Modeling nonheme diiron enzymes: Hydrocarbon hydroxylation and desaturation by a high-valent Fe2O2 diamond core

Full text of DOI:10.1021/ja963578a

J. Am. Chem. Soc. 1997, 119, 3635-3636
Modeling Nonheme Diiron Enzymes: Hydrocarbon
3635
Hydroxylation and Desaturation by a High-Valent
Fe2O2 Diamond Core
Cheal Kim, Yanhong Dong, and Lawrence Que, Jr.*
Department of Chemistry and
Center for Metals in Biocatalysis
UniVersity of Minnesota, 207 Pleasant Street SE
Figure 1. Reactions of [Fe2(µ-O)2(TPA)2]3+ (1) mimicking oxidations
carried out by the diiron centers of methane monooxygenase, fatty acid
desaturases, and ribonucleotide reductase.
Minneapolis, Minnesota 55455
ReceiVed October 15, 1996
Table 1. Hydrocarbon Oxidations by the High-Valent Nonheme
Diiron Complex 1a
Methane monooxygenase (MMO),1,2 stearoyl ACP ∆9-de-
saturase (∆9D),3 and ribonucleotide reductase (RNR)4,5 comprise
a new class of metalloenzymes that activate dioxygen at a
nonheme diiron active site6 to carry out diverse functions such
as the hydroxylation of methane, the desaturation of saturated
fatty acids, and the generation of the catalytically essential Tyr
radical for ribonucleotide reduction. The mechanisms for
dioxygen activation appear to involve high-valent species as
indicated by the spectroscopic properties of intermediates
detected in rapid kinetic studies of O2 and the diiron(II) forms
of MMO (intermediate Q)7-9 and RNR (intermediate X).10-12
However, the absence of a porphyrin requires a mechanistic
paradigm that differs from the high-valent iron-oxo porphyrin
radical commonly implicated as the key oxidant for heme
enzymes.13 Instead, species with a high-valent Fe2(µ-O)2
diamond core have been proposed to serve as the oxidizing
intermediates for this class of enzymes.14 This hypothesis
derives from the recent synthesis of a series of metastable
Fe(III)Fe(IV) complexes with the Fe2(µ-O)2 diamond core using
the tetradentate tripodal ligand TPA and its methylated deriva-
tives, [Fe2(µ-O)2L2]3+ (Figure 1) (1, L ) TPA; 2, L ) 5-Me3-
TPA; 3, L ) 6-Me-TPA);15-17 these complexes represent the
first high-valent nonheme iron-oxo species to be synthesized.
In this paper, we demonstrate that an Fe2(µ-O)2 species can carry
out oxidation reactions corresponding to those associated with
MMO, ∆9D, and RNR.
substrate
products
yieldb
PhCH(CH3)2
(500 mM)
PhCH(CH3)2
(500 mM in air)
PhCH2CH3
(500 mM)
PhC(OH)(CH3)2
PhC(CH3)dCH2
PhC(OH)(CH3)2
PhC(O)CH3
PhCH(OH)CH3
PhCHdCH2
0.17c
0.27c
0.82
0.15
0.14c (kH/kD ) 22 ( 3)d
0.14c (kH/kD ) 28 ( 3)d
trace
PhC(O)CH3
cycloheptane
(120 mM)
no product
a A typical reaction mixture consisted of 2 mM of 1 and substrate
at -40 °C under Ar (except where noted) in acetonitrile with 0.75%
H2O. The lower concentration used for cycloheptane was because of
its low solubility. b Yield given in moles of product/moles of 1. All
products were identified and quantified by gas chromatography. The
1/substrate reaction stoichiometry for alkane oxidation under Ar is 2:1
with a maximum product yield of 50% based on 1, since 1 is a one-
electron oxidant and the products are oxidized by two electrons. In
the presence of O2, the sole function of 1 is to generate the substrate
alkyl radical, so the reaction stoichiometry is 1:1 and the maximum
yield of products under these conditions would be 100%, assuming no
radical chain process. c Ratio unchanged over the course of the reaction.
d Product isotope effects. Reactions were carried out on 1:4 to 1:10
mixtures of ethylbenzene and ethylbenzene-d10 to improve the accuracy
for measuring the amounts of the deuterated products.
