Spectra and kinetic studies of the Compound I derivative of cytochrome P450 119

Article abstract of DOI:10.1021/ja802652b

The Compound I derivative of cytochrome P450 119 (CYP119) was produced by laser flash photolysis of the corresponding Compound II derivative, which was first prepared by reaction of the resting enzyme with peroxynitrite. The UV-vis spectrum of the Compoun

Full text of DOI:10.1021/ja802652b

Published on Web 09/13/2008
Spectra and Kinetic Studies of the Compound I Derivative of
Cytochrome P450 119
Xin Sheng, John H. Horner, and Martin Newcomb*
Department of Chemistry, UniVersity of Illinois at Chicago, 845 West Taylor Street,
Chicago, Illinois 60607
Received April 10, 2008; E-mail: men@uic.edu
Abstract: The Compound I derivative of cytochrome P450 119 (CYP119) was produced by laser flash
photolysis of the corresponding Compound II derivative, which was first prepared by reaction of the resting
enzyme with peroxynitrite. The UV-vis spectrum of the Compound I species contained an asymmetric
Soret band that could be resolved into overlapping transitions centered at ∼367 and ∼416 nm and a Q
band with λmax ≈ 650 nm. Reactions of the Compound I derivative with organic substrates gave epoxidized
(alkene oxidation) and hydroxylated (C-H oxidation) products, as demonstrated by product studies and
oxygen-18 labeling studies. The kinetics of oxidations by CYP119 Compound I were measured directly;
the reactions included hydroxylations of benzyl alcohol, ethylbenzene, Tris buffer, lauric acid, and methyl
laurate and epoxidations of styrene and 10-undecenoic acid. Apparent second-order rate constants, equal
to the product of the equilibrium binding constant (Kbind) and the first-order oxidation rate constant (kox),
were obtained for all of the substrates. The oxidations of lauric acid and methyl laurate displayed saturation
kinetic behavior, which permitted the determination of both Kbind and kox for these substrates. The unactivated
C-H positions of lauric acid reacted with a rate constant of kox ) 0.8 s-1 at room temperature. The CYP119
Compound I derivative is more reactive than model Compound I species [iron(IV)-oxo porphyrin radical
cations] and similar in reactivity to the Compound I derivative of the heme-thiolate enzyme chloroperoxidase.
Kinetic isotope effects (kH/kD) for oxidations of benzyl alcohol and ethylbenzene were small, reflecting the
increased reactivity of the Compound I derivative in comparison to models. Nonetheless, CYP119 Compound
I apparently is much less reactive than the oxidizing species formed in the P450cam reaction cycle. Studies
of competition kinetics employing CYP119 activated by hydrogen peroxide indicated that the same oxidizing
transient is formed in the photochemical reaction and in the hydrogen peroxide shunt reaction.
The ubiquitous cytochrome P450 (CYP or P450) enzymes
are heme-containing enzymes with thiolate from protein cysteine
as the fifth ligand to iron.1 P450s serve in several catalytic roles
in nature, but their major function is to catalyze oxidation
reactions, typically via two-electron oxo-transfer processes. In
humans, the P450s perform both highly specific reactions, such
as oxidation of androgens to estrogens, and broad-spectrum
oxidations of drugs, pro-drugs, and xenobiotics in the liver.2
Much of the interest in P450s derives from the pharmaceutical
impacts of these enzymes and their relationships to disease
states, including cancers and liver disease, that result from
overexpression of P450s.3-5
P450s in the 1960s. Other heme-containing enzymes, such as
chloroperoxidase (CPO) and horseradish peroxidase, react with
hydrogen peroxide to convert the ferric forms of the enzymes
to iron(IV)-oxo porphyrin radical cations that are termed
“Compound I” derivatives,6,7 and it has long been assumed that
the activated transient in a P450 enzyme that effects oxidation
reactions is a Compound I species.1
The oxygen-containing transients in P450s typically are
formed by a reaction sequence unlike that in the peroxidases
(Figure 1).8-10 Following substrate binding, the ferric enzyme
is reduced, and then molecular oxygen binds reversibly; next,
a second reduction occurs to give the peroxoiron species.
Protonation of this species on the distal oxygen gives the
hydroperoxyiron intermediate, which is the last well-character-
ized transient in the P450 reaction sequence, having been
Exceptionally high reactivity is displayed by some P450s in
their ability to oxidize unactivated C-H bonds in substrates,
and the nature of the P450 transient involved in the oxidation
steps has been a subject of study since the initial discoveries of
(6) Dawson, J. H. Science 1988, 240, 433–439.
(7) Sono, M.; Roach, M. P.; Coulter, E. D.; Dawson, J. H. Chem. ReV.
1996, 96, 2841–2887.
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Ortiz de Montellano, P. R., Ed.; Kluwer: New York, 2005.
(2) Guengerich, F. P. In Cytochrome P450 Structure, Mechanism, and
Biochemistry, 3rd ed.; Ortiz de Montellano, P. R., Ed.; Kluwer: New
York, 2005; pp 377-530.
(8) Groves, J. T. In Cytochrome P450 Structure, Mechanism, and
Biochemistry, 3rd ed.; Ortiz de Montellano, P. R., Ed.; Kluwer: New
York, 2005; pp 1-43.
(9) Makris, T. M.; Denisov, I.; Schlichting, I.; Sligar, S. G. In Cytochrome
P450 Structure, Mechanism, and Biochemistry, 3rd ed.; Ortiz de
Montellano, P. R., Ed.; Kluwer: New York, 2005; pp 149-182.
(10) Ortiz de Montellano, P. R.; De Voss, J. J. In Cytochrome P450
Structure, Mechanism, and Biochemistry, 3rd ed.; Ortiz de Montellano,
P. R., Ed.; Kluwer: New York, 2005; pp 183-245.
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6, 780–789.
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13310 J. AM. CHEM. SOC. 2008, 130, 13310–13320
10.1021/ja802652b CCC: $40.75
2008 American Chemical Society

Kinetic Studies of the Compound I Derivative of CYP119
A R T I C L E S
observed by electron paramagnetic resonance (EPR) and
electron-nuclear double-resonance (ENDOR) spectroscopies
when cryogenic conditions were employed.11,12 A second
protonation on the distal oxygen and loss of water by heterolytic
fragmentation of the O-O bond would give an iron-oxo species
with the iron atom in the formal +5 oxidation state, which is
either a transient perferryloxo species that can relax to a
Compound I derivative or the transition state for the direct
formation of the Compound I derivative. The perferryloxo
species or the Compound I derivative can react in two-electron
oxo-transfer reactions to return the ferric enzyme.
Peroxidase enzymes form Compound I species by reactions
with hydrogen peroxide,7 and P450s also can react with
hydrogen peroxide or other hydroperoxy compounds to give
an active oxidant in what is termed a “shunt” reaction (Figure
1). Fast mixing, stopped-flow mixing, and freeze-quench
mixing experiments with P450s and peroxy species have been
attempted for many years13 with limited success. A short-lived
transient with a calculated spectrum resembling that of CPO
Compound I was detected in reactions of P450cam with m-
chloroperoxybenzoic acid (mCPBA),14,15 but freeze-quench
studies of the same enzyme with either mCPBA or peroxyacetic
acid oxidation employing EPR, ENDOR, and Mo¨ssbauer
spectroscopic analyses indicated that a Compound I derivative
did not accumulate to a detectable level.16-19 Moreover,
production of various iron-oxo transients under “cryoreduction”
conditions suggested that the reaction of the active oxidant in
P450cam with substrate is faster than the rate of formation of
this species, which likely precludes its detection in mixing
studies.11,12 Despite the outcome of attempted P450cam oxida-
tions with mCPBA, reactions of another P450 enzyme, cyto-
chrome P450 119 (CYP119), with mCPBA gave promising
results in that a short-lived transient with a calculated spectrum
similar to that of CPO Compound I was formed;20 these results
are discussed in detail below.
