Kinetic solvent isotope effect in steady-state turnover by CYP19A1 suggests involvement of Compound 1 for both hydroxylation and aromatization steps

Article abstract of DOI:10.1016/j.febslet.2014.06.050

CYP19A1, or human aromatase catalyzes the conversion of androgens to estrogens in a three-step reaction through the formation of 19-hydroxy and 19-aldehyde intermediates. While the first two steps of hydroxylation are thought to proceed through a high-valent iron-oxo species, controversy exists surrounding the identity of the reaction intermediate that catalyzes the lyase and aromatization reaction. We investigated the kinetic isotope effect on the steady-state turnover of Nanodisc-incorporated human CYP19A1 to explore the mechanisms of this reaction. Our experiments reveal a significant (～2.5) kinetic solvent isotope effect for the C10-C19 lyase reaction, similar to that of the first two hydroxylation steps (2.7 and 1.2). These data implicate the involvement of Compound 1 as a reactive intermediate in the final aromatization step of CYP19A1.

Full text of DOI:10.1016/j.febslet.2014.06.050

FEBS Letters 588 (2014) 3117–3122
journal homepage: www.FEBSLetters.org
Kinetic solvent isotope effect in steady-state turnover by CYP19A1
suggests involvement of Compound 1 for both hydroxylation and
aromatization steps
1
⇑
Yogan Khatri , Abhinav Luthra, Ruchia Duggal, Stephen G. Sligar
Department of Biochemistry, University of Illinois Urbana-Champaign, 505 S. Goodwin Avenue, Urbana, IL 61801, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
CYP19A1, or human aromatase catalyzes the conversion of androgens to estrogens in a three-step
reaction through the formation of 19-hydroxy and 19-aldehyde intermediates. While the first two
steps of hydroxylation are thought to proceed through a high-valent iron-oxo species, controversy
exists surrounding the identity of the reaction intermediate that catalyzes the lyase and aromatiza-
tion reaction. We investigated the kinetic isotope effect on the steady-state turnover of Nanodisc-
incorporated human CYP19A1 to explore the mechanisms of this reaction. Our experiments reveal
a significant (ꢀ2.5) kinetic solvent isotope effect for the C10–C19 lyase reaction, similar to that of
the first two hydroxylation steps (2.7 and 1.2). These data implicate the involvement of Compound
1 as a reactive intermediate in the final aromatization step of CYP19A1.
Received 6 May 2014
Revised 3 June 2014
Accepted 13 June 2014
Available online 2 July 2014
Edited by Miguel De la Rosa
Keywords:
Human aromatase
CYP19A1
Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Steady-state kinetics
KSIE
C–C lyase
1. Introduction
a non-productive release of peroxide, regenerating the ferric form
of the enzyme (depicted as gray lines in Fig. 1). Alternatively, a sec-
The catalytic cycle of cytochrome P450 has evolved after the
characterization of key intermediates involved in the hydrogen
abstraction and substrate hydroxylation by P450cam [1,2] as
depicted in Fig. 1.
ond protonation of the distal oxygen may occur which reduces the
O–O bond order resulting in cleavage, release of a water molecule
and generation of a higher valent metal-oxo species referred to as
‘‘Compound 1’’ (Cpd 1) [6] which then generates the hydroxylated
substrate.
Human aromatase (CYP19A1) is a membrane-bound microsomal
P450 that catalyzes the three-step conversion of androgens (andro-
stenedione, testosterone and 16-a-hydroxytestosterone) to estro-
gens (estrone, 17-b-estradiol and estriol, respectively) utilizing
three molecules each of NADPH and dioxygen per estrogen mole-
cule. The reaction proceeds through two intermediates, 19-hydroxy
(step I) and 19-aldehyde (step II) compounds, followed by the aro-
matization (step III) of ring A in the same active site (Fig. 2) [3–8].