Complexes 1 and 2 have been formulated to have the Fe2-
(µ-O)2 diamond core on the basis of electrospray ionization mass
spectral, Raman, and EXAFS evidence.16 Their characteristic
intense green color (1, λmax 614 nm, ꢀ 5500 M-1 cm-1; 2, λmax
616 nm, ꢀ 5200 M-1 cm-1) arising from the Fe2O2 core provides
a convenient probe for monitoring oxidation reactions which
are carried out at -40 °C in CH3CN to inhibit self decomposi-
tion. Thus, 1 and 2 can quantitatively oxidize 2,4-di-tert-
butylphenol within seconds to its phenoxy radical (Figure 1),
which dimerizes readily to form the 2,2′-biphenol as analyzed
by NMR. Concomitantly the oxidant is converted to its (µ-
oxo)diiron(III) precursor complex as shown by NMR, demon-
strating that 1 and 2 act as one-electron oxidants.16 Thus, 1
and 2 mimic the role of RNR intermediate X in the assembly
of the (µ-oxo)diiron(III)-Tyr radical cofactor of RNR.10-12
Similarly, 1 oxidizes hydrocarbons and becomes reduced to
its (µ-oxo)diiron(III) precursor, while organic products derive
from hydroxylation or desaturation of the substrate, similar to
the action of MMO and ∆9D, respectively (Figure 1). Cumene
is converted to cumyl alcohol and R-methylstyrene, and ethyl-
benzene is converted to 1-phenylethanol and styrene; but
cycloheptane is not oxidized (Table 1). The desaturation
reaction in particular is a novel result for this nonheme oxidant
(1) Rosenzweig, A. C.; Frederick, C. A.; Lippard, S. J.; Nordlund, P.
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(15) Abbreviations used: R3TACN ) 1,4,7-trialkyl-1,4,7-triazacy-
clononane, TPA ) tris(2-pyridylmethyl)amine, 5-Me3-TPA ) tris(5-methyl-
2-pyridylmethyl)amine, 6-Me-TPA ) N-(6-methyl-2-pyridylmethyl)-N,N-
bis(2-pyridylmethyl)amine.
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Soc. 1995, 117, 11377-11378.
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S0002-7863(96)03578-0 CCC: $14.00 © 1997 American Chemical Society

3636 J. Am. Chem. Soc., Vol. 119, No. 15, 1997
Communications to the Editor
Figure 2. Concentration dependence of the decomposition of 1 (2 mM)
in the presence of ethylbenzene or ethylbenzene-d10 at -40 °C under
Ar in CH3CN.
Figure 3. Proposed mechanism for alkane oxidation by 1.
carbocation. In support, when cumene oxidation is carried out
in the presence of H218O (250 equiv), the cumyl alcohol obtained
is 77% labeled with 18O, consistent with the amount of 18O
incorporated into the bis(µ-oxo)diiron core by H218O exchange.26
The olefin, on the other hand, would derive from the abstraction
of a â hydrogen by the Fe2O2 diamond core or its equivalent
(e.g., electron transfer followed by loss of a â proton). Thus,
the oxidation of substrate requires 2 equiv of 1; for cumene
oxidation, the transformation efficiency is 88%. What governs
the partitioning between hydroxylation and desaturation at this
nonheme diiron center is not currently understood and presum-
ably reflects a competition between C-O bond formation to
form alcohol and loss of the â hydrogen to form olefin, as
manifested by the differing product isotope effects noted above.
Further support for the two-step mechanism derives from
results obtained for the cumene oxidation under O2. Under these
conditions, cumyl alcohol and acetophenone are the reaction
products, and neither product shows any 18O incorporation when
the reaction is carried out in the presence of H218O. These
products derive from the breakdown of cumylperoxy radical
formed by the trapping of the intermediate alkyl radical by O2
(pathway b, Figure 3).
