Benzocycloarene hydroxylation by P450 biocatalysis

Article abstract of DOI:10.1039/b107584p

Experimental and theoretical studies of the hydroxylation of a family of benzocycloarene compounds [benzocyclobutene, benzocyclopentene (indan), benzocyclohexene (tetralin), and benzocycloheptene] by wild type and Y96F mutant P450cam were performed in order to understand the factors affecting product distribution, catalytic rate and cofactor utilization. The products of all reactions except that of benzocycloheptene were regiospecifically hydroxylated in the 1-position. Reaction energetics predominated over active site steric constraints in this case so that quantum mechanical calculations (B3LYP/6-31G*) comparing the energetics of all possible radical intermediates successfully predicted hydroxylation at the 1- and 3-positions of benzocycloheptene, and at the 1-position for the other three compounds. However, the fact that the ratio of 1-alcohol to 3-alcohol changes significantly between wild type and Y96F mutant P450cam indicates that active site geometry and composition also play a significant role in determining BCA7 product regiospecificity. The indan and tetralin reaction products were stereoselective for the R enantiomer (88 and 94%, respectively). Steric constraints of the active site were confirmed by molecular dynamics calculations (locally enhanced sampling dynamics) to control enantiomer distribution for tetralin hydroxylation. NADH coupling, binding affinity, and product turnover rates were dramatically higher for Y96F P450cam, showing that the removal of the active site hydroxyl group on tyrosine makes the enzyme better suited for oxidation of these hydrophobic compounds. NADH coupling, binding affinity and product turnover rate for each enzyme generally increased with arene ring size. For both enzymes, NADH coupling and product turnover rates were correlated with the extent of high-spin shift upon substrate binding as determined by the shift in Soret absorption bands at 417 and 391 nm.

Full text of DOI:10.1039/b107584p

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Benzocycloarene hydroxylation by P450 biocatalysis
Martin P. Mayhew, Adrian E. Roitberg,y Yadu Tewari, Marcia J. Holden,
David J. Vanderah and Vincent L. Vilker*
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg,
MD 20899, USA. E-mail: vilker@nist.gov
Received (in New Haven, CT, USA) 27th July 2001, Accepted 28th September 2001
First published as an Advance Article on the web8th January 2002
Experimental and theoretical studies of the hydroxylation of a family of benzocycloarene compounds
[benzocyclobutene, benzocyclopentene (indan), benzocyclohexene (tetralin), and benzocycloheptene] by wild
type and Y96F mutant P450cam were performed in order to understand the factors affecting product
distribution, catalytic rate and cofactor utilization. The products of all reactions except that of
benzocycloheptene were regiospecifically hydroxylated in the 1-position. Reaction energetics predominated over
active site steric constraints in this case so that quantum mechanical calculations (B3LYP/6-31G*) comparing
the energetics of all possible radical intermediates successfully predicted hydroxylation at the 1- and 3-positions
of benzocycloheptene, and at the 1-position for the other three compounds. However, the fact that the ratio of
1-alcohol to 3-alcohol changes significantly between wild type and Y96F mutant P450cam indicates that active
site geometry and composition also play a significant role in determining BCA7 product regiospecificity. The
indan and tetralin reaction products were stereoselective for the R enantiomer (88 and 94%, respectively). Steric
constraints of the active site were confirmed by molecular dynamics calculations (locally enhanced sampling
dynamics) to control enantiomer distribution for tetralin hydroxylation. NADH coupling, binding affinity, and
product turnover rates were dramatically higher for Y96F P450cam, showing that the removal of the active site
hydroxyl group on tyrosine makes the enzyme better suited for oxidation of these hydrophobic compounds.
NADH coupling, binding affinity and product turnover rate for each enzyme generally increased with arene
ring size. For both enzymes, NADH coupling and product turnover rates were correlated with the extent of
high-spin shift upon substrate binding as determined by the shift in Soret absorption bands at 417 and 391 nm.
Oxygenated benzocycloarenes (fused benzene–cycloalkane
structures) are useful reactants in asymmetric syntheses.1
Toluene and naphthalene dioxygenases, and cytochrome P450
monooxygenases, either in the context of whole cells or
reconstituted enzyme systems, have been investigated as bio-
catalysts for hydroxylation of benzocycloarenes to regio- and
stereospecific alcohols.2–6 Considered as a suite of catalysts,
these three enzymes offer many different possibilities for the
enzymatic insertion of the hydroxyl functionality, including
mono-ol or di-ol, on the benzene ring and for several mono-
oxygenation positions on the fused aliphatic ring. Cytochrome
P450 catalyzes only monooxygenation on the aliphatic ring. In
part, the present work differentiates the catalytic functionality
of one P450 monooxygenase from the dioxygenases that have
been applied to this class of substrates.
The cytochrome P450 superfamily of enzymes is ubiquitous
in nature, existing in a wide variety of organisms, from bac-
teria to humans.7 For the potential impact of P450 biocatalysis
in industrial fine chemical synthesis to become a reality,
methods to identify potential catalytic target compounds and
to improve natural P450 enzymes for use with non-natural
substrates of interest must be developed. Slow reaction rates
and cofactor utilization efficiencies for non-natural substrates
hinder the application of P450 enzymology to large-scale
chemical syntheses.
