as 5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphinatoiron(III) [Fe(III)TMyP] and 5,10,15,20-tetrakis-(2,4,6-trimethyl-3,5-sulfonatophenyl)porphinatoiron(III) [Fe(III)TMPS], have been shown to catalyze the efficient decomposition of ONOO^- to NO₃^- and NO₂^- under physiological conditions. However, the mechanisms of ONOO^- decomposition catalyzed by these water-soluble iron porphyrins have not yet been elucidated. We have shown that there are two different pathways operating in the catalytic decomposition of ONOO^- by FeTMPyP. Fe(III)TMyP reacts rapidly with ONOO^- to produce oxoFe(IV)TMPyP and NO₂ (k ~ 5 x 10⁷ M^-1 s^-1). The oxoFe(IV) porphyrin, which persisted throughout the catalytic decomposition of ONOO^-, was shown to be relatively unreactive toward NO₂ and NO₂^-. This oxoFe(IV) porphyrin was also shown to react with ONOO^- (k = 1.8 x 10⁶ M^-1 s^-1), and it was this oxoFe(IV)-ONOO^- reaction pathway that predominated under conditions of excess ONOO^- with respect to Fe(III)TMPyP. The competition between the two pathways explains the highly nonlinear relationship observed for k(cat) with respect to ONOO^- concentration. Fe(III)-TMPyP is also known to catalyze the dismutation of the ONOO^- precursor superoxide (O^2-.), and using stopped-flow spectrophotometry, the rate of Fe(III)TMPyP-catalyzed O₂^-. dismutation has been determined to be 1.9 x 10⁷ M^-1 s^-1 by direct measurement. A detailed mechanistic understanding of how iron porphyrins function in the catalytic decomposition of both ONOO^- and O₂^-. may prove essential in the exploration of the chemistry and biology of these reactive oxygen species, and in understanding the biological activity of these metalloporphyrins.

Full text of DOI:10.1021/ja980507y

J. Am. Chem. Soc. 1998, 120, 7493-7501
7493
Mechanisms of Iron Porphyrin Reactions with Peroxynitrite
Jinbo Lee, Julianne A. Hunt, and John T. Groves*
Contribution from the Department of Chemistry, Princeton UniVersity, Princeton, New Jersey 08544
ReceiVed February 16, 1998
-
Abstract: Peroxynitrite (ONOO ) is a major cytotoxic agent that has been implicated in a host of
pathophysiological conditions; it is therefore important to develop therapeutic agents to detoxify this potent
biological oxidant, and to understand the modes of action of these agents. Water-soluble iron porphyrins,
such as 5,10,15,20-tetrakis(N-methyl-4′-pyridyl)porphinatoiron(III) [Fe(III)TMPyP] and 5,10,15,20-tetrakis-
(
2,4,6-trimethyl-3,5-sulfonatophenyl)porphinatoiron(III) [Fe(III)TMPS], have been shown to catalyze the efficient
-
-
-
decomposition of ONOO to NO3 and NO2 under physiological conditions. However, the mechanisms of
ONOO decomposition catalyzed by these water-soluble iron porphyrins have not yet been elucidated. We
have shown that there are two different pathways operating in the catalytic decomposition of ONOO by
-
-
-
7
FeTMPyP. Fe(III)TMPyP reacts rapidly with ONOO to produce oxoFe(IV)TMPyP and NO2 (k ≈ 5 × 10
-
1
-1
-
M
s ). The oxoFe(IV) porphyrin, which persisted throughout the catalytic decomposition of ONOO , was
-
shown to be relatively unreactive toward NO2 and NO2 . This oxoFe(IV) porphyrin was also shown to react
-
6
-1 -1
-
with ONOO (k ) 1.8 × 10 M s ), and it was this oxoFe(IV)-ONOO reaction pathway that predominated
-
under conditions of excess ONOO with respect to Fe(III)TMPyP. The competition between the two pathways
-
explains the highly nonlinear relationship observed for kcat with respect to ONOO concentration. Fe(III)-
TMPyP is also known to catalyze the dismutation of the ONOO precursor superoxide (O2 ), and using
stopped-flow spectrophotometry, the rate of Fe(III)TMPyP-catalyzed O2 dismutation has been determined
-
-•
-
•
7
-1 -1
to be 1.9 × 10 M
s
by direct measurement. A detailed mechanistic understanding of how iron porphyrins
-
-•
function in the catalytic decomposition of both ONOO and O2 may prove essential in the exploration of
the chemistry and biology of these reactive oxygen species, and in understanding the biological activity of
these metalloporphyrins.
•
Introduction
or HO , can travel distances of cellular dimensions and should
have free access to cell interiors, even in the presence of
biological membranes. There is mounting evidence that reactive
oxygen and nitrogen species play important roles in the
-
Peroxynitrite (ONOO ) is a potent cytotoxic agent formed
by the direct and rapid combination of nitric oxide and the
-
• 1-3
superoxide anion (O2 ).
A strong one- and two-electron
18
regulation and inhibition of mitochondrial respiration and in
-
oxidant, ONOO has been shown to react with a wide variety
of biomolecules, including proteins (via nitration of tyrosine
and tryptophan residues, oxidation of methionine and sele-
nocysteine residues, and oxidation of metalloenzymes
19,20
-
apoptosis;
recent studies have shown that ONOO in
4
,5
particular is involved in apoptotic cell death in leukemia
6
,7
8
21,22
23
24
cells,
aortic smooth muscle cells, and neural cells. In
9
10,11
),
light of its high reactivity with biological targets, and possibly
connected with its proposed role in the regulation of mitochon-
DNA,1
2,13
lipids, and antioxidants.
