Oxidation of 1,2,4,5-tetramethoxybenzene to a cation radical by cytochrome P450 [15]

Full text of DOI:10.1021/ja000838m

J. Am. Chem. Soc. 2000, 122, 8099-8100
8099
Oxidation of 1,2,4,5-Tetramethoxybenzene to a
Cation Radical by Cytochrome P450
Hideaki Sato and F. Peter Guengerich*
Department of Biochemistry and
Center in Molecular Toxicology
Vanderbilt UniVersity, NashVille, Tennessee 37232-0146
ReceiVed March 8, 2000
Figure 1. Optical absorption spectra of 1•+ formed during oxidation of
1 by rabbit P450 1A2. The incubation contained 2 µM P450 1A2, 4 µM
rat NADPH-P450 reductase, 50 µM L-R-dilauroyl-sn-glycero-3-phos-
phocholine, 0.10 M potassium phosphate (pH 7.4), and an NADPH-
generating system containing 0.05 mM NADP+, 10 mM glucose
6-phosphate, and 2.6 U glucose 6-phosphate dehydrogenase mL-1 in the
presence (A) or absence (B) of 5 mM 1. Spectra were recorded every 40
s at room temperature. Difference spectra for the HRP (C) and P450
1A2 (D) reactions are derived from (A). The concentration was estimated
to be 3.5 µM (ꢀ450 ) 9800 M-1 cm-1). The inset in A (labeled “HRP”)
shows spectra recorded with a mixture of 0.25 µM HRP, 0.44 mM H2O2,
and 0.1 mM 1 in 100 mM potassium acetate buffer (pH 4.0), with spectra
recorded every 1 min at room temperature (full scale absorbance 0.8).
Cytochrome P450 (P450) enzymes are found throughout nature
and are of interest because of their ability to catalyze various
oxidations, including those at chemically inert sites such as
unactivated methyl groups.1 A number of mechanistic possibilities
have been presented, including mobile oxygen species and several
high-valent Fe complexes, but the most generally accepted view
is that an FeIV ) O porphyrin radical is usually the oxidant.1a,d,2
This entity is the same as that generally accepted for peroxidases,
for example, horseradish peroxidase (HRP).3 Peroxidases have
generally been considered to be inefficient at hydrogen atom
abstraction, a process ascribed to P450s, but part of the reason
may be the spatial inaccessibility of substrates to the FeO entity
of peroxidases such as HRP and the tendency to use electron
transfer via the porphyrin edge.4 The view has been expressed
that P450s are capable of hydrogen atom abstraction but not the
1e- oxidation of low E1/2 substrates, for example amines.5
Several lines of evidence indicate that P450s can catalyze 1e-
oxidations under some conditions. The evidence includes observed
rearrangements,6 radicals trapped from 4-alkyl-1,4-dihydro-
pyridines,7 linear free energy relationships,8 several similarities
with the electrochemical oxidation of amines (e.g., product
distribution),8a,9 and low intrinsic kinetic hydrogen isotope
effects,4b,10 However, all of this evidence is indirect. The
production of stable cation radicals has been observed in P450
reactions supported by “oxygen surrogates” such as cumene
hydroperoxide11 and iodosylbenzene.8c However, the relevance
to the normal reaction cycle, supported by NADPH and NADPH-
P450 reductase, can be questioned.
Some polymethoxybenzenes have low E1/2 values and yield
stable cation radicals (at low pH) when oxidized by peroxidases
such as HRP and lignin peroxidase.12 When 1,2,4,5-tetramethoxy-
benzene (1)13 (Scheme 1) was added to an aerobic system
containing rabbit P450 1A2,14 NADPH-P450 reductase, and
NADPH, the formation of a stable spectral intermediate at 450
nm was observed, characteristic of the cation radical and similar
to that seen in the reaction with HRP and H2O2 at pH 4 (Figure
1).15 No band was seen when either P450, the reductase, or 1
was omitted.16,20
The characteristic ESR spectrum of 1•+ was observed in the
HRP reaction,12b which is much faster that that of P450 1A2
(Figure 1). The level of the radical was too low to clearly observe
the ESR spectrum in the P450 1A2 reaction, and a spin trap was
used to accumulate radicals. 5,5-Me2-1-pyrroline N-oxide (DMPO)
* Author for correspondence. Telephone: (615) 322-2261. Fax: (615) 322-
3141, E-mail: guengerich@toxicology.mc.vanderbilt.edu.