active-site residues critical to substrate specificity, product
regiospecificity, and rates of catalysis have been elucidated for
many P450s, including P450cam,8 P450 BM3,9 P450 1A2,10
and P450 2B4.11 P450 enzymes with enhanced stability and
solubility have been made using site-directed mutagenesis and
truncation of hydrophobic tails,12–14 and creation of a bac-
terial–human P450 chimera.15 A thermostable P450, CYP119,
has been isolated from the archaeon Sulfolobus sulfataricus16
and its biocatalytic properties and structure determined.17,18
Several studies have used computer-based algorithms to
investigate the predictability of ligand–P450 interactions.19–24
In early work, the active site of the enzyme was kept rigid, but
later more refined calculations included full dynamical prop-
erties of the system. Loew’s group in particular pioneered the
use of joint dynamical=energetic criteria for prediction and
explanation of observed hydroxylation trends.19,21 These cri-
teria were then used by Loew and others to successfully explain
trends in hydroxylation of camphor, camphane, thiocamphor
and norcamphor.19,23,24 There is a need for additional systemic
experimental information in order to model the pertinent P450
structural and dynamic features that allow for prediction of
novel P450 catalytic activities. This need is in part due to the
fact that there are very few known structures of P450 enzymes
when compared to the number of P450s of immediate scientific
and industrial interest. Presently, these less well-known P450s
are investigated using sequence homology models to enzymes
with known structure.25
Numerous studies have explored ways to improve P450s as
effective biocatalysts. Through site-directed mutagenesis,
This study focuses on P450cam (CYP101), the three-protein
enzyme system that metabolizes camphor in Pseudomonas
putida (ATCC 17 453), and the mutant Y96F P450cam for
which the active site pocket is more hydrophobic. The binding
y Present address: Quantum Theory Project and Department of
Chemistry, University of Florida, Gainesville, Florida. E-mail:
Adrian@qtp.ufl.edu
DOI: 10.1039/b107584p
New J. Chem., 2002, 26, 35–42
35
This journal is # The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2002

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of the native substrate, camphor, to P450cam acts as a redox
gate for the enzyme system, as the redox potential of the
enzyme shifts to enable electron acceptance from putidare-
doxin. The coupling of NADH oxidation to camphor hydro-
xylation is nearly 100%. For non-natural substrates, however,
NADH coupling and turnover rate decrease, as formation of
hydrogen peroxide, superoxide, or water precedes catalytic
turnover.26 Increased water access to the reduced, non-native
substrate-bound active site is believed to cause less efficient
NADH coupling and slower reaction rates. The removal of the
active site hydroxyl group upon replacement of tyrosine 96
with phenylalanine has been shown to improve P450cam cat-
alysis of hydrophobic substrates such as styrene,8,27 alkanes,28
and polycyclic aromatic hydrocarbons.29
In this work, we studied the biocatalysis of wild type and the
Y96F mutant P450cam toward four benzocycloarene com-
pounds of increasing aliphatic ring carbon number: benzocy-
clobutene, benzocyclopentene (indan), benzocyclohexene
(tetralin) and benzocycloheptene. Products were identified, and
kinetic parameters and NADH coupling efficiencies deter-
mined for each substrate. Regiospecific product distributions
were determined, with stereochemistry determined for the
indan and tetralin reaction products. A theoretical protocol
employing both dynamic and thermodynamic criteria was used
to predict the regio- and stereospecificity of these reactions. A
spectrophotometric assay that measures the Soret band shift
upon substrate binding was developed and compared with
NADH coupling and product turnover rates to assess this
assay’s potential as a biocatalyst screening tool.
desalting resin. Protein that eluted from the column was con-
centrated using either a Centricon-10 or Centriprep-10 cen-
trifugal concentrator (Millipore, Inc., Bedford, MA). P450cam
proteins were then passed through another Sephadex G-25
superfine column that was equilibrated with buffer T at 4 ꢀC.
Protein concentrations were quantified from absorption spectra
using Pdx, e455 ¼ 10.4 mMÀ 1 cmÀ 1; PdR, e454 ¼ 8.50 mMÀ 1
cmÀ 1; for P450cam devoid of camphor at 25 ꢀC, e417,WT ¼ 112
mMÀ 1 cmÀ 1 and e417,Y96F ¼ 107 mMÀ 1 cmÀ 1. These data
come from Gunsalus and Wagner,30 and recently performed
measurements from our laboratory for the Y96F mutant.8
The percentage of high-spin state iron in solutions of
P450cam, with and without benzocycloarene substrates, was
determined by a spectrophotometric method.31 Solutions of
100 mM DMSO (see Reaction kinetics and NADH coupling
measurements) and various concentrations of benzocycloarene
in buffer T were allowed to equilibrate in a stirred, thermo-
static spectrophotometer setup (25 ꢀC, 2 min equilibration).
Wild type or Y96F P450cam (5 mM) was added and spectra
from 350 to 600 nm were recorded after an additional 2 min
equilibration period. The same experiment was performed with
P450cam solutions devoid of substrate, and solutions with 0.2
mM camphor added. In each case, various aspects of the
spectra, most notably the isobestic point at 405 nm, were
examined to ensure consistent quality of the spectra.
Syntheses
Benzocycloheptene (BCA7). A solution of 5 g (0.031 mol) 1-
benzosuberone (Aldrich Chemical, St. Louis, MO), 4.93 g
(0.123 mol) NaOH, and 5.16g (0.103 mol) NH NH2 Á H2O in
2
Materials and methodsz
80 mL diethylene glycol was heated to reflux for 1 h. A
receiver flask was then inserted between the reaction flask,
which was covered in aluminum foil, and the condenser and
the temperature was slowly raised to ꢁ235 ꢀC. After removal
of the aluminum foil the temperature was stabilized at
ꢁ225 ꢀC and held for an additional 2 h. After cooling, the
reaction mixture was diluted with H2O and the product was
then extracted into 95% hexanes (2 Â 100 mL). The
combined hexane extracts were washed with brine, dried
(MgSO4), and concentrated under reduced pressure to give
Principal substances
Most substances used in this study were purchased commer-
cially at their highest level of purity and used without further
purification. NADH (CAS 606-68-8), dimethyl sulfoxide
(DMSO; CAS 67-68-5), benzocyclobutene (BCA4; 1,2-dihy-
drobenzocyclobutene; CAS 694-87-1), benzocyclopentene
(BCA5; indan; CAS 496-11-7), and benzocyclohexene (BCA6;
1,2,3,4-tetrahydronaphthalene; tetralin; CAS 119-64-2) were
purchased from Sigma-Aldrich (St. Louis, MO) with purities of
98, 99.5, 99, 95, and 99 wt%, respectively. Reported purities of
the benzocycloarene compounds were confirmed by in-house
GC analysis. Benzocyclohepten-3-ol was kindly provided by
Dr Derek R. Boyd and the structure was confirmed by GC-MS.
