Heme/Hydrogen peroxide reactivity: Formation of paramagnetic iron oxophlorin isomers treatment of iron porphyrins with hydrogen peroxide

Article abstract of DOI:10.1021/ja0016405

Treatment of iron(II) porphyrins, (py)₂Fe^II(OEP), (py)₂Fe^II(EtioP), (py)₂Fe^II(DeuteroP), (pY)₂-FeII(MesoP), and (py)₂Fe^II(ProtoP), where OEP are the dianions of octaethylporphyrin, etio-1 porphyrin, deuteroporphyrin-IX dimethyl ester, mesoporphyrin-IX dimethyl ester, and protoporphyrin-IX dimethyl ester, with hydrogen peroxide in pyridine-d₅ at -30°C in the strict absence of dioxygen is shown to result in clean oxygenation of the heme and the formation of oxophlorin complexes, (py)₂Fe(OEPO), (py)₂Fe(EtioPO), (py)₂Fe-(DeuteroPO), (py)₂Fe(MesoPO), and (py)₂Fe(ProtoPO). Reactions have been monitored by ¹H NMR spectroscopy. The product oxophlorin complexes are stable as long as the samples are protected from exposure to dioxygen. The hyperfine shift patterns and the relative intensities of the individual resonances have been analyzed in terms of a model in which the location of the meso oxygen substituent dominates the pattern of spin density distribution. The resulting ¹H NMR spectra obtained from oxidation of the unsymmetrically substituted hemes, (py)₂Fe^II(DeuteroP), (py)₂Fe^II(MesoP), and (py)₂Fe^II(ProtoP), with hydrogen peroxide have been analyzed. These spectra show that the four isomeric oxophlorin products are formed in a nonrandom fashion. Solvent effects can produce a significant alteration in the regiospecificity of heme oxygenation.

Full text of DOI:10.1021/ja0016405

12478
J. Am. Chem. Soc. 2000, 122, 12478-12486
Heme/Hydrogen Peroxide Reactivity: Formation of Paramagnetic Iron
Oxophlorin Isomers by Treatment of Iron Porphyrins with Hydrogen
Peroxide
Heather R. Kalish,† Lechosław Latos-Graz3yn´ski,‡ and Alan L. Balch*,†
Contribution from the UniVersity of California, DaVis, California 95616, and the Department of Chemistry,
UniVersity of Wrocław, Wrocław, Poland
ReceiVed May 12, 2000. ReVised Manuscript ReceiVed October 12, 2000
Abstract: Treatment of iron(II) porphyrins, (py)2FeII(OEP), (py)2FeII(EtioP), (py)2FeII(DeuteroP), (py)2-
FeII(MesoP), and (py)2FeII(ProtoP), where OEP are the dianions of octaethylporphyrin, etio-1 porphyrin,
deuteroporphyrin-IX dimethyl ester, mesoporphyrin-IX dimethyl ester, and protoporphyrin-IX dimethyl ester,
with hydrogen peroxide in pyridine-d5 at -30 °C in the strict absence of dioxygen is shown to result in clean
oxygenation of the heme and the formation of oxophlorin complexes, (py)2Fe(OEPO), (py)2Fe(EtioPO), (py)2Fe-
(DeuteroPO), (py)2Fe(MesoPO), and (py)2Fe(ProtoPO). Reactions have been monitored by 1H NMR
spectroscopy. The product oxophlorin complexes are stable as long as the samples are protected from exposure
to dioxygen. The hyperfine shift patterns and the relative intensities of the individual resonances have been
analyzed in terms of a model in which the location of the meso oxygen substituent dominates the pattern of
1
spin density distribution. The resulting H NMR spectra obtained from oxidation of the unsymmetrically
substituted hemes, (py)2FeII(DeuteroP), (py)2FeII(MesoP), and (py)2FeII(ProtoP), with hydrogen peroxide have
been analyzed. These spectra show that the four isomeric oxophlorin products are formed in a nonrandom
fashion. Solvent effects can produce a significant alteration in the regiospecificity of heme oxygenation.
Introduction
Scheme 1. Heme Cleavage by Heme Oxygenasea
The reactivity of heme proteins and hemes themselves toward
hydrogen peroxide is a significant process that contributes to
biological utility of hemes.1 Catalases are heme enzymes that
are designed to destroy hydrogen peroxide through dispropor-
tionation. Peroxidases utilize hydrogen peroxide to generate
highly oxidized hemes that are then used in substrate oxidation.2
In both of these enzyme classes it is the Fe(III) state of the
heme that interacts with hydrogen peroxide to generate ferryl
intermediates.3 While cytochrome P450 is designed to utilize
dioxygen to oxidize substrates, it can also use hydrogen peroxide
in a short-circuited path (the peroxide shunt) to effect the same
transformations.4 The reactions of hydrogen peroxide with heme
model compounds, particularly with mesotetra(aryl)porphyrins,
have been utilized to effect olefin epoxidation as well as
hydrocarbon hydroxylation.5 However, reactions of hydrogen
peroxide with model hemes frequently are accompanied by
bleaching and catalyst destruction. The results reported here
investigate the earliest stages in the heme modification by
hydrogen peroxide in cases where the porphyrin meso sites are
not protected by the presence of aryl substituents.
a Key: M ) methyl; V ) vinyl; Pr ) propionate.
Biologically, heme degradation is catalyzed by the protein
heme oxygenase, which utilizes heme as substrate to activate
dioxygen to destroy the heme. This reaction results in the
regiospecific cleavage of heme at the R-meso site as shown in
Scheme 1.6-9 This process generates the green tetrapyrrole,
biliverdin, which is subsequently reduced by biliverdin reductase
to bilirubin, the yellow pigment that is responsible for jaundice
in humans. The iron is released as free iron ion, and the meso
carbon atom is oxidized to carbon monoxide, a neurotransmit-
ter.10
The regiospecificity of the reaction catalyzed by heme
oxygenase is determined by the initial event, which involves
attack of oxygen at the R-meso carbon. It is widely accepted
that the first step in heme degradation by heme oxygenase results
† University of California.
‡ University of Wrocław.
(1) Strukul, G., Ed. Catalytic Oxidations with Hydrogen Peroxide as
Oxidant; Kluwer Academic Publishers: Boston, 1992.
(2) Everse, J.; Everse, K. E.; Grisham, M. B. Peroxidases in Chemistry
and Biology; CRC Press: Boca Raton, FL, 1991: Vols. I and II.
(3) Weiss, R.; Gold, A.; Trautwein, A. X.; Ternier, J. In The Porphyrin
Handbook; Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Academic
Press: NewYork, 2000; Vol. 4, p 65.
