The diagnostic substrate bicyclohexane reveals a radical mechanism for bacterial cytochrome P450 in whole cells

Article abstract of DOI:10.1002/anie.200603282

(Figure Presented) On home ground: The reaction mechanisms of bacterial alkane-oxidizing cytochrome P450s were determined in their native environment using a novel diagnostic substrate probe, bicyclohexane, in whole cells and cell-free extracts (see picture). Purified P450cam also oxidizes bicyclohexane. Clear evidence for a substrate-based radical with a lifetime of 75-250 ps was obtained.

Full text of DOI:10.1002/anie.200603282

Communications
Cytochrome P450
hydroxylation of the novel diagnostic substrate probe,
bicyclo[3.1.0]hexane, with cell-free extracts and whole cells
containing CYP153A1, a bacterial P450 hydroxylase found in
Acinetobacter sp. strain EB104,[16] and CYP153A6 from a
Mycobacterium sp. strain HXN-1500.[17] The results are
compared to those obtained from isolated, purified
P450cam (CYP101). Bicyclohexane has provided unusually
clear evidence for substrate-based radical intermediates that
persists in the enzyme active site for 75–250 ps with all three
CYPs studied.
DOI: 10.1002/anie.200603282
The Diagnostic Substrate Bicyclohexane Reveals
a Radical Mechanism for Bacterial
Cytochrome P450 in Whole Cells**
Rachel N. Austin,* Dayi Deng, Yongying Jiang,
Kate Luddy, Jan B. van Beilen,
Substrates that display mechanistically diagnostic molec-
ular rearrangements have been useful in characterizing
enzymatic oxygen-transfer reactions.[2,3] Cyclopropylcarbinyl
rearrangements have been particularly revealing, in that
radical and cation intermediates have been shown to re-
arrange by different pathways. Further, the rates of the radical
rearrangement have been used to clock the lifetime of
intermediate radicals. Still, various interpretations have
been offered for the array of products obtained for a given
diagnostic substrate, such as norcarane,[8,18] and the apparent
lack of correlation between intrinsic radical rearrangement
rates and the degree of rearrangement observed for some
cases.[19] Possible complicating factors are the participation of
Paul R. Ortiz de Montellano, and John T. Groves*
Few enzymatic transformations have attracted more attention
than the processes by which cytochrome P450 (CYP) acti-
vates molecular oxygen to catalyze the hydroxylation of the
[1–3]
À
inert C H bonds of alkanes.
This reaction cycle elicits
great interest due to the fundamental intellectual challenges
posed by the mechanistic chemistry, the elusiveness of the
reactive intermediates involved, and the significance of the
transformations to human health, global carbon cycling, and
“green” chemical processing. The consensus “oxygen
rebound” mechanism for aliphatic hydroxylation by cyto-
chrome P450, which involves a stepwise process and a short-
lived substrate radical, rests on a body of experimental
observations of substrate rearrangements. This scenario is
supported by comparisons to model studies,[4] experiments
with diagnostic substrates,[5–8] and theoretical approaches.[9–12]
The recent recognition that informative mechanistic informa-
tion can be obtained from whole-cell assays[5,13–15] offers a
means to characterize enzymes of interest that have not yet
been isolated and purified. Here we report results for the
multiple reactive species (Fe O and Fe OOH[20]), divergent
=
À
=
behaviors of different Fe O spin states (the two-state
hypothesis[9]), and dynamical properties intrinsic to the
recombination of reactive species in an active site cage.[21]
Also likely are potential shortcomings in the substrates
themselves and technical difficulties in the analyses. Given
these challenges, we have examined a new diagnostic
substrate which benefits from particular chemical character-
istics that simplify the product analysis and the subsequent
mechanistic interpretation.
Bicyclo[3.1.0]hexane is a diagnostic substrate similar to
norcarane but whose hydroxylated products chromatograph
with exceptional resolution. Like norcarane, bicyclohexane
generates distinct products for a substrate-based radical (3-
hydroxymethyl-cyclopentene, 2) and a substrate-based cation
(cyclohex-2-en-1-ol, 3), a property that permits informative
mechanistic conclusions to be drawn. An overview of possible
reaction routes for bicyclohexane is provided in Figure 1 (see