4.32
g (94.7%) of a liquid product. Flash column
chromatography yielded 3.71 g of pure product. The purity
of the compound was checked by gas chromatography and
its structure was confirmed by IR, proton NMR, and GC-MS.
Benzocyclohepten-1-ol. Sodium borohydride (0.36g, 0.0095
mol) was added in small increments to a solution of 5.625 g
(0.351 mol) 1-benzosuberone in 35 mL absolute methanol.
Vigorous bubbling accompanied this addition and the
temperature rose to near the boiling point of methanol. The
flask was cooled to room temperature, stirring was continued
for an additional 12 h, then the reaction mixture poured into
Protein preparation and absorbance measurements
Protein expression, site-directed mutagenesis, and purification
of putidaredoxin (Pdx), putidaredoxin reductase (PdR), and
cytochrome P450cam were performed as previously described.8
Prior to incubation with substrates, proteins were buffer-
exchanged with Buffer T (50 mM TRIS-HCl, 0.2 mM KCl, pH
7.4 at 22 ꢀC) by passing through Sephadex G-25 superfine
desalting resin (Amersham-Pharmacia Biotech, Piscataway,
NJ) at 4 ꢀC. Wild type (WT) and mutant P450cam proteins
were treated as follows to ensure complete removal of camphor.
P450cam samples were made 50 mM in dithiothreitol (DTT;
Diagnostic Chemicals, Ltd., Oxford, CT) and incubated at
22 ꢀC for 15 min. Next, the proteins were buffer exchanged at
4 ꢀC with 10 mM TRIS-HCl, 1 mM DTT, pH 7.4 at 22 ꢀC using
200 mL of
a 2.5% sodium bicarbonate solution and
extracted with methylene chloride (2 Â 100 mL). The
combined methylene chloride extracts were washed with
brine, dried over magnesium sulfate, and concentrated to
give ꢁ6.0 g of crude solid product. Recrystallization from
ꢁ50 mL of 95% hexanes yielded 3.84 g of purified product.
The melting point of the synthesized benzocyclohepten-1-ol
was measured to be 100–101.7 ꢀC, compared to the reported
value32 of 100–101 ꢀC. The benzocyclohepten-1-ol structure
was confirmed by proton NMR and purity was checked by
gas chromatography.
z Certain commercial equipment, instruments, and materials are
identified in this paper to specify adequately the experimental
procedure. In no case does such identification imply recommendation
or endorsement by the National Institute of Standards and Technol-
ogy, nor does it imply that the material or equipment is necessarily the
best available for the purpose.
Extraction and measurement of reaction products
All reactions in which oxidized products were to be measured
were performed in 1.0 mL glass septum microvials (Kimble,
36
New J. Chem., 2002, 26, 35–42

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Vineland, NJ). Extraction was performed in the same micro-
vial as the reaction in order to reduce systematic and
experimental errors. Internal standards were n-decane (Sigma–
Aldrich, St. Louis, MO) for analysis of BCA4 and BCA5 in
reaction extracts, BCA7 for analysis of BCA6in reaction
extracts, and BCA6for analysis of BCA7 in reaction extracts.
In all cases, the enzyme reaction was quenched by addition of
chloroform. After vigorous agitation followed by centrifuga-
tion, aliquots (1.0 mL) of the chloroform phase were injected
into an HP 5890 GC chromatograph fitted with a packed
column injector (modified for use with capillary columns) and
an FID detector. Different GC columns and temperature set-
tings were used for each substrate to optimize resolution and
sensitivity. All analytes were baseline-resolved. BCA4 and
BCA5 reaction extracts were analyzed using an HP-5 cross-
linked phenylmethyl silicone glass capillary column
(30 m  0.53 mm  0.88 mm film thickness; Hewlett–Packard,
Wilmington, DE). BCA6and BCA7 reaction extracts were
analyzed with a ZB-WAX polyethylene glycol glass capillary
column (30 m  0.53 mm  1.0 mm film thickness; Pheno-
menex, Inc., Torrance, CA). Chromatographic response fac-
tors were measured for all substrates, products, and internal
standards using authentic samples, except for benzocyclobu-
ten-1-ol, which was assumed to have the same response factor
as that of BCA4, and benzocyclobuten-3-ol, which was
assumed to have that of benzocyclobuten-1-ol.
Oxidized product identification was done by GC-MS ana-
lysis (SAIC, Inc., NCI-FCRDC, Frederick, MD). Electron
impact source energy was 70 eV, and GC conditions were
similar to the conditions stated above. Both un-derivatized and
TMS-derivatized samples of each benzocycloarene reaction
extract were analyzed by GC-MS. In each case, mass spectra
for reaction extracts were compared with mass spectra from an
authentic sample of the putative reaction product. Benzocy-
clobuten-1-ol was identified by analysis of the derivatized and
un-derivatized mass spectra of BCA4 reaction extracts.
