Pronounced axial thiolate ligand effect on the reactivity of high-valent oxo-iron porphyrin intermediate

Full text of DOI:10.1021/ja972892h

12008
J. Am. Chem. Soc. 1997, 119, 12008-12009
Scheme 1. S.R. Complex
Pronounced Axial Thiolate Ligand Effect on the
Reactivity of High-Valent Oxo-Iron Porphyrin
Intermediate
Yasuteru Urano, Tsunehiko Higuchi,* Masaaki Hirobe, and
Tetsuo Nagano*
Graduate School of Pharmaceutical Sciences
The UniVersity of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan
differentiating the nature of some oxidative intermediates.
Therefore, we examined the O-demethylation mechanisms of
p-dimethoxybenzene in various iron porphyrin-oxidant systems,
in addition to a microsomes from livers of a phenobarbital
treated rats-NADPH/O2 system4 and an expressed human
CYP1A2-NADPH/O2 system. We used five kinds of meso-
tetraaryl iron porphyrins. Among them, “Swan-Resting” form
porphyrin (S.R. complex, Scheme 1), which was previously
reported by us, is a unique complex which retains its axial
thiolate coordination during catalytic oxidation reactions.5
First of all, we investigated the modes of O-O bond cleavage
mediated by these iron porphyrins by using peroxyphenylacetic
acid (PPAA), which has frequently been used as a probe for
this purpose.5b,6 Though S.R. is an oxido-stable complex
compared to other thiolate-ligated porphyrins,7 the amount of
each peroxy acid used in the reactions below was kept at 1 mol
equiv to the iron porphyrins, because it is necessary to minimize
the decomposition of the S.R. complex and the secondary
oxidation of the formed acid by active species. In the other
four porphyrin systems, the same amount of oxidant was used
to get comparable results. In every iron porphyrin-PPAA
system examined in either solvent, benzene or dichloromethane,
phenylacetic acid was the major product, which indicated the
predominance of heterolytic O-O bond cleavage and compound
I formation.8
ReceiVed August 18, 1997
Among heme enzymes, cytochrome P450 has the strongest
oxidizing ability, e.g. only P450 can hydroxylate nonactivated
alkanes.1 The distinctive structural features of P450 are the
unusual thiolate coordination to the heme and also the extreme
hydrophobicity of its active site,1 and much interest has been
focused on the effect of the axial thiolate ligand on the O-O
bond cleavage step in the catalytic cycle of P450. Recently
Gross and Nimri2 have reported an axial ligand effect on styrene
epoxidation mediated by iron porphyrin-ozone systems, but
they employed halide anion or alkoxy anion as a ligand and
did not use a thiolate or an imidazole ligand, which may be
more relevant to heme-containing enzymes. In addition, little
is known yet about the axial ligand effect on the reactiVity of
the oxidizing intermediate of the heme enzymes. We report
here that a high-valent oxo-iron porphyrin intermediate with
a thiolate ligand has similar reactivity to that of cytochrome
P450.
Oxidative O-dealkylation of alkyl aryl ethers is one of the
major metabolic reactions catalyzed by cytochrome P450.1 There
are two generally accepted mechanisms, that is, the H atom
abstraction mechanism and the ipso-substitution mechanism.3
Next, the KIEs in the O-demethylation of p-dimethoxyben-
zene were examined (Table 1).9,10 In the rat liver microsomes-
and human CYP1A2-NADPH/O2 systems, p-dimethoxyben-
zene was O-demethylated with high KIE values (>10). Among
the porphyrin-oxidant systems, only the S.R.-PPAA system
showed high KIE values. All other iron porphyrin-oxidant
systems gave low KIE values (≈1.0). Further, we have
investigated the 18O incorporation from 18O-enriched oxidants
in the O-demethylation of p-dimethoxybenzene (Table 2), and
again unambiguous results, which are in harmony with those
in the KIE experiments, were obtained. In the rat liver
microsomes-NADPH/O2 system, p-dimethoxybenzene was
Clear differences between these two mechanisms are observed
in the kinetic isotope effects (KIEs) and in the origin of the
oxygen atom of the resulting phenolic hydroxy group. (In the
reaction scheme, the filled O indicates the oxygen atom
originated from the active oxidizing intermediate.) So far, it is
thought that the mechanism which actually operates depends
on the oxidizing system used, namely in the cytochrome P450-
dependent enzymatic reaction and the iron porphyrin-iodosyl-
benzene (PhIO) systems, the former mechanism operates, and
in hydroxyl radical-mediated reactions, the latter does.3c
In a previous paper,4 we have shown that the O-demethylation
mechanisms of p-dimethoxybenzene can be used as a probe for
(5) (a) Higuchi, T.; Uzu, S.; Hirobe, M. J. Am. Chem. Soc. 1990, 112,
7051. (b) Higuchi, T.; Shimada, K.; Maruyama, N.; Hirobe, M. J. Am. Chem.
