Kinetics of two-electron oxidations by the compound I derivative of chloroperoxidase, a model for cytochrome P450 oxidants

Article abstract of DOI:10.1021/ol060762k

Rate constants for two-electron oxidation reactions of Compound I from chloroperoxidase (CPO) with a variety of substrates were measured by stopped-flow kinetic techniques. The thiolate ligand of CPO Compound I activates the iron-oxo species with the resu

Full text of DOI:10.1021/ol060762k

ORGANIC
LETTERS
2006
Vol. 8, No. 13
2731-2734
Kinetics of Two-Electron Oxidations by
the Compound I Derivative of
Chloroperoxidase, a Model for
Cytochrome P450 Oxidants
Rui Zhang,† Nandini Nagraj,† Dharmika S. P. Lansakara-P.,† Lowell P. Hager,§ and
Martin Newcomb*,†
Department of Chemistry, UniVersity of Illinois at Chicago, Chicago, Illinois 60607,
and Department of Chemistry and Biochemistry, UniVersity of Illinois at Urbana,
Urbana, Illinois 61801
men@uic.edu
Received March 29, 2006
ABSTRACT
Rate constants for two-electron oxidation reactions of Compound I from chloroperoxidase (CPO) with a variety of substrates were measured
by stopped-flow kinetic techniques. The thiolate ligand of CPO Compound I activates the iron oxo species with the result that oxidation
oxo porphyrin radical cations containing weaker binding
−
reactions are 2 to 3 orders of magnitude faster than oxidations by model iron(IV)
counterions.
−
Enzyme-catalyzed oxidation reactions represent an area of
considerable contemporary interest, and oxidations of un-
activated C-H bonds to give alcohol products are of special
interest. The ubiquitous cytochrome P450 enzymes (P450s)
hydroxylate unactivated C-H bonds with facility, and these
heme-containing enzymes serve as models for practical
catalysts.1-3 The true oxidants in P450s have not been
observed under natural conditions, however, nor do they
accumulate to observable amounts under cryogenic condi-
tions when the requisite electrons are provided by γ-radi-
olysis4,5 or by radioactive decay of phosphorus-32.6
The oxidant in a P450 enzyme is usually thought to be an
iron(IV)-oxo porphyrin radical cation, termed Compound
I, by analogy to the intermediates formed in peroxidase and
catalase enzymes,7,8 but differences exist between the latter
(4) Davydov, R.; Macdonald, I. D. G.; Makris, T. M.; Sligar, S. G.;
Hoffman, B. M. J. Am. Chem. Soc. 1999, 121, 10654-10655.
(5) Davydov, R.; Makris, T. M.; Kofman, V.; Werst, D. E.; Sligar, S.
G.; Hoffman, B. M. J. Am. Chem. Soc. 2001, 123, 1403-1415.
(6) Denisov, I. G.; Makris, T. M.; Sligar, S. G. J. Biol. Chem. 2001,
276, 11648-11652.
† University of Illinois at Chicago.
§ University of Illinois at Urbana.
(1) Cytochrome P450 Structure, Mechanism, and Biochemistry, 3rd ed.;
Ortiz de Montellano, P. R., Ed.; Kluwer Academic: New York, 2005.
(2) Sono, M.; Roach, M. P.; Coulter, E. D.; Dawson, J. H. Chem. ReV.
1996, 96, 2841-2887.
(3) Denisov, I. G.; Makris, T. M.; Sligar, S. G.; Schlichting, I. Chem.
ReV. 2005, 105, 2253-2277.
(7) Dawson, J. H. Science 1988, 240, 433-439.
(8) Hiner, A. N. P.; Raven, E. L.; Thorneley, R. N. F.; Garcia-Canovas,
F.; Rodriguez-Lopez, J. N. J. Inorg. Biochem. 2002, 91, 27-34.
10.1021/ol060762k CCC: $33.50
© 2006 American Chemical Society
Published on Web 05/24/2006

enzymes and P450s. The oxidants in P450s are formed by a
sequence of reduction, oxygen binding, and protonation steps
instead of reaction with hydrogen peroxide, and P450
enzymes contain thiolate from cysteine as the fifth ligand to
iron as opposed to nitrogen of histidine or oxygen of tyrosine.
