Concerted dismutation of chlorite ion: Water-soluble iron-porphyrins as first generation model complexes for chlorite dismutase

Article abstract of DOI:10.1021/ic801681n

Three iron-5,10,15,20-tetraarylporphyrins (Fe(Por-Ar₄), Ar = 2,3,5,6-tetrafluro-N, N, N-trimethylanilinium (1), N, N, N-trimethylanilinium (2), and p-sulfonatophenyl (3)) have been investigated as catalysts for the dismutation of chlorite (CIO<

Full text of DOI:10.1021/ic801681n

Inorg. Chem. 2009, 48, 2260-2268
Concerted Dismutation of Chlorite Ion: Water-Soluble Iron-Porphyrins
As First Generation Model Complexes for Chlorite Dismutase
Michael J. Zdilla, Amanda Q. Lee, and Mahdi M. Abu-Omar*
Brown Laboratory, Department of Chemistry, Purdue UniVersity, 560 OVal DriVe,
West Lafayette, Indiana 47907
Received September 1, 2008
Three iron-5,10,15,20-tetraarylporphyrins (Fe(Por-Ar4), Ar ) 2,3,5,6-tetrafluro-N,N,N-trimethylanilinium (1), N,N,N-
trimethylanilinium (2), and p-sulfonatophenyl (3)) have been investigated as catalysts for the dismutation of chlorite
(ClO2-). Degradation of ClO2- by these catalysts occurs by two concurrent pathways. One leads to formation of
chlorate (ClO3-) and chloride (Cl-), which is determined to be catalyzed by O)FeIV(Por) (Compound II) based on
stopped-flow absorption spectroscopy, competition with 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonicacid), 18O-
labeling studies, and kinetics. The second pathway is a concerted dismutation of chlorite to dioxygen (O2) and
chloride. On the basis of isotope labeling studies using a residual gas analyzer, the mechanism is determined to
be formation of O)FeIV(Por)· + (Compound I) from oxygen atom transfer, and subsequent rebound with the resulting
hypochlorite ion (ClO-) to give dioxygen and chloride. While the chlorate production pathway is dominant for
catalysts 2 and 3, the O2-producing pathway is significant for catalyst 1. In addition to chlorite dismutation, complex
1 catalyzes hypochlorite disproportionation to chloride and dioxygen quantitatively.
Introduction
regarding water contamination by oxychlorine species, a
wealth of work has been performed on the redox and
decomposition chemistry of chlorine species.7-10
The removal of toxic oxychlorine species (ClOx-) from
drinking water is a problem that has garnered much attention
in recent years because of the accumulation of these species
as a result of their wide use as bleaching agents,1 explosives,2
herbicides,3 and disinfectants.4 Chlorite has been listed by
the EPA as regulated toxic substance,5a and perchlorate is
currently under consideration5b by the agency because of
links to disease5 and acute toxicity toward aquatic micro-
organisms.6 Given the environmental and health concerns
Perchlorate respiring bacteria (PRB) offer an exciting
prospect for bioremediation of oxychlorine species. PRB
possess two enzymes that employ oxychlorine species as
metabolic oxidants for respiration. The first of these enzymes,
perchlorate reductase (PR),11 catalyzes the reduction of
perchlorate to chlorate, and chlorate to chlorite (eq 1). The
second enzyme, chlorite dismutase (Cld),12 catalyzes the
dismutation of chlorite ion to innocuous chloride and
dioxygen (eq 1).
(6) (a) Rosemarin, A.; Lehtinen, K-J.; Notini, M.; Mattsson, J. EnViron.
Pollut. 1994, 85, 3. (b) van Wijk, D. J.; Hutchingson, T. H. Ecotoxicol.
EnViron. Safety 1995, 32, 244. (c) Lehtinen, K-J.; Notini, M.; Mattsson,
J.; Landner, L. Ambio 1998, 17, 387.
(7) For examples of iron-mediated chorite redox chemistry, see (a) Fa´bia´n,
I. Prog. Nucl. Energy 2000, 37, 47. (b) Fa´bia´n, I.; Gordon, G. Inorg.
Chem. 1992, 31, 2144. (c) Lednicky, L. A.; Stanbury, D. M. J. Am.
Chem. Soc. 1983, 105, 3098. (d) Ondrus, M. G.; Gordon, G. Inorg.
Chem. 1972, 11, 985. (e) Shakhashiri, B. Z.; Gordon, G. J. Am. Chem.
Soc. 1969, 91, 1103. (f) Kahn, A. H.; Higginson, W. C. E. J. Chem.
Soc., Dalton Trans. 1981, 2537. (g) Birke, R. L., Jr J. Am. Chem.
Soc. 1969, 91, 3481.
(8) For examples of iron-catalyzed decomposition of chlorite, see (a)
Launer, H. F.; Wilson, W. K.; Flynn, J. H. J. Res. Nat. Bur. Stand.
1953, 51, 237. (b) Launer, H. F.; Tomimatsu, Y. J. Am. Chem. Soc.
1954, 76, 2591. (c) Schmitz, H.; Rooze, H Can. J. Chem. 1985, 63,
975.
* To whom correspondence should be addressed. E-mail: mabuomar@
purdue.edu.
(1) (a) Axegård, P.; Gunnarsson, L.; Malmqvist, A. SV. Papperstidning
1989, 92, 50. (b) Germgård, U.; Tder, A.; Tormund, D. Pap. Puu.
1981, 63, 127.
(2) Hogue, C. Chem. Eng. News 2003, 81, 127.
(3) Åslander, A. J. Agric. Res. 1928, 36, 915.
(4) Harman, D. C.; Frankenberger, W. T. J. J. EnViron. Qual. 1999, 28,
1018.
(5) (a) USEPA. National Primary Drinking Water Regulations; Disinfec-
tants and Disinfection Byproducts Notice of Data Availability;
Proposed Rule. Fed. Regist. 1998, 63, 15674. (b) USEPA. Priority
List of Substances Which May Require Regulation under the Safe
Drinking Water Act. Fed.Regist 1991, 56, 1470. (c) Drinking Water
and Health; Boraks, J. , Ed.; National Research Council, National
Academy Press: Washington, DC, 1987; pp 99-111.
2260 Inorganic Chemistry, Vol. 48, No. 5, 2009
10.1021/ic801681n CCC: $40.75  2009 American Chemical Society
Published on Web 01/12/2009

Model Complexes for Chlorite Dismutase
PR
PR
Cld
ClO-4 98 ClO3- 98 ClO-2 8 O2 + Cl-
(1)
This second enzyme, which is the focus of our study, is a
homotetrameric heme b enzyme with a molecular weight of
113 kD. It has been assumed that the iron porphyrin (a
common motif for biocatalyzed oxidation chemistry) is
directly responsible for the dismutation chemistry.13 While
some activity measurements have been done on native and
recombinant forms of Cld,12 very little is known about the
Figure 1. Water soluble iron porphyrins. TF4TMAP ) 5,10,15,20-
tetrakis(2,3,5,6-tetrafluoro-4-N,N,N-trimethylanilinium)porhyrin; TMAP )
5,10,15,20-tetrakis(4-N,N,N-trimethylanilinium)porhyrin. TPPS ) 5,10,15,20-
tetrakis(4-sulfonatophenyl)porhyrin.
molecular mechanism.12e
,f
molecular O2 in the transfer of O-atoms to substrate. This
can be viewed as the reverse of the Cld reaction, which
involves dioxygen evolution from the substrate, ClO2 .