14
15,16
We have recently
-
demonstrated that ONOO crosses lipid membranes at a rate
significantly faster than the rates of its known decomposition
-
drial respiration and cellular apoptosis, ONOO has been
implicated in a host of disease states, including neurodegen-
17
-
-•
pathways. Thus ONOO , unlike reactive radicals such as O2
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To whom correspondence should be addressed. Phone: (609) 258-
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S0002-7863(98)00507-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/18/1998

7494 J. Am. Chem. Soc., Vol. 120, No. 30, 1998
GroVes, et al.
erative disorders,25-38 heart diseases,
39,40
chronic inflammation
ischemia-reperfusion
Mn(III)TMPyP and its amphiphilic analogues requires the
presence of biological antioxidants, and the concentrations of
these antioxidants might be low under conditions of oxidative
stress, when the levels of prooxidants and antioxidants are not
41-44
45-47
and autoimmune diseases,
injury,
cancer,
1
,48-51
52
and septic shock. This large and rapidly expand-
-
ing body of evidence on the role of ONOO in a wide variety
of human diseases has naturally led to a search for drugs that
can intercept this powerful oxidant.
5
7,58
-
in balance.
Thus, it is highly desirable to develop ONOO
decomposition catalysts that function even in the absence
biological antioxidants.
-
We have recently shown that ONOO reacts rapidly and
5
9
stoichiometrically with the water-soluble synthetic manganese
porphyrin 5,10,15,20-tetrakis(N-methyl-4′-pyridyl)porphinato-
manganese(III) [Mn(III)TMPyP] to generate oxomanganese
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of ONOO in the presence of biological antioxidants, such as
ascorbate, glutathione, and Trolox (a water-soluble analogue
of R-tocopherol). In addition to this “peroxynitrite reductase”
activity, Mn(III)TMPyP has been shown to possess significant
activity for the simultaneous oxidation of superoxide (O2 ) and
reduction of ONOO . Furthermore, amphiphilic analogues
of Mn(III)TMPyP have been prepared which proved to be
Stern et al. have shown that 5,10,15,20-tetrakis(N-methyl-
4′-pyridyl)porphinatoiron(III) [Fe(III)TMPyP] and 5,10,15,20-
tetrakis(2,4,6-trimethyl-3,5-sulfonatophenyl)porphinatoiron-
(III) [Fe(III)TMPS] display profound activity in biological
17,53
intermediates.
-
-
models of ONOO related disease states and have been
-
investigated as therapeutic agents for diseases in which ONOO
has been implicated.
54
59-62
These iron porphyrins were proposed
to possess catalytic “peroxynitrite isomerase” activity, converting
-•
-
-
ONOO to NO3 . However, a consensus has not yet been
-
55
-
reached on the mechanism of ONOO decomposition catalyzed
by these iron porphyrins.
-
effective ONOO decomposition catalysts in liposomal as-
Herein we elaborate on the reactions of Fe(III)TMPyP with
semblies.5 The fast catalytic turnover of both the water-soluble
6
-
ONOO . Significantly, both Fe(III)TMPyP and oxoFe(IV)-
-
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Fe Porphyrin Reactions with Peroxynitrite
J. Am. Chem. Soc., Vol. 120, No. 30, 1998 7495
-
ONOO with horseradish peroxidase has been shown to produce
compound I,1 while it is the compound II analogue that has
0,77
been observed under conditions of catalytic turnover with Fe-
(
III)TMPS.59
-
Our studies of the catalytic mechanism of ONOO isomer-
ization by Fe(III)TMPyP thus began with an investigation of
reactive oxoFe intermediates. The generation of oxoFe inter-
-
mediates by ONOO and analogous peroxides, such as m-CPBA
-
and HSO5 , was explored with use of rapid-mixing stopped-
flow spectrophotometry.
Detection of Reactive Intermediates. The stoichiometric
reaction of 5 µM Fe(III)TMPyP and 5 µM m-CPBA produced
an oxidized iron porphyrin intermediate with a Soret maximum
at 427 nm, as monitored by time-resolved UV-vis spectroscopy
(Figure 1a). This intermediate then decayed slowly back to Fe-
(III)TMPyP (λmax ) 423 nm). The spectrum of the observed
intermediate and the spectral change in the conversion of this
intermediate back to Fe(III)TMPyP are nearly identical to those
of well-studied oxoFe(IV) porphyrin intermediates of other
5
9,78
water-soluble iron porphyrins.
Thus, we formulate the
intermediate as an oxoFe(IV) species.7
9-82
Though isosbestic
points around 410, 448, 522, and 568 nm can be identified in
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resulted in significant destruction of the porphyrin chromophore
(bleaching), with only 62% of the Fe(III)TMPyP recovered after
the reaction (Figure 1a, inset).
-
One equivalent of nitrite (NO2 ) in an otherwise identical
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were better defined in the time-resolved UV-vis spectra when
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Figure 1. (a) Time-resolved UV-vis spectra of the stoichiometric
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equiv of NO2 was present (data not shown). In fact, addition
-
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bleaching than oxoFe(IV); this intermediate can be reduced
(
74) Dawson, J. H.; Eble, K. S. AdV. Inorg. Bioinorg. Mech. 1986, 4,
1
-64.
75) Groves, J. T.; Haushalter, R. C.; Nakamura, M.; Nemo, T. E.; Evans,
B. J. J. Am. Chem. Soc. 1981, 102, 2884-2886.
76) Groves, J. T.; Gross, Z.; Stern, M. K. Inorg. Chem. 1994, 33, 5065-
072.
