Reactions of the peroxo intermediate of soluble methane monooxygenase hydroxylase with ethers

Article abstract of DOI:10.1021/ja050865i

Soluble methane monooxygenase (sMMO) isolated from Methylococcus capsulatus (Bath) utilizes a carboxylate-bridged diiron center and dioxygen to catalyze the conversion of methane to methanol. Previous studies revealed that a di(μ-oxo)diiron(IV) intermediate termed Q is responsible for the catalytic activity with hydrocarbons. In addition, the peroxodiiron(III) intermediate (H_peroxo) that precedes Q formation in the catalytic cycle has been demonstrated to react with propylene, but its reactivity has not been extensively investigated. Given the burgeoning interest in the existence of multiple oxidants in metalloenzymes, a more exhaustive study of the reactivity of H_peroxo was undertaken. The kinetics of single turnover reactions of the two intermediates with ethyl vinyl ether and diethyl ether were monitored by single- and double-mixing stopped-flow optical spectroscopy. For both substrates, the rate constants for reaction with H_peroxo are greater than those for Q. An analytical model for explaining the transient kinetics is described and used successfully to fit the observed data. Activation parameters were determined through temperature-dependent studies, and the kinetic isotope effects for the reactions with diethyl ether were measured. The rate constants indicate that H_peroxo is a more electrophilic oxidant than Q. We propose that H_peroxo reacts via two-electron transfer mechanisms, and that Q reacts by single-electron transfer steps.

Full text of DOI:10.1021/ja050865i

Published on Web 04/27/2005
Reactions of the Peroxo Intermediate of Soluble Methane
Monooxygenase Hydroxylase with Ethers
Laurance G. Beauvais and Stephen J. Lippard*
Contribution from the Department of Chemistry, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139
Received February 9, 2005; E-mail: lippard@mit.edu
Abstract: Soluble methane monooxygenase (sMMO) isolated from Methylococcus capsulatus (Bath) utilizes
a carboxylate-bridged diiron center and dioxygen to catalyze the conversion of methane to methanol.
Previous studies revealed that a di(µ-oxo)diiron(IV) intermediate termed Q is responsible for the catalytic
activity with hydrocarbons. In addition, the peroxodiiron(III) intermediate (Hperoxo) that precedes Q formation
in the catalytic cycle has been demonstrated to react with propylene, but its reactivity has not been
extensively investigated. Given the burgeoning interest in the existence of multiple oxidants in metalloen-
zymes, a more exhaustive study of the reactivity of Hperoxo was undertaken. The kinetics of single turnover
reactions of the two intermediates with ethyl vinyl ether and diethyl ether were monitored by single- and
double-mixing stopped-flow optical spectroscopy. For both substrates, the rate constants for reaction with
Hperoxo are greater than those for Q. An analytical model for explaining the transient kinetics is described
and used successfully to fit the observed data. Activation parameters were determined through temperature-
dependent studies, and the kinetic isotope effects for the reactions with diethyl ether were measured. The
rate constants indicate that Hperoxo is a more electrophilic oxidant than Q. We propose that Hperoxo reacts
via two-electron transfer mechanisms, and that Q reacts by single-electron transfer steps.
Introduction
Multiple oxidizing species also occur in synthetic analogues
of dicopper-containing hydroxylases.1c A (µ-η2:η2)peroxodi-
Mechanistic studies of metalloenzymes have expanded our
understanding of how nature utilizes a small set of metals and
ligands to perform a wide variety of tasks. In the case of
oxygenases, much more complicated catalytic cycles have
emerged than were first envisioned.1 For example, the long
accepted radical rebound mechanism mediated by the FeIVdO
porphyrin cation radical (Cpd I) in cytochrome P450 reactions2
has been challenged by the proposal that an Fe(III)-hydro-
peroxo intermediate (Cpd 0), which precedes formation of Cpd
I, can serve as a second, electrophilic oxidant. Evidence for a
second oxidant derives from product distribution and kinetic
isotope effects for reactions of wild-type P450 enzymes.1d,e,3
An alternative explanation has been proposed for these results,
namely, that Cpd I exists in both low and high spin states, each
of which reacts by a different mechanism.1b,4 An experimental
resolution of this controversy suffers from the lack of an
appropriate spectroscopic technique to monitor the reactions of
the two intermediates, which cannot be deconvoluted by
transient kinetics studies.
copper(II) center is assigned as the active oxidant in biological
systems,5 but small molecule mimics reveal a diverse range of
peroxodicopper(II) and di(µ-oxo)dicopper(III) species.1c,6 The
relative energies of the peroxo and oxo forms can be modulated
by judicious choice of supporting ligands.1c Intermediates in
the activation of dioxygen by dicopper complexes can be ob-
served by stopped-flow optical spectroscopy. A similar situa-
tion obtains for reactions of dioxygen with carboxylate-
bridged diiron proteins, in which peroxodiiron(III), di(µ-oxo)di-
iron(III,IV), and diiron(IV) intermediates have been detected.
Soluble methane monooxygenase (sMMO), an enzyme system
(3) Selected references: (a) Vaz, A. D. N.; Pernecky, S. J.; Raner, G. M.;
Coon, M. J. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4644-4648. (b) Vaz,
A. D. N.; McGinnity, D. F.; Coon, M. J. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 3555-3560. (c) Zakharieva, O.; Trautwein, A. X.; Veeger, C.
Biophys. Chem. 2000, 88, 11-34. (d) Volz, T. J.; Rock, D. A.; Jones, J. P.
J. Am. Chem. Soc. 2002, 124, 9724-9725. (e) Vatsis, K. P.; Coon, M. J.
Arch. Biochem. Biophys. 2002, 397, 119-129. (f) Newcomb, M.; Hollen-
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Newcomb, M.; Aebisher, D.; Shen, R.; Chandrasena, R. E. P.; Hollenberg,
P. F.; Coon, M. J. J. Am. Chem. Soc. 2003, 125, 6064-6065. (h) Jin, S.;
Makris, T. M.; Bryson, T. A.; Sligar, S. G.; Dawson, J. H. J. Am. Chem.
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(1) The role of multiple intermediates in enzyme-catalyzed reactions was
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Chem. 2004, 9, 643. (b) Shaik, S.; de Visser, S. P.; Kumar, D. J. Biol.
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J. H. J. Biol. Inorg. Chem. 2004, 9, 644-653. (f) Que, L., Jr. J. Biol. Inorg.