The enantiomeric excess of the hydroxylated products from
BCA5 and BCA6hydroxylation were analyzed by chiral
HPLC. Poor separation of the BCA4 and BCA7 hydroxylation
products prevented the establishment of the enantiomeric
purity of these compounds. Hexane extracts of reaction solu-
tions were analyzed on an HP Series 1100 HPLC, fitted with a
Chiralcel OB-H cellulose column (46mm  5 mm; Chiral
Technologies, Exton, PA). The isocratic separation was per-
formed in 5% isopropyl alcohol–95% hexane, at 0.5 mL
minÀ 1. Compounds were detected by monitoring absorbance
at 254 nm. The retention times were: BCA5 (7.7 min), BCA6
(7.8 min), (R)-1-tetralol (13.7 min), (R)-1-indanol (16.5 min),
(S )-1-tetralol (26min), ( S )-1-indanol (29 min). Due to a lack
of authentic enantiopure 1-indanol, it was assumed that the R
enantiomer eluted first by analogy with (R)-1-tetralol.
CA). Dichloromethane was used as the extractant instead of
hexane in the benzocyclohepten-1-ol determination.
Reaction kinetics and NADH coupling measurements
P450cam reaction kinetics for hydroxylation of the benzocy-
cloarenes was determined by measuring the disappearance of
NADH. The assay solution contained 10 mM Pdx, 1 mM PdR,
2000 U mLÀ 1 catalase, 160 mM NADH, 100 mM DMSO, and
varying concentrations of benzocycloarene. This DMSO co-
solvent concentration was previously found to be optimal for
6
BCA6(tetralin) P450cam biocatalysis, although it had negli-
gible effect on BCA6solubility. No further attempts were
made to optimize the concentration for the other enzyme-
substrate pairs of this study. Assay solutions were placed into
a quartz cuvette fitted with an electric stirrer (Spectracell,
Oreland, PA), and were allowed to equilibrate for 2 min in a
thermostatic cuvette holder (25 ꢀC). WT (1 mM) or Y96F
(0.1 mM) P450cam was added to initiate the reaction, and the
absorbance monitored at 340 nm to measure NADH dis-
appearance (e340 ¼ 6.22 mMÀ 1 cmÀ 1). Pdx auto-oxidation,
about 0.2 minÀ 1 as measured in trials devoid of P450cam, was
subtracted from the overall NADH utilization rate.
Reactions for NADH coupling determinations were per-
formed in 1.0 mL septum glass micro-vials containing 1 mM
PdR, 2 mM Pdx, and 2000 U mLÀ 1 bovine liver catalase
(Sigma Chemical, St. Louis, MO) in Buffer T. Wild type
P450cam reactions included 10–15 mM P450cam, whereas
Y96F P450cam reactions included 2 mM P450cam. The ben-
zocycloarenes were added as DMSO stock solutions to give
solution concentrations of about 100 mM DMSO and near-
saturation levels for each benzocycloarene. NADH at limiting
concentrations (200–600 mM) was added to initiate the reac-
tion. A low Pdx-to-P450cam ratio was used in order to mini-
mize Pdx auto-oxidation. Reactions were complete within 5
min. Calibration solutions for quantifying oxidized products
were made by adding known amounts of product to solutions
that were identical to the reaction mixtures except that NADþ
was substituted for NADH. Calibration solutions of benzo-
cyclobutene and benzocyclohepten-1-ol were used to estimate
response factors for benzocyclobuten-1-ol and benzocyclo-
hepten-3-ol, respectively. NADH coupling was calculated by
dividing total product by the NADH added at the start of the
reaction.
The rate of total hydroxylated products for each enzyme-
substrate reaction was determined from NADH utilization
rate times NADH coupling efficiency. This assumes constant
proportionality between NADH utilization rate and substrate
hydroxylation rate. The validity of this assumption will be
addressed later. Michaelis–Menten parameters Km and kcat
were obtained by nonlinear regressions (SigmaPlot 4.0, SPSS
Science, Chicago, IL) of the total product rate determinations.
Saturation molality of benzocycloarene compounds
Theoretical calculation of reaction products
The saturation molality of each benzocycloarene was deter-
mined by approaching equilibrium from two different tem-
peratures. Approximately 0.02 g of benzocycloarene was
placed in two 50 ml Erlenmeyer flasks containing 40 g of
Buffer T. One flask was placed in a shaker bath set at 15 ꢀC
and the other in a shaker bath set at 37 ꢀC. After 24 h both
flasks were placed in one shaker bath at 25 ꢀC and allowed to
equilibrate for an additional 4 days. From each flask, 28 g of
the equilibrated aqueous phase was transferred to a 40 ml
Teflon tube, to which 1.3 g of n-hexane and 0.06g of internal
standard (1-decanol in hexane) were added. After vigorous
agitation and centrifugation for 15 min at 3000 g, 1.0 mL of
the hexane phase was injected into a Hewlett Packard (HP)
5890 gas chromatograph equipped with a flame ionization
detector and a fused silica ZB-WAX column (30 m  0.53
mm  1.0 mm film thickness; Phenomenex, Inc., Torrance,
The molecular dynamics runs were performed using the pro-
gram AMBER33 with the standard force field34 used for the
P450cam protein. The force field for the heme group and the
heme–thiolate bond, as well as the iron-oxo species were
described by the parameters found in publications of Loew’s
group.35 BCA6(tetralin) was selected as the model compound
to represent binding and dynamical characteristics of all ben-
zocycloarenes under investigation in this work. The program
Leap36 was used to build the BCA6molecule. A -631G**
Hartree–Fock in vacuo calculation was done, and point char-
ges assigned to classical atoms based on a global fit to the
electrostatic potential.37 Equilibrium values for internal BCA6
degrees of freedom as well as force constants were assigned
based on the same 6-31G** Hartree–Fock calculation. The
initial structure used for the simulation came from the
New J. Chem., 2002, 26, 35–42
37