CYP19A1 also proceeds through the same typical P450 cycle as
depicted in Fig. 1, where the hydroxylation during the step I and II
reactions are believed to proceed through the conventional high-
valent FeIV–O porphyrin cation radical, Cpd 1 [6], characteristic of
most P450s. During these steps, the acid-alcohol pair, Thr310 and
Asp309 conserved in the I-helix of CYP19A1 along with active site
water molecules play an important role in protonating the distal
oxygen atom of the peroxo anion to facilitate O–O bond cleavage
and generate Cpd 1, as described in P450cam and human CYP17A1
[9–14]. However, there has been a considerable debate for the
Binding of the substrate molecule to cytochrome P450 [1]
partially or completely displaces the water molecule on the sixth
coordination site of the heme iron and causes a shift from a
hexa-coordinated low-spin complex to a penta-coordinated high-
spin ferric state [2], thereby resulting in an increase in redox
potential (by 100–130 mV) and facilitating the first electron trans-
fer from its redox partners. Binding of molecular oxygen to the fer-
rous protein [3] generates the ferrous oxygenated (oxy-ferrous)
state [4], a key intermediate in P450 catalysis. The availability of
a second electron to the oxy-ferrous intermediate from the redox
partner yields the ‘‘peroxo anion’’ state [5a]. Proton transfer to
the distal oxygen atom orchestrated by the active site acid-alcohol
pair and bound water molecules results in formation of the
‘‘hydroperoxo’’ state [5b]. Each of these peroxo states may undergo
⇑
Corresponding author. Fax: +1 (217) 265 4073.
E-mail address: s-sligar@illinois.edu (S.G. Sligar).
Current address: Department of Biochemistry, Campus B2.2, Saarland University,
1
66123 Saarbruecken, Germany.
http://dx.doi.org/10.1016/j.febslet.2014.06.050
0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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Y. Khatri et al. / FEBS Letters 588 (2014) 3117–3122
CYP17 for C–C cleavage reactions with aldehyde- or ketone-contain-
ing substrates [20–23], and by nitric oxide synthase [24]. Applica-
tion of density functional theory on a minimal CYP19A1 active site
showed an energetic preference for Cpd 1-mediated hydrogen
abstraction from the C1 of 19-aldo AD leading to estrone formation
rather than direct reaction of the peroxo-ferric species with 19-aldo
AD [17]. Importantly, it has been hypothesized that the carbonylpair
(Thr310-Ala306) of CYP19A1 and the catalytic water molecule could
also be responsible for the H2b abstraction of the 2,3-enolization
processes during the aromatization step, in which the acid-alcohol
pair Asp309 directly participate [7], unlike the indirect role of
Asp251 and Glu244 in hydroxylation by P450cam and P450eryF,
respectively [12]. Recently, Mak et al. characterized the oxy-com-
plexes of AD- and 19-oxo-AD bound CYP19A1 using resonance
Raman (rR) spectroscopy and found no difference in the rR signature
of these two complexes. Their data also suggested the presence of a
H-bonding interaction to the terminal oxygen atom of the Fe–O–O
fragment for both cases, implying the involvement of Cpd 1 in both
reactions [25]. Therefore, the involvement of reactive intermediates
during the final stage of aromatization in the ring A of androgens by
human CYP19A1 remains controversial and lacks an unambiguous
conclusion for the C10–C19 lyase reaction.
Herein, we report investigation of kinetic solvent isotope
effects (KSIEs) on the steady-state turnover of the hydroxylation
(steps I and II) and lyase (step III) reactions by human CYP19A1.
This technique has been shown to be useful in quantitating the
consecutive proton-transfer processes, involved in generating
the high-valent iron-oxo species [6] [26]. Therefore, the effect
of deuterium substitution on the catalytic rates can provide an
important indication of the number and identity of protons
involved in O–O bond heterolysis [13]. As a result, either the
involvement of Cpd 1, which depends on at least two protons
to generate the oxoferryl intermediates ([5a] ? [5b] ? [6]), or
nucleophilic reactivity of a ferric peroxoanion intermediate before
involvement of the proton in O–O bond cleavage could be sug-
gested, as was recently demonstrated for human CYP17A1 [23].
Although several steady-state parameters for the conversion of
AD to estrone have been measured earlier either in human placental
aromatase [6,27–29], recombinant human CYP19A1 expressed in
insects [30,31] or bacteria [32–34], the transient-state kinetic stud-
ies during the conversion of AD to estrone have not been studied
except by the Guengerich group [34]. However, all previous kinetic
studies were performed in detergent solubilized-form and reconsti-
tuted with different stoichiometries of CPR, and the detailed mech-
anism of the lyase reaction was not addressed. In this study, we
employed recombinant human CYP19A1 incorporated in Nanodiscs,
whichprovidesa soluble, homogenous, monodispersed proteinwith
well controlled stoichiometry during reconstitution with its redox
partner CPR and hence mimics the biological integrity in the mem-
brane [35–37].
Fig. 1. Cytochrome P450 catalytic cycle.