Soc. 1993, 115, 7551.
(6) (a) White, R. E.; Sligar, S. G.; Coon, H. J. J. Biol. Chem. 1980, 255,
11108. (b) Traylor, T. G.; Lee, W. A.; Stynes, D. V. J. Am. Chem. Soc.
1984, 106, 755. (c) Traylor, T. G.; Tsuchiya, S.; Byun, Y.-S.; Kim, C. J.
Am. Chem. Soc. 1993, 115, 2775 and references cited therein.
(7) (a) Battersby, A. R.; Howson, W.; Hamilton, A. D. J. Chem. Soc.,
Chem. Commun. 1982, 1266. (b) Collman, J. P.; Groh, E. J. Am. Chem.
Soc. 1982, 104, 1391. (c) Sta¨ubli, B.; Fretz, H.; Piantini, U.; Woggon, W.
D. HelV. Chim. Acta 1987, 70, 1173.
(8) The following papers strongly support the formation of the two-
electron oxidizing active intermediate in dichloromethane. (a) Groves, J.
T.; Watanabe, Y. J. Am. Chem. Soc. 1988, 110, 8443. (b) Watanabe, Y.;
Yamaguchi, K.; Morishima, I.; Takehira, K.; Shimizu, M.; Hayakawa, T.;
Orita, H. Inorg. Chem. 1991, 30, 2582.
(9) Abbreviations used: TPP, meso-tetraphenylporphyrin; TMP, meso-
tetramesitylporphyrin; TPFPP, meso-tetrakis(pentafluorophenyl)porphyrin;
1-MeIm, 1-Methylimidazole.
(10) Since the amount of peroxy acid used in the reactions with
porphyrins was kept at 1 mol equiv to the iron porphyrins, the yields of
p-methoxyphenol were relatively low (ranging from 18 to 80 µM). The
turnover value in the reaction with rat liver microsomes was 16. A small
amount of 2,5-dimethoxyphenol, the aromatic hydroxylation product, was
also detected in every system.
(1) (a) Cytochrome P-450; Ortiz de Montellano, P. R., Ed.; Plenum
Press: New York, 1995. (b) Dawson, J. H. Science 1988, 240, 433. (c)
Woggon, W. D. Top. Curr. Chem. 1997, 184, 29.
(2) Gross, Z.; Nimri, S. Inorg. Chem. 1994, 33, 1731.
(3) (a) Miwa, G. T.; Walsh, J. S.; Lu, A. Y. H. J. Biol. Chem. 1984,
259, 3000. (b) Harada, N.; Miwa, G. T.; Walsh, J. S.; Lu, A. Y. H. J. Biol.
Chem. 1984, 259, 3005. (c) Lindsay Smith, J. R.; Sleath, P. R. J. Chem.
Soc., Perkin Trans. 2 1983, 621.
(4) Urano, Y.; Higuchi, T.; Hirobe, M. J. Chem. Soc., Perkin Trans. 2
1996, 1169.
S0002-7863(97)02892-8 CCC: $14.00 © 1997 American Chemical Society

Communications to the Editor
J. Am. Chem. Soc., Vol. 119, No. 49, 1997 12009
Table 1. Kinetic Isotope Effects on the O-Demethylation of
p-Dimethoxybenzene by Various Iron Porphyrin-Oxidant Systems,
As Well As Rat Liver Microsomes-NADPH/O2 and Human
CYP1A2-NADPH/O2 Systems
Scheme 2. Equilibrium between Two Isoelectronic
Iron-Oxo Structures Involving Thiolate Ligated Heme
iron porphyrin
axial ligand oxidant solvent kH/kD
S.R.