The difficulties in observing a Compound I derivative of a
P450 enzyme in fast-mixing studies prompted us to explore an
alternative entry to these intermediates that has much shorter
temporal resolution than mixing. In principle, photolyses of
iron(IV)-oxo neutral porphyrin complexes, so-called Compound
II derivatives, could give Compound I derivatives by photo-
ejection of an electron from the porphyrin macrocycle, and laser
flash photolysis (LFP) methods with photomultiplier detection
would permit submicrosecond temporal resolution. In practice,
we found that photolyses of Compound II derivatives of a model
Figure 1. Reaction cycles for P450 enzymes. The normal reaction cycle
is shown in black, and the shunt reaction pathway is shown in blue. The
sequence of reactions used to produce Compound I in this work is
highlighted in red. It should be noted that the Compound II derivative is
formulated as a ferrylhydroxy species, on the basis of recent XAS studies
of the CYP119 Compound II species (ref 34).
iron-porphyrin complex, horseradish peroxidase, and myoglo-
bin gave Compound I derivatives.21 Extension of the photo-
oxidation method to the production of a Compound I derivative
of the CYP119 enzyme was subsequently reported in a
communication.22 In the present report, we detail the spectra
and kinetic studies of the CYP119 Compound I derivative.
Results
The enzyme used in this study was CYP119, which was
expressed in Escherichia coli and purified as previously
described.22-24 The CYP119 samples employed were of high
purity, as determined by R/Z values of 1.5 or greater, where
R/Z is the ratio of absorbances at 416 and 280 nm. CYP119
was originally thought to derive from the thermophile Sulfolobus
solfataricus, but a recent study indicated that it came from the
closely related organism Sulfolobus acidocaldarius that con-
taminated an S. solfataricus culture.25
Formation of a Compound II Derivative by Peroxynitrite
Oxidation of CYP119. The photooxidation method for produc-
tion of a Compound I derivative requires that a Compound II
derivative first be produced. The only report of the formation
of a true Compound II derivative of a P450 enzyme prior to
our work was the claim that peroxynitrite (PN) reacted with
cytochrome P450BM3 (CYP102) to give the corresponding
Compound II derivative as a transient and eventually deactivated
the enzyme by nitration of a tyrosine residue.26 Compound II
derivatives of other heme-containing enzymes were known to
be formed by treatment of the resting ferric enzymes with
PN,27-31 and CPO, which is a heme-thiolate enzyme closely
(11) 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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Hoffman, B. M. J. Am. Chem. Soc. 2001, 123, 1403–1415.
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Med. Ger. 1979, 38, 449–458.
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Commun. 1994, 201, 1464–1469.
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Newcomb, M. Org. Lett. 2005, 7, 1193–1195.
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J. Inorg. Biochem. 2002, 91, 586–596.
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Horner, J. H.; Makris, T. M.; Sligar, S. G. J. Am. Chem. Soc. 2006,
128, 4580–4581.
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Interact. 2002, 141, 279–284.
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Biochem. Biophys. Res. Commun. 1998, 252, 166–172.
(24) Maves, S. A.; Sligar, S. G. Protein Sci. 2001, 10, 161–168.
(25) Rabe, K. S.; Kiko, K.; Niemeyer, C. M. ChemBioChem 2008, 9, 420–
425.
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Sligar, S. G.; Trautwein, A. X. J. Biol. Chem. 2004, 279, 10919–
10930.
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J.; Galander, M.; Bottger, L. H.; Richter, M.; Barra, A. L. Biol. Chem.
2005, 386, 1043–1053.
(26) Daiber, A.; Herold, S.; Scho¨neich, C.; Namgaladze, D.; Peterson, J. A.;
Ullrich, V. Eur. J. Biochem. 2000, 267, 6729–6739.
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2002, 277, 9641–9644.
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A R T I C L E S
Sheng et al.
related to the P450s, also gives a transient Compound II
derivative upon reaction with PN.22,26,32
catalyzes the decay of PN, similar to other heme-containing
enzymes.22,28,31,32 Figure S1A in the Supporting Information
shows the spectra of CYP119 resting enzyme and the Compound
II derivative, and Figure S1B in Supporting Information shows
typical results from monitoring the signal at 429 nm (λmax for
the Compound II derivative) as a function of time under two
types of detection conditions. The Compound II derivative
formed in less than 2 s, and PN decayed fully in less than 10 s.
The CYP119 that was present after decay of the Compound
II species was spectroscopically indistinguishable from the
original enzyme, but it was possible that the enzyme was altered
by the PN treatment. For example, PN treatment of CYP102
results in nitration of a tyrosine residue.26 As a control
reaction for our work, we exposed CYP119 to PN at a
concentration typical for the LFP studies, allowed the PN to
decay, and then conducted a shunt reaction (oxidation of
styrene using hydrogen peroxide as a sacrificial oxidant). The
yield of styrene oxide in this reaction was the same as when
untreated CYP119 was used as discussed later, and thus, the
CYP119 behaved the same before and after PN treatment. This
study does not prove that CYP119 was unaltered by PN but
does show that the reactivity of the enzyme was not affected
by the PN treatment at the concentrations used in our studies.
In summary for this section, the reaction of CYP119 with
PN at ambient temperature rapidly formed the Compound II
derivative. This species persisted when excess PN was present
and decayed with a rate constant of ∼0.5 s-1 at ambient
temperature after the PN was depleted. After the PN treatment
and decay of the Compound II species back to ferric enzyme,
the UV-vis spectrum of enzyme was indistinguishable from
that of the original enzyme and the catalytic chemistry of the
CYP119 enzyme when activated in a hydrogen peroxide shunt
reaction was unaffected.
Laser Flash Photolysis Generation of CYP119 Compound
I. The apparatus used for the LFP studies was a commercial
kinetic unit affixed with a stopped-flow mixing unit that
permitted mixing on the millisecond time scale. Samples were
delivered to a sample cell that could be irradiated with laser
light from the bottom and analyzed by an analyzing beam from
one side. The third harmonic of a Nd:YAG laser (355 nm) was
used, with a nominal power of 4 mJ at the reaction cell delivered
in 7 ns. Spectra were acquired with either a diode-array detector
with a minimum integration time of 1.2 ms or photomultiplier
tubes (PMTs) with rise times of ∼2 ns. The analysis beam was
produced by a 150 W xenon lamp at constant power for long
detection times or pulsed to high power for short (<0.1 ms)
detection times.
In a communication, we reported that CYP119 was oxidized
to the corresponding Compound II derivative by PN.22 Subse-
quent to that publication, Green and co-workers33 reported that
CYP102 did not give the Compound II derivative upon reaction
with PN but instead gave a nitrosyl complex. The implications
in the recent paper33 were that the UV-vis spectral changes
were incorrectly interpreted in the original CYP102 study26 and
that PN does not oxidize P450 enzymes to Compound II
derivatives. The dramatically different interpretations26,33 for
the outcome of the PN reaction with CYP102 suggested that
further study of this system was in order, especially since a
short-lived transient (with a lifetime of a few seconds or less)
was detected in both investigations, whereas nitrosyl complexes
of heme proteins typically are quite stable.
For the CYP119 enzyme, the formation of the Compound II
derivative by reaction with PN was further demonstrated with
X-ray absorption spectroscopy (XAS) studies of the Compound
II species that gave a detailed picture of the bonding to iron in
this derivative.34 The edge and near-edge features in the XANES
spectrum supported an iron(IV) oxidation state, and the EXAFS
spectrum gave bond lengths to iron similar to those found for
the CPO Compound II species,35 including a long Fe-O bond
(1.82 Å) implicating a protonated ferryl oxygen species,
Fe(IV)-OH.34 In the XAS project,34 an authentic sample of
the nitrosyl complex of CYP119 was prepared and studied for
comparison to the Compound II derivative. A long-lived
transient (lifetime of hours) was formed by reaction of the
resting enzyme with either NO gas or the NO donor diethy-
lamine diazeniumdiolate, and XAS spectra of the NO complex
differed from those of Compound II. In the X-ray beam at <140
K, photoreduction of the CYP119-NO complex was observed
with multiminute lifetimes, whereas the CYP119 Compound II
derivative showed no apparent photoreduction after hours of
irradiation under the same conditions, leading us to conclude
that the Compound II derivative contained essentially no nitrosyl
adduct as a contaminant.34
In the kinetic studies reported in this work, CYP119 was
treated with 20-25 equiv of PN. Excess PN was required for
efficient conversion of the enzyme to the Compound II
derivative because PN, which is prepared and stored in basic
solutions, is protonated in buffer solutions to give peroxynitrous
acid, which rapidly decomposes in an acid-catalyzed process
to give nitrite and nitrate.36-39 The CYP119 enzyme also
(28) Mehl, M.; Daiber, A.; Herold, S.; Shoun, H.; Ullrich, V. Nitric Oxide
1999, 3, 142–152.