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1591-1595. (b) Yamazaki, S.-i.; Itoh, S. J. Am. Chem. Soc. 2003, 125,
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7370
J. AM. CHEM. SOC. 2005, 127, 7370-7378
10.1021/ja050865i CCC: $30.25 © 2005 American Chemical Society

Reactions of the Peroxo Intermediates of sMMO Hydroxylase
A R T I C L E S
Scheme 1
functional theoretical calculations, which accounts for many of
the observed reactions of MMOH.12,13 The calculations re-
veal that Q reacts by two sequential single-electron transfer
events.12,13
Although the reactivity of Q has been extensively probed,
that of Hperoxo has not been fully explored. Early indications
of multiple oxidizing intermediates in sMMO arose from
“peroxide shunt” reactions, in which Hox is activated by
hydrogen peroxide in the absence of MMOB.14 Different product
distributions are obtained for these reactions compared to those
employing the complete sMMO enzyme system. The greatest
difference occurs for reactions with trans-2-butene, where the
product distribution for the wild-type system revealed 68%
alcohol, whereas the peroxide shunt reaction afforded 97%
epoxide.14 This difference could arise from an increase in the
activity of the peroxo intermediate compared to that of Q. In
addition, sMMO reactions with radical clock substrate probes15
yield products derived from a cationic intermediate, which were
postulated to arise from reaction of a hydroperoxo species with
substrate.12
that converts methane to methanol in methanotrophic bacteria,
is unique among wild-type carboxylate-bridged diiron proteins,
in that both (peroxo)diiron(III) (Hperoxo) and high-valent di(µ-
oxo)diiron(IV) (Q) intermediates have sufficiently long lifetimes
and extinction coefficients to be followed by stopped-flow
optical spectroscopy.7 Thus, sMMO is well suited for studying
the role of multiple oxidants in a biological system.
sMMO isolated from Methylococcus capsulatus (Bath) (Mc)
and Methylosinus trichosporium OB3b (Mt) have been exten-
sively investigated. Substrate hydroxylation occurs at a car-
boxylate-bridged diiron center residing in the R subunit of the
hydroxylase protein MMOH, a 251 kDa R2â2γ2 heterodimer.
In addition to MMOH, two other proteins are required for
activity in vitro, a 38.5 kDa reductase MMOR, which receives
electrons from NADH and then transfers them to MMOH, and
a 16 kDa regulatory protein MMOB necessary for efficient
catalysis.7 A range of spectroscopic techniques revealed the
existence of intermediates in the reaction of reduced MMOH
(Hred) with dioxygen in the presence of 2 equiv of MMOB
(Scheme 1).7 The first observable intermediate is Hperoxo, which
converts rapidly to Q. In the absence of substrates, Q slowly
decays to afford oxidized protein (Hox).
The first direct evidence for Hperoxo reactivity arose from a
stopped-flow optical spectroscopic study carried out in our
laboratory, which revealed the decay rate of the peroxo
intermediate to increase when exposed to increasing concentra-
tions of propylene.16 The data covered only a small range of
substrate concentration because of the limited solubility of
propylene in water, and the differences in the observed rate
constants are not large. Using protein isolated from Mt, others
17
failed to observe reactions of propylene with Hperoxo and
suggested that substrate might increase the rate of Q formation.
At the low protein and substrate concentrations used in their
study, however, the rate constants for reaction with Hperoxo would
be lower or near the value of peroxo conversion to Q, and
consequently, that reaction could not be observed. In addition,
they monitored the absorbance change near 420 nm, whereas it
is essential to follow reactions of Hperoxo at its absorption
maximum of 720 nm to avoid interference from Q.
Studies of MMOH reactions with substrates other than
methane provide valuable information for understanding the
hydroxylation mechanism. Reactions employing isotopically
labeled substrates allow for the determination of kinetic isotope
effects (KIEs), the magnitude of which gives information about
the transition state.8-10 The reactions of Q with CH4, CH3CN,
and nitromethane exhibit KIEs larger than the theoretical limit
for classical mechanisms,8,9,11 indicating that C-H bond break-
ing occurs by tunneling at the transition state and is rate-limiting
for these reactions. For larger substrates, the KIE approaches
1, and it is postulated that substrate binding or product release
becomes rate-limiting.9-11 Reactions with radical clock substrate
probes demonstrate that a discrete radical is not involved in
the hydroxylation mechanism.12 These experimental results,
when coupled with recent theoretical studies, have allowed a
detailed mechanism to be postulated, based on combined
semiclassical molecular dynamics simulations and density
To provide a more definitive assessment of the relative
reactivity of the MMOH intermediates with substrates, we have
investigated the reactions of ethyl vinyl ether and diethyl ether
by single- and double-mixing stopped-flow optical spectroscopy.
(13) (a) Gherman, B. F.; Dunietz, B. D.; Whittington, D. A.; Lippard, S. J.;
Friesner, R. A. J. Am. Chem. Soc. 2001, 123, 3836-3837. (b) Guallar, V.;
Gherman, B. F.; Lippard, S. J.; Friesner, R. A. Curr. Opin. Chem. Biol.
2002, 6, 236-242. (c) Guallar, V.; Gherman, B. F.; Miller, W. H.; Lippard,
S. J.; Friesner, R. A. J. Am. Chem. Soc. 2002, 124, 3377-3384. (d) Baik,
M.-H.; Gherman, B. F.; Friesner, R. A.; Lippard, S. J. J. Am. Chem. Soc.
2002, 124, 14608-14615. (e) Gherman, B. F.; Baik, M.-H.; Lippard, S.
J.; Friesner, R. A. J. Am. Chem. Soc. 2004, 126, 2978-2990. (f) Gherman,
B. F.; Lippard, S. J.; Friesner, R. A. J. Am. Chem. Soc. 2005, 127, 1025-
1037.
(14) (a) Andersson, K. K.; Froland, W. A.; Lee, S.-K.; Lipscomb, J. D. New J.
Chem. 1991, 15, 411-415. (b) Jiang, Y.; Wilkins, P. C.; Dalton, H. Biochim.
Biophys. Acta 1993, 1163, 105-112.
(15) Selected references: (a) Dalton, H.; Golding, B. T.; Waters, B. W.; Higgins,
R.; Taylor, J. A. J. Chem. Soc., Chem. Commun. 1981, 482-483. (b)
Ruzicka, F.; Huang, D.-S.; Donnelly, M. I.; Frey, P. A. Biochemistry 1990,
29, 1696-1700. (c) Liu, K. E.; Johnson, C. C.; Newcomb, M.; Lippard, S.
J. J. Am. Chem. Soc. 1993, 115, 939-947. (d) Valentine, A. M.; LeTadic-
Biadatti, M.-H.; Toy, P. H.; Newcomb, M.; Lippard, S. J. J. Biol. Chem.
1999, 274, 10771-10776. (e) Choi, S.-Y.; Eaton, P. E.; Kopp, D. A.;
Lippard, S. J.; Newcomb, M.; Shen, R. J. Am. Chem. Soc. 1999, 121,
12198-12199. (f) Jin, Y.; Lipscomb, J. D. Biochemistry 1999, 38, 6178-
6186. (g) Jin, Y.; Lipscomb, J. D. Biochim. Biophys. Acta 2000, 1543,
47-59. (h) Newcomb, M.; Shen, R.; Lu, Y.; Coon, M. J.; Hollenberg, P.
F.; Kopp, D. A.; Lippard, S. J. J. Am. Chem. Soc. 2002, 124, 6879-6886.
(16) Valentine, A. M.; Stahl, S. S.; Lippard, S. J. J. Am. Chem. Soc. 1999, 121,
3876-3887.
(7) Merkx, M.; Kopp, D. A.; Sazinsky, M. H.; Blazyk, J. L.; Mu¨ller, J.; Lippard,
S. J. Angew. Chem., Int. Ed. 2001, 40, 2782-2807 and references therein.
(8) Green, J.; Dalton, H. J. Biol. Chem. 1989, 264, 17698-17703.
(9) Nesheim, J. C.; Lipscomb, J. D. Biochemistry 1996, 35, 10240-10247.
(10) Brazeau, B. J.; Lipscomb, J. D. Int. Congr. Ser. 2002, 1233, 229-233.