A more subtle difference is that the P450 enzymes are
activated when substrate is bound in the active site, whereas
substrates diffuse into the active sties of “activated” peroxi-
dase and catalase enzymes. Compound I analogues in
peroxidases and biomimetic porphyrin-iron models are
relatively low reactivity oxidizing species, and the high
reactivity of P450 oxidants typically has been ascribed to a
counterion effect of the thiolate ligand that strongly activates
the Compound I derivative by weakening the iron-oxygen
bond.2
One peroxidase enzyme, chloroperoxidase (CPO) from
Caldariomyces fumago, contains a cysteine thiolate ligand
to iron.9,10 CPO is the only known thiolate-heme enzyme
that gives a well-characterized Compound I species, which
has been studied by visible absorption,11 Mo¨ssbauer,12
resonance Raman,13 EPR,12 ENDOR,14 and XAFS15 spec-
troscopies. Compound I of CPO is often considered to be
the best model available for the putative Compound I in P450
enzymes,16 and it catalyzes two-electron, oxo-transfer oxida-
tion reactions that mimic those catalyzed by P450 en-
zymes.17,18 Limited kinetics of one-electron oxidations by
CPO Compound I were reported,19 and no kinetic information
for two-electron oxidation reactions was available, however.
We report here kinetic studies of reactions of CPO Com-
pound I with various two-electron reductants that provide
benchmark data. Most of the substrates studied are known
to be oxidized to alcohols, epoxides and hypohalides by CPO
under turnover conditions.17,18,20 The general findings were
that the rate constants for the CPO Compound I oxidation
reactions are 2-3 orders of magnitude greater than those of
models.
used. CPO was oxidized to the Compound I derivative in
>95% yield as described11 by using 1.5-2.0 equiv of
commercial peroxyacetic acid (32%).23 The reported rate
constant for oxidation of resting CPO with peroxyacetic acid
is ca. 4 × 106 M-1 s-1,24 and comparable rate constants were
observed here. Figure S1 in the Supporting Information
shows typical UV-visible spectral changes upon oxidation
of resting enzyme to the Compound I species.
The kinetics of CPO Compound I oxidation reactions were
measured with a three-syringe, stopped-flow kinetic unit. The
resting enzyme in 100 mM potassium phosphate buffer (pH
4.8) was mixed with the peroxyacetic acid solution. After a
100 ms delay, the solution containing CPO Compound I was
mixed with a solution containing a large excess of substrate.
Kinetics were monitored at 400 nm (growth of the Soret band
of resting enzyme) or at 690 nm (decay of the Q-band of
Compound I). A typical time-resolved spectrum and kinetic
traces are shown in Figure 1.
Figure 1. (A) Time-resolved UV-vis spectrum for reaction of
CPO Compound I with 0.10 mM styrene over 330 ms. (B) Kinetic
traces at 400 nm for reactions of CPO Compound I with styrene at
(from the bottom) 0, 0.10, 0.20, 0.40, 0.8, and 1.6 mM concentra-
tions.
We isolated CPO from C. fumago and purified it by
reported methods.21,22 The enzyme purity was evaluated from
the R/Z value (A400nm/A280nm), and CPO with R/Z > 1.4 was
In the presence of a large excess of substrate, CPO
Compound I decayed with pseudo-first-order kinetics. Second-
order rate constants were determined from eq 1 , where kobs
(9) Morris, D. R.; Hager, L. P. J. Biol. Chem. 1966, 241, 1763-1768.
(10) Sundaramoorthy, M.; Terner, J.; Poulos, T. L. Structure 1995, 3,
1367-1377.
kobs ) k0 + kox[Sub]
(1)
(11) Palcic, M. M.; Rutter, R.; Araiso, T.; Hager, L. P.; Dunford, H. B.
Biochem. Biophys. Res. Commun. 1980, 94, 1123-1127.
(12) Rutter, R.; Hager, L. P.; Dhonau, H.; Hendrich, M.; Valentine, M.;
Debrunner, P. Biochemistry 1984, 23, 6809-6816.
(13) Egawa, T.; Proshlyakov, D. A.; Miki, H.; Makino, R.; Ogura, T.;
Kitagawa, T.; Ishimura, Y. J. Biol. Inorg. Chem. 2001, 6, 46-54.
(14) Kim, S. H.; Perera, R.; Hager, L. P.; Dawson, J. H.; Hoffman, B.