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adamantane-bound P450cam crystal structure38 (Protein Data
Bank ref. 5cpp), from which adamantane and all water mole-
cules located more than 10 A from the heme iron were
Table 1 Benzocycloarene hydroxylation products from P450cam bio-
catalysis
+
Relative productsa
removed. The bound conformation of BCA6in the active site
was estimated by initial placement of BCA6in alignment
(main axis of rotation) with the original adamantane, followed
by use of the locally enhanced sampling (LES) method,39 as
implemented in AMBER, to over-sample the ligand orienta-
tions within the heme pocket. Ten copies of the ligand were
used, at a temperature of 150 K, for 200 ps. After the MD run,
no correlation was found between the possible ligand posi-
tions, with extensive orientation randomization. A slow cool-
ing protocol produced a single structure with all copies almost
completely overlapped. This structure is designated as the
BCA6bound state. Also, MD runs with two very different
(manually obtained) initial orientations of BCA6inside the
active site were made with a very detailed and slow equili-
bration method, similar to that used by Wade et al.40 With this
method, the system was run for 40 ps at 150 K, with atomic
Substrate
Wild type
Y96F
Benzocyclobutene (BCA4)
Benzocyclopentene (BCA5)
(nd)b
(nd)
(87% R)
(87% R)
harmonic restraints (AHR) using a force constant of 20 kJ
Benzocyclohexene (BCA6)
Benzocycloheptene (BCA7)
+
molÀ 1 A2. It was followed by 10 ps of dynamics at 300 K with
(95% R)
(93% R)
+
AHR and a 20 kJ molÀ 1 A2 force constant, 10 ps with AHR at
+
+
4 kJ molÀ 1 A2, 10 ps at 0.4 kJ molÀ 1 A2, and 10 ps of
unrestrained dynamics. A production run of 1.5 ns was done
for each of the initial structures. Both simulations converged
not only to very similar structures, but also to a conformation
very close to that obtained using the LES protocol.
54% (nd)c
46% (nd)
83% (nd)
17% (nd)
Tyrosine 96was mutated to phenylalanine to give the
mutant Y96F P450cam. A dielectric constant of 1 was used
throughout the simulations. A spherical cutoff of 30 A was
used for the Lennard-Jones non-bonded potentials. The time
step was 2 fs. The non-bonded list was updated every 20 fs.
The bonds that involved an H atom were constrained to their
equilibrium lengths by using the SHAKE method.
Structures for benzocyclopentene, benzocyclohexene, and
benzocycloheptene were optimized with both semi-empirical
(AM1 within Hyperchem) and density functional theory
(B3LYP/6-31G*, within Gaussian 98)41 calculations. The
energetics of the free radicals assumed to form during the
reaction were computed at the same theory level, but under an
unrestricted Hamiltonian, to account for the doublet spin
state. The free radical energies were computed for the geo-
metries of the original molecules, as well as for the locally
minimized doublets.
+
a
Wild type and Y96F benzocycloarene reaction products identified by
GC-MS as described in Materials and methods. For the first three
compounds, only one product was detected under the substrate
b
saturating conditions used for the reactions. Enantiomeric purity
(
2%) indicated by numbers in parentheses (nd ¼ not determi-
c
ned). Numbers without parentheses indicate relative amount of
products ( 3%) found for BCA7 reaction products.
is the predominant reaction product in all cases. BCA7 is the
only substrate that forms two regioisomers: benzocyclohepten-
1-ol and benzocyclohepten-3-ol. The presence or absence of
the active site tyrosine hydroxyl group is seen to affect the
regioisomer product distribution for benzocycloheptene.
There are significant differences and similarities with the
products formed by dioxygenase catalysis of these compounds.
For biocatalysis using toluene dioxygenase (TDO): BCA4 is
hydroxylated at multiple positions including attack on the
benzyl ring3 while P450cam gives only the regioisomer 1-
hydroxybenzocyclobutene; BCA5 is hydroxylated regio- and
stereospecifically to 1-(R)-indanol, the same as for P450cam
hydroxylation; BCA6and BCA7 are not known substrates
while P450cam forms a single regio- and stereospecific product
[1-(R)-tetralol] from BCA6, and the 1-ol and 3-ol from BCA7.
For biocatalysis by naphthalene dioxygenase (NDO): BCA4 is
regiospecifically hydroxylated to the single product ( )-1-
hydroxybenzocyclobutene,43 the same as for P450cam hydro-
xylation; BCA5 goes to multiple mono-ol and di-ol products
but the major product is 1-(S)-indanol,2 opposite to the chir-
Results and discussion
Benzocycloarene reaction products: measurements and
comparisons with dioxygenases
Except for preliminary mass balance studies performed at sub-
saturation concentrations and low substrate conversion, all
reactions with WT and Y96F P450cam were carried out at
saturation levels of benzocycloarene where the likelihood of
generating secondary products from enzymatic attack on the
primary hydroxylation products is minimized.6,8 Saturating
solutions were made using the following solubility data
acquired as part of this study [in the form quantity (standard
error)]: BCA4 ¼ 1580 (20) mM, BCA5 ¼ 690 (20) mM,
BCA6 ¼ 359 (7) mM, and BCA7 ¼ 127 (6) mM. The solubility
obtained for BCA6is in statistical agreement with our pre-
vious result of tetralin solubility as a function of buffer com-
position.42 Preliminary mass balance measurements made at
sub-saturating concentration of BCA5 and low substrate
conversion (< 20 mol%) indicated that only one product is
formed during biocatalysis, similar to our earlier finding with
styrene epoxidation by P450cam Y96F mutant.8
ality given by TDO, and by P450cam where only one product
43
is made; BCA6is known not to be an NDO substrate
BCA7 is not a known substrate.