The CYP119 Compound II derivative is photolabile, and its
decay under constant irradiation from the analysis beam was
noticeably faster when it was irradiated with a full spectrum of
UV-vis light as opposed to low-intensity monochromatic light.
Nonetheless, some diode-array experiments were conducted with
a full range of light in order to characterize the spectra of the
transients. In all of the studies using PMTs, the monochromator
was placed before the sample cell to reduce the exposure of
the sample to light.
The dashed line in Figure 2 shows the difference spectrum
obtained by irradiating samples containing CYP119 Compound
II with 355 nm laser light. This spectrum, which was formed
in the 7 ns time span of the laser pulse, contains a bleached
(29) Exner, M.; Herold, S. Chem. Res. Toxicol. 2000, 13, 287–293.
(30) Boccini, F.; Herold, S. Biochemistry 2004, 43, 16393–16404.
(31) Furtmu¨ller, P. G.; Jantschko, W.; Zederbauer, M.; Schwanninger, M.;
Jakopitsch, C.; Herold, S.; Koppenol, W. H.; Obinger, C. Biochem.
Biophys. Res. Commun. 2005, 337, 944–954.
(32) Gebicka, L.; Didik, J. J. Inorg. Biochem. 2007, 101, 159–164.
(33) Behan, R. K.; Hoffart, L. M.; Stone, K. L.; Krebs, C.; Green, M. T.
J. Am. Chem. Soc. 2007, 129, 5855–5859.
(34) Newcomb, M.; Halgrimson, J. A.; Horner, J. H.; Wasinger, E. C.;
Chen, L. X.; Sligar, S. G. Proc. Natl. Acad. Sci. U.S.A. 2008, 105,
8179–8184.
(35) Stone, K. L.; Behan, R. K.; Green, M. T. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 16563–16565.
(36) Bolzan, R. M.; Cueto, R.; Squadrito, G. L.; Uppu, R. M.; Pryor, W. A.
Methods Enzymol. 1999, 301, 178–187.
(37) Kissner, R.; Koppenol, W. H. J. Am. Chem. Soc. 2002, 124, 234–
239.
(38) Kirsch, M.; Korth, H. G.; Wensing, A.; Sustmann, R.; de Groot, H.
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2470.
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Kinetic Studies of the Compound I Derivative of CYP119
A R T I C L E S
Figure 3. Absorbance spectra (gray lines) and second-derivative spectra
(black lines) for the Soret bands of the Compound I derivatives of (A) CPO
and (B) CYP119. The scales were arbitrarily adjusted to show the signals.
The horizontal lines indicate zero intensity.
Figure 2. UV-vis spectrum of CYP119 Compound I. The dashed line is
the observed difference spectrum obtained by LFP irradiation of the CYP119
Compound II species. A scaled spectrum of CYP119 Compound II was
added to the difference spectrum to produce the spectrum for the Compound
I derivative (solid line). The Q-band region from 600 to 700 nm is an
amplification of an observed difference spectrum with no correction for
bleaching.
are present. The minima of the negative peaks in the second-
derivative spectrum are slightly displaced (by ∼2 nm) from the
maxima of the individual peaks (the short-wavelength band to
shorter wavelength and the long-wavelength band to longer
wavelength) from the values that were used by Egawa and co-
workers42 for their simulation; this displacement is an artifact
of the differentiation. The CYP119 Compound I Soret band also
contains two peaks, which are apparent in the spectrum and
even more obvious in the second-derivative spectrum (Figure
3B). The two peaks in the Soret bands of the Compound I
derivatives of CPO and CYP119 have similar λmax values, but
the relative intensities are reversed in the two spectra. From
this type of analysis, the Soret bands of CPO Compound I and
CYP119 Compound I are seen to be quite similar.
region in which the absorbance of the Compound II derivative
was stronger than the absorbance of the product of the
photochemical reaction and a growth region in which the product
absorbed more strongly than the Compound II derivative. The
difference spectrum is quite similar to those observed when the
Compound II derivatives of a model porphyrin-iron complex,
horseradish peroxidase, and myoglobin were irradiated with 355
nm light to give the corresponding Compound I derivatives.21
Specifically, in each of those cases, negative peaks were red-
shifted from positive peaks because the absorbance maximum
for the Soret band of the Compound II species was at a longer
wavelength than that of the Compound I species.
No P450 Compound I spectrum has been measured previ-
ously, but P450 Compound I spectra have been calculated from
deconvolution of spectral data obtained in rapid-mixing studies.
Egawa et al.14 reported results from rapid mixing of mCPBA
and P450cam in 1994, and similar studies were conducted with
CYP119 by Sligar and co-workers20 in 2002. In both studies,
the spectra for the Soret bands of the Compound I derivatives
calculated from the composite data using global analysis
methods contained highly asymmetric peaks with λmax ≈ 367
nm, similar to that of CPO-I. Deconvolution methods often
cannot give unique simulations but instead yield answers that
depend on the initial guesses for the rate constants. When this
occurs, one must select the “correct” spectrum from among the
possibilities; in the present case, this involved evaluation of the
results using the known spectrum of CPO-I as a guide.40
We repeated the stopped-flow mixing study20 of CYP119 with
mCPBA at 4 °C and obtained the results shown in Figure 4.
For the first 65 ms of the reaction, the signal centered at ∼367
nm grew slightly and the one centered at ∼416 nm decreased
slightly (Figure 4A). After 65 ms, the signals returned to the
original absorbances of the resting enzyme (Figure 4B). The
spectral file (800 spectra) was deconvoluted by global analysis
using a model involving the two steps A + B f C (with a
second-order rate constant kAB) and C f A (with a first-order
rate constant kC), where A is the CYP119 enzyme, B is mCPBA,
and C is the Compound I derivative of CYP119. We used the
same software and kinetic model as used in the previous study.20
Examples of simulated spectra of CYP119 Compound I resulting
from four deconvolutions with different initial guesses for the
rate constants are shown in Figure 4, and 12 additional sets of
spectra and their residuals are given in Table S1 in the
Supporting Information. We emphasize that no constraints were
employed in these deconvolutions.
Addition of a normalized spectrum of the CYP119 Compound
II derivative to the difference spectrum in Figure 2 compensated
for the destroyed Compound II species and gave a spectrum of
the Compound I derivative of CYP119 (the solid line in Figure
2). The spectrum consists of a broad Soret band comprising
two overlapping peaks, the smaller one at ∼360-370 nm and
the larger one at ∼416 nm, and a broad Q band in the range
600-700 nm that is highly characteristic of a porphyrin radical
cation.40
The position of the Q-band absorbance for the CYP119
Compound I species was expected,40 but that for the absorbance
of the Soret band was surprising. On the basis of the UV-vis
spectral results for the heme-thiolate enzyme CPO, P450
Compound I derivatives are predicted to have a Soret band
absorbance with λmax ) 367 nm. The UV-vis spectrum of CPO-
I, first reported in 1980,41 has a highly asymmetric Soret band.
Egawa et al.42 later demonstrated that this band can be resolved
into two overlapping symmetric peaks.