Chlorite and chlorous acid (abbreviated Cl(III)) have been
the focus of much research. Several research groups have
addressed the mechanistic details of the metal-free decom-
position of Cl(III).9 The reaction chemistry of Cl(III) with
transition-metal ions has been reviewed by Fa´bia´n,14 and
much of the work has focused on aqueous nonheme Fe
species as redox agents7 and catalysts for decomposition.8
While a number of metal species are known to catalyze the
formation of oxygen from hypochlorite (OCl-),10a-d syn-
thetic systems that evolve O2 from ClO2- are rare. Collman
and co-workers have recently reported on a manganese-
porphyrin catalyst for alkane oxidation by ClO2- that evolves
O2 as a minor side reaction.15 To our knowledge this is the
only example of metal-catalyzed oxygen-evolving chlorite
decomposition.
Iron porphyrins are ubiquitous in biology as redox agents.
Cytochrome c employs an iron-porphyrin as the electron
carrier in the electron transport chain, peroxidases facilitate
the conversion of dangerous peroxides, and cytochrome P450
catalyzes oxygen atom transfer reactions for drug metabolism
and biological detoxification.16 The latter is of particular
interest to our current discussion of Cld in that it activates
-
The traditional mechanism for cytochrome P450 implicates
an oxoferryl porphryin radical cation species
(O)FeIV(Por) ·+, Compound I) as the oxygen transfer re-
agent.17 However, recent results have implicated the involve-
ment of more than one oxidizing species in cytochrome
P450.18,19 It remains to be seen what species are relevant to
the mechanism of chlorite dismutase.
We have recently communicated the concerted dismutation
of chlorite by an FeIII(TF4TMAP) complex (TF4TMAP )
tetrakis(2,3,5,6-tetrafluro-N,N,N-trimethylanilinium)porphy-
rin).20 In this work, we examine the full scope of this and
several water-soluble iron porphyrins as biomimetic catalysts
for Cld. Reaction kinetics, product distribution, 18O-labeling
studies, and competition with a sacrificial reductant (ABTS)
are employed in determining operative mechanisms for the
models and hence the enzyme.
Results
Evolution of O2. We studied three water-soluble iron
porphyrins (Fe(Por)) at neutral pH as model catalysts for
Cld. These are shown in Figure 1. All three catalyze the
(9) For examples of metal-free decomposition of chlorite, see (a) Leitner,
N. K.V.; Delaat, J.; Dore, M. Water Res. 1992, 26, 1655. (b) Deshwal,
B. R.; Jo, H. D.; Lee, H. K. Can. J. Chem. Eng. 2004, 82, 619. (c)
Cosson, H.; Ernst, W. R. Ind. Eng. Chem. Res. 1994, 33, 1468. (d)
Jia, Z.; Margerum, D. W.; Francisco, J. S. Inorg. Chem. 2000, 39,
2614. (e) Furman, C. S.; Margerum, D. W. Inorg. Chem. 1998, 37,
4321.
(10) For examples of hypochlorite decomposition studies, see (a) Lister,
M. W. Can. J. Chem. 1956, 34, 479. (b) Church, J. A. Ind. Eng. Chem.
Res. 1994, 33, 239. (c) Ayres, G. H.; Booth, M. H. J. Am. Chem. Soc.
1955, 77, 825. (d) Ayres, G. H.; Booth, M. H. J. Am. Chem. Soc.
1955, 77, 828. (e) Adam, L. C.; Gordon, G. Inorg. Chem. 1999, 38,
1299. (f) Lister, M. W Can. J. Chem. 1956, 34, 479.
(11) Kengen, S. W. M; Rikken, G. B.; Hagen, W. R.; van Ginkel, C. G.;
Stams, A. J. M. J. Bacteriol. 1999, 181, 6706. (b) Okeke, B. C.;
Frankenberger, W. T. Microbiol. Res. 2003, 158, 337. (c) Bender,
K. S.; Shang, C.; Cakraborty, R.; Belckik, S. M.; Coates, J. D.;
Achenback, L. A. J. Bacteriol. 2005, 187, 5090.
(12) (a) Van Ginkel, C. G.; Rikken, G. B.; Kroon, A. G. M.; Kengen,
S. W. M. Arch. Microbiol. 1996, 166, 321. (b) Stenklo, K.; Thorell,
H. D.; Bergius, H.; Aasa, R.; Nilsson, T. J. Biol. Inorg. Chem. 2001,
6, 601. (c) Xu, J.; Logan, B. E. J. Microbiol. Meth. 2004, 271, 3539.
(d) Bender, K. S.; O’Connor, S. A.; Cakraborty, R.; Coates, J. D.;
Achenbach, L. A. Appl. EnViron. Microb. 2002, 68, 4820. (e) Streit,
B. R.; DuBois, J. L. Biochemistry 2008, 47, 5271. (f) Lee, A. Q.;
Streit, B. R.; Zdilla, M. J.; Abu-Omar, M. M.; DuBois, J. L. Proc.
Natl. Acad. Sci. U.S.A. 2008, 105, 15654–15659.
-
degradation of ClO2 with formation of O2 at varying
efficiencies. Dioxygen evolution was monitored using an in-
house built residual gas analyzer (RGA) consisting of a
sample cell, the headspace of which is swept with an inert
gas (usually nitrogen) which is carried to a single quadrupole
(17) (a) McLain, J. L.; Lee, J.; Groves, J. T. In Biomimetic Oxidations
Catalyzed by Transition Metal Complexes; Meunier, B., Ed.; Imperial
College Press: London, 2000, pp 91-169. (b) Meunier, B.; Bernadou,
J. Top. Catal. 2002, 21, 47. (c) Watanabe, Y. In The Porphyrin
Handbook; Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Academic
Press: New York, 2000, Vol. 4, pp 97-117.
(18) (a) Song, W. J.; Ryu, Y. O.; Song, R.; Nam, W. J. Biol. Inorg. Chem.
2005, 10, 294. (b) Koppenol, W. H. J. Am. Chem. Soc. 2007, 129,
9686. (c) Machii, K.; Watanabe, Y.; Morishima, I. J. Am. Chem. Soc.
1995, 117, 6691. (d) Vaz, A. D. N.; McGinnity, D. F.; Coon, M. J.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3555. (e) Toy, P. H.;
Dhanabalasingam, B.; Newcomb, M.; Hanna, I. H.; Hollenberg, P. F.
J. Org. Chem. 1997, 62, 9114. (f) Nam, W.; Lim, M. H.; Moon, S. K.;
Kim, C. J. Am. Chem. Soc. 2000, 122, 10805. (g) Collman, J. P.; Zeng,
L.; Decre´au, R. A. Chem. Commun. 2003, 2974. (h) Wang, S. H.;
Mandimutsira, B. S.; Todd, R.; Ramdhanie, B.; Fox, J. P.; Goldberg,
D. P. J. Am. Chem. Soc. 2004, 126, 18.
(19) (a) Nam, W.; Choi, S. K.; Lim, M. H.; Rohde, J.-U.; Kim, I.; Kim, J.;
Kim, C.; Que, L., Jr Angew. Chem., Int. Ed. 2003, 42, 109. (b)
Collman, J. P.; Chien, A. S.; Eberspacher, T. A.; Brauman, J. I. J. Am.