(4) Ortiz de Montellano, P. R., Ed. Cytochrome P450: Structure,
Mechanism and Biochemistry; Plenum Press: New York, 1995.
(5) Meunier, B.; Robert, A.; Pratviel, G.; Bernadou, J. In The Porphyrin
Handbook; Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Academic
Press: New York, 2000: Vol. 4, p 119.
(6) Maines, M. D. Heme Oxygenase: Clinical Applications and Func-
tions; CRC Press: Boca Raton, FL, 1992
(7) Schmid, R.; McDonagh, A. F. In The Porphyrins; Dolphin, D., Ed.;
Academic Press: New York, 1979; Vol. 6, p 258.
(8) Bissel, D. M. In LiVer: Normal Function and Disease; Ostrow, J.
D., Ed.; Marcel Decker, Inc.: New York, 1986; Vol. 4 (Bile Pigments and
Jaundice), p 133.
(9) Ortiz de Montellano; P. R. Acc. Chem. Res. 1998, 31, 543.
(10) Verma, A.; Hirsch, D. J.; Glatt, G. E.; Ronnett, G. V.; Snyder, S.
H. Science 1993, 259, 381.
10.1021/ja0016405 CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/23/2000

Heme/Hydrogen Peroxide ReactiVity
J. Am. Chem. Soc., Vol. 122, No. 50, 2000 12479
Scheme 2
in the meso hydroxylation of the heme.11-15 The major evidence
for initial heme hydroxylation has come from studies that show
that R-hydroxylated heme binds to heme oxygenase and can
replace heme as a substrate for heme oxygenase. However, only
recently has direct spectroscopic evidence been produced that
suggests that a meso-hydroxylated heme is formed by heme
oxygenase.16
The coupled oxidation process, in which heme degradation
is brought about by dioxygen in the presence of a reducing agent
(generally ascorbic acid), has been widely employed as a model
for biological heme catabolism.17-19 The coupled oxidation
procedure can be utilized to oxidize iron porphyrins as model
compounds20,21 or to oxidize intact heme proteins. Coupled
oxidation of heme alone produces a mixture of all four biliverdin
isomers as a result of reaction at all four meso positions.22
Moreover, the four isomers appear to be formed in nearly equal
proportions, and the process does not show the regiospecificity
displayed by heme oxygenase oxidation. However, with a
trifluoromethyl-substituted heme, 7-demethyl-7-(trifluorometh-
yl)mesohemin IX, an unusually high degree of regiospecificity
has been observed, and this specificity has been attributed to
electronic control of the process.23 Notably, coupled oxidation
of myoglobin, as well as an active site variant of myoglobin,
results in cleavage that occurs exclusively at the R-meso
carbon.24,25
which the meso substituent is deprotonated.19,26 This complex
has received considerable study27-30 and has recently been
isolated in pure form in this laboratory.31 (py)2Fe(OEPO) is
conveniently prepared by the addition of pyridine to the dimer,
{FeIII(OEPO)}2,32 in the absence of dioxygen as shown in
Scheme 2.33 While the crystallographic data on this species
indicate that the iron is in a high-spin state, the assignment of
the iron and ligand oxidation states in solutions of this complex
is less clear, since it can be described by a combination of the
three canonical structures shown as 1a, 1b, and 1c in Scheme
2. Spectroscopically, (py)2Fe(OEPO) is readily identified on the
1
basis of its characteristic H NMR spectrum which displays
marked upfield shifts for the two types of meso protons and
both upfield and downfield shifts for the methylene protons.
The regiospecificity of heme catabolism produced by heme
oxygenase and by coupled oxidation of myoglobin has been
ascribed to both steric and electronic factors.34,35 The effect of
meso-methyl versus meso-formyl substitution on the regiospeci-
ficity of HO reaction36,37 and the NMR observation38 of larger
spin density at R-meso position in HO have led to the conclusion
that electronic effects are important in regioselectivity. Recently,
the crystal structure of human heme oxygenase-1 with the
substrate (heme) bound has been reported.39 The heme is
sandwiched between two helical protein segments with the
proximal helix providing histidine 25 as an axial ligand. There
are two molecules of the heme/heme oxygenase complex in the
asymmetric unit. One of these molecules offers more confine-
ment of the heme with the distal helix restricting access to the
For coupled oxidation of (py)2FeII(OEP) in pyridine solution,
meso hydroxylation produces (py)2(FeOEPO), a compound in
(11) Yoshida, T.; Noguchi, M.; Kikuchi, G.; Sano, S. J. Biochem. 1981,
90, 125.
(12) Sano, S.; Sano, T.; Morishima, I.; Shiro, Y.; Maeda, Y. Proc. Natl.
Acad. Sci. U.S.A. 1986, 83, 531.
(26) Balch, A. L. Coord. Chem. ReV. 2000, 200-202, 349.
(27) Sano, S.; Sugiura, Y.; Maeda, Y.; Ogawa, S.; Morishima, I. J. Am.
Chem. Soc. 1981, 103, 2888.
(13) Wilks, A.; Ortiz de Montellano, P. R. J. Biol. Chem. 1993, 268,
22357.
(14) Wilks, A.; Torpey, J.; Ortiz de Montellano, P. R. J. Biol. Chem.
1994, 269, 29553.
(15) Mansfield Matera, K.; Takahashi, S.; Fujii, H.; Zhou, H.; Ishikawa,
K.; Yoshimura, T.; Rousseau, D. L.; Yoshida, T.; Ikeda-Saito, M. J. Biol.
Chem. 1996, 271, 6618.
(28) Morishima, I.; Fujii, H.; Shiro, Y. J. Am. Chem. Soc. 1986, 108,
3858.
(29) Morishima, I.; Shiro, Y.; Hiroshi, F. Inorg. Chem. 1995, 34, 1528.
(30) Balch, A. L.; Noll, B. C.; Reid, S. M.; Zovinka, E. P. Inorg. Chem.
1993, 32, 2610.
(31) Balch, A. L.; Koerner, R.; Latos-Graz˘yn´ski, L.; Noll, B. C. J. Am.
Chem. Soc. 1996, 118, 2760.
(16) Liu, Y.; Moe¨nne-Loccoz, P.; Loehr, T. M.; Ortiz de Montellano, P.
R. J. Biol. Chem. 1997, 272, 6909.
(32) Balch, A. L.; Latos-Graz˘yn´ski; Noll, B. C.; Olmstead, M. M.;
Zovinka, E. P. Inorg. Chem. 1992, 31, 2248.
(33) Balch, A. L.; Noll, B. C.; Reid, S. M.; Zovinka, E. P. Inorg. Chem.
