A highly reactive P450 model compound I

Article abstract of DOI:10.1021/ja903394s

(Graph Presented) The detection and kinetic characterization of a cytochrome P450 model compound I, [OFe^IV-4-TMPyP]⁺ (1), in aqueous solution shows extraordinary reaction rates for C-H hydroxylations. Stopped-flow spectrophotometric monitoring of the oxidation of Fe ^III-4-TMPyP with mCPBA revealed the intermediate 1, which displays a weak, blue-shifted Soret band at 402 nm and an absorbance at 673 nm, typical of a porphyrin π-radical cation. This intermediate was subsequently transformed into the well-characterized OFe^IV-4-TMPyP. Global analysis afforded a second-order rate constant k₁ = (1.59 ± 0.06) × 10 ⁷ M^-1 s^-1 for the formation of 1 followed by a first-order decay with k₂ = 8.8 ± 0.1 s^-1. ¹H and ¹³C NMR determined 9-xanthydrol to be the major product (～90% yield) of xanthene oxidation by 1. Electrospray ionization mass spectrometry carried out in 47.5% ¹⁸OH₂ indicated 21% ¹⁸O incorporation, consistent with an oxygen-rebound reaction scenario. Xanthene/xanthene-d₂ revealed a modest kinetic isotope effect, k_H/k_D = 2.1. Xanthene hydroxylation by 1 occurred with a very large second-order rate constant k₃ = (3.6 ± 0.3) × 10⁶ M^-1 s^-1. Similar reactions of fluorene-4-carboxylic acid and 4-isopropyl- and 4-ethylbenzoic acid also gave high rates for C-H hydroxylation that correlated well with the scissile C-H bond energy, indicating a homolytic hydrogen abstraction transition state. Mapping the observed rate constants for C-H bond cleavage onto the Bronsted- Evans-Polanyi relationship for similar substrates determined the H-OFe ^IV-4-TMPyP bond dissociation energy to be ～100 kcal/mol. The high kinetic reactivity observed for 1 is suggested to result from a high porphyrin redox potential and spin-state-crossing phenomena. More generally, subtle charge modulation at the active site may result in high reactivity of a cytochrome P450 compound I.