77) Floris, R.; Piersma, S. R.; Yang, G.; Jones, P.; Wever, R. Eur. J.
Biochem. 1993, 215, 767-75.
78) Colclough, N.; Smith, J. R. L. J. Chem. Soc., Perkin Trans. 2 1994,
139-1149.
79) Chen, S.-M.; Su, Y. O. J. Chem. Soc., Chem. Commun. 1990, 491-
93.
(
(
5
(
-
rapidly by NO2 , and this reduction protects the porphyrin from
(
oxidative destruction. Thus, the overall reaction, presented in
eq 1, involves oxidation of Fe(III)TMPyP to a reactive porphyrin
1
4
1
(
-
intermediate, which is then reduced by NO2 to an oxoFe(IV)
(80) Rodgers, K. R.; Reed, R. A.; Su, Y. O.; Spiro, T. G. Inorg. Chem.
species.
992, 31, 2688-2700.
(81) Mn(III)/Mn(IV) porphyrin redox potentials are higher than corre-
–
sponding values for iron, cf.: Kaaret, T. W.; Zhang, G.-H.; Bruice, T. C.
J. Am. Chem. Soc. 1991, 113, 4652-4656.
(82) Jeon, S.; Bruice, T. C. Inorg. Chem. 1992, 31, 4843-4848.

7496 J. Am. Chem. Soc., Vol. 120, No. 30, 1998
GroVes, et al.
Figure 3. The decay of the oxoFe(IV) species back to Fe(III)TMPyP
is a first-order process. The kinetic trace shown was obtained from a
double-mixing stopped-flow experiment, in which the oxoFe(IV) species
-
was generated by reaction of 5 µM Fe(III)TMPyP with 5 µM HSO
5
in the first-mixing step, and 50 mM pH 7.4 phosphate buffer was added
in the second-mixing step. The experimental data nicely fit a single-
-
1
exponential curve, giving a first-order rate constant of k ) 0.053 s
(
R ) 0.999). Inset: Slow reduction of the oxoFe(IV) species by
-
1
-1
NaNO
2
,with a second-order rate constant of k ) 73 M
s
(R )
0
.999).
Figure 2. (a) Time-resolved UV-vis spectra of the reaction of 5 µM
over the period of a minute (Figure 2a, inset), as in the m-CPBA
reaction shown above. This observation indicates that the
recombination of oxoFe(IV)TMPyP and NO2 is a slow process,
which cannot be the major contributor to the catalytic turnover
-
Fe(III)TMPyP with 100 µM ONOO in 25 mM phosphate buffer at
pH 7.4, 25 °C. The decay of the oxoFe(IV) intermediate back to Fe-
(III)TMPyP is shown. The reaction was followed by UV-vis spec-
troscopy (100 scans in 30 s, 0.005 s integration time) with a stopped-
flow spectrophotometer (for clarity, every other scan is shown for the
first 10 scans, and thereafter every tenth scan is shown). Inset: Decay
-
in Fe(III)TMPyP catalyzed decomposition of ONOO . When
-
5
µM Fe(III)TMPyP reacted with 100 µM ONOO , the oxoFe-
-
of the oxoFe(IV) species back to Fe(III)TMPyP monitored at 427 nm.
(IV) species persisted throughout the decay of ONOO moni-
tored at 302 nm (compare Figure 2b with Figure 2a, inset). After
ONOO was fully decomposed, the oxoFe(IV) species then
-
(
b) Decomposition of 100 µM ONOO catalyzed by 5 µM Fe(III)-
-
-
TMPyP. The decay of ONOO was monitored at 302 nm.
started its typical slow decay back to Fe(III)TMPyP (Figure
This fast reduction of a reactive iron porphyrin intermediate by
2
a, inset). Again, these results suggest that oxoFe(IV)TMPyP
-
NO2 is highly analogous to the reduction of oxoMn(V)TMPyP
cannot be the transient intermediate in the turnover of Fe(III)-
-
83
by NO2 , which is known to be rapid and quantitative.
-
TMPyP-catalyzed decomposition of ONOO . Thus, we set out
Possible candidates for the reactive iron intermediate thus
include the oxoFe(IV) porphyrin cation radical (as shown in eq
to determine the role of the oxoFe(IV) species in the catalytic
-
decomposition of ONOO .
7
5
1)
or an oxoFe(V) species.
The Role of OxoFe(IV) in Peroxynitrite Decomposition.
-
Similarly, ONOO oxidized Fe(III)TMPyP to the same
-
The oxoFe(IV) intermediate generated by ONOO and m-CPBA
oxoFe(IV) porphyrin species at concentrations as low as 1 equiv,
simultaneously producing 1 equiv of NO2, as monitored by time-
resolved UV-vis spectroscopy (eq 2).
-
was also generated by reaction of Fe(III)TMPyP with HSO5 ,
but at a slower rate. This is consistent with the trend in
oxidation rates we previously observed in the reactions of Mn-
-
- 83
(
III)TMPyP with m-CPBA, ONOO , and HSO5 .
The
–
second-order rate constant for the reaction of Fe(III)TMPyP with
-
5
-1 -1
HSO5 is 2.1 × 10 M s , obtained from a linear plot of the
-
-
apparent first-order rate constants, kobs, against the HSO5
concentrations.
Data for the reaction of Fe(III)TMPyP with excess ONOO are
8
4
The oxoFe(IV) species generated by this
shown in Figure 2a; analogous results were obtained in the
stoichiometric reaction (data not shown). The formation of the
oxoFe(IV) species and NO2 could be achieved either by
reaction was fairly stable, decaying slowly back to Fe(III)-
-
1
TMPyP with a first-order rate constant of 0.053 s (Figure 3).