(1) (a) Ortiz de Montellano, P. R. In Cytochrome P-450; Ortiz de
Montellano, P. R.; Ed.; Plenum Press: New York 1986; pp 217-271. (b)
Guengerich, F. P. Crit. ReV. Biochem. Mol. Biol. 1990, 25, 97-153. (c)
Guengerich, F. P. J. Biol. Chem. 1991, 266, 10019-10022. (d) Ortiz de
Montellano, P. R. In Cytochrome P450: Structure, Mechanism, and Bio-
chemistry; Ortiz de Montellano, P. R., Ed.; Plenum Press: New York 1995;
pp 245-303.
(12) (a) Kersten, P. J.; Tien, M.; Kalyanaraman, B.; Kirk, T. K. J. Biol.
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K. E.; Reinhammar, B.; Kirk, T. K. Biochem. J. 1990, 268, 475-480. (c)
Koduri, R. S.; Whitwam, R. E.; Barr, D.; Aust, S. D.; Tien, M. Arch. Biochem.
Biophys. 1996, 326, 261-265. (d) Teunissen, P. J. M.; Sheng, D.; Reddy, G.
V. B.; Moe¨nne-Loccoz, P.; Field, J. A.; Gold, M. H. Arch. Biochem. Biophys.
1998, 360, 233-238.
(13) (a) Benington, F.; Morin, R. D.; Clark, L. C., Jr. J. Org. Chem. 1955,
20, 102-108. (b) Zweig, A. J. Phys. Chem. 1963, 67, 506-508. (c) See
Supplemental Information for synthetic details and analytical data.
(14) Titration of P450 1A2 with 1 yielded a shift of the Soret band from
390 to 419 nm (“Reverse Type I” shift, transition from high- to low-spin
iron), with a spectrally estimated dissociation constant (Ks) of 1.4 mM.
(15) No apparent cation radical was detected when tert-butyl hydroperoxide
(5 or 15 mM) or H2O2 (0.44 mM) was used in place of NADPH. We also
considered the use of iodosylbenzene as an oxygen surrogate, but it directly
reacted with 1 to form 1•+ at a concentration of 0.2 mM.
(16) The development of a 450 nm spectral band in P450 reactions has
been related to CO produced from trichlorethylene17 or lipid peroxidation.18
However, no unsaturated lipid was present here. Some destruction of the P450
heme occurred during the reaction, as described earlier.19 The loss of the heme
(which obscured observation of the cation radical at <430 nm) could be
attenuated by the addition of catalase and superoxide dismutase; the changes
in the spectra (Figure 1A,D) are not isosbestic.
(2) Groves, J. T.; Nemo, T. E.; Myers, R. S. J. Am. Chem. Soc. 1979, 101,
1032-1033.
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F. P. J. Am. Chem. Soc. 1982, 104, 2050-2052. (b) Guengerich, F. P.; Willard,
R. J.; Shea, J. P.; Richards, L. E.; Macdonald, T. L. J. Am. Chem. Soc. 1984,
106, 6446-6447. (c) Stearns, R. A.; Ortiz de Montellano, P. R. J. Am. Chem.
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(8) (a) Burka, L. T.; Guengerich, F. P.; Willard, R. J.; Macdonald, T. L. J.
Am. Chem. Soc. 1985, 107, 2549-2551. (b) Macdonald, T. L.; Gutheim, W.
G.; Martin, R. B.; Guengerich, F. P. Biochemistry 1989, 28, 2071-2077. (c)
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(9) (a) Shono, T.; Toda, T.; Oshino, N. J. Am. Chem. Soc. 1982, 104, 2639-
2641. (b) Seto, Y.; Guengerich, F. P. J. Biol. Chem. 1993, 268, 9986-9997.