Full text of DOI:10.1021/ja903394s

Published on Web 06/24/2009
A Highly Reactive P450 Model Compound I
Seth R. Bell and John T. Groves*
Department of Chemistry, Princeton UniVersity, Princeton, New Jersey 08544
Received April 27, 2009; E-mail: jtgroves@princeton.edu
The substrate oxygenation reactions mediated by cytochrome
P450 proteins comprise many key transformations in steroid
biosynthesis and drug metabolism. Significant advances in heme-
1
protein enzymology, the characterization of synthetic model
2
3
compounds, and computational approaches have pointed to a
reactive oxoiron(IV)-porphyrin π-radical cation in the consensus
mechanism. Compound I model compounds, such as the thoroughly
IV
+
4,5
characterized [OFe -TMP] , have exhibited a full range of
6
oxygen transfer reactions. However, the low kinetic reactivity of
OFe -TMP] has left unanswered just how the protein manages
IV
+
[
to generate a sufficiently reactive intermediate and what that reactive
7
-10
species is.
We have shown that cationic oxomanganese(V)-por-
V
phyrin complexes such as OMn -4-TMPyP display very high rates
for oxygen transfer reactions in aqueous solution.
report the detection and kinetic characterization of [OFe -4-
TMPyP] , which shows extraordinary rates for C-H hydroxylations.
The reaction of Fe -4-TMPyP with mCPBA was analyzed by
stopped-flow spectrophotometry, as we have previously described.
1
1,12
Here we
IV
+
III
11,13
III
Figure 1. (A) UV-vis transients observed upon mixing of 10 µM Fe -4-
TMPyP with 3 equiv of mCPBA at pH 4.7, 10.0 °C. (B) Data obtained at
As can be seen in the UV-vis spectral time course (Figure 1), the
starting iron(III)-porphyrin spectrum (λmax 421 nm) was completely
transformed within 20 ms into that of a new intermediate, 1,
displaying a weak, blue-shifted Soret band at 402 nm and an
(
a) 402 and (b) 427 nm and the superimposed global fits.
IV
+
Scheme 1. Kinetic Partitioning of [OFe -4-TMPyP] (1) between
IV
Spontaneous Decay to OFe -4-TMPyP (k ) and Substrate
2
III
4
,14
Hydroxylation Resulting in the Formation of Fe -4-TMPyP (k )
3
absorbance at 673 nm, typical of a porphyrin π-radical cation.
IV
+
Accordingly, we assigned the [OFe -4-TMPyP] formulation to
1
. Global analysis afforded a second-order rate constant k
1
) (1.59
7
-1 -1
(
0.06) × 10 M
s
for the appearance of 1. In a slower,
subsequent phase of the reaction (Scheme 1), 1 was transformed
IV
into the well-characterized OFe -4-TMPyP (λmax 427 nm; k )
2
-1
15-17
8
.8 ( 0.1 s ).
Isosbestic points for the conversion of 1 to
IV
OFe -4-TMPyP were apparent at 413 and 570 nm. This trans-
formation was more rapid at higher pH, in contrast to the behavior
of the corresponding OMn -4-TMPyP complex.
obtained by monitoring at 673 nm (Figure 2A). Control experiments
showed that OFe -4-TMPyP had negligible reactivity toward
xanthene over this time course.
Reactions of fluorene-4-carboxylic acid and 4-isopropyl- and
4-ethylbenzoic acid with 1 also gave very high rates for C-H
hydroxylation, with second-order rate constants of (1.80 ( 0.06)
V
11,18
IV
Intermediate 1 was found to be very highly reactive toward C-H
bond hydroxylations. Slow addition of mCPBA to a solution of 5
III
1
mM xanthene and 50 µM Fe -4-TMPyP was monitored by H
1
3
and C NMR spectroscopy. The major product, 9-xanthydrol (H9,
5
3
3
-1 -1
δ 5.75; C9, δ 62), was formed in 90% yield based on added oxidant
× 10 , (9.0 ( 0.2) × 10 , and (7.5 ( 0.1) × 10
M
s ,
(
Figure S1 in the Supporting Information). A small amount of
respectively. The corresponding alcohol products were formed in
each case. These reactions are remarkably fast, and as can be seen
in Figure 2B, the rates correlated well with the scissile C-H bond
dissociation energy (BDE), indicating a homolytic hydrogen
abstraction transition state. Reaction of 1 with bromide ion
afforded hypobromite with k
These results show that [OFe -4-TMPyP] (1), which is
accessed chemically from a peroxide precursor, is orders of
magnitude more reactive than the well-studied tetramesityl
III
xanthone was also formed. The Fe catalyst was recovered with
negligible decomposition. Significantly, when the oxidation was
carried out in 47.5% 18OH
, electrospray ionization mass spec-
2
18
21
trometry of the 9-xanthydrol product indicated 21% O incorpora-
tion (m/z 199/197 ) 0.27). This partial exchange is consistent with
an oxygen-rebound reaction scenario with ∼50% dilution of the
5
-1 -1 13
3
) (3.14 ( 0.04) × 10 M s .
IV +
1
6
18
O-peroxyacid-derived oxygen with O water occupying the
19,20
second axial ligation site.
Xanthene/xanthene-d
2
(1:1) revealed
4,6,7,9
a modest kinetic isotope effect, k
H
/k
D
) 2.1 (m/z 197/198).
analogues.
Mapping the rate constants observed here for C-H
III
Since the oxidation of Fe -4-TMPyP by mCPBA was so fast,
we could conveniently monitor the kinetics of the reaction of 1
with xanthene using a single-mixing stopped-flow experiment
bond cleavage by 1 onto the Brønsted-Evans-Polanyi relation-
2
1,22
ship
for similar substrates (as shown in the Table of Contents
graphic) yielded an estimate of ∼100 kcal/mol for the BDE of
IV
(
Figure 2A). Global analysis of the spectral data produced a rate
H-OFe -4-TMPyP; this is much larger than the values estimated
6
-1 -1
IV
7
III
constant k
3
) (3.6 ( 0.3) × 10 M s . A similar value was
for H-OFe -TMP (92 kcal/mol) and H-OFe -TPFPP (∼86
9
640 9 J. AM. CHEM. SOC. 2009, 131, 9640–9641
10.1021/ja903394s CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
suggest that even subtle charge modulation at the heme active site
of P450 to accomplish a similar tuning of state-crossing energies
or lower stability of the porphyrin π-radical cation could result in
high reactivity of a cytochrome P450 compound I.
Acknowledgment. Support of this research by the National
Institutes of Health (R37-GM36298) and the National Science
Foundation (CHE 0316301) is gratefully acknowledged.
Supporting Information Available: Experimental details and NMR
data (Figure S1). This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(
1) Ortiz de Montellano, P. R.; De Voss, J. J. In Cytochrome P450 Structure,
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3
rd ed.; Ortiz de Montellano, P. R., Ed.; Kluwer Academic/Plenum: New
Figure 2. (A) Observed rate constant kobs at 673 nm vs [xanthene] for its
York, 2005; pp 1-44.
6
-1
oxidation by 1 at pH 4.7, 14.5 °C, yielding k
3
) (2.8 ( 0.3) × 10 M
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s
3
. (B) Correlation of k with C-H bond dissociation energy.
2
3
(5) Abbreviations: TMP, 5,10,15,20-tetramesitylporphyrin; 4-TMPyP, 5,10,15,20-
tetrakis(N-methyl-4-pyridinium)porphyrin; mCPBA, m-chloroperoxybenzoic
acid; TMPS, 5,10,15,20-tetramesityloctasulfonate.
kcal/mol). Indeed, 1 showed a rate constant for ethylbenzoic acid
hydroxylation similar to that reported recently by Newcomb and
co-workers for ethylbenzene with CYP119 and chloroperoxidase
compound I.
The interesting question is why synthetic OFe -porphyrin
π-radical cations should have such a great variability in their
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meso substituents dramatically decrease the reactivity of oxoMn -
2
4
(
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1
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manifolds, stabilization of the porphyrin HOMO, and the unique
2
5
centrosymmetric trans-dioxo ligand arrangement. Clearly, the
situation for iron described here is markedly different. A relatively
high redox potential for porphyrin ring oxidation is expected for
Fe -4-TMPyP. Further, efficient transmission of the electron deficit
to the metal core is assured by the large orbital coefficients on the
meso carbon and the pyrrole nitrogens in the a2u porphyrin HOMO.
Indeed, the pK
.5, while the corresponding acidity of diaquaFe -TMPS is 7.5.
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(
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(
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2
(
III
a
of the axial water in diaquaFe -4-TMPyP is only
1
III
26
5
1
(
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(
(
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(
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2,28
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x2-y2 orbital singly occupied, has been calculated to be only 14
1
3310–13320.
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2
9
state structures. We suggest that the high kinetic reactivity
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(
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JA903394S
J. AM. CHEM. SOC. 9 VOL. 131, NO. 28, 2009 9641