the Supporting Information for additional details).
As bicyclohexane has not been used before to character-
ize an enzyme reaction mechanism, the ring-opening rate for
the 2-bicyclohexyl radical was determined. Barton PTOC
(pyridine-2-thioenoxycarbonyl) esters for both the 2-bicyclo-
hexyl and 2-norcaranyl cases were prepared, and the photo-
[*] Prof. J. T. Groves, D. Deng
Department of Chemistry, Princeton University
Princeton, NJ 08544 (USA)
Fax: (+1)609-258-0348
E-mail: jtgroves@princeton.edu
Prof. R. N. Austin, K. Luddy
Department of Chemistry, Bates College
Lewiston, ME 04240 (USA)
Fax: (+1)207-786-8336
E-mail: raustin@bates.edu
Dr. Y. Jiang, Prof. P. R. Ortiz de Montellano
Department of Pharmaceutical Chemistry
University of California at San Francisco
San Francisco, CA 94143-0446 (USA)
Dr. J. B. van Beilen
Institut für Biotechnologie
ETH Zürich
8093 Zürich (Switzerland)
[**] We thank Dr. Enrico Funhoff for advice with the cell-free extract
methodology, Charlotte Lehmann for technical assistance, and FMC
Corp. (Princeton, NJ) for access to GC-MS instrumentation. We
thank the NSF for funding (CHE-0221978 to R.N.A. and J.T.G.
through the Environmental Molecular SciencesInstitute CEBIC at
Princeton University; CHE-0116233 to R.N.A.), the Camille and
Henry DreyfusFoundation (R.N.A.), and the NIH (GM072506 to
R.N.A., and GM036298 to J.T.G.).
Supporting information for thisarticle isavailable on the WWW
under http://www.angewandte.org or from the author.
Figure 1. Overview of bicyclohex-2-yl rearrangement chemistry.
8192
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Angewandte
Chemie
lytically generated radicals were trapped with thiophenol.[22]
The Barton ester of 2-bicyclohexyl carboxylic acid afforded
25% 3-methyl-cyclopent-1-ene, 73% bicyclohexane, and
1.9% cyclohexene, while that of 2-norcaranyl carboxylic
acid yielded 70% 3-methylcyclohex-1-ene, 0.5% cyclohep-
tene, and 30% norcarane. Accordingly, the rearrangement
rate for the 2-bicyclohexyl radical was determined to be 2.9 ”
107 sÀ1 by comparison to the known rearrangement rate for
the 2-norcaranyl radical (2 ” 108 sÀ1).[8] This result is consistent
with products of the reaction of 2-bicyclohexyl chloride with
tin hydride and experiments with bicyclohexane through the
photochemically induced radical chain decomposition of tert-
butyl hypochlorite.[8,23]
Table 1: Product distribution based on total bicyclohexane products, for
CYP systems examined.
System
Product distribution [%]
1a+1b 2[a] 3[b]
Radical
lifetime [ps]
4
5
CYP153A1 whole cell 95.4
CYP153A1 cell free 99.5
CYP153A6 whole cell 92.9
0.3 nd
0.3 nd
0.2 0.02 4.4 2.5
0.2 nd
0.7 0.5
nd 4.3 107
0.4 nd 104
75
74
CYP153A6 cell free
P450cam
97.8
92.2
2.0 nd
6.3 1.4 252
[a] All entries represent the average of two of more experiments. [b] In
one experiment from P450cam the cationic peak appeared to co-elute
with a compound unrelated to the substrate. An upper limit on the
amount of cyclohexenol was estimated in that case. nd=not detected.
Two bacterial cytochrome P450s, CYP153A1 and
CYP153A6, were cloned into an expression vector for co-
expression with ferredoxin and ferredoxin reductase and
inserted into Pseudomonas putida GPo12, a strain lacking
other alkane-degrading hydroxylases.[24] Bicyclohexane was
oxidized by these enzymes either in whole-cell assays[5,15] or in
cell-free extract experiments.[16] Cytochrome P450cam
(CYP101) was purified and used for substrate oxygenation
as previously described.[8,25]
Bicyclohexane proved to be a good P450 substrate, with
over 50% conversion. Product distributions for the three
P450 systems were remarkably similar, with endo- and exo-2-
bicyclohexanol, 1a and 1b, as the major products. The radical
rearrangement product 3-hydroxymethylcyclopentene (2)
was detected in all cases. This mechanistically significant
product has a distinct mass spectrum and was chromato-
graphically well separated from the other bicyclohexane-
derived product peaks, facilitating its identification and
quantification. Also contributing to the simplicity of the
assay was the apparent lack of secondary oxidation prod-
ucts.[25] Small amounts of the cationic rearrangement product
cyclohex-1-en-2-ol (3) were also detected in the P450cam
samples (0.5%) and in one cell-free extract containing
CYP153A6 (0.02%). 3-Bicyclohexanol (4) and 2-bicyclohex-
anone (5) were also detected. GC-MS data for the reaction