Not only was the recombination of oxoFe(IV) and NO2 a slow
process, as discussed above, but the reduction of oxoFe(IV) by
-
homolysis of the bound ONOO or heterolysis followed by fast
reduction of the oxoFe(IV) porphyrin cation radical [or the
-
-
NO2 was also found to be slow, with a second-order rate
oxoFe(V) species] by NO2 in the solvent cage. Surprisingly,
the oxoFe(IV) porphyrin intermediate persisted even in the
presence of NO2, and then slowly returned to Fe(III)TMPyP
(84) The second-order rate constant for the oxidation of Fe(III)TMPyP
-
-
by HSO5 was determined from the following data points: 50 µM HSO5 ,
-
1
-
-1
-
kobs ) 10.4 s ; 75 µM HSO5 , kobs ) 15.1 s ; 100 µM HSO5 , kobs )
-
1
-
-1
-
(83) Groves, J. T.; Lee, J.; Marla, S. S. J. Am. Chem. Soc. 1997, 119,
20.5 s ; 125 µM HSO5 , kobs ) 26.6 s ; 150 µM HSO5 , kobs ) 31.6
-1
6
269-6273.
s ; R ) 0.999.

Fe Porphyrin Reactions with Peroxynitrite
J. Am. Chem. Soc., Vol. 120, No. 30, 1998 7497
constant of 73 M-1 s-1 (Figure 3, inset).85 The stability of the
-
oxoFe(IV)TMPyP species and its low reactivity toward NO2
are, again, analogous to the stability and low reactivity of the
corresponding oxoMn(IV) porphyrin intermediate,8 which is
3
-
17,54
known to be unable to catalyze decomposition of ONOO .
Thus, we were surprised to discover that the oxoFe(IV)
species generated independently by reaction of Fe(III)TMPyP
-
-
with HSO5 was active in decomposing ONOO . In a double-
mixing experiment, the oxoFe(IV) species was generated by
the stoichiometric reaction of 5 µM Fe(III)TMPyP and 5 µM
-
-
HSO5 in the first mixing step; then 100 µM ONOO was added
in the second mixing step. The kinetic profile for ONOO
-
decay catalyzed by the oxoFe(IV) species was observed to be
-
indistinguishable from that of the ONOO decay in the direct
-
mixing of Fe(III)TMPyP and ONOO (Figure 4a). Thus, the
oxoFe(IV) porphyrin intermediate, and not Fe(III)TMPyP, must
-
be the major species catalyzing the isomerization of ONOO
-
under these conditions. In fact, the rate of ONOO decomposi-
tion was not linearly dependent on the Fe(III)TMPyP concentra-
tion in a series of experiments in which the concentration of
-
ONOO was kept constant while the concentration of Fe(III)-
TMPyP was varied. Instead, an upward parabolic relationship
-
was found (Figure 4b). When the concentration of ONOO
was much higher than the concentration of iron catalyst (reaction
-
under pseudo-first-order conditions), the rate of ONOO decay
was almost linearly dependent on the concentration of Fe(III)-
TMPyP. A catalytic rate constant (kcat) was estimated to be
6
-1 -1
around 1.8 × 10 M
s
under these conditions, consistent
59
with that estimated by Stern et al.
However, the rate of ONOO decomposition became much
faster, and deviated significantly from a linear relationship, when
-
-
the concentration of ONOO was comparable to that of Fe-
(III)TMPyP and the reaction was thus no longer under pseudo-
first-order conditions (Figure 4b). This clearly indicates a
-
change of mechanism for ONOO decomposition as the relative
-
concentrations of ONOO and Fe(III)TMPyP were changed.
Interestingly, this nonlinear relationship is similar to the
observation by Stern et al., for a series of experiments in which
the concentration of Fe(III)TMPS was held constant while the
-
59
-
concentration of ONOO was varied. Further, the catalytic
Figure 4. (a) Overlay of two kinetic traces of ONOO decay monitored
-
-
at 302 nm. The solid trace shows decomposition of 100 µM ONOO
in the presence of 5 µM Fe(III)TMPyP. The dotted trace shows a
double-mixing experiment in which the oxoFe(IV) species was gener-
ated by reaction of 5 µM Fe(III)TMPyP with 5 µM HSO
mixing step, then 100 µM ONOO was added in the second mixing-
step. (b) Dependence of the rate of ONOO decomposition on the
concentration of Fe(III)TMPyP in 25 mM phosphate buffer at pH 7.4,
5 °C. The decay of ONOO was followed at 302 nm by stopped-
rate constant of ONOO decomposition catalyzed by Fe(III)-
7
-1 -1 86
TMPyP in the presence of ascorbate (kcat > 10 M s ) was
significantly faster than that in the absence of ascorbate (kcat ∼
-
5
in the first
6
-1 -1 59
2
.2 × 10 M s ), which again indicates a change of
-
mechanism between these two processes. Finally, the catalytic
decomposition of ONOO at near stoichiometric concentrations
-
-
(
10 µM) was almost 10 times faster than the catalytic decom-
-
2
-
-
position of ONOO under pseudo-first-order conditions (100
flow spectrophotometry. The concentration of ONOO was kept
µM), though the concentrations of the catalyst, Fe(III)TMPyP
constant (50 µM), and the concentration of Fe(III)TMPyP was varied
(1-15 µM). The first-order rate constants, kobs (s ), were estimated
by fitting the kinetic traces into single-exponential expressions.
-
1
(5 µM), were the same in both reactions (Figure 5).