(10) (a) Miwa, G. T.; Walsh, J. S.; Kedderis, G. L.; Hollenberg, P. F. J.
Biol. Chem. 1983, 258, 14445-14449. (b) Hall, L. R.; Hanzlik, R. P. J. Biol.
Chem. 1990, 265, 12349-12355.
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Biochem. Biophys. 1980, 205, 543-553.
(17) (a) Uehleke, H.; Tabarelli-Poplawski, S.; Bonse, G.; Henschler, D.
Arch. Toxicol. 1977, 37, 95-105. (b) Miller, R. E.; Guengerich, F. P.
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(18) Levin, W.; Lu, A. Y. H.; Jacobson, M.; Kuntzman, R. Arch. Biochem.
Biophys. 1973, 158, 842-852.
(19) Guengerich, F. P. Biochemistry 1978, 17, 3633-3639.
(20) No radicals (450 nm) were observed when 1,4-dimethoxybenzene
(E1/2 1.34 V vs SCE) was used instead of 1 (E1/2 0.81 V vs SCE).12b
10.1021/ja000838m CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/08/2000

8100 J. Am. Chem. Soc., Vol. 122, No. 33, 2000
Communications to the Editor
Scheme 1
did not produce a heme iron spin shift suggestive of binding in
the substrate site of P450 1A2 when added at a concentration of
5 mM. This concentration of DMPO inhibited the production of
1
•+ (450 nm band) by 30%. The values of the hyperfine coupling
constants deduced from the six-line ESR spectrum observed under
these conditions for 1•+ are typical for trapping of a carbon-
centered radical21 (Figure 2) and could arise from the adduction
of 1•+ or a derived radical.
Some other P450s also formed 1•+, as judged by the extent of
the change at 450 nm, but concentrations of 1•+ were less than
with rabbit P450 1A2 (see Supporting Information).
Incubation of 1 with P450 1A2 also yielded 2,5-dimethoxy-
1,4-benzoquinone (V ) 1.3 min-1) and 4,5-dimethoxy-1,2-
benzoquinone (V ) 0.25 min-1) (expected for hydrolysis of 1•+ 12
and identified by HPLC retention, UV spectra, and mass spec-
trometry; see Supporting Information) and O-demethylation, as
measured by the formation of formaldehyde23 (V ) 2.8 min-1
)
(at a substrate concentration of 5 mM). Substitution of (d12-O-
methyl) 1 yielded a large kinetic isotope effect (V ≈ 0.1 min-1
)
for the O-demethylation reaction but only slightly decreased (<2-
fold) the magnitude of the 450 nm band ascribed to 1•+ or quinone
formation.24,25
Figure 2. ESR spectra of DMPO-radical adducts produced by the reaction
of P450 1A2 and 1. A reaction (100 µL) similar to that described in
Figure 1 was done in an ESR capillary (except that 30 mM DMPO was
included). The ESR spectra shown here were all recorded 3.0 min after
the reaction started. ESR analyses were carried out on aliquots of the
mixture at room temperature and at X-band (9.81 GHz) microwave
frequency with a Bruker EMX spectrometer operating with 100 kHz
magnetic field modulation. Spectrometer conditions were: modulation
amplitude, 1 G; microwave power, 10 mW; time constant, 0.041 s; scan
time, 42 s; scan range, 100 G; receiver gain, 5 × 105. (A) Complete
reaction system. Control experiments did not include either 1 (B), P450
1A2 (C), NADPH-P450 reductase (D), or the NADPH-generating system
(E) Lower concentrations of what appear to be O2•-, known to be
produced by the reductase under these conditions,22 and carbon-centered
radicals were detected in the absence of 1 (B) or P450 (C). The latter
radicals are attributed to flavin semiquinone radicals in NADPH-P450
reductase.