M. J. Am. Chem. Soc. 2006, 128, 5598-5599.
(15) Stone, K. L.; Behan, R. K.; Green, M. T. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 16563-16565.
is the observed pseudo-first-order rate constant, k0 is the
background first-order rate constant for decay in the absence
of substrate, kox is the second-order rate constant, and [Sub]
is the molar concentration of substrate. Plots of kobs versus
[Sub] typically gave straight lines with near-zero intercepts;
examples are shown in Figure 2. We measured the kinetics
in three or four sets of studies for each substrate with three
independent kinetic runs in each set of studies, and the
second-order rate constants are listed in Table 1.
(16) Van Rantwijk, F.; Sheldon, R. A. Curr. Opin. Biotechnol. 2000,
11, 554-564.
(17) Zaks, A.; Dodds, D. R. J. Am. Chem. Soc. 1995, 117, 10419-10424.
(18) Hager, L. P.; Lakner, F. J.; Basavapathruni, A. J. Mol. Catal. B:
Enzym. 1998, 5, 95-101.
(19) Lambeir, A. M.; Dunford, H. B.; Pickard, M. A. Eur. J. Biochem.
1987, 163, 123-127.
Several interesting features are seen in the second-order
rate constants. Many substrates were oxidized successfully
(20) Wagenknecht, H. A.; Woggon, W. D. Chem. Biol. 1997, 4, 367-
372.
(21) Hollenberg, P. F.; Hager, L. P. Methods Enzymol. 1978, 52, 521-
529.
(23) The concentration of peroxyacetic acid was determined by iodo-
metric titration.
(22) Hashimoto, A.; Pickard, M. A. J. Gen. Microbiol. 1984, 130, 2051-
2058.
(24) Araiso, T.; Rutter, R.; Palcic, M. M.; Hager, L. P.; Dunford, H. B.
Can. J. Biochem. 1981, 59, 233-236.
2732
Org. Lett., Vol. 8, No. 13, 2006

of CPO Compound I. At six pH values in the range pH 3 to
7, we observed no effect on the rates of reaction of CPO
Compound I with either styrene or methanol (see the
Supporting Information).
In general, the CPO Compound I kinetic results indicate
an expected high reactivity of the iron-oxo species due to
the thiolate ligand. As shown by Raman spectroscopy,27 the
Fe-O bond strengths of Compound I species weaken as the
counterion binding strength increases, which results in
increased reactivity of iron-oxo species with stronger
binding anions. The kinetic effect of the thiolate counterion
in CPO Compound I is large. For example, the second-order
rate constants for oxidations of ethylbenzene by the Com-
pound I models 1, 2, and 3 are 1.6, 4.5, and 6.2 M-1 s-1,
respectively,28,29 whereas CPO Compound I reacts with PhEt
with a rate constant that is 2-3 orders of magnitude greater
(entry 2). Similarly, the rate constant for epoxidation of
styrene by CPO Compound I (entry 7) is 600 and 3200 times
greater than the rate constants for oxidation of styrene by 2
and 1, respectively.28 A thiolate-substituted model porphy-
rin-iron complex was reported to oxidize substrates about
2 orders of magnitude faster than tetraphenylporphyrin-iron-
(III) chloride under turnover conditions,30 but those rates of
oxidation might reflect a combination of rates for oxidations
of the catalysts by the sacrificial oxidants and substrate
oxidation reactions.
The kinetic isotope effects observed for oxidations of
nondeuterated and perdeuterated ethylbenzene and methanol
(entries 2-5) reflect the high reactivity and concomitant low
selectivity of the CPO Compound I species. For ethylben-
zene, kH/kD ) 2.6, which can be compared to a value of
kH/kD ) 4.4 for oxidations of ethylbenzene and ethylbenzene-
d10 by the model Compound I species 3.29 For methanol, a
similar small kinetic isotope effect was observed, i.e.,
kH/kD ) 3.2.
Figure 2. (A) Observed rate constants for reactions of CPO
Compound I with styrene (red), cinnamyl alcohol (blue), and
2-methyl-1-heptene (green). (B) Observed rate constants for reac-
tions of CPO Compound I with methanol.
by CPO Compound I, and it is apparent that access to the
active site was possible for all but the larger substrates, cis-
stilbene and diphenylmethane (entries 17 and 18). The
seemingly large substrate 2-vinylnaphthalene displayed no
retarding kinetic effect that might be ascribed to sterics,
however (entry 12). The natural substrates chloride and
bromide (entries 19 and 20) reacted very rapidly as expected.