In this study, we have demonstrated that 1, the first high-
valent nonheme iron-oxo complex to be synthesized,16 can
carry out a range of oxidations analogous to those associated
with the diiron sites of RNR, MMO, and ∆9D. Because it is
an Fe(III)Fe(IV) complex, 1 cannot be as powerful an oxidant27
as MMO intermediate Q or its putative ∆9D analogue, both of
which are formally Fe(IV)Fe(IV). Oxidation of hydrocarbons
with stronger C-H bonds may be expected if 1 could be further
oxidized to the Fe(IV)Fe(IV) state, by analogy to the reactivities
of the oxoiron(IV) porphyrin and its one-electron oxidized
counterpart.18,20,28 The diverse reactivity exhibited by 1 thus
supports a new paradigm in which an Fe2(µ-O)2 diamond core
serves as the common structural unit for the high-valent
intermediates in the oxygen activation mechanisms of these
nonheme diiron enzymes.14
and one that has not been associated with synthetic iron-oxo
porphyrin complexes.18-21
Kinetic studies of 1 in the presence of ethylbenzene in CH3-
CN at -40 °C show that its decomposition is a process that is
first order in 1 and first order in substrate, demonstrating that
the rate-determining step entails a bimolecular collision between
1 and the substrate. The second-order rate constant for
decomposition is 4.8 × 10-3 M-1 s-1 for ethylbenzene and 2.4
× 10-4 M-1 s-1 for ethylbenzene-d10, corresponding to a kH/kD
of 20 (Figure 2); similarly, large isotope effects have been found
for the cleavage of C-H bonds by the related [Cu2(µ-O)2(R3-
TACN)2]2+ (kH/kD ) 26 ( 2 for R ) i-Pr and 40 for R ) benzyl
at -40 °C)22 and by MMO intermediate Q (kH/kD g 20 at 4
°C).23 Consistent with the kinetic results are kH/kD values
derived from product ratios in competition experiments using
a mixture of ethylbenzene and ethylbenzene-d10: 22 ( 3 for
alcohol and 28(3 for styrene. These results demonstrate that
cleavage of the substrate R-C-H bond is a major component
of the rate-determining step for both hydroxylation and desatu-
ration. However, cleavage of the â-C-H bond for the latter
has a significantly smaller isotope effect (ca. 1.3), indicating
that the desaturation involves asynchronous scission of the two
C-H bonds, as has been observed for yeast ∆9D.24
A proposed mechanism for hydrocarbon oxidation with
cumene as an example is shown in Figure 3. In the first and
rate-determining step, 1 abstracts a hydrogen atom from the
substrate generating an intermediate alkyl radical which then
reacts in a fast step with a second equivalent of 1 to yield alcohol
or olefin (pathway a). The alcohol would result either from
the capture of the alkyl radical by the Fe2O2 diamond core,
analogous to the proposed “oxygen rebound” step in heme-
catalyzed hydroxylations,25 or from electron transfer between
the alkyl radical and 1 and nucleophilic trapping of the resulting
(22) Mahapatra, S.; Halfen, J. A.; Tolman, W. B. J. Am. Chem. Soc.
1996, 118, 11575-11586.
(23) Nesheim, J. C.; Lipscomb, J. D. Biochemistry 1996, 35, 10240-
10247.
Acknowledgment. This work was supported by the National
Institutes of Health (GM-38767).
(24) Buist, P. H.; Behrouzian, B. J. Am. Chem. Soc. 1996, 118, 6295-
6296.
(25) Groves, J. T. J. Chem. Educ. 1985, 62, 928-931.
(26) It has not been possible to determine the extent of H218O exchange
into 1 by mass spectrometry, due to its instability and the presence of
interfering features from other components in the reaction mixture. However,
the mass spectral analysis of the more stable 2 (0.8 mM in CH3CN) treated
with 250 equiv of H218O showed that 68% of the molecules were doubly
18O-labeled and 27% of the molecules were singly 18O-labeled. Transfer of
one of the oxo groups to cumene would then afford about 80% yield of
labeled cumyl alcohol.
JA963578A
(27) While it would be highly desirable to quantify the hydrogen affinity
of 1 along the lines described by Gardner and Mayer (Gardner, K. A.; Mayer,
J. M. Science 1995, 269, 1849-1851), a meaningful estimate cannot be
obtained with the information currently available.
(28) Liu, M.-H.; Yeh, C.-Y.; Su, Y. O. J. Chem. Soc., Chem. Commun.
1996, 1437-1438.