and
Benzocycloarene reaction products: theoretical calculations
Our calculations of the regio- and stereospecifity of P450 cat-
alysis, largely based on the work of Harris and Loew,19,21,35
introduce both geometric and thermodynamic criteria using
Table 1 shows the products generated by WT or Y96F
P450cam catalysis from each benzocycloarene. The 1-alcohol
38
New J. Chem., 2002, 26, 35–42

View Article Online
the assumption that P450cam hydroxylation is rate-limited by
H-atom abstraction (and the formation of a free radical), with
a subsequent, extremely fast rebound mechanism. The reactive
intermediate at the heme is assumed to be compound I, radical
cation ferryl oxygen. The geometric criterion was then
extracted from a molecular dynamics (MD) run with appro-
priate parameters. The distances (and angles and torsions)
between a number of H atoms in the substrate and the O atom
in the ferryl oxygen heme species were monitored versus time,
and a record was made of the number of close contacts seen
during the dynamics run. The percentage of close approaches
between the reactive species was assumed to correlate directly
with the reaction probability at a given H site.
Although Loew et al. have computed the energetics of free
radicals after local optimization of the spin doublet,19 doubts
persist as to the relevance of the calculation to the hydro-
xylation and hydrogen rebound. There are a number of pub-
lications dealing with the correct time scale for hydroxylation.
Newcomb and Toy44 reviewed the use of hypersensitive radical
probes used as radical clocks for P450 catalyzed hydroxylation
reactions. One can see that the time scale for the reaction in the
enzyme is somewhere in the nano- to picosecond range.
Therefore, our calculations needed to consider if during that
time the free radical generated during the H-atom abstraction
has time to relax to its low energy conformation. We per-
formed the following model calculation to decide on the time
Table 2, it is predicted that the thermodynamic criteria pre-
sented above will favor 1-hydroxylations of BCA4, BCA5, and
BCA6, and 1- and 3-hydroxylations of BCA7 upon biocata-
lysis with P450cam. The fact that the ratio of 1-alcohol to 3-
alcohol changes significantly between wild type and Y96F
P450cam indicates that active site geometry and composition
also play a significant role in determining BCA7 product
regiospecificity.
To provide the geometric criteria necessary for establishing
active site effects on reaction regio- and stereospecificity, MD
of a benzocycloarene compound in the P450cam active site was
performed. First, the structure of P450cam bound with the
benzocycloarenes had to be predicted. The result of these
calculations is the bound conformation of BCA6in Y9F6
P450cam as depicted in Fig. 1. In this conformation, BCA6is
in close contact (< 3 A) with 12 residues of the protein. The
+
aromatic section is in contact with F87, F96and F98, and the
aliphatic region points towards the heme group. This picture
supports the experimental fact that P450cam hydroxylates
only the non-aromatic region of BCA6. Next, data from the
dynamics was used to monitor distances from each non-
aromatic carbon to the heme ferryl oxygen. Such an approach
shows no discrimination between C-1 and C-2 for BCA6, with
both atoms approaching the ferryl oxygen the same percentage
of the dynamics runs, within statistical bounds. Therefore,
active site interactions place BCA6(and likely the other ben-
zocycloarenes) in a conformation amenable to non-aromatic
scale of the rearrangement:
a semi-empirical molecular
dynamics run was done with an AM1 Hamiltonian, starting
from the geometry right after H abstraction, with no geometry
relaxation. Following the change in time from sp3 to sp2
hybridization by monitoring the out-of-plane angle for the
remaining H atom, allows us to estimate the time scale for
rearrangement to be 40 fs. Even if this calculation is wrong by
a factor of 100, the time scale for the reaction in vitro is quite
comparable with the theoretical time scale for rearrangement.
The comparison of energies for the un-optimized free radicals
is not enough to provide a proper picture. Our results for both
the optimized and un-optimized free radicals are shown in
Table 2. Even these calculations can be misleading and should
only be used for trends. A proper (but very expensive) calcu-
lation should account for differential energies inside the active
site of the enzyme, and should provide for the possibility of
free radical migration from the H-atom abstraction site to a
neighboring carbon atom.
The results in Table 2 show that position 1 is always pre-
ferred for H-atom extraction, regardless of the ring size. The
case of BCA7 (7-membered ring) is of particular interest.
Table 2 shows that the energy difference between sites 1 and 3
is not as large as that as between sites 1 and 2. This effect arises
from hyperconjugation between the C-3 free radical and the
aromatic ring via the C-1 atom. An energy difference of 6kJ
molÀ 1 is small enough to rationalize a significant population
of products hydroxylated in position 3. From the data in
Fig. 1 The bound state of benzocyclohexene (BCA6, tetralin) in the
active site of Y96F P450cam. Line widths of the amino acid residues
represent the third spatial dimension.
Table 2 Relative energeticsa for free radicals of the 5-, 6- and 7-membered non-aromatic ring benzocycloarenes
Semiempirical AM1
Un-optimized sp3
Density functional theory
Ring size
Position
Optimized sp2
Un-optimized sp3
Optimized sp2
5
6
7
1
2
1
2
1
2
3
0
10.5
0
17.63251.6.4
0
15.5
2.1
0
27.7
0
45.5
0
18.0
0
14.7
0
0
40.6
0
0
18.9
6
0
23.9
4.63.8
.3
a
Energies in kJ molÀ 1 are relative to lowest energy value (position 1) for the appropriate set. Non-optimized energies averaged over pro-R and
pro-S hydrogen abstraction sites.
