A R T I C L E S
Rivera et al.
reduction of the ferrous dioxygen complex of HO (FeII-O2) to
produce an intermediate that was identified by EPR spectroscopy
as corresponding to the FeIII-OOH complex.11 Upon warming,
this intermediate was converted into the corresponding R-meso-
hydroxyheme complex, thus confirming a ferric hydroperoxide
intermediate as a precursor of R-meso-hydroxyheme.
The EPR spectrum corresponding to the FeIII-OOH complex
of HO displays g-values of 2.37 (or 2.38, depending on
treatment), 2.19, and 1.93 at 77 K.11 The sum of the squares of
the principal g-values (∑g2) for the hydroperoxy complex of
HO is about 14.1. It is interesting to consider this value in the
context of recently reported studies of low-spin Fe(III)
porphyrinates.12-15 These reports demonstrated the presence of
a novel electronic configuration, (dxz,dyz)4(dxy)1, where the
unpaired electron resides in the dxy orbital. Interestingly, all
model hemes known to possess the (dxz,dyz)4(dxy)1 electron
configuration (hereafter abbreviated as dxy) displayed EPR
spectra with ∑g2 < 14. By comparison, low-spin Fe(III) hemes
possessing the more common (dxy)2(dxz,dyz)3 electron configu-
ration (hereafter abbreviated as dπ) display EPR spectra with
Figure 1. Representation of the 3a2u(π) porphyrin orbital. The sizes of the
circles are proportional to the calculated electron density.
alkyl peroxide,19 as well as heme proteins with histidine21,22 or
cysteinate23 sixth ligands, yield very similar EPR spectra with
compressed g anisotropy (∑g2 ≈ 14). These spectra are very
similar to those obtained for the FeIII-OOH complexes of various
heme protein enzymes, which were prepared by cryoreduction
and then annealing of the corresponding FeII-O2 complexes.11,24
The g-values of the complexes of Tajima and co-workers (2.32,
2.16, 1.95, methoxide, tert-butylperoxide;21 2.25, 2.15, 1.96, bis-
tert-butyl-peroxide;21 2.32, 2.19, 1.94, imidazolate, hydroper-
oxide20) are very similar to those of annealed hemoglobin-
hydroperoxide (2.31, 2.18, 1.94),11 heme oxygenase-hydroperoxide
(2.37, 2.19, 1.93),11 and cytochrome P450-hydroperoxide (2.29,
2.16, 1.96).24 We thus reasoned that magnetic resonance
investigation of the Tajima model complexes could provide
important information concerning the orbital of the unpaired
electron, and hence the likely conformation of the porphyrinate
ring of these model complexes, which could thus yield insights
into the electronic and molecular structure of the catalytically
active hydroperoxide complex of heme oxygenase. As will
be shown below, we find that at 8 K the unpaired electron of
[TPPFe(OCH3)(OOtBu)]-, [TPPFe(OOtBu)2]-, and [TPPFe-
(OCH3)2]- resides in one of the dπ orbitals, while at physi-
ological temperatures the unpaired electron of those complexes
that are stable enough to investigate is indeed in the dxy orbital.
2
2
2
the typical gxx + gyy + gzz ≈ 16.14,16
On the basis of these arguments, it was possible to speculate
that the electronic configuration of the FeIII-OOH complex of
HO might have an unpaired electron residing in the dxy orbital.
What is noteworthy about a dxy electronic configuration is that
it places a large amount of π-spin density on the porphyrin meso-
carbons.12-16 To delocalize spin density from the dxy orbital into
the porphyrin π system, the macrocycle has to ruffle signifi-
cantly, so that the nodal planes of the pz orbitals of the
macrocycle are no longer in the xy plane; the components
(projections) of these pz orbitals in the xy plane have the proper
symmetry to interact with the dxy orbital.12 The porphyrin orbital
that has the proper symmetry to interact with the dxy orbital in
this ruffled macrocycle conformation is the 3a2u(π) orbital12
shown in Figure 1. It is evident from the relative sizes of the
circles in the schematic representation of the 3a2u(π) orbital that
the meso-carbons possess large electron density. Large spin
density at the meso positions, in turn, may explain the attack
of the FeIII-OOH intermediate on a heme meso-carbon, as
discussed in more detail later in this work. Consequently, the
main object of the investigations reported herein is to determine
the electron configuration of hydroperoxide or alkyl peroxide
complexes of FeIII porphyrinates.