1993, 32, 2610.
(34) O’Carra, P. In Porphyrins and Metalloporphyrins; Smith, K. M.,
Ed.; Elsevier Scientific Pub. Co.: New York, 1975; p 123.
(35) Brown, S. B.; Chabot, A. A.; Enderby E. A.; North A. C. T. Nature
1981, 289, 93.
(36) Torpey, J.; Ortiz de Montellano, P. R., J. Biol. Chem., 1996, 271,
26067.
(37) Torpey, J.; Ortiz de Montellano, P. R., J. Biol. Chem., 1997, 272,
22008.
(38) Herna´ndez, G.; Wilks, A.; Paolesse, R.; Smith K. M.; Ortiz de
Montellano, P. R.; La Mar, G. N. Biochemistry 1994, 33, 6631.
(39) Schuller, D.; Wilks, A.; Ortiz de Montellano, P. R.; Poulos T. Nature
Struct. Biol. 1999, 6, 861.
(17) Warburg, O.; Negelein, E. Chem. Ber. 1930, 63, 1816.
(18) Lagarias, J. C. Biochim. Biophys. Acta 1982, 717, 12.
(19) St Claire, T. N.; Balch, A. L. Inorg. Chem. 1999, 38, 684.
(20) Balch, A. L.; Latos-Graz˘yn´ski, L.; Noll, B. C.; Olmstead, M. M.;
Szterenberg, L.; Safari, N. J. Am. Chem. Soc. 1993, 115, 1422.
(21) Balch, A. L.; Latos-Graz˘yn´ski, L.; Noll, B. C.; Olmstead, M. M.;
Safari, N. J. Am. Chem. Soc. 1993, 115, 9056.
(22) Bonnett, R.; McDonagh, A. F. J. Chem. Soc., Perkin Trans. 1 1973,
881.
(23) Crusats, J.; Suzuki, A.; Mizutani, T.; Ogoshi, H., J. Org. Chem.
1998, 63, 602.
(24) O’Carra, P.; Colleran, E. FEBS Lett. 1969, 5, 295.
(25) Hildebrand, D. P.; Tang, H.; Luo, Y.; Hunter, C. L.; Smith, M.;
Brayer, G. D.; Mauk, A. G. J. Am. Chem. Soc. 1996, 118, 12909.

12480 J. Am. Chem. Soc., Vol. 122, No. 50, 2000
Kalish et al.
Scheme 3
â-, γ-, and δ-meso carbon atoms while the other molecule
provides less steric constraint with both the R- and δ-meso
carbon atoms accessible to attack. There are no specific basic
or acidic residues in proximity to the R-meso carbon to
participate in the meso hydroxylation step. While the crystal
structure does support the notion of steric control of the
regiochemistry of the initial hydroxylation, electronic factors
may also play a role in directing this step, especially with non-
1
natural heme substrates. Additionally, recent H NMR studies
have revealed that a new species can be detected soon after
substrate binding and it appears that heme seating within HO
may be subject to dynamic effects that complicate the structural
picture and the role of steric effects in determining regioselec-
tivity.40
Figure 1. 500-MHz 1H NMR spectra taken at 20 °C from the reaction
of (py)2FeII(OEP) (trace A) and (py)2FeII(EtioP) (trace B) in pyridine-
d5 with hydrogen peroxide added at -30 °C with subsequent warming
to 20 °C. The inset to trace A was recorded at -30 °C to obtain the
best resolution. Methylene resonances are designated by 1-4, methyl
resonances of ethyl groups are designated by 1*-4*, and pyrrole methyl
resonanes are designated by 1**-4**. In trace B, the methyl resonances
of the ethyl groups are indicated by *.
Previously, Bonnett and Dimsdale were able to isolate metal-
free octaethyloxophlorin in low yield from the reaction of
hydrogen peroxide with (py)2FeII(OEP) and reported that an
olive-green intermediate was formed.41 Here, we report observa-
tions regarding the direct oxidation of hemes by hydrogen
peroxide in pyridine solution in the absence of dioxygen or of
a reducing agent. Thus, these reactions differ significantly from
previous work on heme degradation that utilizes dioxygen with
heme oxygenase or coupled oxidation conditions. By monitoring
the reaction directly by 1H NMR spectroscopy, the regiospeci-
ficity of the initial heme modification process can be examined
without the problems inherent in separation and isolation of
products.
Scheme 4
Results and Discussion
1H NMR Investigations of the Reaction of Hydrogen
Peroxide with Symmetrical Iron Porphyrins, (py)2FeII(OEP)
and (py)2FeII(EtioP). To characterize the species formed during
the reactions of iron(II) porphyrins with hydrogen peroxide, we
have examined the reactions of the symmetric iron porphyrins,
(py)2FeII(OEP) and (py)2FeII(EtioP), with hydrogen peroxide at
-30 °C in the absence of dioxygen. Scheme 3 shows the
structures and transformations involved. Pyridine-d5 solutions
of these complexes were prepared under a dinitrogen atmosphere
to exclude any oxidation by dioxygen and cooled to -30 °C,
at which point a dioxygen-free hydrogen peroxide (50% in
water)/pyridine-d5 solution (1:60 v/v) was introduced into the
sample. The red solutions of the FeII porphyrins turned brownish
green upon addition of the hydrogen peroxide, and the 1H NMR
spectra showed the presence of new sets of resonances due to
the conversion of the low-spin, diamagnetic starting materials
into new, paramagnetic products. After reaction, there was no
evidence for the formation of any precipitate, and so the spectral
data are indicative of the entire course of the reaction. Once
formed at -30 °C, the resulting samples are stable as long as
The results of oxidation of (py)2FeII(OEP) are shown in trace
A. The characteristic resonances of (py)2Fe(OEPO) are clearly
observed with two meso resonances in a 2:1 intensity ratio with
marked upfield hyperfine shifts and with two of the four
methylene resonances observed outside of the diamagnetic
region (0-10 ppm).27 The other resonances (two methylene
resonances, four methyl resonances) of (py)2Fe(OEPO) are
observed in the -5 to +10 ppm region of the spectrum as seen
in the inset to trace A. Note that no other resonances are present
and consequently the reaction proceeds cleanly without the
formation of other potential paramagnetic oxidation products:
[(py)2FeIII(OEP)]+, (py)FeIV)O(OEP), or (OEP)FeIIIOFeIII-
(OEP).
A complete assignment of the spectrum of (py)2Fe(OEPO)
has been made through the use of 1D NOE difference spectra.