Chloride and bromide also are natural substrates for myelo-
peroxidase, and the rate constants for their reactions with
myeloperoxidase Compound I at pH 7 are k ) 2.5 × 104
and 1.1 × 106 M-1 s-1, respectively.25
Table 1. Second-Order Rate Constants for Reactions of CPO
Compound I with Two-Electron Reductantsa
entry
substrate
kox (M-1 s-1
)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
toluene
(5.5 ( 0.2) × 102
(9.6 ( 0.2) × 102
(3.7 ( 0.5) × 102
(3.2 ( 0.1) × 103
(9.9 ( 0.1) × 102
(2.3 ( 0.1) × 104
(6.1 ( 0.5) × 104
(4.0 ( 0.3) × 104
(6.1 ( 0.4) × 104
(6.7 ( 0.6) × 104
(8.3 ( 0.5) × 104
(1.1 ( 0.1) × 105
(4.3 ( 0.4) × 103
(3.6 ( 0.2) × 103
(6.4 ( 0.5) × 103
NDb
ethylbenzene
ethylbenzene-d10
methanol
methanol-d4
cinnamyl alcohol
styrene
4-chlorostyrene
4-fluorostyrene
4-methylstyrene
4-methoxystyrene
2-vinylnaphthalene
2-methyl-1-heptene
2-methyl-2-heptene
10-undecenoic acid
lauric acid
A further reflection of the high reactivity of CPO
Compound I is seen in the linear Hammett plot for oxidations
(25) Furtmu¨ller, P. G.; Burner, U.; Obinger, C. Biochemistry 1998, 37,
17923-17930.
cis-stilbene
diphenylmethane
chloride
NDb
NDb
(26) Green, M. T.; Dawson, J. H.; Gray, H. B. Science 2004, 304, 1653-
(2.5 ( 0.1) × 104
1656.
bromide
(3.8 ( 0.1) × 105
(27) Czarnecki, K.; Nimri, S.; Gross, Z.; Proniewicz, L. M.; Kincaid, J.
R. J. Am. Chem. Soc. 1996, 118, 2929-2935.
a Reactions at 22 ( 1 °C in 100 mM phosphate buffer (pH 4.8). In the
(28) Pan, Z.; Zhang, R.; Newcomb, M. J. Inorg. Biochem. 2006, 100,
524-532.
absence of substrate, the Compound I derivative decayed with a first-order
rate constant of ca. 0.5 s-1 b Not detected; no acceleration in the rate of
.
(29) Zhang, R.; Chandrasena, R. E. P.; Martinez, E., II; Horner, J. H.;
Newcomb, M. Org. Lett. 2005, 7, 1193-1195.
decay of Compound I was observed with these substrates; see the Supporting
Information.
(30) For studies with thiolate-ligated iron-porphyrin models, see: (a)
Higuchi, T.; Uzu, S.; Hirobe, M. J. Am. Chem. Soc. 1990, 112, 7051-
7053. (b) Higuchi, T.; Shimada, K.; Maruyama, N.; Hirobe, M. J. Am. Chem.
Soc. 1993, 115, 7551-7552. (c) Urano, Y.; Higuchi, T.; Hirobe, M.; Nagano,
T. J. Am. Chem. Soc. 1997, 119, 12008-12009. (d) Suzuki, N.; Higuchi,
T.; Nagano, T. J. Am. Chem. Soc. 2002, 124, 9622-9628.
Recent studies suggest that Compound II of CPO is
relatively basic.26 We evaluated pH effects on the reactivity
Org. Lett., Vol. 8, No. 13, 2006
2733

of the series of substituted styrenes in entries 7-11 (Figure
3). For the model oxidant species 1 with a weakly binding
P450cam suggest that functionalization of the unactivated
C-H bond in the substrate has a small activation energy.4-6
An apparent attenuated reactivity of CPO Compound I in
comparison to the P450 oxidants is indicated in our kinetic
results in general. The second-order rate constants we
obtained for reactions of CPO Compound I clearly are not
diffusional rate constants, which would be found if the
oxidation reactions were so fast that substrate could not leave
the active site. If that were the case, then the measured
second-order rate constants would be much greater than
found here, substrates of similar size would react with the
same rate constants, the slope of the Hammett plot in Figure
3 would be zero, and kinetic isotope effects of kH/kD ) 1.0
would be found. In contrast, the catalytic sequence for P450
enzymes is triggered only after the substrate is bound in the
active site, and the substrate does not escape from the active
site after the oxidant is formed.