-
Clearly the Fe(III)TMPyP-catalyzed decomposition of ONOO
is a complicated process. However, all the results presented
above are consistent with the simplified mechanism shown in
Scheme 1, in which two interconnected catalytic processes are
proposed: A fast pathway (path 1) involves Fe(III)TMPyP as
a stoichiometric reactant, while a 10-fold slower pathway (path
NO ; subsequent rapid dimerization of NO to N O (k ) 9 ×
2
2
2
4
8
-1 -1 87
10 M s ) would be followed by hydrolysis to 1 equiv of
-
-
5
-1 -1
87b
NO2 and 1 equiv of NO3 (eq 3), 7 × 10 M
s
overall.
–
–
2
) involves an oxoFe(IV) species as the active catalyst.
Mechanism of Fe(III)TMPyP-Catalyzed ONOO Decom-
-
Once the oxoFe(IV) species is formed, the second and slower
pathway (Scheme 1, path 2) would become the active catalytic
process, resulting in the conversion of ONOO to NO3 . This
second catalytic pathway would explain the observation that
position. In the first and faster process (Scheme 1, path 1),
ONOO reacts with Fe(III)TMPyP to produce oxoFe(IV) and
-
-
-
(85) As observed by Stern et al. (ref 59), unlike the oxoFe(IV) species
of the cationic porphyrin FeTMPyP, the corresponding oxoFe(IV) species
of the anionic porphyrin FeTMPS is rapidly reduced to Fe(III) by NO2 .
We thank M. K. Stern for a gift of FeTMPS.
(87) (a) Forni, L. G.; Mora-Arellano, V. O.; Packer, J. E.; Willson, R.
L. J. Chem. Soc., Perkin Trans. 2 1986, 1-5. (b) Cape, J. N.; Storetonwest,
R. L.; Devine, S. F.; Beatty, R. N.; Murdoch, A. Atmos. EnViron. 1993,
27, 2613-21.
-
(86) Stern, M. K. Personal communication.

7498 J. Am. Chem. Soc., Vol. 120, No. 30, 1998
GroVes, et al.
Figure 6. Formation and decay of reactive intermediate I (see Scheme
-
2
) in the reaction of 5 µM Fe(III)TMPyP and 10 µM ONOO ,
monitored at 427 nm. Fitting the decay to a single exponential gave
-
1
-
6
2.2 ( 0.8 s (R ) 0.993). Inset: Simultaneous decay of ONOO ,
monitored at 302 nm, in the same reaction.
-
dominant in the fomer case, and thus more NO3 was
5
9,85
produced.
-
When ONOO is under pseudo-first-order conditions relative
to the concentration of Fe(III)TMPyP, the two catalytic pro-
cesses depicted in Scheme 1 could collapse into an apparent
single pathway in which the oxoFe(IV) catalytic pathway (path
Figure 5. Decomposition of (a) 10 µM and (b) 100 µM ONOO-
2
) predominates. The initial reservoir of Fe(III)TMPyP would
catalyzed by 5 µM Fe(III)TMPyP in 25 mM phosphate buffer at pH
-
be quickly depleted by the first equivalent of ONOO during
the dead time of our stopped-flow instrument, as was observed.
In the subsequent catalytic reaction, the oxoFe(IV) intermediate
would thus be the active species that isomerized the bulk of
-
7
.4, 25 °C. The decay of ONOO was followed at 302 nm.
Scheme 1. A Simplified Mechanism of the Fe(III)TMPyP-
Catalyzed Decomposition of ONOOj, Which Consists of
Two Catalytic Cyclesa
-
-
the ONOO to NO3 . This would explain why the two traces
in Figure 4a overlay so well, and why the correlation in Figure
4
b at low Fe(III)TMPyP concentrations is nearly linear with an
6
-1 -1
estimated kcat ) 1.8 × 10 M
s
for path 2. However, the
contribution of the fast pathway (path 1) would become
significant when the concentrations of Fe(III)TMPyP and
-
ONOO are near stoichiometric (Figure 5a). Further, as shown
in Scheme 1, the presence of ascorbate would short-circuit the
slow pathway (path 2), making the fast pathway dominant by
reducing oxoFe(IV) back to Fe(III). The rate of oxoFe(IV)
reduction by ascorbate was estimated to be approximately 2 ×
8
-1 -1
1
0 M
s
(data not shown), which is similar to the rate of
5
4,55
oxoMn(IV) reduction by ascorbate.
kcat for the catalyzed ONOO decomposition in the presence
Not surprisingly, the
-
of physiologically significant concentrations of ascorbate has
7
-1 -1 86
been estimated to be >10 M s , which is much faster than
that in the absence of ascorbate which can reduce both oxoFe-
(
IV) and intermediate I. Also consistent with the mechanism
-
shown in Scheme 1, the yield of NO2 increased significantly
under these reducing conditions.
8
6
Intermediates and Mechanisms of Fast and Slow Path-
ways. The simplified mechanism in Scheme 1 gives an overall
-
picture of Fe(III)TMPyP-catalyzed decomposition of ONOO .
However, the detailed mechanisms require further elaboration,
since it became apparent during the course of our investigation
that additional intermediates were involved.
a
The fast cycle (path 1) involves Fe(III) as the active catalyst; the
slower cycle (path 2) involves oxoFe(IV) species as the active catalyst.