The data are presented in Figure S1 in the Supporting Informa-
tion. The pattern of connectivity obtained from the NOE
difference peaks is set out in Scheme 4. Irradiation of the far
upfield meso resonance (Hγ) produces difference NOE peaks
corresponding only to resonances 4 and 4* in trace A of Figure
1. Hence, methyl resonance 4* and methylene resonance 4
originate from the ethyl group protons in positions Et-4 in the
structure of (py)2Fe(OEPO) shown in Scheme 3. Irradiation of
the other meso resonance (Hâ) produces difference NOE peaks
1
they are protected from dioxygen. Relevant H NMR spectra
obtained at 20 °C are shown in Figure 1.
(40) Yeh, S.; La Mar, G. N.; Wilks, A.; Ortiz de Montellano, P. R.,
personal communication.
(41) Bonnett, R.; Dimsdale, M. J. J. Chem. Soc., Perkin Trans 1 1972,
2540.

Heme/Hydrogen Peroxide ReactiVity
J. Am. Chem. Soc., Vol. 122, No. 50, 2000 12481
corresponding to methylene resonances 2 and 3 and methyl
resonances 2* and 3*. Hence, these resonances belong to the
ethyl protons of positions Et-2 and Et-3 that flank Hâ (see
Scheme 3). Irradiation of the downfield methylene resonance
(2) produces a strong NOE difference peak corresponding to
methyl resonance 2* and weaker peaks to resonances 1 and 1*.
These data allow us to assign resonances 2 and 2* to the ethyl
groups in position Et-2 in Scheme 3, resonances 1 and 1* to
the ethyl groups in position Et-1 in the same diagram, and
resonances 3 and 3* to the ethyl groups in position Et-3.
Scheme 5
Trace B of Figure 1 shows the 1H NMR spectrum that results
from treatment of (py)2FeII(EtioP) with hydrogen peroxide. The
spectral pattern is consistent with the formation of (py)2Fe-
(EtioPO). The resonances of (py)2Fe(EtioPO) are readily as-
signed through their relative intensities and comparison with
the spectrum of (py)2Fe(OEPO), which is shown in trace A.
Thus, the 1H NMR spectrum of (py)2Fe(EtioPO) contains four
new resonances that are not observed in the spectrum of (py)2Fe-
(OEPO). These four new resonances, which are observed at 55,
3, -2, and -9 ppm, are assigned to the four pyrrole methyl
groups in (py)2Fe(EtioPO). As usual for heme systems, pyrrole-
bound methyl groups display greater hyperfine shifts than do
the corresponding pyrrole-bound methylene groups,42,43 and the
methyl and methylene resonances can be grouped in pairwise
fashion: methyl at 55 with methylene at 25 ppm, methyl at -9
with methylene at -3 ppm, methyl at -2 with methylene at
4.5 ppm, and methyl at 3 with methylene at 2 ppm. Otherwise
the spectrum of (py)2Fe(EtioPO) contains all of the resonances
found for (py)2Fe(OEPO), and all of these resonances have
hyperfine shifts that are nearly identical to those found for
(py)2Fe(OEPO). Consequently, the substitution of methyl groups
for ethyl groups does not produce a major perturbation of the
electronic structure and spin distribution within the complex.
The characteristic resonances of the meso protons of (py)2Fe-
(EtioPO) are observed with marked upfield hyperfine shifts,
although the differences between symmetry-inequivalent â- and
δ-meso resonances are not resolved.
Further assignments of the spectrum of (py)2Fe(EtioPO) have
been obtained from 1D NOE difference spectra. Relevant data
are given in Figure S2 of the Supporting Information, and the
connectivity pattern is set out in Scheme 4. Irradiation of the
downfield meso resonance that originates from the protons (Hâ
and Hδ) produces NOE difference peaks that correspond to the
resonances labeled 2, 2**, 3, and 3** in trace B of Figure 1.
Hence, these resonances must originate from the groups in
positions Me-2, Et-2′, Et-3, and Me-3′ in the structure of
(py)2Fe(EtioPO) given in Scheme 3. Irradiation of the methyl
resonance at 55 ppm (resonance 2**) results in an NOE
difference peak corresponding to the methylene resonance at
-3 ppm (resonance 1). This is consistent with assigning
resonance 2** to methyl group Me-2 and resonance 1 to the
methylene group of Et-1. Irradiation of the methylene resonance
at 25 ppm (resonance 2) results in a NOE difference peak at
-9 ppm (resonance 1**). This is consistent with assigning
resonance 2 to the methylene group of ethyl group Et-2′ and
resonance 1** to the methyl group Me-1′. Thus, resonances 3
and 3**, which have NOE difference peaks that connect them
to Hâ and Hδ, must originate in Me-3′ and Et-3. The remaining
resonances, 4 and 4′, can then be assigned to Me-4 and to the
methylene resonances of Et-4′. This set of assignments is also
completely in accord with the assignments made for (py)2Fe-
(OEPO).
Reaction of Hydrogen Peroxide with Unsymmetrical Iron
Porphyrins. (1) Spectroscopic Results. The reactions of the
unsymmetrical hemes, (py)2FeII(DeuteroP) and (py)2FeII(MesoP),
and (py)2FeII(ProtoP), which are shown in Scheme 5, with
hydrogen peroxide have been examined under the conditions
used for the reaction of hydrogen peroxide with (py)2FeII(OEP)
and (py)2FeII(EtioP).
Figure 2 shows spectra for each of these unsymmetrical hemes
in the upfield region where the meso resonances are expected
to occur. The spectrum obtained by oxidation of (py)2FeII-
(DeuteroP) with hydrogen peroxide is shown at 20 °C in trace
A. This temperature gives the best resolution of all of the
resonances observed. The corresponding spectra obtained by
treating (py)2FeII(MesoP) and (py)2FeII(ProtoP) with hydrogen
peroxide are shown in traces B and C of Figure 2. These spectra
were recorded at -30 °C where again the resolution is best. In
each case, the overall pattern of resonances is similar to that
seen for (py)2Fe(OEPO) in the upfield region of Figure 1.
However, it is also abundantly clear that multiple resonances
are present in each of the three traces in Figure 2. Consequently,
several different species are formed upon oxidation. Unfortu-
nately, the resolution within this region of each spectrum is not
sufficient to allow a thorough analysis of the species present.
