Communications
DOI: 10.1002/anie.200801184
Enzyme Mechanisms
Cage Escape Competes with Geminate Recombination during Alkane
Hydroxylation by the Diiron Oxygenase AlkB**
Rachel N. Austin,* Kate Luddy, Karla Erickson, Marilla Pender-Cudlip, Erin Bertrand,
Dayi Deng, Ryan S. Buzdygon, Jan B. van Beilen, and John T. Groves*
The alkane hydroxylase AlkB of Pseudomonas putida GPo1 is
typical of a large class of membrane-spanning diiron oxygen-
ases that catalyze hydroxylation, epoxidation, and desatura-
tion reactions.[1,2] These enzymes are of considerable interest
due to their impact on global hydrocarbon metabolism,[3] their
potential for practical biocatalytic application, and the
resulting inspiration for the design of synthetic biomimetic
catalysts.[4] Although the three-dimensional structures of
AlkB or any closely related proteins are unknown, topology
modeling has predicted a structure comprised of six mem-
brane-spanning helices with the catalytic iron diad appended
to the cytoplasmic termini of the helix bundle.[5] Mössbauer
data and alanine scanning have suggested that the diiron
binding site is histidine-rich,[6] as found in hemerythrin, and in
contrast to the predominantly carboxylate binding motifs
found in the diiron hydroxylases sMMO[7] and T4MOh.[8]
Through protein side-chain mutations, a long, hydrophobic
substrate-binding channel within the bundle has been iden-
tified that is tuned to accept medium-length alkanes.[5] AlkB
was the first alkane hydroxylase shown to generate a long-
lived substrate carbon radical during catalysis, as revealed by
diagnostic skeletal rearrangements of the hydrocarbon probe
norcarane.[9]
phenylcyclopropane.[12] Significantly, the ratios of rearranged
and unrearranged products (R/U) found for the three most-
slowly rearranging substrates were all in the range of unity
even though their rearrangement rates differed widely. To
account for these unusual results we propose a new diffu-
sional model of AlkB hydroxylation that involves radical
cagelike active-site dynamics of the type observed for heme-
and cobalamin-containing metalloproteins.
AlkB from P. putida GPo1 was expressed in P. putida
GPo12 in the manner we have previously described.[13] GPo12
is a receptacle clone that has been stripped of its innate
hydroxylases and dehydrogenases. This approach has the
advantages of producing unambiguous protein expression and
high activity for only the inserted hydroxylase gene, while
showing otherwise negligible background oxidation. Sub-
strates were oxidized in resting whole cells and in cell-free
extracts because all attempts to isolate and purify AlkB to
date have led to loss of activity. Results for the AlkB-
mediated oxygenation of the three alkane substrates, bicyclo-
[4.1.0]heptane (norcarane, 1), bicyclo[3.1.0]hexane (2), and
bicyclo[2.1.0]pentane (3), are presented in Table 1. These
simple alkanes were chosen because of their similar size,
nearly spherical shape, highly analogous structures, and
similar rearrangement chemistry (Scheme 1). The data for
all three substrates showed large amounts of rearrangement
products (> 50%) consistent with the involvement of discreet
radical intermediates during the hydroxylation process.
Further, the ratios of primary to secondary alcohols formed
from norcarane and bicyclohexane are similar to the partition
ratios observed for bona fide radical reactions for these
substrates (ca. 2 and 10%, respectively).
It is striking, however, that the ratios of rearranged
products to unrearranged products (R/U) for these three
substrates do not correlate with the 100-fold change in the
radical rearrangement rate constants for bicyclo[2.1.0]pent-2-
yl (kr = 2 109 sÀ1),[14] 2-norcaranyl (kr = 2 108 sÀ1), and
bicyclo[3.1.0]hex-2-yl (kr = 2.8 107 sÀ1).[11] We found the
average R/U values for the three substrates remarkably
constant (1.6, 1.6, and 4.7), corresponding to apparent radical
lifetimes of 0.78, 7.8, and 170 ns, respectively. Indeed,
bicyclohexane, with the slowest rearrangement rate, displayed
the most rearranged product and by far the longest radical
lifetime. The same effect was observed when norcarane and
bicyclohexane were oxidized as a mixture. By contrast, the
ultrafast rearranging probe trans-1-methyl-2-phenylcyclopro-
pane (kr = 1011 sÀ1) was confirmed to afford only rearranged
products.[9,15] Clearly, there is a discrepancy here between the
observed results for the more-slowly rearranging substrates
and expectations based on Arrhenius-type kinetic behavior
Herein we report results for the AlkB hydroxylation
reaction using a panel of radical-clock substrates that display
intrinsic rearrangement rates spanning five orders of magni-
tude, from a moderately slow 2.8 107 sÀ1 for bicyclo-
[3.1.0]hexane[11] to an ultrafast 1011 sÀ1 for trans-1-methyl-2-
[*] D. Deng, R. S. Buzdygon, Prof. J. T. Groves
Department of Chemistry, Princeton University
Princeton NJ08544 (USA)
Fax: (+1)609-258-0348
E-mail: raustin@bates.edu
Prof. R. N. Austin, K. Luddy, K. Erickson, M. Pender-Cudlip,
E. Bertrand
Department of Chemistry, Bates College
Lewiston ME 04240 (USA)
Fax: (+1)207-786-8336
E-mail: jtgroves@princeton.edu
Dr. J. B. van Beilen
Department of Plant Molecular Biology, University of Lausanne
(Switzerland)
[**] We thank FMC, Princeton, for access to GC-MS instrumentation and
NSF (CHE-0221978 (R.N.A., J.T.G.) and CHE 0616633 (J.T.G.)), NIH
(GM 072506 (R.N.A.) and 2R37M036298 (J.T.G.)), the Henry
Dreyfus Foundation (R.N.A.), and the Howard Hughes Foundation
(R.N.A.) for support of this work.
Supporting information for this article is available on the WWW
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ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 5232 –5234