J. Am. Chem. Soc. 1997, 119, 9949-9955
9949
Regio- and Stereoselectivity of Particulate Methane
Monooxygenase from Methylococcus capsulatus (Bath)
Sean J. Elliott, Mei Zhu, Luke Tso, H.-Hoa T. Nguyen, John H.-K. Yip, and
Sunney I. Chan*
Contribution from the A. A. Noyes Laboratory of Chemical Physics, 127-72,
California Institute of Technology, Pasadena, California 91125
X
ReceiVed April 3, 1997
Abstract: The regiospecificity and stereoselectivity of alkane hydroxylation and alkene epoxidation by the particulate
methane monooxygenase from Methylococcus capsulatus (Bath) was evaluated over a range of substrates. Oxidation
products were identified by conventional GC analysis, and the stereoselectivity of oxidation was determined by a
1
combination of chiral GC and HPLC methods, as well as H NMR analysis of the corresponding (R)-2-acetoxy-2-
phenylethanoate ester derivatives in the case of alkanol products. Alkane hydroxylation was found to proceed favoring
attack at the C-2 position in all cases, and the stereoselectivity for n-butane and n-pentane was characterized by an
enantiomeric excess of 46% and 80%, respectively, with preference for the (R)-alcohol noted for both substrates.
Epoxides were formed with smaller stereoselectivities. Together, the regio- and stereoselectivity results suggest that
an equilibrium of competing substrate binding modes exists. A simple substrate-binding model that incorporates
preferential C-2 oxidation with the observed stereoselectivity of alkane hydroxylation is proposed, and hypotheses
for the general mechanism are suggested and discussed.
The particulate methane monooxygenase (pMMO) from
in a subset of methanotrophic organisms, and it is expressed in
Methylococcus capsulatus (Bath) catalyzes the dioxygen-
copper-free, iron-rich growth conditions only. Further, while
sMMO has demonstrated a wide range of substrate specificity,
including straight-chain, branched, and aromatic C-H bonds,1
pMMO mediates hydroxylation and epoxidation for a small set
of straight-chain alkane and alkene substrates solely.3
1
-3
dependent, two-electron oxidation of methane to methanol.
The enzyme is a copper-containing membrane protein comprised
0-13
1
,3
of three subunits of 47-, 23-, and 20-kDa molecular mass.
,14
Similar to its soluble counterpart (sMMO), pMMO has garnered
increased attention of late due to its ability to activate dioxygen
and hydrocarbon C-H bonds. In cell cultures, the copper(II)
Determining the mechanism of C-H bond activation in
15,16
biological systems is of great interest currently.
To this end,
4
content has been correlated to pMMO activity, as well as the
the mechanism of sMMO-mediated hydroxylation has been
active repression of sMMO transcription.5 The addition of
copper(II) has been shown to stimulate pMMO activity in a
cell-free membrane system, while other divalent transition metals
such as nickel(II) and zinc(II) do not.6 Further, in cell-free
assays, the inhibition of pMMO activity by the suicide substrate
acetylene has been correlated to the total concentration of
copper(II) ions in the assay.6 The active site of pMMO is
17
studied with radical spin-traps as well as radical clock substrate
1
8
probes. Several mechanisms, which have been suggested to
account for the observations, invoke alkyl free radical and/or
carbocation structures as potential intermediate species1
or a concerted process involving direct oxygen insertion into a
3,18-20
2
1,22
substrate carbon-iron bond.
Unfortunately, while pMMO
warrants similar consideration due to its pervasive presence in
methanotrophs, analogous studies using pMMO have been
hindered by difficulty in maintaining the activity of the purified
pMMO, as well as the limited substrate range of the enzyme.1
However, the hydroxylation mechanisms of sMMO from
Methylosinus trichosporium (OB3b) and pMMO from M.
capsulatus (Bath) have recently been investigated by the use
1
surmised to contain a trimeric copper cluster, whereas the
7
sMMO active site contains a diiron, hydroxyl-bridged cluster.
The hydroxylase component of sMMO exists as an R2â2γ2 dimer
which has been crystallized recently, and its three-dimensional
8
,9
structure has been determined by X-ray crystallography.
pMMO is expressed under copper-containing growth conditions
in all known methanotrophs, while sMMO has been found only
1
9,23,24
of cryptically chiral ethanes.
The determination of the
X
Abstract published in AdVance ACS Abstracts, October 1, 1997.
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