Examination of the most upfield region in trace A, where the
resolution of the meso resonances is best, does show that three
individual meso resonances are present and that these have
unequal intensities. Comparison with the data given in Figure
1 allows these resonances to be assigned to the single meso
protons opposite to the oxygenated site in the porphyrin. Thus,
(42) If the spin density at the â-pyrrole carbon positions is identical,
π-contact shifted pyrrole-bound methyl groups display greater hyperfine
shifts than do the corresponding pyrrole-bound methylene group. Theoreti-
cally, the contact shift of the individual CH proton is proportional to ACH R
2
) QCH RF/2S ) (B0 + B2 cos2θF/2S), where QCH R, K, Bo, and B2 are
2
2
constants and F is π-spin density at a carbon atom.43, Typically B0 is
44
very small; thus, ACH R ) QCH RF/2S ) B2 cos2θF/2S. The orientation of
2
2
the CH proton is conveniently described by a dihedral angle θ between the
plane determined by the pz axis of the â-pyrrole carbon (Câ) and the Câ-
Cmethyl bond and the Câ-Cmethyl-H bonds. For the methyl group rotating
between three positions of the idealized geometry (θ ) 0, 120, 240), the
averaged contact shift will be proportional to 0.5(B2FK/2S). Once the
â-methyl group is replaced by the ethyl group, the bulky methyl fragment
of the â-ethyl group tends to be located furthest away from the porphyrin
plane (θ ) 0°). Thus, this arrangement sets the lowest limit for the estimated
shift of the methylene resonances as equal to 0.25(B2FK/2S).
(43) Pignolet, L. H.; La Mar, G. N. In NMR of Paramagnetic Molecules;
La Mar, G. N., Horrocks, W. D., Jr., Holm, R. H., Eds.; Academic Press:
New York, 1973; p 357.

12482 J. Am. Chem. Soc., Vol. 122, No. 50, 2000
Kalish et al.
Figure 3. Portions of the 500-MHz 1H NMR spectrum recorded at 20
°C from the reaction of (py)2FeII(DeuteroP) in pyridine-d5 with hydrogen
peroxide added at -30 °C and subsequently warmed to 20 °C. Inset A
shows an expansion of the 35-25 ppm region (propionate methylene
resonances) at 20 °C, inset B shows an expansion of the 14.5-12.5
ppm region at 20 °C, and inset C shows an expansion of the -14 to
-16 ppm region at 0 °C. Resonances are identified by three characters
The first character indicates the isomer (R, â, γ, or δ) that produces
the resonance. The second character identifies the position site (1, 2,
3, or 4) of the functional group (see Scheme 5). The final character
specifies the chemical nature of the functional group, M methyl-H, m,
methylene-H, or p, pyrrole-H.
1
Figure 2. Far-upfield region of the 500-MHz H NMR spectra from
resonances. Resonances in the 25-30 ppm region (downfield
methylene region) are assigned to the protons of pyrrole-bound
methylene groups. Resonances in the -10 to -20 ppm region
are similarly assigned to another pyrrole methyl group. This
region will be referred to as the upfield methyl region.
Resonances in the 0 to -10 ppm region are assigned to
remaining pyrrole-bound methylene and methyl groups.
To assign resonances to individual isomers, we proceed under
the proposition that the pattern of the spin density distribution
is dominated by the location of the oxygenated meso site and
that this functional group imposes a basic 2-fold electronic
symmetry on the porphyrin (as shown in structure 1 in Scheme
6) which is only moderately perturbed by the presence of other
functional groups. Thus, the unpaired spin densities at the pairs
of positions, 1 and 1′, are expected to be similar. Additionally,
the spin densities at positions 2 and 2′ are also anticipated to
be similar, and so on. Thus, the δ-isomer, which has two methyl
groups at positions 1 and 1′, is expected to exhibit two equally
intense resonances with very similar chemical shifts for these
methyl protons. Likewise, the γ-isomer, with methyl groups in
the positions 2 and 2′, is expected to display a pair of equally
intense resonances with similar chemical shifts for these methyl
groups.
The published spectrum12 of the individual R-isomer of
(py)2Fe(ProtoPO) allows certain key features of all of the spectra
to be understood and facilitates the interpretation of the data
shown in Figure 5. For our assignment purposes, the most
significant features in the spectrum of the R-isomer of (py)2Fe-
(ProtoPO) are the single, widely dispersed methyl resonances
at 46 and -12 ppm which are differentiated by the sign of the
isotropic shifts. These resonances must arise from methyl groups
on the A and A′ pyrrole rings (positions 1 and 2). The two
methyl groups on pyrrole rings B and B′ in positions 3 and 3′
should produce a pair of resonances with identical sign and
similar values for the hyperfine shifts. (There are no methyl
the reaction of (A) (py)2FeII(DeuteroP), (B) (py)2FeII(MesoP), and (C)
(py)2FeII(ProtoP) with hydrogen peroxide (added at -30 °C) in pyridine-
d5 at -30 °C. The spectrum in trace A was recorded with the sample
at 20 °C, while the spectra in traces B and C were obtained from
samples at -30 °C.
at least three of the four possible isomeric complexes shown in
Scheme 4 are present in the sample of (py)2Fe(DeuteroPO) and
their abundances are unequal.
The 75 to -40 ppm region of the 1H NMR spectra obtained
by reaction of (py)2FeII(DeuteroP), (py)2FeII(MesoP), and (py)2-
FeII(ProtoP) with hydrogen peroxide are shown in Figures 3-5,
respectively. These spectra are shown at the temperatures at
which each has the best resolution. The various insets show
additional details of particular features. In some cases, these
insets were recorded at a different temperature where that
particular feature shows enhanced resolution. Assignments of
the individual resonances to the possible isomeric products are
also made. The procedure used to identify the resonances and
assign them to specific isomers is given in the next section.
1
(2) Analysis of the H NMR Spectra and Assignment of
Resonances to Specific Isomeric Products. General Consid-
erations. Two issues need to be addressed in interpreting the
spectra shown in Figures 2-5. Resonances need to be assigned
to specific functional groups within each product, and they also
need to be assigned to individual isomers, where possible. The
data shown in Figure 1 for the symmetrical hemes allows certain
1
regions of the H NMR spectra shown in Figures 2-5 to be
associated with particular functional groups. Thus, all of the
resonances seen in Figure 2 are assigned to meso protons, and
those resonances with the most negative chemical shifts are
assigned to the meso protons opposite the site of oxygenation.
In Figures 3-5, the most downfield resonances, those with
chemical shifts greater than 45 ppm, are assigned to a pyrrole
methyl group and will be termed the downfield methyl

Heme/Hydrogen Peroxide ReactiVity
J. Am. Chem. Soc., Vol. 122, No. 50, 2000 12483
1
Figure 4. Portions of the 500-MHz H NMR spectrum taken at -30 °C from the reaction of (py)2FeII(MesoP) in pyridine-d5 with hydrogen
peroxide (added at -30 °C). Inset A shows an expansion of the 59-51 ppm region (pyrrole methyl resonances) at 20 °C, inset B shows an
expansion of the 41-36.5 ppm region (propionate methylene resonances) at 60 °C, and inset C shows an expansion of the -9 to -11 ppm region
(propionate methylene resonances) at 20 °C. Resonance assignments follow those used in Figure 3.