Fig. 2. Three-step sequential oxidation of steroid catalyzed by human CYP19A1.
mechanism of the third step i.e. C10–C19 lyase reaction, in which
two hypotheses have been proposed. In the first postulated mecha-
nism, Cpd 1 is the key reactive species. Aromatization results from
C10–C19 scission of 19-oxo-AD intermediate and subsequent
release of C19 as formic acid, initiated by 1b-hydrogen abstraction
of A-ring of the steroid molecule by Cpd1 [15–17]. Alternatively,
the direct involvement of nucleophilic peroxo-ferric intermediate
with the substrate’s aldehyde functionality resulting in the decom-
position of peroxo hemiacetal intermediate to yield the observed
products has also been proposed [18].
It was shown earlier that in AD-bound CYP19A1 the first interme-
diate that accumulates during the low temperature oxygenation and
subsequent cryoreduction experiment is an unprotonated peroxo-
ferric heme [19]. This suggested that the proton delivery pathway
is more hindered in CYP19A1 in the presence of AD. AD, however,
is the substrate for the hydroxylation step of CYP19A1. 19-oxo-AD,
the substrate for the lyase step of CYP19A1 was not investigated in
this study. The involvement of peroxo-ferric species as the reactive
intermediate has also been suggested in CYP2B4, CYP51 and
2. Materials and methods
2.1. Human CYP19A1 expression, purification and Nanodisc
incorporation
The expression and purification of CYP19A1 and cytochrome
P450 oxidoreductase (CPR) was performed as described [19,38].
The self assembly of CYP19A1 Nanodiscs was performed as
described [37].
2.2. NADPH oxidation
Incorporation of CPR into preformed and purified human
CYP19A1 Nanodiscs was made by direct addition of oligomeric

Y. Khatri et al. / FEBS Letters 588 (2014) 3117–3122
3119
CPR at 1:4 CYP19A1 (200 pmol)/CPR (800 pmol) molar ratio, as
described [39]. Briefly, 1 ml of CYP19A1 and CPR solution in
100 mM potassium phosphate buffer, pH 7.4, containing 50 mM
NaCl and 50 lM substrate (AD, 19-OH-AD, or 19-Oxo-AD) was
brought to 37 °C in a stirred quartz cuvette, path length of
pH/pD 7.4. Deuterated samples were prepared by exhaustive
exchange of the proteins in corresponding D2O buffers.
In order to determine the reaction time for the substrates AD,
19-OH-AD and 19-oxo-AD, firstly time-dependent substrate con-
version was performed and the 10 min incubation time was chosen
for the catalytic activity, during which the catalytic activity was in
linear phase (Fig. 3).
0.4 cm. The sample was incubated for 3 min and the reaction was
initiated by addition of 300
NADPH was monitored by recording the absorbance at 340 nm
for 10 min. The reaction was stopped by adding 50 l of 9 M sulfu-
lM of NADPH. The consumption of
Using AD as the starting substrate, 19-OH-AD and 19-oxo-AD
were detected in the reaction mixture under steady state
conditions. Similarly, 19-oxo-AD and estrone were obtained
when 19-OH-AD was used as the starting substrate for turnover
experiments. Starting with AD, 19-OH-AD and 19-oxo-AD
were obtained with the individual rates of 1.63 0.04 minÀ1 and
l
ric acid to bring the pH below 4.0. The sample was removed from
the cuvette, flash frozen in liquid nitrogen, and stored at À80 °C
until product analysis. The optical measurements were performed
on Hitachi U-3300 spectrophotometer supplied with temperature
controller and built-in magnetic stirrer. The rate of NADPH oxida-
tion was determined from the slope of absorption at 340 nm dur-
ing the first three minutes using an extinction coefficient of
6.22 cmÀ1mMÀ1. A ratio of 1:4 of P450:CPR was chosen based on
previously established optimal ratio of reductase to P450 for
in vitro turnover experiments using the Nanodisc system [39]. In
this approach the reductase, in a dynamic equilibrium with reduc-
tase molecules in solution, inserts into the P450 containing Nano-
disc through its membrane anchoring tail thereby allowing the
formation of an efficient electron transfer complex.
4.06 0.30 minÀ1, respectively, (total rate of 5.70 0.34 minÀ1
)
when the reaction was carried out in a protiated buffer system.
Rates of formation of both these products showed substantial
slowing upon H/D substitution, yielding a KSIE for the step-I
hydroxylation reaction of kH/kD = 2.7. In both the cases, the rate
of second product formation was almost 2.5 times faster, suggest-
ing a distributive nature of the enzyme. It is noteworthy that both
the rates of formation of individual products (0.62 0.07 minÀ1
and 1.52 0.15 minÀ1 for 19-OH-AD and 19-oxo-AD, respectively)
as well as total product (2.14 0.22 minÀ1), displayed an isotope
effect of P2.6 (Fig. 4A).