The second derivative of the Soret-band spectrum provides
another demonstration that the “peak” of the Soret band of
CPO-I actually comprises overlapping peaks. Figure 3A shows
a spectrum of CPO-I obtained in a previous study in our
laboratory43 along with its second-derivative spectrum, in which
the two observed minima clearly demonstrate that two peaks
(40) Makris, T. M.; von Koenig, K.; Schlichting, I.; Sligar, S. G. J. Inorg.
Biochem. 2006, 100, 507–518.
(41) Palcic, M. M.; Rutter, R.; Araiso, T.; Hager, L. P.; Dunford, H. B.
Biochem. Biophys. Res. Commun. 1980, 94, 1123–1127.
(42) Egawa, T.; Proshlyakov, D. A.; Miki, H.; Makino, R.; Ogura, T.;
Kitagawa, T.; Ishimura, Y. J. Biol. Inorg. Chem. 2001, 6, 46–54.
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M. Org. Lett. 2006, 8, 2731–2734.
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A R T I C L E S
Sheng et al.
Figure 5. Kinetic traces. (A) Growth at 430 nm and decay at 405 nm for
reaction of CYP119 Compound I with 1.5 mM styrene; the rate constants
obtained from the two traces were the same. (B) Kinetic traces (gray lines)
and fits (black lines) at 430 nm for reactions of CYP119 Compound I with
benzyl alcohol at substrate concentrations of (bottom to top) 0.0, 0.6, and
2.2 mM.
Chart 1
Figure 4. Results from stopped-flow mixing of 14 µM CYP119 with 8.6
µM mCPBA at 4 °C. (A) Observed spectra at 1.0, 7.0, 13, 26, and 65 ms
after mixing. (B) Observed spectra at 65, 180, 290, 390, and 700 ms after
mixing. (C-F) Simulated spectra of the intermediate obtained from
unconstrained minimizations using different initial guesses for the rate
constants. The program-derived final values for kC in the deconvolutions
were (C) 17.4, (D) 9.2, (E) 6.7, and (F) 2.3 s-1. The Supporting Information
provides additional information.
As noted earlier, the Soret band of CYP119 Compound I can
be resolved into two peaks, one centered at 367 nm and the
other at 416 nm. The deconvolutions weight these peaks
differently depending on the values for the rate constants. The
simulated spectrum associated with kC ) 17.4 s-1 (Figure 4C)
is essentially the same as the simulated spectrum for CYP119
Compound I reported previously20 and similar to the CPO
Compound I spectrum.41 As the value of kC decreases, the
simulated spectra display an increasing component of the 416
nm peak and a decreasing component of the 367 nm peak.
Eventually, when kC ) 2.3 s-1 (Figure 4F), the simulated
spectrum closely matches the experimental spectrum of the
CYP119 Compound I species (Figure 2). Importantly, a kC value
of 2 s-1 for decay of the transient at 4 °C is consistent with the
measured rate constant for decomposition of the CYP119
Compound I derivative in the absence of substrate, whereas a
decay rate constant of 17 s-1 is too large (see below).
In summary for this section, laser flash photolysis of the
CYP119 Compound II derivative gave the CYP119 Compound
I derivative. The experimental spectrum of this species was
obtained from the LFP experiments using the difference
spectrum (i.e., the difference of spectra recorded before and
immediately after the flash). Stopped-flow mixing results for
oxidation of CYP119 with mCPBA gave the same calculated
UV-vis spectrum for the Soret band of CYP119 Compound I
as obtained in the LFP experiments when an appropriate rate
constant for decay of this transient was found in the spectral
deconvolution. The Soret-band absorbance of CYP119 Com-
pound I contains two overlapping transitions, much like that of
CPO Compound I42 but with a different ratio of the component
peaks. The Q band displays a broad, weak absorbance between
600 and 700 nm, as expected for a porphyrin radical cation
structure.40
Kinetics of CYP119 Compound I Reactions. No direct kinetic
studies of reactions of a P450 Compound I derivative with
substrate have been reported previously, but such studies are
now possible. Using 2 ns risetime PMTs, first-order rate
constants as large as 2 × 108 s-1 can be measured, and
diffusion-controlled bimolecular reactions are readily monitored.
Decay of CYP119 Compound I to the ferric enzyme resting
state resulted in decreasing absorbance at wavelengths in the
range 390-420 nm and increasing absorbance in the range
425-450 nm. The results obtained by following the kinetics in
the two wavelength ranges were the same (Figure 5A). In
practice, we generally followed signal growth at ∼430 nm.
The kinetic studies involved the oxidation reactions shown
in Chart 1. Characterizations of the products from these
oxidation reactions are discussed below. All of the kinetic studies
9
13314 J. AM. CHEM. SOC. VOL. 130, NO. 40, 2008

Kinetic Studies of the Compound I Derivative of CYP119
A R T I C L E S
Figure 6. Kinetic results for reactions of CYP119 Compound I in 25 mM phosphate buffer (pH 7.4) at 22 °C: (A) benzyl alcohol (top) and styrene
(bottom); (B) methyl laurate (top) and lauric acid (bottom). The lines are regression solutions based on (A) eq 1 and (B) eq 3. The error bars on the
pseudo-first-order rate constants represent one standard deviation.
Table 1. Apparent Second-Order Rate Constants for Reactions of
CYP119 Compound I with Organic Substratesa
were performed with large excesses of substrate to ensure that
secondary oxidations were not important.
substrate
kapp (M-1 s-1
)
CPO-I kapp (M-1 s-1 b
)
For reactions with substrates, the substrate of choice was
present in the enzyme solution prior to mixing that solution with
the PN solution. A series of reactions was conducted with
varying concentrations of substrate while maintaining pseudo-
first-order reaction conditions. Figure 5B shows typical results
obtained with benzyl alcohol. Each trace was fit using a double-
exponential function in which the major process (80-90%)
varied with the concentration of substrate and the minor process
was constant in successive experiments. Because the CYP119
Compound I derivative reacts with excess PN, the laser flash
was delayed until ∼5 s after mixing to permit PN decay. In all
cases for a series of studies with a given substrate, the reaction
conditions were maintained such that the background rate
remained constant. For example, a series of studies with a given
substrate employed one solution of enzyme, one solution of PN,
and a constant delay time after mixing before the laser flash.
First-order rate constants for reaction of CYP119 Compound
I without added substrate varied in the range 2-8 s-1 at 20 °C,
depending on the amount of excess peroxynitrite present in the
solution (with larger rate constants obtained when the concen-
tration of PN was greater). PN is stored in basic solutions and
decays rapidly in an acid-catalyzed reaction upon mixing with
the buffered solutions of enzyme. Delaying the laser irradiation
for 5 s after mixing allowed most of the PN to decay, as
determined by UV-vis spectroscopy, yielding small, relatively
consistent rate constants for decay of CYP119 Compound I.
Details of the background decay reaction for the Compound I
derivative should be revealed with further study, and this
“reaction” might actually be a sum of several reactions,
including reaction with PN, reaction of the Compound I
derivative with nitrite, which is a major product of PN decay,36,38
and self-reaction of the Compound I moiety with an oxidizable
portion of the protein. In any event, the first-order decay reaction
of Compound I introduced a constant kinetic effect that was
subtracted from the observed rate constants.
benzyl alcohol
benzyl alcohol (pH 6.4)c
benzyl alcohol-d7
ethylbenzene
ethylbenzene-d10
styrene
(2.70 ( 0.07) × 104
(1.97 ( 0.08) × 104
(1.17 ( 0.06) × 104
(2.54 ( 0.12) × 103
(1.6 ( 0.4) × 103
(7.64 ( 0.18) × 103
(9.5 ( 0.3) × 103
(2.5 ( 0.2) × 102
7.2 × 102
9.6 × 102
3.7 × 102
6.1 × 104
6.4 × 103
10-undecenoic acid
Tris buffer (pH 7.4)d
lauric acide
methyl lauratee
1.2 × 103
a Reactions conducted at 22 ( 1 °C in 25 mM phosphate buffer (pH
7.4) unless otherwise noted. Errors are 1σ. b Apparent second-order rate
constants for reactions of CPO Compound I derivative, from ref 43.