thiolate
Cl-
PPAA C6H6
PPAA CH2Cl2
PPAA C6H6
PPAA CH2Cl2
PPAA C6H6
PPAA CH2Cl2
PPAA CH2Cl2
PPAA C6H6
PPAA CH2Cl2
11.7
5.5
1.0
1.0
1.2
1.0
1.2
1.1
1.0
1.9
proceeded by the H atom abstraction mechanism. In other iron
porphyrin-oxidant systems with a chloride anion or an imid-
azole ligand, low KIE values and high 18O incorporations were
observed, which showed that the reaction proceeded in the ipso-
substitution manner. The thiolate ligand has been believed to
affect the O-O bond cleavage step in the catalytic cycle of
P450. However, considering that in every porphyrin system
PAA was the major product from PPAA, our data in this paper
provide evidence that the two-electron oxidizing iron-oxo
porphyrin intermediate of thiolate-ligated porphyrin has different
reactivity to that of chloride anion- or imidazole-ligated por-
phyrin. Champion11 has suggested on the basis of the resonance
Raman spectroscopy that the iron-oxo π cation complex is not
preferred, but rather the oxygen radical configuration is favored
as an oxidative active intermediate of P450s (Scheme 2), because
of the strong π-electron-donating effects of thiolate ligand. On
the other hand, in the histidine-ligated heme proteins, the iron-
oxo π cation radical intermediate is favored. Champion
speculated that the difference of reactivity between P450 and
peroxidases might arise from the isoelectronic states of the
oxidative intermediates. The results presented in this paper are
the first experimental evidence to support this speculation.
Furthermore, it is suggested that the oxygen radical type
intermediate can abstract the hydrogen atom from the substrate
more easily than the iron-oxo π cation radical intermediate.
In conclusion, we have established that the thiolate ligand
has a marked influence on the reactivity of the high-valent iron-
oxo porphyrin intermediate. The high-valent iron-oxo inter-
mediate of thiolate-ligated porphyrin, such as P450s and S.R.,
has a stronger hydrogen-atom-abstracting ability than that of
chloride anion- or imidazole-ligated porphyrins. These findings
imply that the preferred structure of the active oxidizing
intermediate of thiolate-ligated porphyrins is of the oxygen
radical type. Thiolate ligation plays an important role in the
characteristic oxidizing ability of P450s, and thus, our findings
are of great significance for both chemical and biological studies
of P450s.
Fe(TPP)Cl
Fe(TMP)Cl
Cl-
Fe(TMP)(1-MeIm)2ClO4
Fe(TPFPP)Cl
imidazole
Cl-
Fe(TPP)Cl
Cl-
PhIO
CH2Cl2
Rat liver microsomes
Human CYP1A2
thiolate
thiolate
O2
O2
H2O
H2O
11.9
11.6
a The reactions with porphyrins were carried out at room temperature
under argon for 10 min. [Fe(Por)] ) 1.0 mM; [PPAA] ) 1.0 mM;
[substrate] ) 0.10 M. The reaction with rat liver microsomes or human
CYP1A2 was carried out at 37 °C under air for 10 min. Rat liver
microsomes were prepared according to ref 4. Human CYP1A2 was
purchased from GENTEST Corp. Kinetic isotope effects were deter-
mined by GC/SIM from the M+ peak area ratio of the products.
Table 2. 18O Incorporations from 18O-Enriched Oxidants in the
O-Demethylation of p-Dimethoxybenzene by Various Iron
Porphyrin-Oxidant Systems and the Rat Liver
Microsomes-NADPH/O2 System
iron porphyrin
S.R.
axial ligand oxidant solvent 18O incorp (%)
thiolate
PPAA
mCPBA C6H6
C6H6
<1%
1
Fe(TPP)Cl
Fe(TMP)Cl
Fe(TPFPP)Cl
Rat liver microsomes
Cl-
PPAA
PPAA
PPAA
O2
C6H6
C6H6
C6H6
H2O
90
51
89
1
Cl-
Cl-
thiolate
a The reaction conditions were identical to those described in Table
1, except that 18O-enriched oxidants were used. 18O incorporations were
determined by GC/SIM from the M+ peak area ratio.
O-demethylated with low 18O incorporation. Among the
porphyrin-oxidant systems, such low 18O incorporations were
observed only in the S.R.-PPAA and S.R.-m-chloroperoxy-
benzoic acid (mCPBA) systems. In other iron porphyrin-
oxidant systems, high 18O incorporation values were obtained.
From the results above, it is clear that in the thiolate-ligated
heme-containing systems, i.e., S.R. and P450 systems, p-
dimethoxybenzene was O-demethylated with high KIE values
and with low 18O incorporation, which showed that the reaction
Acknowledgment. This study was supported by JSPS Research
Fellowships for Young Scientists and also partly by a research grant
from the Ministry of Education, Science, Sports and Culture of Japan.
JA972892H
(11) Champion, P. M. J. Am. Chem. Soc. 1989, 111, 3433.