(11) Ambundo, E. A.; Friesner, R. A.; Lippard, S. J. J. Am. Chem. Soc. 2002,
124, 8770-8771.
(12) Baik, M.-H.; Newcomb, M.; Friesner, R. A.; Lippard, S. J. Chem. ReV.
2003, 103, 2385-2419.
(17) Brazeau, B. J.; Lipscomb, J. D. Biochemistry 2000, 39, 13503-13515.
9
J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005 7371

A R T I C L E S
Scheme 2
Beauvais and Lippard
Scheme 3
These molecules have increased solubility and greater nucleo-
philicity than simple alkanes or alkenes, such as methane and
propylene. We derive analytical equations for analyzing the
kinetic data obtained from transient kinetic studies of the reac-
tions of Hperoxo and Q with these substrates. Activation param-
eters of the relative rate constants for the two intermediates
provide significant insight into the reaction mechanisms, al-
lowing us to conclude unambiguously that Hperoxo is reactive,
and we propose that it prefers two-electron transfer steps.
Reactions time courses were monitored at 420 or 720 nm using a
photomultiplier detector. Data collection and analysis were performed
by using KinetAsyst 3 (Hi-Tech Scientific) and KalediaGraph v. 3.51
(Synergy Software) software.
Analysis of Stopped-Flow Data. Transient-state kinetic data are
typically fit with multiple exponentials and not with analytically derived
equations. This procedure yields observed rate constants and amplitudes,
each of which is typically assigned to one process. These amplitudes
can be complex functions of rate constants, extinction coefficients, and
intermediate concentrations. It can be difficult to extract meaningful
parameters from them without the use of a full model for the reaction
and corresponding mathematical analysis.20 Therefore, a model based
on an A f B f C f D mechanism21 was modified to account for the
ability of B (Hperoxo) and C (Q) to react with substrates to give D and
products. It is well established that two exponentials differing by a
factor of 7 or less cannot be distinguished.20 Double-mixing experiments
were therefore utilized to determine rate constants for the reactions of
Hperoxo and Q, to obtain initial parameters for use in fitting the single-
mixing data for the reactions.
Experimental Section
General Considerations. The regulatory (MMOB) and reductase
(MMOR) proteins were prepared recombinantly in Escherichia coli
and purified as described elsewhere.18 Distilled water was deionized
with a Milli-Q filtering system. Diethyl ether was saturated with argon
and purified by passing through an activated alumina column under
argon. Other reagents were of commercial origin and used as received.
MMOH Purification. Purification of the hydroxylase (MMOH)
from native Methylococcus capsulatus (Bath) as previously described19
affords protein that is sometimes contaminated with a small amount of
cytochrome that exhibits an absorption band at 410 nm. Protein obtained
from this two-step purification procedure typically has a specific
activity, as measured for propylene oxidation, in the range of
200-400 mU/mg at 45 °C. Further purification with an Amersham
Biosciences MonoQ column allows for separation of MMOH from the
cytochrome. Protein eluted from the S-300 size-exclusion column was
desalted and concentrated to ca. 150 µM. Between 250 and 350 mg of
this concentrated protein was loaded onto a MonoQ column (1 × 8
cm) equilibrated in 25 mM MOPS (pH 7.0) containing 10% glycerol.
After the sample was loaded, the column was washed with 10 mL of
a 50 mM NaCl solution followed by a linear 200 mL gradient of 0-200
mM NaCl in the same buffer. The hydroxylase eluted from the column
at approximately 100 mM NaCl. Hydroxylase-containing fractions were
identified by absorbance at 280 nm and SDS-PAGE assays. Pooled
fractions were concentrated with an Amicon YM-100 membrane, flash-
frozen in liquid nitrogen, and stored at -80 °C. The resulting protein
had a specific activity at 45 °C of 400-750 mU/mg, as determined for
propylene oxidation.
Stopped-Flow Visible Spectroscopy. Single turnover kinetics
experiments were performed on a Hi-Tech Scientific (Salisbury, U.K.)
SF-61 DX2 stopped-flow spectrophotometer as described in detail
elsewhere.16 The hydroxylase was reduced with dithionite and methyl
viologen in the presence of 2 equiv of MMOB in 25 mM MOPS (pH
7.0) solution, as previously reported.16 For experiments monitoring
reactions of Hperoxo, the concentration of Hred:2B in the optical cell was
50 µM; otherwise, the concentration was 25 µM. Single-mixing
experiments were performed by rapidly mixing the reduced protein
solution with a dioxygen-saturated buffered solution. For single-mixing
studies in the presence of substrate, the latter was dissolved in the
dioxygen-saturated solution. Hperoxo and Q are generated by rapidly
mixing a fully reduced, anaerobic buffered hydroxylase (Hred) solution
containing 2 equiv of MMOB with O2-saturated buffer in the initial
push of a double-mixing experiment. After a specified time delay that
coincides with maximal concentration of either Hperoxo or Q, substrate-
containing buffer is introduced in a second push to initiate the reaction.
Single-Mixing Experiments. Single-mixing experiments were em-
ployed to determine rate constants for the formation and decay of the
intermediates and their extinction coefficients. In the absence of
substrate, the absorbance versus time (Absλ(t)) curves for reaction of
Hred:2B with a large excess of O2 at λ ) 420 nm were fit to an A f
B f C f D model (Scheme 2) by using eq 1, where [Hred]0 is the
initial concentration of hydroxylase, ꢀ is extinction coefficient for the
designated species, and k1′, k2, and k3 are rate constants for Hperoxo
formation, Hperoxo conversion to Q, and Q conversion to Hox, respec-
tively. A full derivation of eq 1 is supplied as Supporting Information.
In the presence of substrates that react with Hperoxo and Q, Absλ(t)
can be fit to eq 2 based on the model shown in Scheme 3, where k′ )
(k2 + kR[S]), the sum of the rate constants for peroxo conversion to Q
(k2) and its reaction with substrate (kR[S]), and k′′ ) (k3 + kâ[S]), the
sum of the rate constants for Q conversion to Hox (k3) and its reaction
with substrate (kâ[S]). A full derivation of eq 2 is given in Supporting
Information. It is apparent from eq 2 that determination of the rate
constants for the reaction of substrate with Hperoxo and Q by single-
mixing experiments would be a challenge. However, since the absorp-
tion maxima for Hperoxo and Q are well separated in energy, and each
reaches a maximum concentration at distinct times, it is possible to
study the reactions using double-mixing stopped-flow techniques.
(18) (a) Coufal, D. E.; Blazyk, J. L.; Whittington, D. A.; Wu, W. W.;
Rosenzweig, A. C.; Lippard, S. J. Eur. J. Biochem. 2000, 267, 2174-
2185. (b) Kopp, D. A.; Gassner, G. T.; Blazyk, J. L.; Lippard, S. J.
Biochemistry 2001, 40, 14932-14941.
(19) Gassner, G. T.; Lippard, S. J. Biochemistry 1999, 38, 12768-12785.
(20) (a) Pettersson, G. Acta Chem. Scand. B 1978, 32, 437-446. (b) Fisher, H.
F. In Enzymatic Mechanisms; Frey, P. A., Northrup, D. B., Eds.; IOS
Press: Amsterdam, 1999; pp 264-277.
(21) Szabo´, Z. G. In The Theory of Kinetics; Bamford, C. H., Tipper, C. F. H.,
Eds.; Elsevier: New York, 1969; Vol. 2, pp 20-24.