When Fe(III)TMPyP was allowed to react with near sto-
-
ichiometric amounts of ONOO , a new reactive intermediate,
I, was detected (Figure 6). As determined by monitoring the
Soret λmax at 427 nm, intermediate I was formed and reached a
steady state during the course of ONOO decomposition (Figure
NO3- is the main product in the iron porphyrin-catalyzed
-
59
-
-
decomposition of ONOO . In fact, the NO2 /NO3 ratio was
found to be lower for the more reactive porphyrin, Fe(III)-
TMPyP, than for a less reactive porphyrin, Fe(III)TMPS,
presumably because the oxoFe(IV) pathway (path 2) was more
-
6
). The formation rate of intermediate I was very rapid, with
7
-1
an estimated second-order rate constant faster than 5 × 10 M
-
1
s
. Interestingly, the half-life of intermediate I (≈10 ms)

Fe Porphyrin Reactions with Peroxynitrite
J. Am. Chem. Soc., Vol. 120, No. 30, 1998 7499
Scheme 2. Proposed Mechanisms and Intermediates in the
Fast (path 1) and Slow (path 2) Cycles of the
Fe(III)TMPyP-Catalyzed Decomposition of ONOOj
Figure 7. (a) Formation of reactive intermediate II (see Scheme 2) in
-
the reaction of 5 µM Fe(III)TMPyP and 100 µM ONOO , monitored
at 427 nm. Inset: Kinetic profile of the formation of intermediate II
fitted to a single-exponential curve, providing a first-order rate constant
-
1
of 33.7 ( 0.7 s (R ) 0.994). (b) Formation of reactive intermediate
II in a double-mixing stopped-flow experiment, in which the oxoFe-
(
IV) species was generated in the first-mixing step by reaction of 5
-
-
µM Fe(III)TMPyP with 5 µM HSO , and 100 µM ONOO was added
5
in the second mixing step.
(
IV), which would be captured by excess ONOO- to form
coincides with that of ONOO- (≈8 ms; Figure 6, inset, and
Figure 5a), suggesting that the decay of I is the rate-limiting
step under these conditions. A likely candidate for intermediate
intermediate II. Thus, the rate of formation of intermediate II
should be limited by the decay of I. Indeed, the rate of
intermediate II formation was estimated to be about 33.7 s
under these conditions (Figure 7a, inset), which was comparable
to but slower than the decay rate observed for intermediate I
-
1
-
I is a 1:1 adduct of Fe(III)TMPyP and ONOO (see Scheme 2,
-
path 1). Ligand exchange to form the Fe(III)TMPyP-ONOO
-
1
adduct is expected to be fast, and cleavage of the adduct to
produce oxoFe(IV) and NO2 would thus be rate-limiting. The
apparent first-order rate constant for the decay of intermediate
I, determined by fitting of the trace shown in Figure 6 to a
(62.2 s ).
To confirm that intermediate II was produced from a reaction
-
of oxoFe(IV) and ONOO , the oxoFe(IV) species was prepared
independently by the stoichiometric reaction of Fe(III)TMPyP
(5 µM) and HSO5 (5 µM). Subsequent addition of ONOO
(100 µM) under pseudo-first-order conditions afforded the same
intermediate II (Figure 7b), with exactly the same kinetic
behavior as the intermediate in the direct reaction of Fe(III)-
-
1
-
-
single-exponential decay, was 62.2 s .
A second intermediate, II, was detected when excess ONOO
-
(100 µM) reacted with Fe(III)TMPyP (5 µM) under pseudo-
first-order conditions (Figure 7a). The formation and decay of
-
intermediate II were much slower than those of intermediate I.
TMPyP and ONOO .
-
Significantly, intermediate II reached a steady state as ONOO
The exact identity of intermediate II is not clear from the
information at hand, though several possible candidates are
shown in Scheme 2 (structures A, B, and C). Structure A is a
ligation adduct of ONOO and oxoFe(IV), and the similarity
of the visible spectrum of II and oxoFe(IV) supports this
structural assignment for II. Adduct A could decompose to an
oxoFe(IV) porphyrin cation radical species and NO by ho-
molysis of the oxoFe(IV)-bound ONOO . Subsequent rebound
of NO with the oxoFe(IV) porphyrin cation radical would
produce NO3 and oxoFe(IV), thus completing the catalytic
cycle of path 2.
The second candidate for intermediate II is structure B, the
oxoFe(IV) porphyrin cation radical itself [or oxoFe(V)], possibly
formed by homolysis of the O-O bond in structure A, with
concurrent formation of an equivalent of NO2. However, the
was disappearing via the slower path 2, and this steady state
was followed by the decay of II to the oxoFe(IV) species.
Estimation of the decay rate of II back to oxoFe(IV) was not
straightforward, as the kinetics of this decay overlapped with
the decay of oxoFe(IV) to Fe(III)TMPyP. Nevertheless, under
these pseudo-first-order conditions, the decay of intermediate
-
2
-
-
II was almost as fast as the disappearance of ONOO , which
-
1
had an apparent first-order rate of 13.1 s
under these
2
-
conditions (see Figure 5b for a representative trace). Therefore,
the turnover of the catalytic cycle (path 2) appeared to be limited
by the decay rate of intermediate II.
-
Under conditions of excess ONOO , intermediate I would
be fully formed during the dead time of our stopped-flow
instrument. Intermediate I would then decay rapidly to oxoFe-

7500 J. Am. Chem. Soc., Vol. 120, No. 30, 1998
GroVes, et al.
similarity of the visible spectrum of II and oxoFe(IV) makes
structure B a less likely intermediate than A or C. Reduction
-
of intermediate II-B by NO2 to give the oxoFe(IV) porphyrin
and NO2 is expected to be very fast, by analogy to the rapid
-
83
-
reduction of oxoMn(V) by NO2 . Thus, NO2 (generated via
eq 3) could be recycled to NO2 during the catalytic turnover,
-
-
and this NO2 would then generate more NO2 and NO3 .