Chem. Soc. 2000, 122, 11098. (c) Nam, W.; Lim, M. H.; Lee, H. J.;
Kim, C. J. Am. Chem. Soc. 2000, 122, 6641.
(20) Zdilla, M. J.; Lee, A. Q.; Abu-Omar, M. M. Angew. Chem., Int. Ed.
2008, 47, 7697–7700.
(13) Hagedoorn, P. L.; Geus, D. C.; Hagen, W. R. Eur. J. Biochem. 2002,
269, 4905.
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(15) Slaughter, L. M.; Collman, J. P.; Eberspacher, T. A.; Brauman, J. I.
Inorg. Chem. 2004, 43, 5198.
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Inorganic Chemistry, Vol. 48, No. 5, 2009 2261

Zdilla et al.
solvent demonstrated a significant incorporation of 18O into
the O2 product (Table 1, entry 12).
Analysis of Solution Species by Ion Chromatography
(IC). Ion chromatography was used to identify and quantify
anionic products resulting from iron-porphyrin-catalyzed
decomposition of chlorite and hypochlorite. IC indicates the
-
complete consumption of reactant ClO2 to give Cl- and
Figure 2. Diagram of the RGA. The sample head space is swept with an
inert gas, which is pulled through a 1 m long capillary using a rough vacuum
pump. A small portion of the remaining gas is pulled into the high-vacuum
region by a turbo pump and analyzed by quadrupole mass spectrometry.
The instrument allows the simultaneous monitoring of numerous masses
for each experiment.
-
ClO3 as the major ionic products in an approximate 1:2
-
ratio for Fe(TPPS) and Fe(TMAP). The ratio of ClO3 : Cl-
is about 1:1.5 for Fe(TF4TMAP). When chlorite decomposi-
tion was carried out in the presence of a 10-fold excess of
-
ABTS, ClO3 formation was suppressed, and the sole
observed anionic product was Cl-. In the case of hypochlorite
disproportionation catalyzed by Fe(TF4TMAP), Cl- is the
sole anionic product of the reaction. Exact yields of ions
are summarized in Table 1. Ion chromatograms are available
in the Supporting Information.
mass spectrometer (electron impact). The instrument is
illustrated in Figure 2. Typical yields of O2 from dismutation
by catalytic amounts of Fe(Por) complexes 1-3 are given
in Table 1. Other studies have implicated chlorine dioxide
(ClO2) as an important species in chlorite redox chemistry
and decomposition.9 However, we did not observe ClO2 via
RGA or EPR21 under our conditions. Given the sensitivity
of EPR and of our mass spectrometer RGA (10-9 Torr), it
is compelling to conclude that ClO2 is not formed/ac-
cumulated by our catalytic reactions to any appreciable
extent.
Mass Spectrometry Analysis of Chlorate Product. To
obtain mechanistic information on the source of oxygen in
the chlorate product, we employed isotopically labeled H218O
as solvent in Fe(Por) catalyzed decomposition of chlorite.
Product solutions were analyzed by ESI mass spectrometry
-
to determine the degree of 18O incorporation into ClO3 .
Dioxygen formation from ClO2- was monitored in phos-
phate buffer (pH 7.14) prepared using 18O-labeled water as
the solvent to test for incorporation of oxygen from water
into produced O2. A small amount of 18O incorporation was
seen for Fe(TF4TMAP) (of the total O2 formed, 1.4%
18O-16O, 0.4% 18O-18O), and no incorporation was observed
for Fe(TPPS) or Fe(TMAP). These results are summarized
in Table 1.
Isotopically labeled chlorite ions were employed in a
double-crossover experiment to probe for scrambling of
oxygen atoms in product O2. An isotopically enriched (77%)
sample of chlorite was disproportionated with an equivalent
amount of 16O chlorite. The result shows predominantly mass
peaks at 32 (16O2) and 36 (18O2) with a smaller peak at 34
(16O18O) consistent with the incompletely enriched 18O
sample (Table 1, entries 9 and 10).
Using Fe(TPPS) as a catalyst, 57% 18O incorporation was
observed in the chlorate product as Cl16O218O-, entry 7, Table
1, (spectrum available in the Supporting Information).
Therefore, only a single 18O was incorporated. Multiple
-
-
incorporation products (Cl16O18O2 , Cl18O3 ) were not
detected. In the case of Fe(TF4TMAP) as a catalyst, no 18
incorporation (from H218O solvent) was observed (Table 1,
entry 6).
O
Absorption Spectroscopy and Kinetics. Disappearance
of ClO2- was monitored by following its absorption band at
260 nm. Kinetic experiments were designed to keep the
catalyst concentration at a minimum such that the UV charge
transfer (CT) bands due to the Fe(Por) are of much lower
intensity than that of chlorite. This condition is not suitable
for Fe(TPPS) and Fe(TMAP), as these catalysts are easily
bleached by large excesses of chlorite. Fe(TF4TMAP),
however, is quite resistant to bleaching and hence suitable
for kinetics analysis.
2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS) scavenges FeIV(Por) ·+ (Compound I) and FeIV(Por)
(Compound II) species to form FeIII(Por) and the stable
ABTS·+ cation radical.22 The effect of ABTS on oxygen
formation was tested by including a 1.4-fold excess of ABTS
When a solution of chlorite (2.4-24 mM) was monitored
in the presence of 0.05 mol % Fe(TF4TMAP), an induction
period was seen before the approximate first-order disap-
pearance of the chlorite band at 260 nm (Figure 3). The
induction period is approximately 0.5-1.0 s depending upon
-
over ClO2 during catalysis. Fe(TF4TMAP) and Fe(TPPS)
were tested as catalysts. Presence of ABTS resulted in a
modest increase in the yield of O2 for both catalysts (Table
1, entries 4 and 5).
-
initial concentration of ClO2 (Figure 3). Similar behavior
Hypochlorite was tested as a substrate for oxygen forma-
tion. While use of Fe(TF4TMAP) as a catalyst resulted in
quantitative conversion to O2, neither Fe(TPPS) nor Fe(T-
MAP) resulted in formation of O2 from ClO- (Table 1).
Examination of the oxygen product formed in H218O as
was also seen for Fe(TPPS) as a catalyst; however, complete
ClO2- consumption was not achieved under these conditions
because of bleaching of the catalyst (vide supra). On the basis
of rapid repeat scans by stopped-flow spectroscopy on the
Fe(TF4TMAP) catalyst, this induction period corresponds to
the formation of Compound II (Figure 4), which remains as
a steady state intermediate throughout the reaction and
accounts for the bulk of the catalyst. This identification was
made based on comparison of the spectrum with known
(21) Quiroga, S. L.; Churio, M. S.; Perissinotti, J. L. Appl. Magn. Reson.
2002, 22, 115.