9
7372 J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005

Reactions of the Peroxo Intermediates of sMMO Hydroxylase
A R T I C L E S
Scheme 4
Scheme 5
Double-Mixing Experiments. In the absence of substrate, Hperoxo
decays to Q with rate constant k2. In the presence of substrate, the
data at 720 nm can be fit well with two exponentials, as shown in eq
3, corresponding to the kinetic model of Scheme 4, where A1 and A2
are time-independent constants for the corresponding exponentials, kobs1
) k2 + kR[S] and kobs2 ) k3 + kâ[S]. Reactions of Hperoxo with ethyl
vinyl ether display a hyperbolic dependence on [S] at low tempera-
ture. This behavior may result from a change in protein conforma-
tion that precedes substrate oxidation or from the reversible formation
of an intermediate:substrate complex. Similar behavior has been
observed for the reactions of Q with acetonitrile and nitromethane,11
and mixed quantum mechanics/molecular mechanics calculations
indicate that the behavior is consistent with the formation of a Q:S
complex.13f Substrate can bind reversibly to Hperoxo, forming a complex
with the dissociation constant Kd. The complex can react to afford
oxidized product with rate constant ksat. At high [S], the peroxo
intermediate exists almost entirely as the enzyme-substrate com-
plex. In this limit, kobs1 becomes equivalent to ksat. At low [S], the
observed rate constant approaches k2. Simultaneously, Hperoxo is
converting to Q, which then decays with rate constant k′′. Thus, the
observed data are fit as a sum of two exponentials, one of which
corresponds to the rate constant for Q decay and can be determined in
a separate experiment. The other exponential corresponds to Hperoxo
decay. This explanation is expressed in the model shown in Scheme 5,
and the data can be fit to eq 4, where ksat is the rate constant for the
reaction of Hperoxo with S, and Kd is the dissociation constant for
substrate binding to Hperoxo. A derivation of eq 4 is given in Supporting
Information.
values to the standard entropy of activation (∆Sq°), defined at [S] ) 1
M, using eq 6.
-∆Gq
RT
kBT
k )
exp
(5)
(6)
(
)
h
∆Sq° ) ∆Sq[S] - R ln [S]
Product Determination. Product formation from steady-state oxida-
tions of diethyl ether and ethyl vinyl ether were monitored by GC and
LC-MS, respectively. Solutions of each of the three protein compo-
nents ([MMOH] ) 10 µM; [MMOB] ) 20 µM; [MMOR] ) 5 µM),
NADH, and substrate were prepared in air-saturated 25 mM MOPS
(pH 7.0), and the reactions were carried out as described below.
Diethyl Ether. The products of diethyl ether oxidation by sMMO
under steady-state conditions using soluble cell extracts were previously
determined to be ethanol and acetaldehyde.22 The use of purified protein
components affords the same products, as determined by GC analysis
employing a Hewlett-Packard 5890 gas chromatograph. The reactions
were carried out in a total volume of 1 mL with 5 mM NADH and 5
mM diethyl ether at 45 °C. After 0 and 15 min, aliquots of the reac-
tion solution (5 µL) were injected onto an Alltech Porapak Q column
1
(6’ × /8”) equilibrated at 60 °C. After 10 min, the temperature was
ramped at 10 °C/min to 160 °C. Under these conditions, authentic
samples of acetaldehyde, ethanol, and diethyl ether exhibited retention
times of 15.9, 19.6, and 21.4 min, respectively. Under the same
conditions, in the absence of NADH, no ethanol or acetaldehyde was
detected. Representative GC traces are shown in Figure S1 (Supporting
Information).
Absλ(t) ) A1e-k t + A2e-k t + c
(3)
(4)
obs1
obs2
ksat[S]
kobs1 ) k2 +
Kd + [S]
Ethyl Vinyl Ether. GC analysis of ethyl vinyl ether reactions is
complicated because the substrate decomposes on the column. There-
fore, analysis of the reaction was carried out in a manner similar to
that used for the reaction of p-nitrophenyl vinyl ether with microsomal
P450.23 The steady-state reaction was carried out in 0.5 mL containing
2 mM NADH and 2 mM ethyl vinyl ether at 20 °C. After 20 min, the
reaction was terminated by addition of 100 µL of 6 mM 2,4-
dinitrophenylhydrazine (2,4-DNPH) dissolved in 6 M HCl. The solution
was heated at 100 °C for 30 min to ensure conversion of the aldehyde
products to their corresponding hydrazones, which were extracted into
benzene (200 µL). Aliquots (100 µL) of the benzene solution were
dried, and the solid residue was dissolved in 100 µL of methanol and
analyzed by HPLC. The analysis was performed using an Agilent 1100
series LC/MSD trap equipped with a VYDAC C-18 reverse-phase
column with methanol:water (3:2) as the mobile phase. Under these
conditions, 2,4-DNPH, glycolaldehyde-2,4-dinitrophenylhydrazone, and
acetaldehyde-2,4-dinitrophenylhydrazone eluted at 7.1, 10.0, and 13.3
min, respectively. The identity of each substance was determined by
The value of kâ can be determined by double-mixing experi-
ments of Q with substrates at 420 nm. In this case, only a single
exponential, which corresponds to kobs2 from the 720 nm data, is required
to fit the data. After determining the values for the extinction
coefficients, k1′, k2, and k3, from single-mixing experiments and the
values of kR and kâ from double-mixing experiments, it is possible to
fit the data obtained from single-mixing experiments in the presence
of substrate.
Determination of Activation Parameters. For a first-order rate
constant, the activation parameters for a reaction can be deter-
mined by using the Eyring equation (eq 5). The enthalpy of activation
(∆Hq) for the formation and decay of the intermediates was deter-
mined from the slope of a plot of ln(k/T) versus 1/T, and the entropy
of activation (∆Sq) from the slope of a plot of T‚ln(k/T) versus T,
where k is k1′, k2, or k3. For reactions of the intermediates with
substrates, kR[S] and kâ[S] were extracted from the observed rate
constants, kobs1 and kobs2, by subtracting k2 and k3, respectively, and fit
to the Eyring equation to obtain ∆Hq° and ∆Sq[S], where the entropy of
activation is dependent on substrate concentration. To compare activa-
tion entropies for second-order reactions, it is necessary to convert the
(22) Colby, J.; Stirling, D. I.; Dalton, H. Biochem. J. 1977, 165, 395-402.
(23) Isobe, M.; Sone, T.; Takabatake, E. J. Phamacobio-Dyn. 1985, 8, 614-
622.
9
J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005 7373

A R T I C L E S
Beauvais and Lippard
Table 2. Activation Parameters for Formation and Decay of
Table 1. Extinction Coefficients for Intermediates Observed in the
Reaction of Hred:2B with Dioxygen
Intermediates Observed in the Reaction of Hred:2B with Dioxygen
in 25 mM MOPS (pH 7)
1
species
ꢀ420 (M-1 cm-
)
b
b
rate
constanta
∆
Hq
∆
Hq
∆
Sq
(cal/mol
∆
Sq
H
red:2B
Hperoxo
Q
3200
4600
9850
(kcal/mol)
(kcal/mol)
‚
K)
(cal/mol‚K)
k1′
k2
k3
27.9 ( 0.1
28.7 ( 0.3
13.7 ( 0.7
22 ( 4
29 ( 1
14 ( 1
43 ( 2
43.9 ( 0.5
-18 ( 2
21 ( 10
45 ( 3
-15 ( 3
Hox
3400 ( 200
a Values of k1′, k2, and k3 refer to Hperoxo formation, Hperoxo conversion
comparison to hydrazones prepared from authentic glycolaldehyde
dimer and acetaldehyde.
to Q, and Q conversion to Hox, respectively. b Values from ref 16.