c This reaction was run in 25 mM phosphate buffer (pH ∼6.4). d The
reaction was conducted in 25 mM phosphate buffer (pH 7.4) using Tris
buffer as the substrate; Tris stands for tris(hydroxymethyl)-
aminomethane, (HOCH2)3CNH2, which is mainly protonated at pH 7.4.
e For lauric acid and methyl laurate, the apparent second-order rate
constants were calculated from the measured values of Kbind and kox
discussed in the text.
results for this group of substrates. The rate constants are
described by eq 1:
kobs - k0 ) kapp[substrate]
(1)
where kobs is the observed pseudo-first-order rate constant, k0
is the background first-order rate constant, kapp is an apparent
second-order rate constant for reaction with the substrate, and
[substrate] is the substrate concentration. Plots of (kobs - k0)
versus [substrate] had slopes of kapp, as shown in Figure 6A,
and values of kapp determined for the reactive substrates are listed
in Table 1. We note that the apparent second-order rate constant
for reaction in an enzyme’s active site is a composite value
consisting of two factors, an equilibrium constant for binding
substrate (Kbind, units of M-1) and a first-order rate constant
for the oxidation reaction of the bound substrate (kox, units of
s-1), as shown in eq 2:
For reactions of CYP119 Compound I with most of the
substrates studied, we observed a linear relationship between
the pseudo-first-order rate constant for reaction of Compound I
and the concentration of substrate.44 Figure 6A shows typical
kapp ) Kbindkox
(2)
For reactions of lauric acid and methyl laurate with CYP119
Compound I, the kinetic behavior was different than that found
with other substrates. We observed saturation kinetics, with the
rate of reaction approaching a limiting value at high substrate
concentrations (Figure 6B). When saturation kinetics is ob-
served, the rate constants are described by eq 3:
(44) In the initial communication of this work (ref 22), we reported that
lauric acid and styrene had no effect on the kinetics of CYP119
Compound I decay. The substrate concentrations used in those studies
were too small to give measurable kinetic effects.
9
J. AM. CHEM. SOC. VOL. 130, NO. 40, 2008 13315

A R T I C L E S
Sheng et al.
K
bindkox[substrate]
before it relaxes to a Compound I species by internal electron
transfer. This controversial conjecture is supported by recent
evidence for the production of perferryloxo porphyrins and
corroles that are several orders of magnitude more reactive in
oxidation reactions than their iron(IV)-oxo ligand radical cation
isomers48,49 and by results of thermodynamic cycles indicating
that a perferryloxo intermediate in a P450 enzyme is energeti-
cally accessible.50
kobs - k0 )
(3)
Kbind[substrate] + 1
Nonlinear regression analysis of the data according to eq 3 gave
values for both Kbind and kox. For reactions at 22 °C in 25 mM
phosphate buffer (pH 7.4), the results for lauric acid were Kbind
) 900 ( 300 M-1 and kox ) 0.8 ( 0.1 s-1 and those for methyl
laurate were Kbind ) 370 ( 90 M-1 and kox ) 3.3 ( 0.3 s-1
.
Because kapp is the product of Kbind and kox (eq 2), we were
able to calculate kapp for lauric acid and methyl laurate; the
values are included in Table 1 for comparison to those for
the other substrates studied. Table 1 also contains some rate
constants measured for reactions of the Compound I derivative
of the heme-thiolate enzyme CPO.43
The rate constants found for oxidations of ethylbenzene,
styrene, and 10-undecenoic acid by CYP119-I are in general
agreement with those obtained for reactions of the same
substrates with the Compound I derivative of CPO. CPO-I has
long been available from hydrogen peroxide oxidation of the
enzyme and has served as a model for P450 Compound I in a
variety of spectroscopic studies.35,41,42,51,52 Apparent second-
order rate constants for two-electron oxo-transfer reactions of
CPO-I also are available.43,53 The similarity in the rate constants
for CYP119-I and CPO-I oxidations of common substrates
reinforces the conclusion that Compound I derivatives of P450
enzymes are well modeled by CPO-I, but it must be remembered
that the observed rate constants for oxidations by CYP119-I
and CPO-I are apparent second-order rate constants that contain
both equilibrium binding constants and first-order oxidation rate
constants. More meaningful comparisons can be made when
these composite rate constants are separated into their constituent
parts.
It is possible to crudely estimate the Kbind and kox values for
substrates that gave only apparent second-order rate constants.
The binding constant for lauric acid by CYP119-I measured in
this work was 900 M-1. For CYP119 resting enzyme, the lauric
acid binding constant is nearly 400 times larger than the styrene
binding constant.54 The assumption that the same differential
exists for complexation of these two substrates by CYP119-I
yields an estimate of Kbind ≈ 2 M-1 for styrene, and the value
for the oxidation rate constant would be kox ≈ 4000 s-1. This
estimated first-order rate constant for styrene oxidation at
ambient temperature gives ∆Gq ) 12.4 kcal/mol, which appears
to be in reasonable agreement with ∆Gq values of ∼15 kcal/
mol for styrene oxidations by simple Compound I models that
should be less reactive than P450 Compound I species45 if one
factors in an expected accelerating effect for the thiolate ligand
in CYP119-I. If similarly small Kbind values are assumed for
the other aromatic substrates studied here, namely, ethylbenzene
and benzyl alcohol, then the kox values for these substrates are
in the 1000 and 20 000 s-1 ranges, respectively, which again
appear to be in line with rate constants for reactions of simple
Compound I models.45
Perhaps the most noteworthy kinetic result is the small rate
constant for oxidation of unactivated C-H bonds in lauric acid
(kox ) 0.8 s-1). If the binding constant for lauric acid were not
large enough to result in saturation of the activated enzyme,
then we would not have been able to measure the kinetics of
this reaction. Nonetheless, the measured rate constant is
consistent with the observed reactivities of a number of
iron(IV)-oxo porphyrin radical cations, which are known to
be too low in activity to oxidize unactivated C-H bonds in
hydrocarbons efficiently.8,45 P450 enzymes do oxidize unacti-
vated C-H bonds rapidly under natural conditions, of course,
and it is commonly assumed that the requisite reactivity arises
from the electron-donating effect of the cysteinate ligand.7,10
For CYP119-I, the cysteinate ligand does appear to increase
reactivity somewhat (see below) but not enough to match the
apparent reactivity for C-H oxidations displayed by some P450s
in nature.
A paradox involving Compound I reactivities and P450
oxidations is that Compound I derivatives in models are not
reactive enough to explain the oxidation strength of P450
enzymes. When P450cam with substrate present in the active
site was allowed to react with radiolytically generated electrons
and oxygen under cryogenic conditions, the last intermediate
in the sequence observable by EPR and ENDOR spectroscopy
was the hydroperoxyiron species (see Figure 1).11,12 Extrapola-
tion of the low-temperature results to ambient temperature
suggests that the hydroperoxyiron species would decay at room
temperature with a rate constant of k ≈ 1000 s-1. Because no
subsequent iron-oxo transient was observed, the transient that
effected the oxidation must have reacted with the unactivated
C(5)-H bond of camphor with a rate constant greater than that
of its formation. Thus, if the estimate for the decay of the
hydroperoxyiron species is correct, then the oxidation reaction
Another manifestation of the kinetic acceleration afforded by
the thiolate ligand in CYP119-I is seen in the kinetic isotope
effect (KIE) results. For benzyl alcohol and ethylbenzene, kH/
kD ) 2.3 and 1.6, respectively, under the assumption that the
substrate binding constants were unaffected by isotopic substitu-
at ambient temperature would have a rate constant of kox
>
1000 s-1; in turn, if that approximation is correct, then camphor
C(5)-H oxidation by activated P450cam is more than 3 orders
of magnitude faster than the lauric acid C-H oxidation by
CYP119 Compound I. In principle, the large difference in
reactivities might be due to very poor “tuning” of the reaction
in CYP119, which displays maximal activity at ∼70 °C.25,46,47
Alternatively, it is possible that P450s can produce a more
reactive transient than Compound I, a perferryloxo species (see
Figure 1) that has a finite lifetime and can effect oxidations
(48) Harischandra, D. N.; Zhang, R.; Newcomb, M. J. Am. Chem. Soc.