Scheme 6
positions 3 and 3′, which would have to produce a doublet with
the same sign for the hyperfine shifts. Consequently, in the
spectra shown in Figures 3-5, the methyl resonances in the
downfield methyl region (chemical shift greater than 45 ppm)
and in the upfield methyl region at -10 to -20 ppm are
assigned to methyl groups on pyrrole rings A and A′ in each
isomer.
This set of considerations is also entirely consistent with the
1
Figure 5. Portions of the 500-MHz H NMR spectra obtained from
assignment of resonances in the spectra of (py)2Fe(OEPO) and
the reaction of (py)2FeII(ProtoP) in pyridine-d5 with hydrogen peroxide
(py)2Fe(EtioPO) that have been obtained using NOE difference
at -30 °C. Inset A shows an expansion of the -12 to -15 ppm region
spectra. Specifically for (py)2Fe(EtioPO), the downfield methyl
(vinyl resonances) at -30 °C, and inset B shows an expansion of the
resonance has been assigned to Me-2 and the upfield methyl
-17 to -19 ppm region (propionate methylene resonances). All data
resonance to Me-1′ and these functional groups are on pyrrole
rings A and A′, respectively.
were obtained with the sample at -30 °C. Resonance assignments
follow those used in Figure 3 but with v indicating resonances arising
from heme vinyl protons.
For (py)2FeII(DeuteroP). Portions of the 1H NMR spectrum
obtained by treatment of (py)2FeII(DeuteroP) with hydrogen
peroxide are shown in Figures 2 and 3. Resonance assignments
have been greatly assisted by comparison of the data shown in
Figure 3 with data obtained from a sample of (py)2FeII-
groups in positions 4 and 4′.) Since the resonances observed at
46 and -12 ppm are single lines with opposite signs for the
hyperfine shifts, they cannot come from the methyl groups in

12484 J. Am. Chem. Soc., Vol. 122, No. 50, 2000
Kalish et al.
Table 1. Distribution of Isomers in Pyridine-d5
(DeuteroP), which was specifically deuterated in the two pyrrole
C-H positions. That sample lacked the resonances indicated
by an asterisk in Figure 3. (Also these pyrrole C-H resonances
are absent from the spectrum obtained from a sample of
(py)2FeII(MesoP) that was treated with hydrogen peroxide; see
Figure 4.) There are eight pyrrole C-H resonances seen in the
spectra shown in Figure 3. (Two of these resonances have
upfield chemical shifts and these resonances will be described
as lying within the upfield pyrrole-H region.) Hence, all four
isomers of (py)2Fe(DeuteroPO) are present and all eight of the
expected pyrrole C-H resonances are observed. However, the
variation in intensities of these eight resonances indicates that
the four isomers are present in a nonrandom population. This
is a very important piece of information because of its analytical
applications. Careful comparison of the relative intensities allows
the selection of four pairs of resonances, which belong to the
four regioisomers shown in Scheme 4. The internal consistency
of the integrated intensities for each set has been carefully
checked. Resonances that belong to one isomer have a set of
relative intensities that reflect the molecular structure. However,
the intensity ratios between resonances that belong to different
isomers reflect the relative populations of those isomers as
determined by the regiospecificity of the reaction. Since the four
isomers are present in a nonrandom population, these consid-
erations are efficient in identifying the resonances of individual
isomers.
Substitution of a methyl group by a proton is expected to
give rise to a resonance that is subject to a hyperfine shift of
opposite sign but a magnitude comparable to that of the
corresponding methyl group if π-spin delocalization dominates
the hyperfine shifts.44 Consequently, the resonances in Figure
3 with the largest hyperfine shifts, those in the downfield methyl
region and those in the upfield pyrrole C-H region, must
originate from a common site (i.e., from a site with similar value
and sign of the spin density) within the product molecule.
Likewise, when methylene groups and methyl groups occupy
analogous sites, these groups will produce resonances with
hyperfine shifts of the same sign but the magnitude of the
hyperfine shift of the methyl group will be greater. Conse-
quently, the resonances in the downfield methyl region, the
downfield methylene region, and the upfield pyrrole-H region
must arise from substituents in either the 1 and 1′ or in the 2
and 2′ positions on pyrrole rings A and A′ in Scheme 5.
heme
R
â
γ
δ
(py)2Fe(DeuteroPO)
(py)2Fe(MesoPO)
(py)2Fe(ProtoPO)
nonselective
31
18
20
25
24
18
22
25
25
36
35
25
20
28
23
25
the resonances in the region at -10 to -20 ppm arise from
methyl substituents in the 1 and 1′ positions, and resonances in
the 25-30 ppm region arise from pyrrole protons in the 1 and
1′ positions.
Following the above analysis, it is possible to assign
individual resonances in Figure 3. The following three-character
scheme is used. The first character indicates which isomer, R,
â, γ, or δ, produces the resonance. The second character
identifies the position (site 1, 2, 3, or 4 as in Scheme 5). We do
not differentiate between 1 and 1′, etc., of the functional group
within the product. The final character specifies the chemical
nature of the functional group: M, methyl-H; m, methylene-H;
and p, pyrrole-H (v, vinyl-H). Thus, the most downfield
resonance in the spectrum shown in Figure 3 is labeled R2M,
indicating that it arises from a methyl group in the 2 or 2′
position in the R-isomer of (py)2Fe(DeuteroPO).
Four resonances appear in the downfield methyl region. Two
have identical intensity and hence are due to the γ isomer, which
has two methyl groups in the 2 and 2′ positions, The other two
resonances are due to the R- and â-isomers. In the upfield methyl
region between -5 and -20 ppm, there are also four resonances,
a pair with equal intensity, which come from the δ-isomer, and
two resonances whose intensities parallel those seen in the
downfield methyl region. The resonances can be assigned to
the R- and â-isomers, and they can be paired with partners in
the downfield methyl region on the basis of their relative
integrated intensities.
The resonances of the R- and, by default, the â-isomers, have
also been differentiated by comparison of the intensities of the
downfield (∼27 ppm) and upfield (∼-34 ppm) shifted pyrrole
resonances. This analysis selected two pyrrole resonances (of
equal intensity but opposite hyperfine shifts) which must belong
to the R-isomer. Once these resonances are identified, they serve
as a convenient starting point for the identification of the other
resonances of the R-isomer through analysis of the relative
integrated intensities of other resonances.