Results
Analysis of the products from the steady-state reaction of
ethyl vinyl ether reveals ethanol, glycolaldehyde, and acetal-
dehyde. The acetaldehyde is formed by acid-catalyzed decom-
position of ethyl vinyl ether, which also produces ethanol. The
epoxidation product is unstable, and its decomposition yields
ethanol and glycolaldehyde; thus, both intermediates epoxidize
the double bond. The instability of epoxides of alkyl vinyl ethers
in water and their decomposition to glycolaldehyde and the
corresponding alcohol have been previously reported.24 The
other possible products are ethylene glycol vinyl ether, which
was not observed by GC analysis, and the hemiacetal resulting
from hydroxylation at the R-carbon atom of the ethyl group,
which would rapidly decompose to afford 2 equiv of acetalde-
hyde.
Figure 1. Plot of kobs versus ethyl vinyl ether concentration for the decay
of Hperoxo (circles) and Q (squares) obtained from double-mixing experiments
at 4 °C and pH 7. A linear fit to the data for reaction with Q reveals a
second-order rate constant of 88.6 ( 0.3 M-1 s-1, and a fit of the data for
the reaction with Hperoxo to eq 4 reveals ksat ) 18.1 ( 0.8 s-1 and Kd ) 11
( 1 mM.
The products of the steady-state oxidation of diethyl ether
were previously determined by whole cell assays to be ethanol
and acetaldehyde, produced by decomposition of the hemiacetal
formed upon hydroxylation at the R-carbon of diethyl ether.
Analysis of the products from steady-state reactions employing
purified enzyme components reveals ethanol and acetaldehyde,
as previously reported.22 The other possible product, ethylene
glycol ethyl ether, was not observed by GC analysis.
Intermediate Formation and Decay in the Absence of
Substrates. The optical spectra of the intermediates observed
upon reaction of Hred:2B with dioxygen have been reported
previously.16 The peroxo intermediate exhibits broad absorption
bands centered at 420 nm (ꢀ ) 4600 M-1 cm-1) and 720 nm (ꢀ
≈ 2000 M-1 cm-1), and intermediate Q displays a broad
absorption centered at 420 nm (ꢀ ) 9850 M-1 cm-1) that tails
into the NIR with an extinction coefficient of ꢀ ≈ 1000 M-1
cm-1 at 720 nm. The extinction coefficient for Hox:2B was
determined by using a known concentration of protein, and the
values for Hred, Hperoxo, and Q were determined by fitting to eq
1 (Table 1). The activation parameters for the formation of
we monitored the reactions of substrates at 4 °C because the
smaller rate constants allowed for the collection of more data
points and reduced noise in double-mixing stopped-flow visible
spectroscopic experiments.
Reactions with Ethyl Vinyl Ether. Previous work revealed
that the decay rate of Q is accelerated in the presence of
substrates,16,25 and that the rate constant for this process is equal
to that for substrate loss,26 but only one such example has been
provided for Hperoxo, its reaction with propylene.16 In this double-
mixing experiment, the decay rate of Hperoxo increased with
increasing concentrations of propylene; however, the change
in peroxo decay rates was small because of the limited solubility
of propylene. Thus, we decided to explore reactions with the
more soluble, electron-rich olefin ethyl vinyl ether. The second-
order rate constant at 4 °C obtained for reaction of ethyl vinyl
ether with Q is 88.6 ( 0.3 M-1 s-1 (Figure 1). The plot of kobs1
versus [S] for reaction with Hperoxo at 4 °C exhibits a hyperbolic
dependence on [S], and for all substrate concentrations, the
observed rate constant for reaction with Hperoxo is greater than
that for Q, providing convincing evidence that Hperoxo does react
with olefins. The data were fit to eq 4 using the model shown
in Scheme 5, with k2 set at 0.4 s-1 to give a rate constant ksat of
18.1 ( 0.8 s-1 and dissociation constant Kd of 11 ( 1 mM. At
20 °C, reactions with both intermediates display linear depend-
encies on [S], yielding second-order rate constants of 1500 (
100 M-1 s-1 for Hperoxo and 223 ( 10 M-1 s-1 for intermediate
Q. These rate constants and those for the reaction of other
substrates with both intermediates are reported in Table 3. The
time traces for the reactions at 20 °C are presented in Figures
S2 and S3 (Supporting Information).
Hperoxo, conversion of Hperoxo to Q, and conversion of Q to Hox
were determined from fitting the reaction time courses obtained
at 420 and 720 nm to an A f B f C model (Table 2); the
results agree well with published values.16 In the absence of
substrate, formation and decay of the peroxo intermediate were
determined to be 9.8 and 10 s-1 at 20 °C and 1.6 and 0.40 s-1
at 4 °C, respectively. The formation of Q, monitored at 420
nm, is equal to the rate of peroxo decay, and the rate constant
for Q decay is 0.12 and 0.05 s-1 at 20 and 4 °C, respectively.
Correspondingly, the concentration of Hperoxo maximizes at ca.
40 ms and 1 s at 20 and 4 °C, respectively, and the concentration
of Q at ca. 500 ms and 8 s at 20 and 4 °C, respectively. Thus,
(25) Liu, Y.; Nesheim, J. C.; Lee, S.-K.; Lipscomb, J. D. J. Biol. Chem. 1995,
270, 24662-24665.
(26) Muthusamy, M.; Ambundo, E. A.; George, S. J.; Lippard, S. J.; Thorneley,
R. N. F. J. Am. Chem. Soc. 2003, 125, 11150-11151.
(24) Sone, T.; Isobe, M.; Takabatake, E. Chem. Pharm. Bull. 1989, 37, 1922-
1924.
9
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Reactions of the Peroxo Intermediates of sMMO Hydroxylase
A R T I C L E S
Table 3. Rate Constants for the Reactions of Ethers and
Propylene with Hperoxo and Q Obtained from Double-Mixing
Experiments
1
1
substrate
species
T (°
C)
k (M-1 s-
)
ksat (s-
)
Kd (mM)
propylenea
Hperoxo 20.0
3400 ( 200
Q
20.0 12000
ethyl vinyl ether Hperoxo
Q
4.0
4.0
18.1 ( 0.8 11 ( 1
88.6 ( 0.3
Hperoxo 20.0 1,500 ( 200
Q
Hperoxo
Q
20.0
4.0
4.0
4.0
4.0
223 ( 10
17 ( 1
2.2 ( 0.1
8.7 ( 0.1
1.59 ( 0.5
diethyl ether
diethyl ether-d10 Hperoxo
Q
a Data taken from ref 16.
Figure 3. Plot of the evolution in absorbance at 420 nm after mixing
Hred:2B with buffer containing dioxygen and 5.7 mM ethyl vinyl ether at
20 °C and pH 7. The blue line is a fit to eq 2 using the parameters given
in Figure 2. The green line is a fit to the data using eq 1, where k2 is allowed
to vary. The value of 39 s-1 is nearly twice that observed in double-mixing
experiments. The red line is generated by fitting the data to eq 1 with k2 set
at 18 s-1
.