New J. Chem., 2002, 26, 35–42
39

View Article Online
TDO substrates, one can speculate that the TDO active site is
too tight for the larger BCA molecules, while being too loose
for BCA4, which can adopt multiple conformations in the
active site and hence is subject to multiple reaction sites.
NADH coupling and kinetic characterizations
In order to study the effects of arene ring size and the Y96F
mutation on P450cam biocatalysis, extensive kinetic analyses
were performed on each enzyme-substrate reaction. The results
are summarized in Table 3. While NADH coupling to WT
P450cam is poor and uncorrelated with substrate size, coupling
is markedly increased for Y96F P450cam on all of the
benzocycloarenes. In addition, for the Y96F mutant, increas-
ing the arene ring size also results in better NADH coupling.
These trends are consistent with the hypothesis that poor
NADH coupling arises from water access to the active site
during catalysis.38 Removing the active site hydroxyl of ty-
rosine makes water access less energetically favorable, and the
binding of larger substrates more effective blockers of heme
iron electrophilic attack by water.
The kinetic characterization showed that Y96F is superior
to WT P450cam enzyme in both substrate binding affinity and
product turnover rate for all of the benzocycloarenes. The Km
values for Y96F are an order of magnitude smaller than for
WT, and kcat values are increased two- to fivefold, depending
on the substrate. A clear trend of increasing binding affinity
with increasing arene ring size is observed for WT, providing
further evidence that larger ring sizes allow for a tighter fit into
the active site. In addition, as suggested above, the absence of
the active site hydroxyl in Y96F P450cam results in an order of
magnitude greater binding affinity for each substrate. Why
would the removal of the active site hydroxyl cause a reduced
dependence of binding affinity on arene ring size? It could be
rationalized that since both substrate and active site are almost
completely hydrophobic in mutant Y96F, the increase in arene
ring size promotes minor stabilization to binding. In contrast,
for the WT enzyme, where substrates are stabilized by
hydrophobic interactions within the active site cavity and
destabilized by the active site hydroxyl, addition of more
hydrophobic surface area to the substrate by increasing arene
ring size provides a larger incremental stabilization.
Fig. 2 Histogram of the distances between the ferryl oxygen atom
and the pro-R or pro-S hydrogen atom at the C-1 position in benzo-
cyclohexene (BCA6, tetralin). Data compiled from a 1.5 ns molecular
dynamics run.
hydroxylation, but do not provide a rational for determining
regiospecificity on the non-aromatic portion of BCA6(or
smaller benzocycloarenes). Since BCA7 is larger than BCA6, it
is not possible to infer active site effects on benzocycloheptene
regio- and stereospecificity from the BCA6MD runs.
We also computed the distances between the ferryl oxygen
atom and the pro-R and pro-S hydrogen atoms at the C-1
position in BCA6to predict whether BCA6hydroxylation
would be stereospecific. A histogram of such distances is
shown in Fig. 2. These data are subject to uncertainties asso-
ciated with not knowing the effects of high flexibility in the
active site and in the choice of distance for defining a close
encounter. Nevertheless, to a first approximation they suggest
a significantly closer approach of the pro-R atom to the ferryl
oxygen, and therefore that the pro-R hydrogen spends con-
siderably more time close to the ferryl oxygen than does the
pro-S hydrogen.
For dioxygenase enzymes, no theoretical studies of this type,
or even simple MD, have been done. This is probably because
only recently has a structure become available for NDO45
(there is no TDO structure yet published), and there is almost
no mechanistic understanding of the dioxygenase catalytic
cycle. As it is shown here, the regioselectivity of hydroxylation
by P450cam can be rationalized by the relative energies of the
free radicals formed in the H-rebound mechanism, along with
steric constraints revealed through the MD simulations. Under
this light, no possibility of hydroxylation on the aromatic ring
exists. However, TDO seems to be able to hydroxylate BCA4
in both rings. A possible explanation includes a mechanism
that does not have a free radical formed at any point. Based on
the facts that the 5-membered aliphatic ring of BCA5 gives the
same products with TDO and P450cam, that toluene is the
natural TDO substrate, and that BCA6and BCA7 are not
Although product turnover rates (kcat) are larger for the
Y96F mutant compared to WT, there is no correlation with
substrate size for either enzyme. Binding affinity of a substrate
has little correlation with its associated product turnover
rate.26,46
Soret shift assay to predict P450 catalysis
All P450s are heme-thiolate proteins with a large Soret
absorbance band at approximately 420 nm, corresponding
to the predominant low-spin resting state. Upon substrate
Table 3 NADH coupling and kinetic characterization for WT and Y96F P450cama
Wild type
Y96F
Substrate
C (%)
Km/mM
kcat/sÀ 1
C (%)
Km/mM
kcat/sÀ 1
BCA4
BCA5
10
15
2
2
310 70
250 40
1.5 0.2
2.1 0.2
35
46
3
3
13
19
3
6
3.3 0.2
6.6 0.9
2
BCA66130 20
BCA7
1.2 0.1
52
3
9.8 0.8
1.0 0.1
5.4 0.2
12
2
55 10
70
5
6
1
4.6 0.2
a
NADH coupling (C), measured at saturating benzocycloarene and limiting NADH concentrations, calculated by dividing total product
concentration after complete NADH exhaustion by initial NADH concentration. Michaelis–Menten parameters Km and kcat determined by
nonlinear regression (SigmaPlot 4.0) of total product rates (product formation rate equals NADH utilization rate times NADH coupling
efficiency).