If the resonances in these regions are produced by protons
in the 1 and 1′ positions, then the γ-isomer will produce a
doublet with equally intense resonances in the downfield
methylene region. No other isomer will produce a doublet
methylene resonance in this region. Inspection of this region
of the spectrum shows two resonances with clearly unequal
intensity; consequently, this assignment is untenable. On the
other hand, if the resonances in the downfield methyl region
and the upfield pyrrole-H region come from groups in the 2
and 2′ positions, then two unequally intense resonances are
excepted in the downfield methylene region. These resonances
arise from the single methylene group in the â (2′-CH2)- and
in the δ (2-CH2) -isomers. The R- and γ-isomers do not have
methylene groups in the 2 and 2′ positions and hence cannot
contribute to this region of the spectrum. Indeed, two unequally
intense methylene resonances are observed in this region (see
inset A in Figure 3). Consequently, the resonances in the
downfield methyl region, the downfield methylene region, and
the upfield pyrrole-H region are assigned to positions 2 and 2′,
Further analysis of the integrated intensities of the resonances
in the spectra obtained from (py)2FeII(DeuteroP) indicates that
the four isomers shown in Scheme 4 are formed in the following
percentages: R, 31; â, 24; γ, 25; and δ, 20. Table 1 shows
these percentages in a format that facilitates comparison with
the corresponding percentages obtained from the reaction of
(py)2FeII(MesoP) and (py)2FeII(ProtoP) with hydrogen peroxide.
Attempts to utilize NOE difference spectra to obtain additional
confirmatory assignments of this spectrum have not been
successful. The inability to detect NOE difference peaks is
caused by two factors: the low symmetry of each isomer and
the fact that four different isomers are produced. As a result,
the spectral intensity is lower and the cross-peaks are too weak
to detect. We were unable to improve the signal intensity by
increasing solute concentration due to the need to keep all
species in solution during the reaction at -30 °C.
To examine this reaction at lower temperatures where
intermediates might be detected, the reactions of hydrogen
peroxide with (py)2FeII(DeuteroP) were examined in mixtures
of dichloromethane and pyridine. Although working at low
temperature in this solvent mixture did not reveal the presence
(44) Walker, A. In The Porphyrin Handbook; Kadish, K. M., Smith, K.
M., Guilard, R., Eds.; Academic Press: New York, 2000; Vol. 5, p 82.

Heme/Hydrogen Peroxide ReactiVity
J. Am. Chem. Soc., Vol. 122, No. 50, 2000 12485
Table 2. Distribution of Isomers for (py)2Fe(DeuteroPO) in
Different Solvent Mixtures
open to consideration. However, the present results are consistent
with the notion that a heme-bound peroxide ligand may be
involved in the attack upon the meso sites within the porphyrin.9
In contrast to the results reported here, addition of hydrogen
peroxide to the heme proteins, myoglobin and horseradish
peroxidase, results in the formation of ferryl, (FedO)2+, forms
of these proteins.45,46 In these proteins, the iron is present in
five-coordinate form that may facilitate coordination of the
peroxide and its eventual cleavage to produce the ferryl complex.
However, the reaction of the heme/heme oxygenase complex,
which also contains a five-coordinated heme, with hydrogen
peroxide does not produce an observable ferryl form; rather,
the heme is converted by hydrogen peroxide into verdoheme.13
However, a ferryl complex is formed by the reaction of the
heme/heme oxygenase complex with m-chloroperoxybenzoic
acid or tert-butyl hydroperoxide.13 Although spectroscopically
detectable amounts of ferryl hemes can be obtained at low
temperatures by reacting iron porphyrins under carefully
controlled conditions with dioxygen,47 iodosyl benzene,48 or
m-chloroperoxybenzoic acid, we are not aware of any experi-
ments where the reaction of hydrogen peroxide with a iron
porphyrin complex results in the formation of a spectroscopically
detected ferryl compound.49
The spectra obtained from oxidation of the unsymmetrically
substituted hemes, (py)2FeII(DeuteroP), (py)2FeII(MesoP), and
(py)2FeII(ProtoP), indicate that the four isomeric oxophlorin
products are formed in arrays that are somewhat nonselective
but do not show a pronounced regiospecificity. Only in the case
of oxygenation of (py)2FeII(DeuteroP) in pyridine-d5 solution
is the R-isomer of the iron oxophlorin the major isomer that is
produced, and the specificity is small. For the other two hemes
studied, (py)2FeII(MesoP) and (py)2FeII(ProtoP), the γ-isomer
is the most abundant and the R-isomer is the least abundant.
Significantly, alteration of the solvent composition for pyri-
dine/dichloromethane mixtures does result in alteration of the
percentages of the four isomers of (py)2Fe(DeuteroPO) that are
produced as seen in Table 1. However, the percentage of the
R-isomer of the iron oxophlorin that forms is reduced when
such mixtures are employed as the reaction medium. Neverthe-
less, these results are important because they demonstrate that
the regiospecificity of this oxidative process is under the control
of environmental factors. Thus the protein environment provided
by heme oxygenase directs the specificity of the initial oxidative
attack upon the heme by control of a combination of steric and
electronic effects. Very recent work has demonstrated that the
regiospecificity of heme oxygenase can be altered by mutage-
nisis of a conserved arginine residue.50
solvent
pure pyridine-d5
pyridine-d5/dichloromethane-d2 (9:1)
pyridine-d5/dichloromethane-d2 (1:1)
pyridine-d5/dichloromethane-d2 (1:5)
nonselective
R
â
γ
δ
31
19
21
28
25
24
14
13
22
25
25
40
39
29
25
20
27
27
21
25
of any new intermediates, these studies did reveal that the
regioselectivity of this oxidation is solvent dependent. As seen
in Table 2, the relative proportions of the four isomers formed
changes as the solvent composition is altered.
For (py)2FeII(MesoP). 1H NMR spectra obtained from
samples prepared by treatment of (py)2FeII(MesoP) with hy-
drogen peroxide are shown in Figures 2 and 4. As expected,
the spectra of (py)2Fe(MesPO) lack the eight pyrrole C-H
resonances seen in Figure 3 for (py)2Fe(DeuteroPO). The
resonance assignments shown in Figure 4 have been made by
utilizing the information obtained and analyzed in the preceding
sections.
For oxidation of (py)2FeII(MesoP) in pyridine solution, the
four isomers of (py)2Fe(MesoPO) shown in Scheme 4 are
formed in the percentages shown in Table 1.