Eventually, in this scenario, all the NO2 produced from the
decomposition of intermediate II would be converted to NO3 ,
-
as is observed.
The third candidate for intermediate II, structure C, could
-
be produced by cyclic addition of ONOO to oxoFe(IV) from
the metal-oxo face of the porphyrin. Structure C is a complex
-
-
of the Fe(IV) porphyrin and peroxynitrate (O2NOO ); O2NOO
8
8
is a known compound that has been prepared previously.
Further, an analogous bidentate nitrato iron porphyrin complex
89
has been characterized by X-ray crystallography. Decomposi-
-
tion of the O2NOO -Fe(IV) porphyrin complex C would
-
produce NO3 and oxoFe(IV). Interestingly, this pathway
would be available to heme proteins such as myeloperoxidase,
which are known to produce oxoFe(IV) intermediates in which
1
0,77
one axial iron position is blocked by imidazole ligation.
Significantly, all three of the postulated candidates for inter-
-
mediate II would lead to the production of NO3 as the major
product, consistent with the observed results.59
Fe(III)TMPyP as Superoxide Dismutase Mimics. Fe(III)-
TMPyP has been shown to possess modest SOD activity by
-
•
Figure 8. Decay of O
(
2
in 25 mM phosphate buffer at pH 7.4, 25 °C
a) in the absence of porphyrin catalyst and (b) in the presence of 5
7
-1 -1 90-92
µM Fe(III)TMPyP. The solid lines overlaid on the two traces are the
nonlinear least-squares fitting of the experimental data to (a) second-
order kinetics (R ) 0.999) and (b) eq 4 (R ) 0.998). Inset: Linear
least-squares fit of the pseudo-first-order rate constants vs Fe(III)TMPyP
indirect assay, with kcat ∼ 10 M s .
We have directly
evaluated the SOD activity of Fe(III)TMPyP for the first time
-
•
by following the decay of O2 using a specialized four-syringe
setup of the stopped-flow spectrophotometer that we have
7
-1 -1
concentration gives kcat ) 1.9 × 10 M
s
(R ) 0.989).
54,55,83
recently described.
Reproducibly, the self-dismutation of
O2 in pH 7.4 phosphate buffer was found to be second order
in O2 , with a rate constant around 7.3 × 10 M
apparatus, as expected from published results (Figure 8a). Fe-
III)TMPyP at low concentrations significantly accelerated the
dismutation of O2 (Figure 8b). As we have recently described
for Mn(III)TMPyP, the apparent first-order rate constants (kobs)
of Fe(III)TMPyP-catalyzed dismutation of O2 were extracted
by nonlinear least-squares fitting of the experimental data into
eq 4, taking into consideration both self-dismutation and Fe-
-
•
55
As we have recently described for Mn(III)TMPyP, the
powerful reducing ability of O2 offers the opportunity for O2
not only to reduce Fe(III)TMPyP to the Fe(II) species, but also
to very rapidly reduce the oxoFe(IV) species to Fe(III). This
rapid reduction of the oxoFe(IV) species by O2 would short-
circuit path 1 of Fe(III)TMPyP-catalyzed decomposition of
ONOO (see Schemes 1 and 2), by analogy with the ascorbate
reduction described above. Thus, the process for simultaneous
removal of ONOO and O that we have described for Mn-
-
•
5
-1 -1
-•
-•
s
in this
55
(
-•
-•
55
-
•
-
-
-•
2
5
5
(III)TMPyP-catalyzed dismutation, which significantly improved
(III)TMPyP would also be available to Fe(III)TMPyP. The
known Fe(III)-Fe(II) dismutation cycle would thus be supple-
mented by the oxoFe(IV)-Fe(III) ONOO reductase cycle,
the fit of the data.
-
-
[
O ] k exp(-kobst)
-
2 0 obs
shown in eq 5.
[
O ] )
(4)
2
-
kobs + kself[O ] (1 - exp(-kobst))
2
0
-•
The rate of Fe(III)TMPyP-catalyzed dismutation of O2 was
7
-1 -1
estimated to be 1.9 × 10 M s , obtained from the slope of
a linear plot of kobs vs Fe(III)TMPyP concentrations (Figure
More significantly, the fast reduction of oxoFe(IV) species by
8
b, inset). This rate is in good agreement with the results
-•
-
O2 would make the fast path for ONOO reaction (path 1,
see Schemes 1 and 2) the major contributor to the activity of
90-92
obtained by indirect assay.
The mechanism of Fe(III)-
-
•
TMPyP-catalyzed dismutation of O2 is similar to that of Mn-
III)TMPyP: reduction of Fe(III)TMPyP to Fe(II)TMPyP by
-
Fe(III)TMPyP as a ONOO decomposition catalyst.
(
-
•
O2 is rate limiting, followed by fast oxidation of Fe(II) back
Conclusions
-
•
to Fe(III) by O2 at a near diffusion-controlled rate (∼4.2 ×
-
9
-1 -1 93
The reactions of Fe(III)TMPyP with ONOO are much more
1
0 M s ).
complex than the analogous reactions of Mn(III)TMPyP.5
4,55
(
(
88) Løgager, T.; Sehested, K. J. Phys. Chem. 1993, 97, 10047-10052.
89) Philippi, M. A.; Baenziger, N.; Goff, H. M. Inorg. Chem. 1981,
We have discovered that two different processes operate in
-
FeTMPyP-catalyzed decomposition of ONOO . A fast and
2
1
2
0, 3904-3911.
90) Pasternack, R. F.; Halliwell, B. J. Am. Chem. Soc. 1979, 101, 1026-
031.