2005, 127, 13776–13777.
(49) Pan, Z. Z.; Zhang, R.; Fung, L. W. M.; Newcomb, M. Inorg. Chem.
2007, 46, 1517–1519.
(50) Koppenol, W. H. J. Am. Chem. Soc. 2007, 129, 9686–9690.
(51) Rutter, R.; Hager, L. P.; Dhonau, H.; Hendrich, M.; Valentine, M.;
Debrunner, P. Biochemistry 1984, 23, 6809–6816.
(45) Pan, Z. Z.; Zhang, R.; Newcomb, M. J. Inorg. Biochem. 2006, 100,
524–532.
(52) Kim, S. H.; Perera, R.; Hager, L. P.; Dawson, J. H.; Hoffman, B. M.
J. Am. Chem. Soc. 2006, 128, 5598–5599.
(46) Puchkaev, A. V.; Wakagi, T.; Ortiz de Montellano, P. R. J. Am. Chem.
Soc. 2002, 124, 12682–12683.
(53) Osborne, R. L.; Coggins, M. K.; Terner, J.; Dawson, J. H. J. Am.
Chem. Soc. 2007, 129, 14838–14839.
(47) Puchkaev, A. V.; Ortiz de Montellano, P. R. Arch. Biochem. Biophys.
2005, 434, 169–177.
(54) Koo, L. S.; Immoos, C. E.; Cohen, M. S.; Farmer, P. J.; Ortiz de
Montellano, P. R. J. Am. Chem. Soc. 2002, 124, 5684–5691.
9
13316 J. AM. CHEM. SOC. VOL. 130, NO. 40, 2008

Kinetic Studies of the Compound I Derivative of CYP119
A R T I C L E S
Table 2. Results of Competition Shunt Oxidations by CYP119a
tion. These KIE values are relatively small; for example, an
H/D KIE value of 4.4 for ethylbenzene-d0 and -d10 was found
for oxidation by the Compound I derivative of 5,10,15,20-
tetrakis(pentafluorophenyl)porphyrin-iron.45 The small KIE
values for CYP119-I suggest a large, negative value of ∆G°
for oxidation of these substrates relative to the value in the
models, leading to an early transition state and a small difference
in the ∆Gq values (and small KIEs) for reactions of the
nondeuterated and perdeuterated substrates.
b
substrate A
substrate B
kA/kB
LFP ratioc
benzyl alcohol
ethylbenzene
benzyl alcohol
styrene
benzyl alcohol-d7
ethylbenzene-d10
styrene
lauric acid
lauric acid
2.65 ( 0.14
1.95 ( 0.14
3.25 ( 0.14
0.16 ( 0.01
0.52 ( 0.04
2.3 ( 0.3
1.6 ( 0.8
3.5 ( 0.2
10.6
38
benzyl alcohol
a Competition reactions between substrates A and B at equal initial
concentrations in 50 mM phosphate buffer (pH 7.0) at 22 ( 1 °C.
b Ratio of rate constants from eq 4; errors are for 95% confidence.
c Ratio of rate constants from LFP studies (Table 1); errors are for
∼95% confidence.
We performed some experiments to crudely evaluate the
effect of the buffer on the Compound I reaction kinetics. Most
of the oxidations in Table 1 were conducted in phosphate buffer
at pH 7.4. For benzyl alcohol, the value of kapp was reduced by
∼25% when the reaction was conducted in phosphate buffer at
a pH of ∼6.4. Until it is possible to separate the constituent
factors in kapp, we are not able to determine whether the kinetic
pH effect is due to reduced binding or to a change in the
oxidation rate constant (or both), but the noteworthy point is
that one should take care to monitor the pH closely in kinetic
studies of P450 Compound I oxidations.
We performed CYP119-hydrogen peroxide shunt reactions
for comparison to the LFP kinetic studies. In the shunt reactions,
CYP119 in buffer with a mixture of two substrates was treated
with aliquots of hydrogen peroxide. Following the reactions,
the products were extracted into an organic solvent, after which
a standard was added and the mixtures were analyzed by GC
for quantification and GC-MS for product identification. The
relative rate constants for reactions of the two substrates were
determined using eq 4, which is the equation for competitive
kinetics in reactions of substrates A and B when the competing
reactions are second-order processes:57
We also performed a cursory study using tris(hydroxymethyl)-
aminomethane (Tris) buffer as a substrate for CYP119-I because
the hydroxymethyl groups of Tris were expected to be reactive
and some oxidations by CYP119 have been conducted with bis-
Tris buffer.54,55 We did not attempt to isolate products from
the oxidation of Tris because they undoubtedly are highly water-
soluble, but it is most likely that the oxidation gave a geminal
diol at one carbon that could dehydrate to an aldehyde group.
The point of the study was to determine if Tris would react
rapidly with CYP119 Compound I, and this was accomplished
by using a phosphate buffer system and adding Tris in varying
amounts as if it were a typical substrate. When the reaction
was studied in this manner, we obtained a reasonably large kapp
value of 250 M-1 s-1 for Tris. This result indicates the need
for caution with respect to the use of Tris or bis-Tris buffers in
Compound I oxidation studies. For example, bis-Tris buffer has
been used at a concentration of 50 mM in studies of styrene
oxidation by CYP119-I under H2O2 shunt conditions.55 If bis-
Tris reacts with the same rate constant as Tris, then the reaction
of CYP119-I with the buffer had a pseudo-first-order rate
constant of k ≈ 12 s-1, which is ∼6 times as large as that for
the decay in the presence of phosphate buffer.
kA ln([A]0/[A]F)
)
(4)
kB ln([B]0/[B]F)
In eq 4, [X]0 is the initial concentration of substrate X and [X]F
is the concentration of substrate X at the end of the reaction.
Table 2 lists the results of the competition kinetics studies.
The ratios of rate constants for the competing reactions in the
shunt experiments are listed as kA/kB in column 3. Column 4 in
the table contains the ratios of rate constants obtained in the
LFP studies for the same substrates. The ratios of rate constants
typically should not be the same for the two types of studies
because the shunt reactions studied here involve competitions,
and a substrate with a large binding constant might be a
competitive inhibitor of a substrate with a small binding
constant. Nonetheless, if there is an equality in the equilibrium
binding constants for the two substrates, then the ratios of rate
constants for the two types of studies should be equal. That
condition can be assumed to hold for the KIE studies because
isotopic substitution should have at most only a minor effect
on substrate binding. For both benzyl alcohol and ethylbenzene,
we found the same KIE values in the competition studies as in
the LFP studies, indicating that the oxidizing species is the
Compound I derivative in both types of oxidation experiments.
The ratio of rate constants for the competition study involving
benzyl alcohol and styrene and the corresponding ratio of LFP
rate constants for these substrates also were the same. This
suggests either that the two aromatic substrates have similar
equilibrium binding constants or that the binding constants for
both were too small to saturate the enzyme. In either case, the
kapp values found in the LFP studies would control the relative
reactivities in the competition studies.
Comparisons of CYP119 Shunt Oxidation Reactions to
CYP119-I Reactions. For most P450 enzymes, the sequence of
reactions that produces active oxidant involves reduction of the
ferric enzyme, reversible binding of oxygen, a second reduction
step, and protonation reactions (see Figure 1). Many P450
enzymes can be “shunted” with peroxides such as hydrogen
peroxide in reactions that likely resemble those of the peroxidase
enzymes. Although there is limited evidence for the formation
of a Compound I derivative in these shunt reactions, P450
enzymes have been known for many years to catalyze
oxidizations of substrates under shunt conditions,56 and the
presence of a Compound I transient generally has been
assumed. In the specific case of CYP119, hydrogen peroxide
and alkyl hydroperoxide shunt reactions are known to give
oxidized substrates.25,46,47,54,55
The competition results with lauric acid were much different
than those involving two aromatic substrates. In the LFP studies,
lauric acid reacted with a kapp value that was much smaller than
those measured for benzyl alcohol and styrene (see Table 1),
but lauric acid was oxidized more efficiently than either of the
aromatic substrates in the competition study. Thus, lauric acid
(55) Koo, L. S.; Tschirret-Guth, R. A.; Straub, W. E.; Moenne-Loccoz,
P.; Loehr, T. M.; Ortiz de Montellano, P. R. J. Biol. Chem. 2000,
275, 14112–14123.