1
For (py)2FeII(ProtoP). Portions of the H NMR spectra of
(py)2Fe(ProtoPO), which was obtained by hydrogen peroxide
oxidation of a pyridine-d5 solution of (py)2FeII(ProtoP), are
shown in Figures 2 and 5. Spectral analysis follows the course
outlined above. A set of resonances that are not present in the
spectra shown in Figures 3 and 4 are assigned to the protons of
the vinyl groups in (py)2Fe(ProtoPO). In Figure 5, these vinyl
resonances are denoted by a v in the three character designation.
Reaction of (py)2FeII(ProtoP) with hydrogen peroxide pro-
duces all four isomers of (py)2Fe(ProtoPO) in the percentages
shown in Table 1.
Conclusions
The results reported here demonstrate that hydrogen peroxide
reacts cleanly with iron(II) porphyrins in pyridine solution under
an inert atmosphere to form the corresponding oxophlorin
complexes, which are stable in the absence of dioxygen. This
process mimics the initial stage of heme degradation that is
catalyzed by heme oxygenase. It is important to note that the
process described here is distinct from the process of coupled
oxidation in which a heme or heme protein is treated with
dioxygen in the presence of a sacrificial reducing agent, which
is generally either ascorbic acid or a hydrazine such as phenyl
hydrazine.19
The general similarity in the 1H NMR spectral patterns seen
for all of the complexes produced here, (py)2Fe(OEPO), (py)2Fe-
(EtioPO), (py)2Fe(DeuteroPO), (py)2Fe(MesoPO), and (py)2Fe-
(ProtoPO), indicates that all of these complexes have similar
electronic structures. Consequently, all of these complexes need
the set of resonance structures shown in Scheme 2 to describe
the possible distribution of electrons between the ligands and
iron center.
No evidence for the formation of intermediates in the process
of conversion of (py)2FeII(P) into (py)2Fe(PO) through the
reaction with hydrogen peroxide has been found. Other potential
paramagnetic oxidation products, the one-electron oxidation
product [(py)2FeIII(OEP)]+, the ferryl complex (py)FeIVd
O(OEP), or the ubiquitous µ-oxo dimer (OEP)FeIIIOFeIII(OEP),
which could have formed, are not detected in these experiments.
Thus, aspects of the mechanism for this hydroxylation remain
Experimental Section
Materials. IronIIIoctaethylporphyrin chloride was purchased from
Mid Century. Etio-1 porphyrin, deuteroporphyrin IX dimethyl ester,
protoporphyrin IX dimethyl ester, and meso porphyrin IX dimethyl
ester were purchased from Porphyrin Products. Deuteroporphyrin IX-
(45) Balch, A. L.; La Mar, G. N.; Latos-Graz˘yn´ski, L.; Renner, M. W.;
Thanabal, V. J Am. Chem. Soc. 1985, 107, 3003.
(46) Poulos, T. L. In The Pophyrin Handbook; Kadish, K. M., Smith,
K. M., Guillard, R., Eds; Academic Press: New York, 2000; Vol. 4, p
189.
(47) Chin, D. H.; Balch, A. L.; La Mar, G. N. J. Am. Chem. Soc. 1980,
102, 1446.
(48) Groves, J. T.; Haushalter, R. C.; Nakamura, M.; Nemo, T. E.; Evans,
B. J. J. Am. Chem. Soc. 1981, 103, 2884.
(49) Weiss, R.; Gold, A.; Trautwein, A. X.; Terner, J. In The Pophyrin
Handbook; Kadish, K. M., Smith, K. M., Guillard, R., Eds.; Academic
Press: New York, 2000; Vol. 4, p 189.
(50) Zhou, H.; Migita, C. T.; Sato, M.; Sun, D.; Zhang, X.; Ikeda-Saito,
M.; Fujii, H.; Yoshida, T. J. Am. Chem. Soc. 2000, 122, 8311.

12486 J. Am. Chem. Soc., Vol. 122, No. 50, 2000
Kalish et al.
d2 dimethyl ester was prepared via a standard route using resorcinol-
d2.51 Iron(III) was inserted into these porphyrins by a standard
procedure.52 Bis(pyridine)ironIIporphyrins were prepared by reduction
of the appropriate iron(III) complex with zinc amalgam in pyridine
solution in a controlled atmosphere drybox under purified dinitrogen.
Hydrogen peroxide, 50 wt % in water, was purchased from Aldrich.
Dioxygen was removed from the hydrogen peroxide samples by five
freeze/pump/thaw cycles.
500.1100 MHz). The spectra were recorded over a 100-kHz bandwidth
with 32K data points and a 5-µs 90° pulse. For a typical spectrum,
between 500 and 1000 transients were accumulated with a 250-ms
repetition time. The residual 1H resonances of the solvent, pyridine-d5,
were used as a secondary chemical shift reference.
1D NOE difference spectra were acquired by interleaving eight scans
in which the peak of interest was irradiated with eight scans off
resonance and using phase cycling to generate a difference spectrum.
A total of 3840 scans per spectrum were collected with a 150-ms
irradiation time and a recycle time of 320 ms. Data were apodized
with 40 Hz line broadening.
Reactions with H2O2. In a controlled atmosphere (glove) box filled
with dinitrogen, a dioxygen-free pyridine-d5 solution of the bis-
(pyridine)ironIIporphyrin (3-4 µmol) was placed in an NMR tube and
sealed with a rubber septum and Parafilm. The solutions were cooled
to -30 °C in an ethanol/liquid dinitrogen bath. Hydrogen peroxide/
pyridine-d5 solutions (1:60, v/v) were added via a microsyringe at -30
°C to the porphyrin solutions to give hydrogen peroxide concentrations
in the range of 4-6 µmol. The progress of the reaction was followed
Acknowledgment. We thank the NIH (Grant GM-26226,
A.L.B.) and the Foundation for Polish Science (L.L.-G.) for
financial support and the NSF (Grant OSTI 97-24412) for partial
funding of the 500-MHz NMR spectrometer.
1
by H NMR spectroscopy. Mixed solvent samples were prepared in
the above manner.
1
Instrumentation. H NMR and 1D NOE difference spectra were
Supporting Information Available: 1D NOE difference
spectra of (py)2FeII(OEP) and (py)2FeII(Etio-1P) (PDF). This
material is available free of charge via the Internet at
http://pubs.acs.org.
recorded on a Bruker Avance 500 FT spectrometer (1H frequency is
(51) Fuhrhop, J.-H.; Smith, K. M. In Porphyrins and Metalloporphyrins;
Smith, K. M., Ed.; Elsevier Scientific Publishing Co.: New York, 1975; p
65.
(52) Adler, A. D.; Longo, F. R.; Kampas, F. J. Inorg. Nucl. Chem. 1970,
32, 2443.
JA0016405