(22) (a) Zipplies, M. F.; Lee, W. A.; Bruice, T. C. J. Am. Chem. Soc. 1986,
108, 4433. (b) Huenig, S.; Balli, H.; Conrad, H.; Schott, A. Justus
Liebigs Ann. Chem. 1964, 676, 32,36, 52.
2262 Inorganic Chemistry, Vol. 48, No. 5, 2009

Model Complexes for Chlorite Dismutase
Table 1. Results of Catalytic Decomposition of ClO2 and ClO- by Fe(Por) Model Complexesa
-
entry
substrate
catalyst
special conditions
products (% yield or % enrichment)
-
-
1
2
3
4
5
6
7
8
ClO2-
Fe(TF4TMAP)
Fe(TPPS)
Fe(TMAP)
Fe(TF4TMAP)
Fe(TPPS)
Fe(TF4TMAP)
Fe(TPPS)
Fe(TF4TMAP)
Fe(TF4TMAP)
Fe(TF4TMAP)
Fe(TF4TMAP)e
Fe(TF4TMAP)
O2 (18), Cl- (36),ClO3 (56)
O2 (3), Cl- (31),ClO3- (63)
-
ClO2-
ClO2-
ClO2-
ClO2-
ClO2-
ClO2-
ClO2
O2 (7), Cl- (32),ClO3 (62)
O2 (30), Cl-
ABTSb e
,
ABTSb e
O2 (5), Cl-
,
H218O
16O2 (98.2), 16O18O (1.4),18O2 (0.4), Cl16O3 (100)c
-
H218O
16O2 (100), Cl16O3 (43), Cl16O218O- (57)c
-
d
-
KHSO5
O2 (15), Cl- (35),ClO3 (57)
16O2 (6%), 16O18O (34%), 18O2 (60%)c
16O2 (54%), 16O18O (17%), 18O2 (29%)c
O2 (100), Cl-
-
9
Cl18O2-
H216O
H216O
-
10
11
12
Cl18O2 and Cl16O2 1:1
,
ClO-
ClO-
H218O
16O2 (22), 16O18O (45), 18O2 (33)c
a All yields are calculated based on chlorite. Yields of oxygen were determined from integration of the RGA trace at m/z ) 32 calibrated with O2
standards. Yields of ions were determined using ion chromatography by integration of ion signals, and by comparison to a standard curve. b O2 evolution
was measured with an excess of ABTS over chlorite. c % in these entries refers to % of total product content. The value represents the level of incorporation
of 18O into the product, not the % yield. The reactions were analyzed by mass spectrometry, electron-impact for gases (RGA), and ESI for the chlorate ion.
d
-
-
.
e Ions were identified but not quantified. However, Cl- was
HSO5 was added to the catalyst to convert it to Compound II before the addition of ClO2
the only chlorine containing species by IC.
Kinetic traces fit well to a single exponential equation (i.e.,
first order in chlorite, the limiting reagent) after the induction
period. The curvature of the plot of kψ versus catalyst (Figure
5A) demonstrates that the order in Fe(TF4TMAP) is complex.
Additionally, a plot of kψ versus initial chlorite concentration
(Figure 5B) demonstrates that, while the chlorite disappear-
ance fits well to a single exponential, the order in chlorite is
actually larger than first order (positive slope) rather than
the expected horizontal line if indeed chlorite dependence
is strictly first order. To get a more detailed look at chlorite
dependence, an instantaneous rates method was used in the
analysis of the chlorite dependent data (Figure 5C).24
Concentration dependences (Figure 5, panels A,C) fit best
to a mixed 1st-2nd order rate equation.25
Figure 3. Absorption of chlorite at 260 nm after addition of Fe(TF4TMAP)
-
catalyst. Solid lines: [Fe(TF4TMAP)] ) 6.2 × 10-6; [ClO2
] ) 0.012,
0
0.0096, 0.0072, 0.0048, 0.0024, and 0.0012 M. Dotted line: [Fe(TF4TMAP)]
Generation of Compound II before Addition of
-
) 3.1 × 10-6 M; [ClO2
]
0
) 0.012 M. Inset: [Fe(TF4TMAP)] ) 6.2 ×
-
ClO2 . To test if Compound II is the active form of the
10-6; [ClO2-]0 ) 0.012; chlorite solution added to FeIII form of the catalyst
(solid line), and chlorite solution added to Compound II form of the catalyst
(dashed line), which was prepared in situ by the addition of HSO5 to
-
catalyst for O2 formation from ClO2 , we generated
-
(TF4TMAP)FeIV(O) in situ by the addition of KHSO5 to
Fe(TF4TMAP).
-
FeIII(TF4TMAP). HSO5 and other oxidants are known to
result in compound I ((por ·+)FeIV(O)), which quickly con-
proportionates with [FeIII(Por)(H2O)]+ to generate 2 equiv
of compound II (eq 2).23
Decomposition of chlorite was carried out as described
above with Compound II as the starting form of the catalyst.
It should be noted that while a 3-fold excess of KHSO5 over
Figure 4. Absorption spectra showing FeIII(TF4TMAP) (solid line),
O)FeIV(TF4TMAP) 1 s after addition of ClO2- (dashed line), and the return
of catalyst to the FeIII form about 30 min after reaction with some bleaching
(dotted line).
(24) Reaction rates were determined by fitting a line through the inflection
point (determined by 2nd derivative method) of the kinetic traces in
Figure 3, the slope of which corresponds to the instantaneous rate
under steady-state conditions. The rate is normalized to catalyst by
dividing the instantaneous rate by [Fe(TF4TMAP)]T. In Fig. 5C,
-
concentration of ClO2 was determined using the molar absorptivity
forms of Compound II of analogous porphyrins.23 At low
concentrations of chlorite, a slight rise in absorbance is seen
because of absorbance of compound II at 260 nm. At higher
concentrations of chlorite, this absorbance change is too small
to observe and an induction period instead is seen.
(ε) at 260 nm based on the absorbance value at the inflection point in
each plot. Total concentration of the catalyst Fe(TF4TMAP) was
assumed constant for each plot under steady-state conditions, i.e.,
catalyst degradation was negligible over the course of reaction.
(25) A detailed derivation of the rate law is given in the Supporting
Information. While our 1st order approximation used in the treatment
of kinetic data is not strictly mathematically correct (since dependences
are really mixed-order), the treatment is sufficient to invalidate the
possibility that the dependences are 1st order and to demonstrate the
existence of a more complex kinetic profile.
(23) Bell, S. E.; Cooke, P. R.; Inchley, P.; Leanord, D. R.; Smith, J. R. L.;
Robbins, A. J. Chem. Soc., Perkin Trans. 2 1991, 549.
Inorganic Chemistry, Vol. 48, No. 5, 2009 2263

Zdilla et al.
Figure 5. Kinetics of chlorite consumption. Fits for A and C are to mixed 1st-2nd order rate equation of the general form: d[A]/dt ) (a[A]2 + b[A])/(c
+ [A]).25 Fit for B is to a line. (A) Dependence of kψ on catalyst concentration. Values of kψ determined from exponential fits of kinetic traces. [ClO2
]
0
-
) 0.038 M; R ) 0.999. (B) Dependence of kψ on initial chlorite concentration. Values of kψ determined from exponential fits of kinetic traces. R ) 0.987.