Figure 2. Plot of the evolution in absorbance at 420 nm after mixing
red:2B with buffer containing dioxygen and ethyl vinyl ether at 20 °C and
H
pH 7. The concentration of ethyl vinyl ether was 0 (cyan), 1.14 (red), 5.7
(blue), and 13.2 (green) mM. For [S] ) 0, the data were fit to eq 1 using
the extinction coefficients listed in Table 1 to afford k1′ of 40, k2 of 9.8,
and k3 of 0.12 s-1. These rate constants were then used to fit the data
obtained in the presence of substrate to eq 2. This procedure yielded values
of kR[S] ) 10.37 ( 0.01, 15.8 ( 0.1, and 26.3 ( 0.6 s-1 and the values of
kâ[S] ) 0.906 ( 0.006, 2.81 ( 0.02, and 2.81 ( 0.6 s-1 for [S] ) 1.14,
5.7, and 13.2 mM, respectively.
Figure 4. Eyring plots for reaction of Hperoxo (circles) and Q (squares)
with 4.16 mM ethyl vinyl ether obtained from double-mixing experiments
at pH 7.
Single-mixing experiments in the presence of substrate were
carried out to verify that the dependence of kobs1 on [S] was
not due solely to an increased value for k2. Fits of the evolution
of the absorbance at 420 nm after mixing Hred:2B with dioxygen
and increasing concentrations of substrate fit to the model shown
in Scheme 3 using eq 2 reveal that Hperoxo is reacting with
substrate and converting directly to Hox instead of Q. This
behavior results in the maximum value of the absorbance
decreasing, and the maximum occurring at an earlier time, with
increasing substrate concentration (Figure 2). If the dependence
of kobs1 on [S] were due solely to an increased value for k2, a
similar shift would be observed and the single-mixing data could
be fit to eq 1. However, the results from such a fit require a
value of k2 twice that observed in double-mixing experiments
(Figure 3). In addition, the fit using eq 1 affords larger residuals
compared to the fit using eq 2. If the value of k2 was set to the
value of kR[S] determined from double-mixing experiments, the
fit was even less satisfying. A dramatic decrease in the extinction
coefficient for Q is required because eq 1 does not account for
the conversion of Hperoxo directly to Hox, which reduces the
absorption maximum at 420 nm due to the lower extinction
coefficients for these species compared to that for Q. The
percentage of Hperoxo converting to Q is equal to (k2/kobs1) such
that, at high values of [S], very little Q is formed. Thus, the
Table 4. Activation Parameters for the Reactions of Hperoxo and Q
with Ethers
a
[S]
∆
Hq
(kcal/mol)
∆
Sq
(cal/mol
∆ °
Sq
(cal/mol
substrate
(mM) intermediate
‚
K)
‚
K)
ethyl vinyl ether 4.16 Hperoxo
4.16
75.0
75.0
4.8 ( 0.8 -40 ( 10 -30 ( 7
15 ( 1 -6.5 ( 0.7 3.9 ( 0.4
Q
diethyl ether
Hperoxo 12.6 ( 0.9 -12 ( 1 -7.1 ( 0.6
Q
19.5 ( 0.4
14 ( 3
8.7 ( 0.2 13.6 ( 0.3
-8 ( 2 -3.1 ( 0.8
diethyl ether-d10 75.0
Hperoxo
Q
75.0
21.0 ( 0.3 13.8 ( 0.2 18.7 ( 0.3
a Calculated using eq 6.
increased rate constant for Hperoxo decay cannot be ascribed to
a larger value of k2 alone. The temperature dependence of Hperoxo
and Q decay in the presence of substrate was monitored at 720
and 420 nm, respectively. Linear Eyring plots (Figure 4) were
obtained for both intermediates, and the extracted activation
parameters are given in Table 4.
Reactions with Diethyl Ether. For the reaction with diethyl
ether, both intermediates exhibit linear dependencies on [S]
(Figure 5), and the second-order rate constants are 17 ( 1 and
2.2 ( 0.1 M-1 s-1 for Hperoxo and Q (Table 3), respectively.
For reactions with perdeuterated diethyl ether, the second-order
9
J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005 7375

A R T I C L E S
Beauvais and Lippard
Figure 7. Plot of the evolution in absorbance at 420 nm after mixing
Figure 5. Substrate concentration dependence of the kinetics of the optical
decay for Hperoxo at 720 nm (circles) and for Q at 420 nm (squares) obtained
from double-mixing experiments with diethyl ether (open) and perdeuterated
diethyl ether (filled) at pH 7 and 4 °C.
Hred:2B with buffer containing dioxygen and 51 mM diethyl ether at 4 °C
and pH 7. The blue line is a fit to eq 2 using the parameters given in Figure
6. The green line is a fit to the data using eq 1, where k2 is allowed to vary.
The value of 2.12 s-1 is nearly twice that observed in double-mixing
experiments. The red line is generated by fitting the data to eq 1 with k2 set
at 1 s-1
.
Figure 6. Plot of the evolution in absorbance at 420 nm after mixing
Hred:2B with buffer containing dioxygen and diethyl ether at 4 °C and pH
7. The concentration of diethyl ether was 0 (cyan), 23 (red), 51 (blue), 107
(green), and 196 (purple) mM. In the absence of diethyl ether, the data
were fit to eq 1 using the extinction coefficients listed in Table 1 to afford
k1′ of 1.6, k2 of 0.46, and k3 of 0.05 s-1. These rate constants were then
used to fit the data obtained in the presence of substrate to eq 2. This
procedure yielded values of kR[S] ) 0.192, 0.5, 2.12, and 4.04 s-1 and the
values of kâ[S] ) 0.0384, 0.069, 0.107, and 0.17 s-1 for [S] ) 23, 51, 107,
and 196 mM, respectively.
Figure 8. Eyring plots for reaction of Hperoxo (circles) and Q (squares)
with 75 mM diethyl ether (open symbols) and diethyl ether-d10 (filled
symbols) obtained from double-mixing experiments at pH 7.
oxygenated intermediates in MMOH agree well with those found
in earlier work.16,27 The large entropy of activation for conver-
sion of peroxo to Q means that the activation free energy is
highly temperature-dependent and highlights the importance of
studying reactions of Hperoxo at 4 °C.
The structure of Hperoxo is not established because it has not
been possible to obtain its resonance Raman spectrum. Recent
calculations from our group and others indicate an η2:η2 butterfly
structure to be energetically most favorable,12,13,28 although
others prefer a µ-1,2 structure based on theoretical studies and
comparison with other diiron proteins.29 For example, the
resonance Raman spectroscopy reveals that the peroxo inter-
mediates observed for stearoyl-acyl carrier protein ∆9-desatu-
rase,30 ferritin,31 and mutants of ribonucleotide reductase
R229,32 have µ-1,2 bridging peroxo ligands. However, it is not
rate constants are 8.7 ( 0.1 and 1.59 ( 0.05 s-1 for Hperoxo and
Q, respectively. The values for the KIE, kH/kD, are 2.0 ( 0.2
and 1.4 ( 0.3 for Hperoxo and Q, respectively. The time traces
for the reaction of Hperoxo with diethyl ether are presented in
Figure S4 (Supporting Information).