(56) Nordblom, G. D.; White, R. E.; Coon, M. J. Arch. Biochem. Biophys.
1976, 175, 524–533.
(57) Newcomb, M. Tetrahedron 1993, 49, 1151–1176.
9
J. AM. CHEM. SOC. VOL. 130, NO. 40, 2008 13317

A R T I C L E S
Sheng et al.
was a competitive inhibitor of the aromatic substrates in the
competition studies. The binding constant for lauric acid found
in the LFP studies, Kbind ) 900 M-1, is the equilibrium constant
for complexation in the active site of the Compound I derivative.
In the competition studies, where the substrate concentrations
were 0.2 mM, a Kbind value of 900 M-1 would not result in
saturation of the activated enzyme. For the resting CYP119
enzyme, however, the reported binding constant for lauric acid
concentrations are listed) in a vessel held in a 0 °C bath. After
10 s, the reaction vessel was removed from the bath, placed in
a photochemical reactor at room temperature, and irradiated for
∼1 min with five 15 W bulbs emitting light centered at 350
nm. The reaction mixture was then worked up and analyzed
using GC and GC-MS to quantify and identify the products.
For styrene and ethylbenzene as substrates, we found styrene
oxide and 1-phenylethanol in ∼70 and ∼65% yield, respectively,
based on enzyme. We estimate that under the conditions
employed for production of the Compound II species in these
experiments, 80-90% of the enzyme was converted to the
Compound II derivative. Thus, the total efficiency for the two
processes (photochemical production of Compound I and its
reaction with substrate) was very high (in the 70-90% range),
given that “self-decay” of the CYP119 Compound I derivative
occurred at a measurable rate on the time scale of the bulk
photolysis studies. In a series of control reactions with styrene
as the substrate, we found no oxidization product when PN,
CYP119, or light was omitted from experiments otherwise
identical to the styrene reaction described above.
The origin of the oxygen atoms in the oxidation products was
established by performing two sets of bulk photochemical oxidation
reactions with 18O-labeled species. In the first set, we used doubly
labeled peroxynitrite (NaN16O18O2) synthesized from H218O2 that
had previously been prepared from 18O2. In the second set, we
used unlabeled peroxynitrite in a 1:1 mixture of unlabeled and 18O-
labeled water (H216O/H218O ) 1:1). The reactions were conducted
by addition of the substrate to the enzyme followed by addition of
PN and then photolysis. The products were analyzed by GC-MS
using single-ion monitoring (SIM) for the molecular ions. In
the case of styrene, the molecular ion of the unlabeled styrene
oxide product appears at m/z 120; in the study conducted with
doubly labeled PN, the ratio of integrals for the m/z 120 and
122 peaks was 1:2, while the integral ratio using unlabeled PN
in labeled water was >99:1. Similar results were obtained for
oxidation of ethylbenzene, in which the unlabeled product
alcohol has a molecular ion at m/z 122; for the reaction with
doubly labeled PN, the ratio of integrals for m/z 122 and 124
was 1:2, whereas as the ratio found in the reaction with
unlabeled PN in labeled water was >99:1.
is ∼8 × 105 M-1 54
,
which would result in saturation of the
resting enzyme with 0.2 mM lauric acid. More detailed studies
are needed to understand the kinetic effects fully, but the
tentative conclusion is that CYP119 saturation by lauric acid
before activation of the enzyme by reaction with H2O2 resulted
in limited access of the aromatic substrates to the active site of
the activated enzyme.
Products from Oxidation Reactions. For the substrates studied
in this work, the products of oxidations by P450 enzymes in
general are known to be epoxidation products for alkenes and
hydroxylation products for hydrocarbons.10 In the specific case
of CYP119, oxidation reactions of lauric acid and styrene have
been studied under shunt conditions as well as under a more
natural reaction sequence.25,46,47,54,55 The major product from
lauric acid oxidation is 11-hydroxylauric acid, and the 12-, 10-,
and 9-hydroxylated products also are formed. The major product
from styrene oxidation is styrene oxide.
In the present work, shunt reactions similar to the competition
kinetics studies were conducted, and the products were analyzed
using GC and GC-MS. The oxidation products were identified
by comparison of their GC retention times and mass spectral
fragmentation patterns to those of authentic samples of the
products benzaldehyde (from benzyl alcohol), styrene oxide
(from styrene), 1-phenylethanol (from ethylbenzene), and 10,11-
epoxyundecanoic acid (from 10-undecenoic acid). For lauric
acid, the product mixture from reaction with CYP119 is known
to contain 11-hydroxylauric acid as the major product, 12-
hydroxylauric acid as the second most abundant product, and
other hydroxylauric acids as minor products.54 We converted
the lauric acid products to the corresponding methyl esters by
reaction with diazomethane. The mixture of products from this
sequence was compared to the mixture obtained from oxidation
of methyl laurate in order to identify the methyl laurate oxidation
products; these were found to be the same as those from
oxidation and subsequent methylation of lauric acid, although
the ratios of products differed slightly.
The labeling results demonstrate that the Compound I
oxidations involved reaction via insertion of the oxygen from
the iron-oxo species rather than electron transfer followed by
water capture of the radical cations. The formation of oxidation
products via radical cations had already been judged to be highly
unlikely because it is known that styrene radical cation reacts
largely to give benzaldehyde58 and electron-transfer oxidations
of ethylbenzene and other alkylarenes give good yields of phenol
products,59 but neither benzaldehyde nor ethylphenols were
found in our product mixtures.
The yields of products from some of the shunt reactions were
determined using GC by addition of a calibrated internal
standard to the product mixture from the oxidations. The yields
of oxidation products from styrene and ethylbenzene were
8-10% based on hydrogen peroxide as the limiting reagent.
Similar yields for hydrogen peroxide shunt reactions with
CYP119 were reported previously.54
Conclusions
In order to evaluate the effect of PN on the CYP119 enzyme,
we conducted a shunt reaction identical to the one with styrene
except that the CYP119 enzyme was first treated with 20 equiv
of PN. The yield of styrene oxide formed in this shunt reaction
was the same as that found with untreated CYP119. Thus, the
PN reaction using 20 equiv of PN appears to have no effect on
the CYP119 enzyme.
The LFP study involved production of subnanomole amounts
of reactive transients, but we also performed bulk photolysis
reactions that were designed to simulate the LFP studies on a
somewhat larger scale. In these reactions, 200 µM PN was added
to a mixture of 10 µM CYP119 and 100 µM substrate (final
Photoejection of an electron from the P450 119 Compound
II derivative produced the P450 Compound I species (CYP119-
I), which is widely regarded as the active transient in P450-
catalyzed oxidation reactions. The UV-vis spectrum of CYP119-I
was thus directly observed (as opposed to calculated) and found
to be similar to the UV-vis spectrum of chloroperoxidase
(58) Ren, Y.; Che, Y.; Ma, W.; Zhang, X.; Shen, T.; Zhao, J. New J. Chem.
2004, 28, 1464–1469.
(59) Bartoli, J.-F.; Lambert, F.; Morgenstern-Badarau, I.; Battioni, P.;
Mansuy, D. C. R. Chim. 2002, 5, 263–266.
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Kinetic Studies of the Compound I Derivative of CYP119
A R T I C L E S
the resulting mixture had a pH of 7.4. After a delay of 5 s, the
laser was fired. For kinetic studies, the substrate at the desired
concentration was added to the CYP119 solution.
Compound I, consisting of two overlapping peaks in the Soret-
band region and a long-wavelength Q-band absorbance.