(C). Chlorite dependence determined by computing Rateinfl/[Fe(TF4TMAP)]0, where Rateinfl and [ClO2 ]infl refer to the reaction rate and chlorite concentration
-
at the curve’s inflection point, [Fe(TF4TMAP)]0 ) 6.2 × 10-6 M; R ) 0.999.
Fe was used to ensure that the majority of the catalyst is in
the form of Compound II, the ClO2- added subsequently is
-
in a 740-fold excess over HSO5 . Thus, the majority of
-
-
oxidant in the system is ClO2 and not HSO5 .
-
When ClO2 is added to FeIV(O)(TF4TMAP) (Com-
pound II) the distribution of products by IC is very similar
to that starting with FeIII(TF4TMAP) form of the catalyst
(Table 1), suggesting the general reaction profile is not
-
affected. However, monitoring the disappearance of ClO2
at 260 nm by stopped-flow shows the disappearance of
the induction period (Figure 3, inset). More importantly,
O2 formation is inhibited when Compound II form of the
catalyst is used. Figure 6 shows comparative traces for
the evolution of O2 measured by RGA. It can be seen from
Figure 6 that the yield of O2 is decreased when
(TF4TMAP)FeIV(O) is the starting catalyst versus
FeIII(TF4TMAP).
Figure 6. Plots of total O2 production from chlorite decomposition.
[Fe(TF4TMAP)] ) 2.4 mM; [ClO2]0 ) 91 mM. Reaction started with FeIII
-
form of the catalyst (b) and after addition of 7.1 mM HSO5 to convert
catalyst to Compound II, O)FeIV(TF4TMAP) (O).
not afford dioxygen. For this reason, it is not straightforward
to address the issue of reaction stoichiometry for the
formation of O2. An easier issue to address first is the bulk
reaction, which according to the product distribution for
Discussion
The simplest stoichiometry one can envision for chlorite
dismutation is that described in eq 1. One chlorite ion
dismutes to one chloride ion and one molecule of dioxygen:
-
catalysts 2 and 3 (2:1 ClO3 /Cl-) is as follows:
3ClO-2 cat. 2 or 38 Cl- + 2ClO-3
(3)
-
ClO2 f Cl- + O2. In our model systems, O2 formation
constitutes only one of the products based on the complete
consumption of chlorite; see yield of O2 in Table 1, 18% in
the case of Fe(TF4TMAP), and less than 10% in the case of
the other two catalysts. In the latter two cases, catalysts 2
and 3, chlorite is largely decomposed via a pathway that does
Reaction 3 is the dominant one for Fe(TPPS) and Fe(T-
MAP), which give low yields of O2. On the other hand, while
reaction 3 does occur for Fe(TF4TMAP), it is not the only
significant pathway (vide infra).
2264 Inorganic Chemistry, Vol. 48, No. 5, 2009

Model Complexes for Chlorite Dismutase
-
Scheme 1. Reaction Mechanism for ClO2 Dismutation to Cl- and
Scheme 2. Oxygen Exchange with Solvent in Porphyrin FedO
Complexes
-
ClO3
The presence of an induction period in the kinetic profiles
for chlorite consumption indicates that the active form of
the catalyst is not present at the onset of reaction. Rapid
scan absorption spectroscopy implicates the iron(IV) oxo
compound II (O)FeIV(Por)) as the active catalyst since the
appearance of this spectrum coincides with the induction
period. Compound II is known to form in water-soluble
porphyrin systems from the conproportionation of Compound
I and FeIII(Por)(OH2), eq 2.23
therefore expected to undergo 18O exchange and incorporate
the labeled isotope into the products.
Table 1 shows that Fe(TF4TMAP) does not facilitate 18
O
incorporation into chlorate, but this is attributed to the fact
that the TF4TMAP porphyrin is known to undergo tautomeric
exchange much more slowly than other water-soluble por-
phyrins.29 The Fe(TPPS) catalyst, however, shows significant
(57%) incorporation of a single 18O into the chlorate product
(Cl16O218O-) as determined by negative mode ESI mass
spectrometry (see Supporting Information). No evidence of
The activity of (Por)FeIV(O) as the catalyst for chlorite
consumption was confirmed by generating Compound II
initially using HSO5- as an oxidant, and then adding chlorite.
This test resulted in disappearance of the induction period.
We therefore propose reaction Scheme 1 for reaction 3
catalyzed by FeII/FeIV redox couple.
The last two steps of catalysis represent the catalytic
formation of chlorate from chlorite and hypochlorite. While
this reaction is spontaneous without a catalyst,26 it is slow
under our conditions, and would result in accumulation of
hypochlorite without the invocation of the catalyst. Such
accumulation of OCl- would result in O2 formation in the
presence of Fe(TF4TMAP). This mechanism for O2 formation
will be considered shortly.
-
-
double (Cl16O18O2 ) or full (Cl18O3 ) incorporation is seen,
suggesting that there is a direct transfer of 18O to chlorite
ion without scrambling of the oxygen atoms on chlorine.
Additionally, control experiments demonstrate that ClO3- and
ClO2- do not exchange their oxygen atoms with H218O fast
enough at neutral pH to explain 18O incorporation without
invoking participation of the iron porphyrin.
While the 18O incorporation is seen in chlorate, the
dioxygen product does not show 18O incorporation, which
suggests that Compound II, which exchanges its oxo with
bulk water, is not responsible for the O2 production pathway.
Further evidence that Compound II is not the active catalyst
for O2 production comes from the data in Figure 6, which
demonstrates that starting with Compound II, O)FeIV(Por),
as the catalyst results in less dioxygen formation. Some
oxygen is still produced in this reaction because Compound
II can revert to Fe(III) as demonstrated by the spectra in
Figure 4. This most likely occurs via conproportionation of
(Por)FeIV(O) with FeII(Por).
We tested the validity of the mechanism in Scheme 1 using
ABTS, 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonicacid),
as a scavenger for FeIV)O species. ABTS reduces Com-
pounds I and II in a fast reaction, which should, in theory,
destroy catalytically active Compound II species, and return
the oxidation state to Fe(III). Additionally, ABTS is reactive
toward ClO- in the presence of other ions27 and could
potentially remove these species from the catalytic cycle as
well. When the reaction is carried out in the presence of
excess ABTS, the sole anionic product is indeed Cl-
[OdFeIV(Por)] + [FeII(Por)] + 2H+ f 2[FeIII(Por)]+ + H2O
(4)
-
according to IC with no formation of ClO3 . This result
Lastly, addition of ABTS to the reaction catalyzed by
FeIII(TF4TMAP), 3, increases O2 yield (entry 4 vs 1 in Table
1). This result provides additional support to the notion that
FeIII, not O)FeIV(Por), is responsible for O2 formation. ABTS
acts to destroy Compound II and regenerate the FeIII form
of the catalyst, which encourages dioxygen formation.
Hence, possible mechanisms for dioxygen formation are
outlined in Scheme 3. In pathway A, accumulated hypochlo-
rite from chlorite decomposition is catalytically decomposed
to O2 via an oxoferryl intermediate. In this mechanism,
hypochlorite acts as a nucleophile toward the oxo ligand,
resulting in a proposed short-lived peroxyhypochlorite ad-
suggests that ABTS successfully scavenges Compound II
since it is this species that gives rise to chlorate. Chloride is
produced from the reduction of ClO- by kf in Scheme 1 and
possibly by reaction of ClO- with ABTS.