Single-mixing experiments in the presence of substrate were
performed in a manner similar to that for ethyl vinyl ether, and
the same general features were observed (Figure 6). A fit of
the evolution of the absorbance at 420 nm to eq 2 supports the
proposed reaction scheme (Scheme 3). Again, eq 1 does not
adequately fit the data (Figure 7). The temperature dependence
of Hperoxo and Q decay in the presence of substrate was
monitored at 720 and 420 nm, respectively. Linear Eyring plots
(Figure 8) were obtained for both intermediates, and the
extracted activation parameters are given in Table 4.
(27) Recent work in our lab indicates that MOPS is a substrate for MMO and
that the reported activation parameters may be those for reaction of the
intermediates with MOPS. Further studies are underway to determine the
role of buffer in transient studies of MMOH. Beauvais, L. G.; Lippard, S.
J., unpublished work.
Discussion
(28) (a) Siegbahn, P. E. M. J. Biol. Inorg. Chem. 2001, 6, 27-45. (b) Siegbahn,
P. E. M. Chem. Phys. Lett. 2002, 351, 311-318.
Intermediates in the MMOH Catalytic Cycle. The activa-
tion parameters obtained for formation and decay of the
(29) Skulan, A. J.; Brunold, T. C.; Baldwin, J.; Saleh, L.; Bollinger, J. M., Jr.;
Solomon, E. I. J. Am. Chem. Soc. 2004, 126, 8842-8855.
9
7376 J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005

Reactions of the Peroxo Intermediates of sMMO Hydroxylase
A R T I C L E S
surprising that the structure of the peroxo intermediate would
be different for MMOH compared to these other diiron proteins
because MMOH is the only system in which a diiron(IV)
intermediate has been observed. Thus, the side-on bridging
butterfly structure may be necessary for homolytic O-O bond
cleavage to afford Q, whereas the µ1,µ2 end-on bridged structure
might lead to heterolytic O-O bond cleavage. In fact, recent
work on diiron model complexes that form (µ-1,2-peroxo)-
diiron(III) intermediates suggests that these species are not very
reactive and may not be involved in the dioxygen activation
pathway of MMOH.33 Moreover, butterfly peroxo structures are
common for dicopper enzymes and react in an electrophilic
manner, whereas di(µ-oxo)dicopper(III) species prefer one-
electron transfer mechanisms.34 These studies revealed that the
(µ-η2:η2-peroxo)dicopper(II) complexes are much more elec-
trophilic than the oxo species. The barrier to conversion between
these two intermediates can be very small, such that they are
in equilibrium in solution.34,35 In contrast, the free energy of
Hperoxo in MMOH is calculated to be 9.8 kcal/mol higher than
that of Q and 54.5 kcal/mol higher than that of Hox;12,13d,e thus,
there is a large driving force for Hperoxo both to react with
substrates and to convert to Q.
Figure 9. Proposed mechanism for the reactions of Q (top) and Hperoxo
(bottom) with olefins. Note that the reaction with Q proceeds via two single-
electron transfer steps, and the reaction with Hperoxo occurs via two-electron
transfer steps to afford a cationic intermediate. The oxonium intermediate
stabilizes the transition state for the reaction with Hperoxo and leads to a
much lower activation energy compared to that for Q.
reactions would have a higher energy barrier. The stabilization
of an oxonium transition state would be consistent with the lower
activation enthalpy observed for Hperoxo versus Q, and the more
ordered transition state would explain the large negative entropy
of activation.
Hydroxylation of Diethyl Ether. The second-order rate
constant for reaction of diethyl ether with Hperoxo is larger than
that observed for Q. The homolytic C-H bond dissociation
energies for the methylene and methyl C-H bonds of diethyl
ether are 89 and 112 kcal/mol, respectively, as determined from
the C-H stretching overtone spectra. Given these values, it is
not surprising that sMMO reacts only at the R-C-H bond.37
Additionally, the gas-phase heterolytic bond dissociation energy
D(R+H-) of diethyl ether has been calculated to be 214 kcal/
mol,38 indicating that abstraction of hydride ions from this
substrate is facile. A more useful parameter for solution phase
experiments is the solution hydride affinity ∆Ghydride(R+)s, which
can be estimated as 95 kcal/mol using eq 7.39 Thus, H• and H-
abstractions from diethyl ether have comparable energetic bar-
riers. Indeed, gas-phase reactions of diethyl ether with FeH+
occur via hydride abstraction,38 and the mechanism for the
oxidation of ethers with bromide, permanganate, and Hg(II) has
been assigned to a hydride transfer mechanism.40 More impor-
tantly, reactions of aqueous Fe(IV) species with ethers are
believed to proceed by both hydrogen atom and hydride
abstraction.41
Epoxidation of Ethyl Vinyl Ether. It was previously
determined that the second-order rate constant for reaction of
Hperoxo with propylene to form exclusively propylene oxide was
ca. 5 times less than that with Q.16 Upon switching to the more
electron-rich olefin ethyl vinyl ether investigated here, the
reaction is even faster with Hperoxo than with Q. By what
mechanism can these results be rationalized? Mayr and co-
workers determined a scale of nucleophilicity (N) for a wide
range of compounds according to their reactions with various
electrophiles, where a larger value of N indicates greater
nucleophilicity. On this scale, the N values are -2.41 and 3.92
for propylene and diethyl ether, respectively.36 Hperoxo thus
appears to be a more electrophilic oxidant because it reacts with
a rate constant significantly larger than that of Q with the more
nucleophilic substrate.
Proposed mechanisms for olefin epoxidation are depicted in
Figure 9. Intermediate Q is expected to react by two single-
electron transfer steps, according to our previous analysis, to
afford a bound radical intermediate.12,13e,f Hperoxo could react
by a concerted mechanism or by two-electron transfer to afford
a cationic intermediate, which would be stabilized for ethyl vinyl
ether by the oxonium resonance structure (Figure 9). Such
stabilization would not be available for propylene, and thus its
∆Ghydride(R+)s ) 0.904D(R+H-) - 98.7
(7)
(30) (a) Broadwater, J. A.; Ai, J.; Loehr, T. M.; Sanders-Loehr, J.; Fox, B. G.
Biochemistry 1998, 37, 14664-14671. (b) Broadwater, J. A.; Achim, C.;
Mu¨nck, E.; Fox, B. G. Biochemistry 1999, 38, 12197-12204. (c) Lyle, K.
S.; Moe¨nne-Loccoz, P.; Ai, J.; Sanders-Loehr, J.; Loehr, T. M.; Fox, B. G.
Biochemistry 2000, 39, 10507-10513.
(31) Moe¨nne-Loccoz, P.; Krebs, C.; Herlihy, K.; Edmondson, D. E.; Theil, E.
C.; Huynh, B. H.; Loehr, T. M. Biochemistry 1999, 38, 5290-5295.
(32) (a) Moe¨nne-Loccoz, P.; Baldwin, J.; Ley, B. A.; Loehr, T. M.; Bollinger,
J. M., Jr. Biochemistry 1998, 37, 14659-14663. (b) Baldwin, J.; Voegtli,
W. C.; Khidekel, N.; Moe¨nne-Loccoz, P.; Krebs, C.; Pereira, A. S.; Ley,
B. A.; Huynh, B. H.; Loehr, T. M.; Riggs-Gelasco, P. J.; Rosenzweig, A.
C.; Bollinger, J. M., Jr. J. Am. Chem. Soc. 2001, 123, 7017-7030.