The LFP method for production of CYP119-I permitted direct
kinetic studies of reactions of this transient with a variety of
organic substrates. The measured rate constants for many
substrates were apparent second-order rate constants, which are
products of a binding constant times a first-order rate constant
for the oxidation reaction, that were similar in magnitude to
those for oxidations of the same substrates by the Compound I
derivative of chloroperoxidase. For lauric acid and methyl
laurate, saturation kinetics were observed, and values of both
Kbind and kox were determined. The rate constant for oxidations
of unactivated C-H positions in lauric acid was 0.8 s-1 at
ambient temperature, which is more than 3 orders of magnitude
smaller than the kox value estimated for oxidation of the
unactivated C(5)-H bond in camphor by the oxidizing transient
formed in P450cam. Possible explanations for the differences in
reactivities are that CYP119, which is from a thermophile, is
not optimized for reactions at ambient temperature and that a
different, more highly reactive type of oxidant can be formed
in the natural reaction sequence of P450 enzymes.
By comparing relative reactivities in competition studies
involving CYP119 and hydrogen peroxide with the ratios of
rate constants from the LFP studies of the same substrates, we
conclude that the same transient is produced in both types of
experiments. Furthermore, product studies, including oxygen-
18 labeling studies, demonstrated that the known products of
P450-catalyzed oxidations were formed from photochemically
generated CYP119-I by insertion of the oxygen atom of the
iron-oxo group, the expected reaction pathway for Compound
I derivatives.
Competition Shunt Reactions. A 2 mL solution containing 20
µM CYP119 and 0.2 mM of the desired substrates in phosphate
buffer (pH 7.0) was stirred at room temperature as aliquots of H2O2
were added over 0.5 h (total addition of 0.4 mM). The mixture
was extracted with methylene chloride, and the organic phase was
washed with brine, dried (MgSO4), filtered, and analyzed by GC
and/or GC-MS on low-polarity columns (DB-5 or equivalent).
Relative rate constants were determined using eq 4.
Product Identifications and Yields in Shunt Reactions.
Reactions were conducted as described above for shunt reactions
with the exception that one substrate was employed. Following the
reaction, the products were analyzed by GC and GC-MS using
low-polarity columns (DB-5 or equivalent) for all of the substrates
except lauric acid and methyl laurate. Products were identified by
comparison of GC retention times and mass spectral fragmentation
patterns to those of authentic samples, most of which were
commercial samples. A sample of 10,11-epoxyundecanoic acid was
prepared by mCPBA oxidation of 11-undecenoic acid as previously
reported.61 Yields were determined relative to an internal standard
added after the product workup. For lauric acid, the product mixture
was converted to a mixture of methyl esters by reaction with
diazomethane, and the mixture was analyzed on high-polarity
columns (Carbowax or equivalent). Products from lauric acid
oxidation by CYP119 and H2O2 were reported,54 and a commercial
sample of 12-hydroxylauric acid was available for comparison. The
methyl laurate product mixture also was analyzed on high-polarity
columns, and the products were identified by comparison to the
products from the lauric acid oxidations.
Product Yields in Bulk Photolyses. A 1.0 mL solution
containing 10 µM CYP119 and 0.1 mM substrate (styrene or
ethylbenzene) in 50 mM phosphate buffer (pH 7.0) was cooled in
a 0 °C bath. To this solution was added 5 µL of 100 mM PN
solution. After 10 s, the mixture was removed from the ice bath
and irradiated in a photochemical reactor containing five 15 W
fluorescent bulbs (300-400 nm irradiation band) for 1 min. The
reaction mixture was worked up by extraction into CHCl3, and an
internal standard was added, after which the mixture was analyzed
by GC for yield as described above. In a series of control reactions
for the styrene oxidation, the reaction was repeated with the
exception that the CYP119 enzyme, the PN, or the irradiation step
was omitted; no styrene oxide was detected in these reactions.
Oxygen-18 Labeling Studies. Doubly labeled hydrogen perox-
ide, H218O2, was prepared from 18O2 as previously described.62,63
The H218O2 was then used for the preparation of doubly labeled
sodium peroxynitrite, NaN16O18O2, from isoamyl nitrite by the
method of Uppu and Pryor.60 Experiments were performed with
doubly labeled PN in unlabeled water and with unlabeled PN in
labeled water (1:1 H216O/H218O). In these studies, solutions of
CYP119 in 50 mM phosphate buffer (pH 7.0) were prepared in a
cell placed in a temperature-regulated chamber cooled to 0 °C.
Substrate followed by PN solution was added, and the UV-vis
spectrum was measured with a fiber-optic diode-array UV-vis
spectrometer. The final concentrations were 10 µM CYP119, 0.1
mM substrate, and 0.5 mM PN. The samples were irradiated with
15 pulses of 320-490 light (1 J delivered in 0.1 s per pulse).
UV-vis spectroscopy confirmed that the Compound II species was
depleted by the photolysis. The samples were worked up as above
and analyzed by GC-MS in SIM mode.
The methods developed in this work are expected to be useful
for studies of Compound I derivatives of other P450 enzymes.
Further insights into the nature and reactivity of the active
oxidants in P450s should be forthcoming.
Experimental Section
The preparation and isolation of CYP119 followed the methods
previously described.22-24 The enzyme used in these studies had
an R/Z ratio (A416/A280) of >1.5. All of the substrates used in the
oxidation studies were commercial samples of nominally high purity
that were checked by NMR spectroscopy or GC for purity. Basic
solutions containing PN were prepared by the method of Uppu and
Pryor.60
LFP Studies. The spectroscopic and kinetic studies of CYP119
were conducted in a similar manner using a stopped-flow mixing
unit (SX-18, Applied Photophysics) affixed to an LKS-60 laser flash
photolysis kinetics unit (Applied Photophysics). The laser light used
was the third harmonic (355 nm) from a Quantel Brilliant B Nd:
YAG laser that delivered ∼4 mJ to the base of a 2 mm × 10 mm
quartz reaction flow cell that was charged by the mixing unit. A
150 W xenon lamp under continuous irradiation conditions was
used for most studies; the lamp was pulsed during acquisition of
the data used for the CYP119 Compound I spectrum. PMTs with
monochromatic light or a PDA 1 diode-array detector (Applied
Photophysics) with broad spectrum light were used for detection.
When monochromatic light was used, the monochromator was
placed before the reaction cell to ensure that low-intensity light
impinged on the sample.
mCPBA Mixing Study. The experiment followed the procedure
reported by Kellner et al.20 Thus, thermostatted (4 °C) solutions of
CYP119 and mCPBA were mixed in the stopped-flow kinetic unit
to give 14 µM CYP119 and 8.6 µM mCPBA. Data acquisition over
In a typical LFP study, 10 µM CYP119 in 50 mM phosphate
buffer (pH 7.0) was placed in one syringe of the mixing unit, and
0.25 mM PN solution was placed in a second syringe. Equal
volumes of the two syringes were mixed in the reaction cell, and
(61) Yao, Z. J.; Wu, Y. L. J. Org. Chem. 1995, 60, 1170–1176.
(62) Sawaki, Y.; Foote, C. S. J. Am. Chem. Soc. 1979, 101, 6292–6296.
(63) Groziak, M. P.; Chern, J. W.; Townsend, L. B. J. Org. Chem. 1986,
51, 1065–1069.
(60) Uppu, R. M.; Pryor, W. A. Anal. Biochem. 1996, 236, 242–249.
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A R T I C L E S
Sheng et al.
2 s gave 800 spectra. Deconvolution was achieved with global-
analysis software from Applied Photophysics for the two-step model
A + B f C and C f A. The results from the global analysis
program were functions of the initial guesses of the rate constants.
Supporting Information Available: Results of PN mixing
studies (Figure S1) and additional examples of deconvolutions
of spectra from stopped-flow mixing of CYP119 with mCPBA
(Table S1). This material is available free of charge via the
Internet at http://pubs.acs.org.
Acknowledgment. This work was supported by a grant from
the National Institutes of Health (GM-48722). We thank Prof. S. G.
Sligar for providing the DNA for cloning of CYP119.
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