The kinetic results for CPO Compound I two-electron
oxidations of organic substrates provide kinetic and energetic
information that is relevant to the P450 oxidants. The thiolate
ligand in CPO Compound I results in acceleration of
oxidation reactions in comparison to Compound I models
with weaker binding counterions,28 but the reactivity of this
transient apparently is not as great as that required for the
true oxidants in P450s. The CPO results are similar to the
finding in a recent study where a cytochrome P450 Com-
pound I species produced via a photooxidation reaction from
the Compound II species was found not to react with lauric
acid even though laurate is a substrate for the P450 under
catalytic turnover conditions.34 One possible explanation for
these results is that the active oxidant in P450 enzymes is
not a Compound I species but a high-energy isomer of
Compound I, i.e., a highly reactive iron(V)-oxo species.35-37
Figure 3. Hammett plots for reactions of para-substituted styrenes
with the model Compound I species 1 (triangles), the model
Compound I species 2 (circles), and CPO Compound I (squares).
The data for species 1 and 2 are from ref 28.
counterion perchlorate, the F+ value was -1.96,28 which is
similar to the values found with other Compound I species
with weak binding counterions.31,32 For model oxidant species
2 with the stronger binding counterion chloride, F+
)
-0.89,28 again a value similar to that for other chloride
complexed Compound I species.32,33 For CPO Compound I,
however, F+ ) -0.28, which is the smallest F+ value yet
found for oxo transfer reactions from porphyrin-metal-oxo
species.
One of the more noteworthy aspects of the CPO Com-
pound I kinetics is the fact that no reaction was apparent
with lauric acid (entry 16). The successful oxidation of 10-
undecenoic acid with a rate constant similar to that for
oxidations of other alkenes (entries 13-15) suggests that the
long hydrocarbon chain in this substrate, and by analogy the
hydrocarbon chain of lauric acid, readily accessed the CPO
Compound I active site. We observed no increase in the rate
of decay of CPO Compound I species in the presence of
lauric acid, however. A pseudo-first-order rate constant of
kobs ) 1 s-1, which would result from a second-order rate
constant for reaction of 1 mM laurate of kox ) 500 M-1 s-1
as found for toluene, would have been measured readily in
these studies. Thus, the limit for the free energy of activation
for the oxidation of lauric acid is ∆Gq > 13.5 kcal/mol. In
contrast, low-temperature studies of camphor oxidations by
Acknowledgment. A portion of this work was supported
in part by a grant from the National Institutes of Health
(GM48722 to M.N.).
Supporting Information Available: Experimental de-
tails, time-resolved UV-visible spectrum of the formation
of CPO Compound I from reaction of the resting enzyme
with peroxyacetic acid, and results of background control
experiments and pH effect studies. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL060762K
(34) Newcomb, M.; Zhang, R.; Chandrasena, R. E. P.; Halgrimson, J.
A.; Horner, J. H.; Makris, T. M.; Sligar, S. G. J. Am. Chem. Soc. 2006,
128, 4580-4581.
(31) Groves, J. T.; Watanabe, Y. J. Am. Chem. Soc. 1986, 108, 507-
508.
(32) Song, W. J.; Ryu, Y. O.; Song, R.; Nam, W. J. Biol. Inorg. Chem.
2005, 10, 294-304.
(33) Lindsay Smith, J. R.; Sleath, P. R. J. Chem. Soc., Perkin Trans. 2
1982, 1009-1015.
(35) Newcomb, M.; Chandrasena, R. E. P. Biochem. Biophys. Res.
Commun. 2005, 338, 394-403.
(36) Harischandra, D. N.; Zhang, R.; Newcomb, M. J. Am. Chem. Soc.
2005, 127, 13776-13777.
(37) Pan, Z.; Zhang, R.; Fung, L. W.-M.; Newcomb, M., Submitted for
publication.
2734
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