(33) Kryatov, S. V.; Taktak, S.; Korendovych, I. V.; Rybak-Akimova, E. V.;
Kaizer, J.; Torelli, S.; Shan, X.; Mandal, S.; MacMurdo, V. L.; Mairata i
Payeras, A.; Que, L., Jr. Inorg. Chem. 2005, 44, 85-99.
(34) (a) Pidcock, E.; Obias, H. V.; Zhang, C. X.; Karlin, K. D.; Solomon, E. I.
J. Am. Chem. Soc. 1998, 120, 7841-7847. (b) Henson, M. J.; Mukherjee,
P.; Root, D. E.; Stack, T. D. P.; Solomon, E. I. J. Am. Chem. Soc. 1999,
121, 10332-10345. (c) Shearer, J.; Zhang, C. X.; Hatcher, L. Q.; Karlin,
K. D. J. Am. Chem. Soc. 2003, 125, 12670-12671.
(35) Cramer, C. J.; Smith, B. A.; Tolman, W. B. J. Am. Chem. Soc. 1996, 118,
11283-11287.
(36) Mayr, H.; Kempf, B.; Ofial, A. R. Acc. Chem. Res. 2003, 36, 66-77.
The proposed mechanism is shown in Figure 10. Intermediate
Q reacts by two single-electron transfers, which gives a bound
radical intermediate as for methane.12,13e,f Comparing the
activation parameters for diethyl ether with those for these other
substrates, and their solution phase ionization potentials, reveals
Et2O to have a much higher enthalpy of activation (Table 5).
Given the low calculated solution phase ionization potentials,
it may first transfer an electron and then a proton, in contrast
(37) Fang, H. L.; Meister, D. M.; Swofford, R. L. J. Phys. Chem. 1984, 88,
410-416.
(38) Halle, L. F.; Klein, F. S.; Beauchamp, J. L. J. Am. Chem. Soc. 1984, 106,
2543-2549.
(39) This relationship was originally determined for aromatic species but should
yield a decent approximation for nonaromatic systems. Cheng, J.-P.;
Handoo, K. L.; Parker, V. D. J. Am. Chem. Soc. 1993, 115, 2655-2660.
(40) Barter, R. M.; Littler, J. S. J. Chem. Soc. B 1967, 205-210.
(41) Pestovsky, O.; Bakac, A. J. Am. Chem. Soc. 2004, 126, 13757-13764.
9
J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005 7377

A R T I C L E S
Beauvais and Lippard
Table 5. Comparison of Activation Parameters for Reactions of Q
with Various Substrates and Their Calculated Solution Phase
Ionization Potential (I′)
For example, hydride abstraction from benzyl alcohols by
methyltrioxorhenium has a KIE of 3.2.44 In addition, a hydride
abstraction mechanism would explain the presence of products
arising from cationic intermediates found for the steady-state
reaction of radical clock substrate probes with sMMO.12,13
substrate
∆
Hq (kcal/mol)
∆
Sq
°
b (cal/mol
‚K)
I′
c (eV)
methanea
ethanea
8.5 ( 0.3
-9.6 ( 0.4
12 ( 0.7
15.1 ( 0.2
13.3 ( 0.1
11.7 ( 0.1
11.1 ( 0.2
11.2 ( 0.1
14.3 ( 0.2
14.3 ( 0.2
14.1 ( 0.2
19.5 ( 0.4
21.0 ( 0.3
a
ethane-d6
6.5 ( 0.4
-6.2 ( 0.2
Conclusions and Predictions
MeCNa
9.35
8.64
7.25
6.79
Stopped-flow experiments have provided insight into the
reactivity of Hperoxo and Q with olefins and diethyl ether. The
data indicate that the peroxo intermediate of sMMO is more
electrophilic than Q and suggest that Hperoxo reacts by a two-
electron transfer mechanism. The greater reactivity of the peroxo
intermediate is consistent with its higher calculated free energy.
Density functional theoretical work indicates a (µ-η2:η2-peroxo)-
a
MeCN-d3
-13.5 ( 0.4
-10.7 ( 0.3
-4.4 ( 0.3
3.5 ( 0.4
2.7 ( 0.3
13.6 ( 0.3
18.7 ( 0.3
a
MeNO2
a
MeNO2-d3
CH3OHa
CD3ODa
Et2O
Et2O-d10
a Data from ref 11. b Calculated using eq 6. c Values from ref 42.
diiron(III) structure to be the preferable geometry for Hperoxo
,
and the related species is also highly electrophilic for related
dicopper(II) systems. The greater charge of a diiron(III)
compared to that of a dicopper(II) center may further activate
Hperoxo, increasing its electrophilicity. In addition, the oxo-
bridged dimetallic intermediates in sMMO and dicopper models
prefer one-electron transfer steps.
The present work suggests that Hperoxo should react with
alcohols and aldehydes, which have low heterolytic bond
dissociation energies. Further work with reporter substrates
capable of differentiating between one- and two-electron reac-
tions, such as norcarane, cyclobutanol, and methylcyclopropane,
is required to test this hypothesis. Moreover, we propose that
the electrophilicity of Hperoxo can be further revealed by
examination of additional olefins of varying nucleophilicity.36
Finally, this study demonstrates that the reactions of multiple
intermediates in an enzyme system can be deconvoluted using
stopped-flow optical spectroscopy, and extensions to other
bacterial multicomponent monooxygenases would be of interest
for systems in which the intermediates can be accessed.
Figure 10. Proposed mechanism for the hydroxylation of diethyl ether by
Q (top) and Hperoxo (bottom). Note that the intermediate for the reaction
with Hperoxo has two resonance forms, a 3c-2e- species and an oxonium
species.
to substrates having higher ionization potentials.42 This mech-
anism would explain the low value of the KIE because electron
transfer would be the rate-limiting step. Alternatively, the true
KIE would be obscured if substrate binding were rate-limiting.
The peroxo intermediate could react by two-electron transfer
to afford either a cationic or a 3c-2e- intermediate, which
would be stabilized for diethyl ether due to the oxonium
resonance structure (Figure 10). Low values of KIE for hydride
transfer reactions are not uncommon, and they typically arise
from the geometry of the transition state.43 Thus, the value of
2.0 does not preclude hydride transfer as the rate-limiting step.
Acknowledgment. This work was supported by NIH Grant
GM32134. L.G.B. is a Postdoctoral Trainee under NCI Cancer
Training Grant CA09112. We thank J. Bautista for experimental
assistance, and Prof. J. P. Sadighi, Prof. R. A. Friesner, Mr. L.
J. Murray, and Dr. D. Rinaldo for helpful discussions.
Supporting Information Available: Derivations of eqs 1, 2,
and 4, and Figures S1-S4 as described in the text (PDF). This
material is available free of charge via the Internet at
http://pubs.acs.org.
JA050865I
(42) Pearson, R. G. J. Am. Chem. Soc. 1986, 108, 6109-6114.
(43) (a) Kwart, H. Acc. Chem. Res. 1982, 15, 401-408. (b) McLennan, D. J.;
Gill, P. M. W. J. Am. Chem. Soc. 1985, 107, 2971-2972.
(44) Zauche, T. H.; Espenson, J. H. Inorg. Chem. 1998, 37, 6827-6831.
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7378 J. AM. CHEM. SOC. VOL. 127, NO. 20, 2005