mixture are shown in Figure 2, and the product distributions
are reported in Table 1.
of 1010 sÀ1. Thus, the whole-cell and cell-free extracts give
evidence for a substrate-based radical generated by these
bacterial P450s that persists for tens of picoseconds for
bicyclohexane. These are very similar values to those reported
by us for other cyclopropylcarbinyl substrates, norcarane,[8]
bicyclopentane,[5] and thujone.[25,26]
By contrast, when trans-2-phenylmethylcyclopropane,
with a rearrangement rate of 1.8 ” 1011 sÀ1,[27] was used as a
substrate probe with CYP153A1 and CYP153A6 in the
whole-cell and cell-free assays, the lifetime of the substrate-
based radical was found to be 0.3–0.4 ps. A substrate-based
radical lifetime of 1.4 ps for trans-phenylmethylcyclopropane
and P450cam has been reported by Newcomb and co-
workers,[27] while Woggon and Sbaragli used this substrate
probe with a biomimetic P450 metalloporphyrin and observed
a substrate-based radical lifetime of 0.63 ps.[28] These very
similar results with an isolated P450, whole-cell and cell-free
=
extracts containing CYP153, as well as a model O Fe
porphyrin/iodosylbenzene system argue that similar processes
are being probed in each case. Thus, the 10- to 50-fold lower
amounts of rearrangement with the trans-phenylmethylcyclo-
propane probe derive from intrinsic properties of the ferryl
heme reaction with this substrate as we have discussed.[2,21]
Several conclusions regarding the mechanism of substrate
oxygenation by bacterial P450s can be drawn from this work.
Bicyclo[3.1.0]hexane is a useful and robust probe molecule
that confirms the presence of a short-lived radical intermedi-
ate in the CYP reaction cycle. This information was obtained
with a much simplified, and arguably more lifelike, whole-cell
assay approach, thus presenting opportunities for more facile
surveys of the emerging class of alkane-oxidizing bacterial
P450s[29–32] that do not require isolation of the proteins of
interest. Further, the radical lifetime criterion of simple
hydrocarbon probes could be used to characterize unknown
hydroxylases in novel cell cultures.
The product ratios observed are consistent with a stepwise
radical process for these hydroxylation reactions, with a
rebound rate for the [FeIV OH + CR] radical pair in the range
À
Experimental Section
For whole-cell experiments, cells were grown to an optical density
(OD) of 1 (600 nm) in MSB (mineral salts based) medium with octane
as the sole carbon source as we have previously described.[15] In the
“growing cell” approach, diagnostic substrate was simply provided in
lieu of octane and cells were allowed to grow for another 2–3 h. The
samples were centrifuged at 8000 ” g, and the supernatant was
extracted with ethyl acetate, dried, and concentrated. In the “resting
cell” approach, cells were centrifuged at OD = 1 and resuspended in
Figure 2. Ion chromatogram of CYP153A6 whole-cell oxidation of
bicyclo[3.1.0]hexane for m/e 96, 80, 79, 67, and 54.
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8193

Communications
50 mm phosphate buffer (pH 7.2). The substrate was then provided,
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and the cells were incubated for 2—3 h. The workup was the same as
described above. Both protocols yielded comparable results.
For cell-free extract experiments, growing cells were centrifuged
at 8000 ” g, and the cell pellet was resuspended in 50 mm potassium
phosphate buffer (pH 7.4) with 5% glycerol, 1 mm DTT (dithiothrei-
tol), and 200 mm PMSF (phenylmethylsulfonyl fluoride) at a concen-
tration of 20 g cell dry weight per liter (100 g cell wet weight/L). The
cells were sonicated on ice water and then centrifuged at 100000 ” g
for 20 min. 2 mL of substrate and DTT (1 mm) were added to 1 mL of
decanted supernate, and the mixture was stirred for 2 min. NADH
(12.5 mm) was added, and the reaction was shaken in a 378C
incubator for 2 h. The reaction was quenched with the addition of
1 mL CH2Cl2, and the mixture was then vortexed and centrifuged.
The CH2Cl2 layer was removed and dried with anhydrous sodium
sulfate.
Received: August 10, 2006
Published online: November 14, 2006
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Keywords: alkanes· cytochromes· oxidation ·
radical ions· reaction mechanisms
.
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