2.3. Catalytic turnover
These KSIE data are very similar to the reported values for other
well studied P450 systems catalyzing hydroxylation chemistry
[13,40,41]. This was expected since the first two steps of CYP19A1
catalysis i.e. AD to 19-OH-AD and 19-OH-AD to 19-oxo-AD are
standard P450 hydroxylations and are thought to go through the
classical Cpd 1 mediated H-rebound mechanism [42].
When 19-oxo-AD was used as the C10–C19 lyase substrate, the
rate of estrone formation was 2.5 times slower in D2O (i.e.
7.1 0.8 minÀ1 in H2O versus 2.8 0.8 minÀ1 in D2O) (Fig. 4C). This
corresponds to the KSIE of kH/kD = 2.53, similar to that seen when
AD was used as the starting substrate. This suggests the involve-
ment of same reactive intermediate Cpd 1 during catalysis. In con-
trast, the involvement of unprotonated peroxo-ferric intermediate
in the catalysis of C17–C20 lyase step by CYP17A1 resulted in a
large inverse solvent isotope effect kH/kD = 0.4 arising from compe-
tition between catalysis through a proton-independent intermedi-
ate, viz. the peroxo anion, and uncoupling via proton-dependent
uncoupling pathway(s), viz. the peroxide and the oxidase shunt
[23].
The conversion of AD, 19-OH and 19-oxo-AD to estrone was
analyzed by HPLC (Waters). Briefly, 1 ll of 18 mM progesterone
solution in methanol was added to 1 ml each of the reaction sam-
ple, as an internal standard, and vortexed for 30 s. 2 ml of chloro-
form was added to each aliquots and vortexed for 30 s. The
organic phase was removed and dried under the stream of nitro-
gen. The dried sample was dissolved in 100 l of methanol and 30
l was injected onto C18-HPLC column, using a 150 Â 2.1 mm,
3 lm (ACE-111-1502) with the mobile phase of 45% each of meth-
anol and acetonitrile in water and a flow rate of 0.2 ml/min. The
19-hydroxylated and 19-Oxo product of AD was separated in the
linear gradient of methanol and acetonitrile from 20% to 80% in
30 min, and detected at 240 nm. The formation of estrogen was
detected at 280 nm. Peak integration was performed with GRAM/
32 software (Thermo Fischer Scientific).
3. Results and discussion
The conversion of 19-OH-AD to its products by CYP19A1 is unu-
sual. Use of 19-OH-AD, substrate for the second hydroxylation
step, as the starting substrate also yielded two products. In H2O,
19-oxo-AD and estrone were obtained at a rate of 2.3 0.2 minÀ1
3.1. Steady-state kinetic turnover by CYP19A1 in protiated and
deuterated solvent systems
and 1.8 0.3 minÀ1 respectively giving
a total rate of 4.1
CYP19A1 and CPR self-assembled in Nanodiscs was used to
quantitate product formation and NADPH oxidation rates in the
presence of saturating concentrations of AD and 19-OH-AD for
hydroxylation and 19-oxo-AD for the lyase reaction at 37 °C and
0.5 minÀ1. The second hydroxylation step of CYP19A1 also showed
the same distributive nature of the enzyme and gave two products.
However, the rate of second product formation was slower in this
Fig. 3. Time dependent total conversion of AD (A), 19-OH-AD (B) and 19-oxo-AD (C) by CYP19A1.

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Y. Khatri et al. / FEBS Letters 588 (2014) 3117–3122
kH/kD = 1.2 (Fig. 4B). Formation of estrone showed a KSIE of 2
(1.8 minÀ1 in H2O versus 0.91 minÀ1 in D2O). However, the forma-
tion of 19-oxo-AD showed no solvent isotope effect bringing the
net KSIE of product formation down to 1.2. Conversion of 19-OH-
AD to 19-oxo-AD goes through a hydroxylation at the C19 to
produce a gem-diol that then undergoes dehydration to yield the
19-oxo-AD intermediate (Fig. 5). It is plausible that the rate of
dehydration of the gem-diol intermediate is partially rate limiting
resulting in a diminished KSIE for 19-oxo-AD formation and giving
rise to a smaller observed KSIE with respect to total product for
step II as compared to steps I and III of CYP19A1 catalysis. It is
noteworthy that the formation of estrone from 19-OH-AD shows
a KSIE >2, which is consistent with other data. Table 1 summarizes
the individual and total product formation rates using AD, 19-OH-
AD and 19-oxo-AD as starting substrates in protiated and deuter-
ated solvents.
Fig. 4. Steady-state kinetic solvent isotope effects observed for hydroxylase (A and
B) and lyase (C) CYP19A1 catalysis. The number on the bar represents the value of
the activity. The error bar represents the standard deviation of 3–5 independent
measurements.