40
New J. Chem., 2002, 26, 35–42

View Article Online
binding, the Soret band for most P450s is blue-shifted by about
20 nm, corresponding to a predominant high-spin form of the
heme iron.31 This low-spin to high-spin transition adjusts the
redox potential of P450 to allow for electron exchange with
iron-sulfur or flavin protein redox partners, thereby modulat-
ing catalytic activity.47 Since the four benzocycloarenes tested
in this study displayed a wide range of catalytic activity
between the WT and Y96F mutant enzymes, we sought to
learn if a Soret shift measurement could be developed that
would correlate with these different catalytic activities. Turn-
over of native and non-native substrates by several different
P450s has been reported along with Soret band absorbance
data27,48 that indicate this might be an expedient optical
method to screen for P450 activity. Also, compounds that
induce low-spin Soret band shifts are usually hydroxylation
products of P450 catalysis, or potent P450 inhibitors.49
Substrate concentration is a critical parameter in the design
of the Soret shift assay. The Soret shift is largest at the sub-
strate concentration where the enzyme is completely saturated
by the substrate (but not over-saturated.50,51) However at
saturating concentrations, the assay loses its usefulness to
distinguish between substrate–enzyme pairs with more or less
affinity for binding and causing transition to the catalytically
active high-spin form. Furthermore, enzyme-saturating levels
of substrate in free solution may not always be achievable due
to solubility limitations, as was true for the benzocycloarene
compounds used here. Therefore, we adjusted the substrate
concentration to a single value for all benzocycloarene–enzyme
pairs so that the Soret shift was readily measured, but not
optimized for any single substrate–enzyme combination. This
concentration, 50 mM, is well below the solubility limit of all
the benzocycloarenes used here, but provided an easily mea-
sured Soret shift that could be interpreted as a relative measure
of binding affinity and extent of high-spin shift.
Fig. 3 shows absorbance spectra for wild type P450cam
without camphor, which is the reference state for the com-
pletely low-spin state, and with bound camphor, which is the
reference state for the completely high-spin state. The high-
spin and low-spin states exhibit the characteristic Soret
absorption peaks at 417 and 391 nm, respectively. The spin
state of unbound P450cam is taken to be 0% HS, and the spin
state of wild type P450cam with 0.2 mM camphor is taken to
be 100% HS.31 Since Y96F does not exhibit a complete shift
to high spin upon binding camphor, the WT þ camphor was
also used to represent 100% HS for measurements involving
the mutant Y96F P450cam. Also shown in Fig. 3 are the
spectra for the BCA6-enzyme pairs: WT þ tetralin and
Y96F þ tetralin. For each benzocycloarene-enzyme pair, the
percent high spin (%HS) was computed from:
"
ꢀ
ꢁ
ꢀ
ꢁ
AS391 À AS405 À AU391 À A4U05
1
2
ꢀ
ꢁ
ꢀ
ꢁ
%HS ¼
AC391 À AC405 À AU391 À A4U05
#
ꢀ
ꢁ
ꢀ
ꢁ
ꢁ
AS405 À AS417 À AU405 À A4U17
ꢀ
ꢁ
ꢀ
þ
AC405 À AC417 À AU405 À A4U17
where A refers to the absorbance value at the wavelength
denoted by the subscript (in nm) and the superscript refers to
the ligand added to the P450cam solution (U for unbound, C
for camphor, and S for benzocycloarene). The %HS calculated
from this formula is taken to be the average of the increase in
the 391 nm peak and the decrease in the 417 nm peak. The
isobestic point is used as the reference to minimize effects of
baseline drift on the calculated %HS. The %HS values for WT
enzyme [BCA4 ¼ (5 2) %HS, BCA5 ¼ (10 2) %HS,
BCA6 ¼ (10 2) %HS, BCA7 ¼ (18 2) %HS] were found
to be well below the values for Y96F P450cam
[BCA4 ¼ (31 1) %HS, BCA5 ¼ (53 2) %HS, BCA6 ¼
(58 2) %HS, BCA7 ¼ (61 2) %HS].
Fig. 4 Compares the %HS values with the normalized
NADH coupling efficiencies and maximum product turnover
rates. The calculated %HS values show a clear correlation to
both NADH coupling and product turnover. This result sug-
gests that by using sub-saturating concentrations of potential
substrates, a simple fast spectroscopic method might be
developed for high-throughput screening of P450 biocatalysis
targets.
Conclusions
P450cam monooxygenation generally yields fewer products
from each substrate than does the equivalent catalysis per-
formed by either toluene or naphthalene dioxygenases.
P450cam is the only demonstrated catalyst for oxygenating
this class of substrates when the alkane ring size is 6carbons or
more. There are also differences in the regio- and stereo-
specificity of the products formed by each of the three
enzymes. The observed regiospecificity for P450 products can
be rationalized in terms of the relative radical stabilities of each
potential hydroxylation site. Molecular dynamics simulations
were in agreement with the above finding in that each substrate
was mobile in the active site. NADH coupling efficiencies,
binding affinities, and product turnover rates for the benzo-
cycloarenes were found to be much higher for the Y96F
mutant enzyme, indicating that the removal of the active site
Fig. 3 Absorbance spectra (5 mM protein) for WT P450cam with and
without camphor (WT þ camphor, WT Unbound, respectively); with
BCA6(WT þ tetralin); and for mutant Y96F P450cam with BCA6
(Y96F þ tetralin). The spectra with camphor (0.2 mM camphor) or
without substrate are in buffer T. Spectra with BCA6contain 50 mM
BCA6in 100 mM DMSO.
Fig. 4 Comparison of normalized Soret shift assay values (%HS)
with NADH coupling and product turnover rates for each P450cam-
substrate pair.
New J. Chem., 2002, 26, 35–42
41

View Article Online
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NADH coupling, binding affinity, and product turnover rate
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A simple spectro-
photometric assay that takes advantage of the Soret band shift
induced by substrate binding qualitatively predicted the levels
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tool for identifying potential targets of P450 catalysis in a high-
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hepten-3-ol.
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