(
stoichiometric pathway involving Fe(III)TMPyP as the active
-
species will dominate when the ONOO concentration is
(91) Pasternack, R. F.; Skowroneck, W. R. J. Inorg. Biochem. 1979, 11,
61-267.
(93) Faraggi, M. O2- dismutation catalyzed by water soluble porphyrins.
A pulse radiolysis study; Bors, W., Saran, M.; Tait, D., Eds.; Walter de
Gruyter: Berlin, 1984; pp 419-430.
(92) Ilan, Y.; Rabani, J.; Fridovich, I.; Pasternack, R. F. Inorg. Nucl.
Chem. Lett. 1981, 17, 93-96.

Fe Porphyrin Reactions with Peroxynitrite
J. Am. Chem. Soc., Vol. 120, No. 30, 1998 7501
comparable to or lower than that of Fe(III)TMPyP. A 10-fold
slower catalytic pathway involves oxoFe(IV)TMPyP as the
concentrations after mixing. The porphyrin solutions were buffered
with 50 mM sodium phosphate, pH 7.4. The same reactions were also
-
2
performed in the presence of NaNO in the case of m-CPBA as the
active species when ONOO is in excess (Schemes 1 and 2).
-
oxidant.
Which pathway dominates the ONOO decomposition is highly
Reaction Kinetics. Reaction kinetic profiles were collected in the
photomultiplier mode on the HI-TECH SF-61 DX2 stopped-flow
spectrophotometer (1 ms dead time) by using single- or double-mixing
modes. The kinetic profiles of Fe(III)TMPyP-catalyzed decomposition
dependent on the reaction conditions (e.g., the relative concen-
-
trations of iron catalyst and ONOO , the presence or absence
-•
of reducing agents such as biological antioxidants or O2 ). The
two pathways are certainly connected by oxoFe(IV) intermedi-
ates; however, it is not clear if they are also interconnected by
any other means.
-
of ONOO in 25 mM phosphate pH 7.4 buffer at 25 °C were monitored
at 302 nm. The generation and the decay of intermediates were
followed at 427 nm.
Further, the presence of antioxidants in vivo and O2-• under
oxidative stress conditions may allow the faster Fe(III) pathway
The reduction rates of oxoFe(IV) species by NaNO and ascorbate
2
were measured directly by performing double-mixing experiments: The
(
path 1) to be the dominant mode of ONOO- decomposition
oxoFe(IV) intermediates were fully generated during the first mixing
-
step by the stoichiometric reaction of Fe(III)TMPyP (5 µM) and HSO
5
catalyzed by Fe(III)TMPyP. Thus, the activity of Fe(III)TMPyP
in vivo would be expected to be higher than that in vitro when
reducing agents are absent.
Finally, the SOD-mimetic activity that we have measured for
Fe(III)TMPyP allows this catalyst to perform simultaneously
(
(
5 µM), followed by the addition of NaNO (0.5-50 mM) or ascorbate
2
5 µM) in the second mixing step. To examine the reactivity of the
-
oxoFe(IV) species toward ONOO , the oxoFe(IV) was similarly
prepared in the first mixing step, followed by addition of ONOO (100
-
µM) in the second mixing step.
-
•
-
as a O2 dismutase and a ONOO isomerase. Given the
Superoxide Dismutation. A DMSO solution of KO
2
(∼2 mM) was
-•
excellent reducing ability of O2 , the concerted removal of both
94
-•
prepared following the published procedure. The dismutation of O
2
-
•
-
-•
O2 and ONOO , mediated by the potential O2 -coupled
was monitored directly at the O2-• absorbance (245 nm), using a special
-
55
ONOO reductase activity of Fe(III)TMPyP, may be a
significant component of the biological activity that this and
setup of the four-syringe, double-mixing sample handling unit to reduce
changes in the refractive index due to the mixing of DMSO and buffer.
59
-
In this stopped-flow setup, the KO /DMSO solution was loaded in
other iron porphyrins have shown in ONOO -related disease
states.
2
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
Experimental Section
Materials. 5,10,15,20-Tetrakis(N-methyl-4′-pyridyl)porphinatoiron-
(III) chloride [Fe(III)TMPyP(Cl)] was purchased from Mid-Century
2
contents of syringes C and D. A 12-fold dilution of the KO /DMSO
Chemical. Anhydrous monosodium phosphate and anhydrous disodium
solution was achieved with this two-stage mixing, thus minimizing the
interference due to DMSO/water interaction. In the SOD activity
assays, iron porphyrin solutions were loaded into syringe C.
phosphate were purchased from Sigma. Sodium l-ascorbate, potassium
-
2
superoxide (KO ), potassium peroxymonosulfate (HSO
5
), and 3-chlo-
roperoxybenzoic acid (m-CPBA) were obtained from Aldrich. Perox-
ynitrite was prepared from the reaction of acidic H with sodium
2
O
2
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. J.A.H. is the
recipient of an NIH NRSA fellowship (GM18490).
5
3
nitrite following the published procedure. 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).
Time-Resolved Visible Spectra. All the time-resolved visible
spectra were recorded on a HI-TECH SF-61 DX2 stopped-flow
spectrophotometer with use of the photodiode array fast-scan mode.
The spectral resolution was about 1 nm. Reactions between Fe(III)-
JA980507Y
-
TMPyP and oxidants (m-CPBA, ONOO ) were single-mixing experi-
(94) Riley, D. P.; Rivers, W. J.; Weiss, R. H. Anal. Biochem. 1991, 196,
344-349.
ments, and the concentrations presented in all cases are the final