H218O is commonly used to test the involvement of
accumulated FedO species in cytochrome P450 model
reactions because it exchanges with FedO via a tautomeric
mechanism (Scheme 2).28 Accumulated FedO species are
(26) (a) Lister, M. W Can. J. Chem. 1956, 34, 465. (b) Gordon, G.;
Tachiyashiki, S. EnViron. Sci. Technol. 1991, 25, 468.
(27) Pinkernell, U.; Nowack, B.; Gallard, H.; Von Gunten, U. Wat. Res.
2000, 34, 4343.
(28) Primus, J.-L.; Teunis, K.; Mandon, D.; Veeger, C.; Rietjens, I. M. C. M.
Biochem. Biophys. Res. Commun. 2000, 272, 551.
(29) La, T.; Miskelly, G. M. J. Am. Chem. Soc. 1995, 117, 3613.
Inorganic Chemistry, Vol. 48, No. 5, 2009 2265

Zdilla et al.
Scheme 3. Possible Mechanisms for Dioxygen Formation from
Chlorite
Nevertheless, mechanism D can be rejected on the basis
of an 18O-labeling crossover experiment using a 1:1 mixture
-
-
of ClO2 and isotopically labeled Cl18O2 . The crossover
experiment gave a majority of 16O2 and 18O2 based on mass
spectrometry. The intensity of the smaller 16O18O mass peak
-
is consistent with the incomplete enrichment of the Cl18O2
starting sample. This result is contrary to the expectation of
mechanism D, which would result in a 1:2:1 binomial
distribution of 16O2, 16O18O, and 18O2, respectively. Therefore,
this double-crossover result confirms that formation of O2
from chlorite is concerted, each oxygen in the O2 product
originates from the same chlorite ion. While mechanisms B
and D both involve the same intermediate at iron (Compound
I), the intermediate in mechanism B is an ion pair that
undergoes rebound faster than competing bimolecular reac-
tions. Rebound reactions in iron dependent oxygenases have
been shown to have rate constants g108 s-1.17a,33
duct, which rapidly decomposes to O2 and Cl-. This
mechanism is proposed based on the observation that
Fe(TF4TMAP) quantitatively forms dioxygen and Cl- from
ClO- (entry 11, Table 1). In mechanism B, chlorite transfers
an oxygen atom to iron, and then rebounds in a subsequent
fast step to give O2 and chloride. The quantitative formation
of dioxygen from hypochlorite suggests that this mechanism
is feasible as long as hypochlorite rebounds with Compound
I faster than diffusing into solution. Similar to mechanism
A, the nucleophilic attack of ClO- on the oxoferryl is
proposed to give the intermediate peroxyhypochlorite adduct.
Mechanism C is formation of O2 from rearrangement of a
chlorite adduct. This mechanism is kinetically indistinguish-
able from mechanism B and would likely result in the same
Fe-O2Cl moiety as A and B. Finally mechanism D is similar
to mechanism B except ClO-, formed from reaction of
chlorite with FeIII, escapes into solution, and Compound I
requires a second chlorite molecule to give dioxygen.
Pathway A can be excluded based on isotope labeling
experiments for hypochlorite dismutation. OCl- dismutation
in H218O results in substantial 18O incorporation into the O2
product (entry 12, Table 1). This occurs because of the rapid
exchange of oxygen atoms between water and the hypochlo-
rite ion30 prior to reaction with the catalyst. This result is in
contrast to chlorite dismutation carried out in H218O, which
shows no 18O incorporation into product oxygen (entries 6
and 7, Table 1). If O2 formation from chlorite was occurring
because of accumulated ClO- as in pathway A, 18O
incorporation would have been observed into the O2 product.
The remaining mechanisms cannot be distinguished by
kinetic analysis. Speciation of the catalyst into Compound
II complicates the theoretical rate equations (see Supporting
Information for rate law derivation) and any of the mecha-
nisms B, C, and D satisfy the complex kinetic profiles (Figure
5). It should be noted that while condensation of FeIII(Por)
complexes to form dinuclear µ-oxo complexes is known,31
and could further complicate kinetics analysis, the
Fe(TF4TMAP) porphyrin used for kinetic studies herein is
resistant to dinuclear complex formation.32a
Discrimination between mechanisms B and C by experi-
mental means is daunting because of their indistinguishable
kinetic form, identical expectations for 18O-labeling experi-
ments, and the fact that the intermediate state of either
mechanism would be short-lived. This admitted, one could
argue that the observation of Compound II (Figure 4)
demonstrates the feasibility of formation of oxoferryl from
-
FeIII(Por) and ClO2 via mechanism B. Deactivation of the
O2-evolving catalyst would thereby occur via escape of
hypochlorite from the ion-pair to give Compound I, which
relaxes to Compound II (via conproportionation with FeIII).
Conversely, while some coordination complexes of chlorite
are known,14 there is no chemical precedent for metal-bound
chlorite rearrangement on a metal ion without changes in
the chlorine oxidation state, as in mechanism C. Given the
observation of ferryl species in the system, and the lack of
precedent for molecular rearrangement of chlorite bound to
a metal, mechanism C is disfavored, though not rigorously
discarded.
The energetics of chlorite ion degradation to either
dioxygen and chloride or chlorate and chloride are favorable:
-
-
ClO2 f Cl- + O2 (∆G° ) -149 kJ mol-1); 3ClO2
f
34
-
Cl- + 2ClO3 (∆G° ) -189 kJ mol-1). It is interesting
to note that the enzyme chlorite dismutase has evolved an
exquisite selectivity for the thermodynamically less favorable
dioxygen evolving pathway. On the other hand, degradation
of the chlorite ion to chlorate and chloride is overwhelmingly
dominant for the model complexes investigated herein
especially catalysts 2 and 3; only in the case of the fluorinated
porphyrin, catalyst 1, does the oxygen evolving pathway
(31) (a) Harris, F. L.; Toppen, D. L. Inorg. Chem. 1978, 17, 71. (b)
Kobayashi, N.; Koshiyama, M.; Osa, T.; Kuwana, T. Inorg. Chem.
1983, 22, 3608. (c) Trondeau, G. A.; Wilkings, R. G. Inorg. Chem.
1986, 25, 2745. (d) Miskelly, G. M.; Webley, W. S.; Clark, C. R.;
Buckingham, D. A. Inorg. Chem. 1988, 27, 3773. (e) Bell, S. E. J.;
Hill, J. N.; Hester, R. E.; Shwcross, D. R.; Lindsay Smith, J. R.
J. Chem. Soc., Faraday Trans. 1990, 4017.
(32) (a) La, T.; Richards, R.; Miskelly, G. M. Inorg. Chem. 1994, 33, 3159.
(b) Kadish, K. M.; Araullo-McAdams, C.; Han, B. C.; Franzen, M. M.
J. Am. Chem. Soc. 1990, 112, 8364.
(33) (a) He, X.; Ortiz de Montellano, P. R. J. Biol. Chem. 2004, 279, 39479.