Experimental Section
Some years ago Tajima and co-workers showed that synthetic
hemes in the presence of alkyl peroxides and a variety of sixth
ligands, including methoxide,17-19 imidazolate,20 or a second
Reagents. Tetramethylammonium hydroxide (TMAOH) 25% (w/w)
in methanol and 70% (w/w) aqueous tert-butylhydroperoxide (tBuOOH)
were purchased from Alfa Aesar. TMAOH was used as received,
t
whereas BuOOH was extracted into methylene chloride by swirling
(11) Davydov, R. M.; Yoshida, T.; Ikeda-Saito, M.; Hoffman, B. M. J. Am.
Chem. Soc. 1999, 121, 10656-10657.
2.5 mL of the aqueous peroxide solution with 6 mL of dichloromethane
in a separatory funnel. The organic phase was separated and then dried
over anhydrous MgSO4 before being filtered into a brown glass
container. Dichloromethane solutions of tBuOOH were prepared before
each experiment. Chloroiron(III) tetraphenylporphyrin (TPPFeCl) and
meso-13C-TPPFeIIICl were purchased from Porphyrin Products (Logan,
UT) and used without further purification. 13C labeled perchlorato-
(12) Safo, M. K.; Walker, F. A.; Raitsimring, A. M.; Walters, W. P.; Dolata,
D. P.; Debrunner, P. G.; Scheidt, W. R. J. Am. Chem. Soc. 1994, 116,
7760-7770.
(13) Walker, F. A.; Nasri, H.; Torowska-Tyrk, I.; Mohanrao, K.; Watson, C.
T.; Shkhirev, N. V.; Debrunner, P. G.; Scheidt, W. R. J. Am. Chem. Soc.
1996, 118, 12109-12118.
(14) Walker, F. A. Coord. Chem. ReV. 1999, 185-186, 471-534.
(15) Simonneaux, G.; Schu¨nemann, V.; Morice, C.; Carel, L.; Toupet, L.;
Winkler, H.; Trautwein, A. X.; Walker, F. A. J. Am. Chem. Soc. 2000,
122, 4366-4377.
(16) Walker, F. A. Proton NMR and EPR Spectroscopy of Paramagnetic
Metalloporphyrins. In The Porphyrin Handbook; Kadish, K. M., Smith,
K. M., Guilard, R., Eds.; Academic Press: San Diego, 2000; pp 81-183.
(17) Tajima, K.; Ishizu, K.; Sakurai, H.; Nishiguchi-Ohya, H. Biochem. Biophys.
Res. Commun. 1986, 135, 972-978.
(20) Tajima, K.; Oka, S.; Edo, T.; Miyake, S.; Mano, H.; Mukai, K.; Sakurai,
H.; Ishizu, K. J. Chem. Soc., Chem. Commun. 1995, 1507-1508.
(21) Tajima, K. Inorg. Chim. Acta 1990, 169, 211-219.
(22) Jinno, J.; Shigematsu, M.; Tajima, K.; Sakurai, H.; Ohya-Nishiguchi, H.;
Ishizu, K. Biochem. Biophys. Res. Commun. 1991, 176, 675-681.
(23) Tajima, K.; Edo, T.; Ishizu, K.; Imaoka, S.; Funae, Y.; Oka, S.; Sakurai,
H. Biochem. Biophys. Res. Commun. 1993, 191, 157-164.
(18) Tajima, K.; Jinno, J.; Ishizu, K.; Sakurai, H.; Ohya-Nishiguchi, H. Inorg.
Chem. 1989, 28, 709-715.
(19) Tajima, K.; Tada, K.; Jinno, J.; Edo, T.; Mano, H.; Azuma, N.; Makino,
K. Inorg. Chim. Acta 1997, 254, 29-35.
(24) Davydov, R.; Macdonald, I. D. G.; Makris, T. M.; Sligar, S. G.; Hoffman,
B. M. J. Am. Chem. Soc. 1999, 121, 10654-10655.
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6078 J. AM. CHEM. SOC. VOL. 124, NO. 21, 2002