We propose that the present work with 1•+ is the first report
of direct observation of formation of a stable radical by a P450
in the normal catalytic system. These results have several
mechanistic implications. P450s are definitely capable of 1e-
transfer whenever the steric factors and the E1/2 (actually a partial
function of steric factors)8b,28 are appropriate. This conclusion
should apply not only to aromatic systems but also amines,4b,6a,b,d,8a,c
strained cycloalkanes,6c and other low E1/2 substrates, including
several major polycyclic aromatic hydrocarbons (PAHs).29 The
detection of purine N7- and C8-PAH adducts in DNA has been
used as evidence for roles of P450s in PAH 1e- oxidation because
model 1e- oxidation systems generate the same adducts (other
mechanisms are also plausible).30
(21) (a) Janzen, E. G.; Liu, J. I.-P. J. Magnet. Res. 1973, 9, 510-512. (b)
Rota, C.; Barr, D. P.; Martin, M. V.; Guengerich, F. P.; Tomasi, A.; Mason,
R. P. Biochem. J. 1997, 328, 565-571. (c) Barr, D. P.; Martin, M. V.;
Guengerich, F. P.; Mason, R. P. Chem. Res. Toxicol. 1996, 9, 318-325.
(22) Aust, S. D.; Roerig, D. C.; Pederson, T. C. Biochem. Biophys. Res.
Commun. 1972, 47, 1133-1137.
The significance of 1e- oxidation and rate-limiting events in
P450 catalysis are under further investigation.
(23) Nash, T. Biochem. J. 1953, 55, 416-421.
Acknowledgment. This work was supported in part by U. S. Public
Health Service Grants R35 CA44353 and P30 ES00267. We thank Drs.
A. Beth and E. Hastedt for the use of the ESR spectrometer.
(24) When 1 was added to a reconstituted P450 1A2 system, the rate of
NADPH oxidation increased from 26 min-1 to 57 min-1 (61 min-1 when d12-1
was used). The electron uptake is presumed to be involved in the cyclic
formation of 1•+ and its subsequent reduction.
(25) The rate of 1e- oxidation (k1) at pH 7.4 (k2, a zero order constant)
may be estimated from the first-order decomposition constant26 and the
Supporting Information Available: Analytical data for methoxy-
benzenes synthesized for use in this work, table of steady-state concentra-
tions of 1•+ formed by various P450 enzymes, and HPLC/UV identifi-
cation of quinone products (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
concentrations of P450 1A2 and 1•+ 17b,27
:
[1•+] ) k1/k2 [P450](1-e-k t). Using
2
data from Figure 1 at t ) 2 min, k1 ≈ 19 min-1
.
(26) The 1 cation radical is relatively stable at pH 3.12b 1•+ was generated
in the HRP/H2O2 reaction, and the kinetics of the stability of 1•+ were examined
upon pH changes. The estimated first-order rate constants of its decay were
0.008 min-1 (pH 0.3), 0.57 min-1 (pH 4.0), 0.83 min-1 (pH 5.0), 4.3 min-1
(pH 6.4), and 14 min-1 (pH 7.4). 1•+ (generated with HRP/H2O2) was also
found to be less stable in the presence of NADPH.
JA000838M
(27) Hess, B.; Wurster, B. FEBS Lett. 1970, 9, 73-77.
(30) In previous work we had attempted to use H218O trapping as a means
of distinguishing between P450 hydrogen atom transfer/oxygen rebound and
1e- transfer pathways in the oxidation of 9-methylanthracene but were unable
to clearly discriminate between the mechanisms.31
(31) Anzenbacher, P.; Niwa, T.; Tolbert, L. M.; Sirimanne, S. S.;
Guengerich, F. P. Biochemistry 1996, 35, 2512-2520.
(28) Eberson, L. AdV. Free Radical Biol. Med. 1985, 1, 19-90.
(29) (a) Cavalieri, E. L.; Rogan, E. G.; Devanesan, P. D.; Cremonesi, P.;
Cerny, R. L.; Gross, M. L.; Bodell, W. J. Biochemistry 1990, 29, 4820-
4827. (b) Cremonesi, P.; Stack, D. E.; Rogan, E. G.; Cavalieri, E. L. J. Org.
Chem. 1994, 59, 7683-7687.