(b) Jin, Y.; Libscomb, J. D. Biochim. Biophys. Acta 2000, 1543, 47.
(34) Shriver, D. F.; Atkins, P. Inorganic Chemistry, 3rd ed.; W.H. Freeman
and Co.: New York, 2003; p 700.
(30) Robert, A.; Meunier, B. C. R. Acad. Sci. II C 2000, 3, 771.
2266 Inorganic Chemistry, Vol. 48, No. 5, 2009

Model Complexes for Chlorite Dismutase
Scheme 4
pH 7 with aqueous sodium hydroxide. UV-vis spectra were
recorded on a Shimadzu UV-2501PC scanning spectrophotometer.
Absorption kinetics experiments were performed using an Applied
Photophysics SX.18MV Stopped-Flow Analyzer. Gas evolution was
analyzed using an in-house built RGA Mass Spectrometer. Typi-
cally, the reaction solution (1-2 mL) was stirred in a custom-made
glass RGA cell with a minimum (1-2 mL) head space. An inert
carrier gas (Ar or N2) was drawn over the reaction head space at 2
mL/min by a Varian model SH 100 vacuum pump and analyzed
by a Stanford Research Systems RGA 100 mass spectrometer
equipped with an Alcatel ATH31 Series turbopump. ESI mass
spectra were obtained using a FinniganMAT LCQ mass spectrom-
eter system or a Finnigan LTQ Linear Ion Trap Mass spectrometer
in negative ion mode. Sample was introduced by direct infusion
from a syringe pump. Ion chromatography was performed on a
Dionex DX-500 Liquid Chromatography System equipped with a
Dionex LC25 Chromatography Oven, a Dionex ED40 Electro-
chemical Detector, and a Dionex Ion-Pac AS9-HC ion exchange
column. Nine millimolar Na2CO3 was used as eluant. Chromatog-
raphy calibration standards were prepared in the 0.5-60 mM
concentration range. Peaks were identified by comparison to
standard samples and quantified by comparison of the integrals of
the signals to standard curves for the corresponding ion.
[Fe(TF4TMAP)][OTf-]5 was prepared from 5,10,15,20-tetrak-
is(pentafluorophenyl)porphine (Frontier) according to the procedure
of Miskelly et al.32a
become somewhat competitive accounting for 1/5 of the
product distribution.35
Conclusion
We have reported herein the first functional model system
for the enzyme chlorite dismutase. The mechanistic results
are summarized in Scheme 4. The initial step of the reaction
is the formation of Compound I and hypochlorite via oxygen
atom transfer. Rebound of the intermediate hypochlorite with
Compound I results in O2 and Cl- formation in a biomimetic
model reaction for chlorite dismutase. The catalyst deacti-
vates when hypochlorite diffuses into solution before re-
bound, resulting in conproportionation of the resulting
Compound I with an equivalent of FeIII(Por) to form
Compound II, at which point the catalyst enters a new cycle
for which the FeII/FeIV(O) cycle disproportionates chlorite
Na3[Fe(TPPS)] was used as received from Mid-Century chemical
company or prepared from (TPPS)H2 (Strem) according to the
procedure of Fleischer et al.36
[Fe(TMAP)]Cl5 was used as obtained from Mid-Century chemi-
cal company.
-
into Cl- and ClO3 in 1:2 molar ratio. This form of the
RGA Calibration. Yields of oxygen were determined by
integration of the RGA signal and comparison to a calibration curve
prepared by the injection of known volumes of oxygen gas. The
calibration plots were obtained in the following way: For a 4 mL
RGA glass cell, 2 mL of water was added into the cell and stirred
to simulate a typical reaction. 50, 125, 250, 375, and 500 µL of air
were injected into the RGA cell through a rubber septum. Partial
pressure (Torr) of oxygen was monitored against time (seconds)
for each calibration point. Integration of the partial pressure versus
time graph is plotted against moles of oxygen injected (assuming
20.95% of air is oxygen).
catalyst can be “rescued” back to the oxygen formation cycle
by the one electron reductant ABTS, which converts
Compound II to FeIII. In comparison, the biological enzyme
chlorite dismutase catalyzes the decomposition of chlorite
exclusively to chloride and dioxygen without formation of
chlorate. A recent mechanistic probe of the enzyme con-
cluded that the enzyme must impede hypochlorite diffusion
from the active pocket to solution by forcing its rebound
with transient Compound I.12f While the described model
system exhibits a biomimetic rebound reaction, the absence
of protein permits the ClO- anion to diffuse into solution
-
Yield of Oxygen Evolution from ClO2 Catalyzed by
-
Fe(TF4TMAP). [NaClO2] ) 91 mM and catalyst concentration of
2.4 mM were used. Partial pressure of O2 at m/z ) 32 was recorded
with respect to time via RGA using Ar as a carrier gas. For reaction
starting with compound II, (TF4TMAP)FeIV(O) was generated by
resulting in catalyst deactivation and ClO3 formation. In
an effort to develop environmentally useful bioinspired
catalysts, future models will employ ligand functionalities
designed to retain ClO- at the catalytic site.
-
adding HSO5 to Fe(TF4TMAP) in the RGA cell before injecting
a small volume of NaClO2. Final concentrations of each reagent
were as follows: 7.1 mM KHSO5, 2.4 mM Fe(TF4TMAP), and 91
mM NaClO2. O2 was quantified by integration of the RGA signal
and comparison to standard calibration plots (vide supra).
Oxygen Formation in the Presence of ABTS. Fe(TPPS):
Oxygen evolution was monitored in the RGA for reaction of 50
mM NaClO2, 70 mM ABTS, and 2.2 mM catalyst in 0.1 M sodium
phosphate buffer, pH 7.1. Fe(TF4TMAP): Oxygen evolution was
monitored by RGA for reaction of 50 mM NaClO2, 70 mM ABTS,
and 2.2 mM catalyst in 0.1 M sodium phosphate buffer, pH 7.1.
Test for 18O Incorporation into Oxygen Product. Chlorite
dismutation was carried out in oxygen-18 enriched water (95%).
Oxygen evolved from the reaction was analyzed using the RGA
Experimental Section
General Procedures. All reactions were carried out in deionized
water obtained from a Millipore Milli-Q Academic TC water
purification system. Reagents were used as obtained from Fisher,
Baker, Acros, Sigma-Aldrich, GFS, Mid-Centruy, Frontier, and
Strem. Sodium chlorite was obtained from GFS chemical as a 98%
pure solid. Ion chromatographic analysis suggests that this material
is ∼95% pure, with a 2.5% chlorate, and a 2.5% unidentified
anion(s) impurity. Phosphate buffers were prepared by dissolving
of mono- and dibasic sodium phosphate. Ammonium acetate buffers
were prepared by dissolution of ammonium acetate and titration to
(35) Comparison of the thermodynamics of oxo transfer for the employed
catalysts and/or the enzyme is not possible because the potentials for
Compounds I and II are not available.
(36) Fleischer, E. B.; Palmer, J. M.; Srivastava, T. S.; Chatterjee, A.
J. Am.Chem. Soc. 1971, 93, 3162.
Inorganic Chemistry, Vol. 48, No. 5, 2009 2267

Zdilla et al.
set up as described above, except the carrier gas used was N2 instead
of Ar because of the interfering argon isotope 36Ar. Fe(TPPS): 0.9
M NaClO2, 4.6 mM catalyst, and 1 M sodium phosphate buffer,
pH 7.1 were all prepared in oxygen-18 enriched water (95%). 0.1
M NaClO2, 2.6 mM Fe(TF4TMAP) or Fe(TMAP) and 0.1 M
sodium phosphate buffer, pH 7.1 were all prepared in oxygen-18
enriched water (95%).
For hypochlorite dismutation, Fe(TF4TMAP) (2.8 mM, 2 mL)
was prepared in 100 mM phosphate buffer (pH ) 7.1) in H218O. A
concentrated solution of hypochlorite (1.55 M) was prepared in
normal H216O to prevent exchange of the hypochlorite oxygens with
18O prior to mixing. To initiate reaction, 30 µL hypochlorite solution
was added to the solution of catalyst in 2 mL H218O phosphate
buffer.
Test for 18O Incorporation into Chlorate Product. Solutions
were prepared in 50 mM ammonium acetate buffer, pH 7.1 in
H218O. A 0.25 mL volume of a 2.5 mM Fe(Por) solution was added
to 0.25 mL of NaClO2 (40 mM). These solutions were stirred about
20 h and analyzed by ESI mass spectrometry. Control experiments
with NaClO3 were performed as well. A 0.25 mL volume of a 2.5
mM Fe(Por) solution was added to 0.25 mL of NaClO3 (40 mM)
and stirred 20 h before analysis by ESI mass spectrometry.
Kinetic Measurements on Chlorite Consumption. Kinetic
measurements were carried out using a stop flow analyzer by
monitoring the absorbance of chlorite at 260 nm (ε260 ) 154 M-1
cm-1). Each trace was repeated 3-5 times to ensure reproducibility.
One of either chlorite or Fe(TF4TMAP) was held constant, while
the other was varied in the obtainment of kinetic plots. All
concentrations noted here represent post-mixing values. For
Fe(TF4TMAP) dependence, initial [ClO2-] was 0.038 M, and
Fe(TF4TMAP) was varied between from 2.5 × 10-5 to 2.5 × 10-4
M. For ClO2- dependence, Fe(TF4TMAP) was 6.2 × 10-6 M, and
ClO2- was varied from 0.0012 to 0.012 M. These profiles exhibited
a sigmoidal shape (see Supporting Information) with an induction
period.
The plot in Figure 5C was generated using the instantaneous
rate method in the following way: An instantaneous rate under
steady state conditions was calculated by fitting a line through the
inflection point of the trace (inflection point determined by second
derivative method). This rate was divided by the reagent being held
constant, [Fe(TF4TMAP)]Total, and the resulting normalized rates
were plotted versus the concentration of the reagent being varied
(chlorite) at the inflection point. The concentration of chlorite at
inflection was determined from the absorbance value at the
inflection point.
NaClO2- was added 1 mL of a 4 mM solution of Fe(Por) (each in
50 mM pH 7.14 phosphate buffer) and stirred for 30 min. This
solution was diluted 100× and quantified by IC. For ABTS runs,
a 10-fold excess of ABTS over chlorite was used in the solution.
Preparation of Isotopically Labeled NaCl18O2. Isotopically
labeled chlorite was prepared and used in situ based on a modified
protocol for the production of sodium chlorite from sodium
chlorate.37 In a thick-walled Schlenk tube, 2.1 g (20 mmol) NaClO3
was stirred in 5 mL of 95% enriched H218O. To this mixture, 0.83
mL of concentrated sulfuric acid was added, and the mixture was
capped and stirred for 1 h at 70 °C. After this period, the tube was
removed from heat, cooled to room temperature, and frozen in liquid
N2. The tube was opened to air, and 1.1 g (9 mmol) of solid Na2SO3
was added to the frozen mixture. The tube was sealed and warmed
to melt the solution, and the mixture was placed back in the heating
bath and stirred for 1.5 h in the dark to afford the appearance of
yellow ClO2 gas. This gas was bubbled through an ice-cold solution
of NaOH (9 mmol), 1.1 mL of 30% H2O2 (9.7 mmol), and 2 mL
of H2O. The resulting solution was stirred with 0.5 g of MnO2 to
disproportionate unreacted H2O2 for 2 h and then filtered to remove
MnO2. Analysis of this solution by IC reveals a chlorite concentra-
tion of 79.8 ( 0.8 mM, and an approximate 1.5-fold contamination
by chloride. Before use in further experiments, the solution was
titrated to neutrality with 3 M H3PO4 (typically 1-1.5 mL). The
resulting solution is 77% 18O-enriched ClO2 (based on ESI MS
-
and on the enrichment of resulting O2 when the compound is
dismuted) in phosphate buffer (0.6 M). Further details are available
in the Supporting Information.
Chlorite Dismutation - Double Crossover. One milliliter of a
mixture of unlabeled chlorite and 18-O chlorite (ca. 75 mM in each)
in 0.3 M phosphate buffer, pH ) 7.2, was disproportionated by
the addition of 0.1 mL of 1 mM Fe(TF4TMAP). The resulting O2
was analyzed by RGA mass spectrometer using N2 as a carrier gas.
Acknowledgment. This work was supported by a grant
from the National Science Foundation (CHE-0502391 and
0749572). We gratefully acknowledge Professor Dale Mar-
gerum, Dr. Katya Albert, Professor Paul Shepson, and Ms.
Chelsea Thompson for their assistance with ion chromatog-
raphy experiments. We thank Professor Hilkka Kenta¨maa,
Mr. Stephen Habicht, and Dr. Karl Wood for assistance with
ESI mass spectrometry. We gratefully acknowledge the
efforts of Alan Ronemus for design and implementation of
the RGA, as well as for generation of schematics for the
instrument published here.
In addition to the instantaneous rate method, curve fitting was
used to analyze the kinetic traces. This analysis leads to the same
conclusions as the use of instantaneous rates (see discussion
section).
For kinetic measurement using O)FeIV(TF4TMAP) (compound
II) as the initial form of catalyst, compound II was generated by
the addition of 3 equiv of KHSO5 to 6.2 × 10-6 M Fe(TF4TMAP).
This solution was used as catalyst in the decomposition of a 0.012
M solution of chlorite. The resulting plot was similar to those in
the absence of HSO5- except that the induction period disappeared.
Ion Chromatography on Reaction Products. A typical IC
determination is as follows. To 9 mL of a 50 mM solution of
Supporting Information Available: Ion chromatograms, EI
mass spectra of O2 products formed in 18O-water, ESI mass spectra
of ClO3- products formed in 18O-water, sample kinetic profiles and
derivation of complex rate equations via steady state approximation,
data on preparation of isotopically enriched Cl18O2-, and schematic
diagram of in-house built RGA (PDF). This material is available
free of charge via the Internet at http://pubs.acs.org.
IC801681N
(37) Zhou, C.; Long, J.; Xu, P. Huaxue Shijie 1993, 34, 38.
2268 Inorganic Chemistry, Vol. 48, No. 5, 2009