3.2. NADPH oxidation rates
The overall rate of NADPH oxidation in the presence of various
substrates of CYP19A1 was also studied. The total rates of conver-
case. The ratÀe1s of 19-oxo-AD and estrone decreased to
2.39 0.14 min and 0.91 0.09 minÀ1, respectively (the total of
sion of AD and 19-OH in H2O are 37 4.5 minÀ1 and 37 4 minÀ1
,
3.30 0.23 minÀ1
)
in D2O, which gave an overall KSIE of
and in D2O are 14 1.5 minÀ1 and 8.00 1.3 minÀ1, respectively.
Table 1
Rates of formation of CYP19 products and product intermediates in a reconstituted system under steady state turnover conditions.
Product
Substrate
19-OH AD
19-oxo-AD
Estrone
Product 2/
product 1
KSIE w.r.t
product 1
KSIE w.r.t
product 2
Total product
Overall KSIE
À1
À1
(min
)
(min
)
(minÀ1
)
(minÀ1
)
AD
1.63
0.62
4.06
1.52
2.30
2.39
2.49
2.45
0.78
0.38
–
2.6
1.0
2.5
2.7
2.0
5.69
2.14
4.10
3.30
7.10
2.80
2.7
1.2
2.5
19-OH AD
19-oxo-AD
1.80
0.91
7.10
2.80
–
H2O/D2O.
Fig. 5. Conversion of 19-OH-AD to 19-oxo-AD by CYP19A1 going through dehydration of a gem-diol intermediate.
Fig. 6. Comparison of the rates of NADPH oxidation (nmol NADPH/min/nmol p450) (solid bar) and coupling efficiency (%) (open bar) during CYP19A1 catalysis in H2O (A) and
D2O (B). The error bar represents the standard deviation of 3–5 independent measurements.

Y. Khatri et al. / FEBS Letters 588 (2014) 3117–3122
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[5] Braselton, W.E., Engel, L.L. and Orr, J.C. (1974) The flux of intermediates and
products in aromatizaton of C19 steroids by human placental microsomes.
Eur. J. Biochem. 48, 35–43.
[6] Thompson Jr., E.A. and Siiteri, P.K. (1974) The involvement of human placental
microsomal cytochrome P-450 in aromatization. J. Biol. Chem. 249, 5373–
5378.
[7] Ghosh, D., Griswold, J., Erman, M. and Pangborn, W. (2010) X-ray structure of
human aromatase reveals an androgen-specific active site. J. Steroid Biochem.
Mol. Biol. 118, 197–202.
[8] Lo, J., Di Nardo, G., Griswold, J., Egbuta, C., Jiang, W., Gilardi, G. and Ghosh, D.
(2013) Structural basis for the functional roles of critical residues in human
cytochrome p450 aromatase. Biochemistry 52, 5821–5829.
[9] Poulos, T.L., Finzel, B.C., Gunsalus, I.C., Wagner, G.C. and Kraut, J. (1985) The
2.6-A crystal structure of Pseudomonas putida cytochrome P-450. J. Biol. Chem.
260, 16122–16130.
These data correspond to the ‘‘normal’’ KSIE observed in the prod-
uct formation rate (Fig. 6). Though both the conversions were
highly uncoupled, the coupling efficiency was higher in D2O (16%
and 41%) compared to 15% and 11% in H2O for the substrates AD
and 19-OH-AD, respectively. Likewise, when the lyase substrate
19-oxo-AD was used, the rate of NADPH oxidation in D2O was also
decreased by 83%, from 35 1 minÀ1 to 6 1.2 minÀ1 (Fig. 6). A
higher level of coupling in deuterated solvents is due to slowing
of the proton dependent uncoupling pathways, predominantly
the peroxide and oxidase shunt pathways. The higher coupling effi-
ciency observed in the presence of 19-OH-AD and 19-oxo-AD may
be attributed to the polar nature of these substrates thereby mak-
ing possible presence of additional water molecules and/or
enhanced stabilization of the existing hydrogen-bonding network
at the active site of the enzyme.
[10] Raag, R., Martinis, S.A., Sligar, S.G. and Poulos, T.L. (1991) Crystal structure of
the cytochrome P-450CAM active site mutant Thr252Ala. Biochemistry 30,
11420–11429.
[11] Imai, Y. and Nakamura, M. (1989) Point mutations at threonine-301 modify
substrate specificity of rabbit liver microsomal cytochromes P-450 (laurate
(x
-1)-hydroxylase and testosterone 16
a-hydroxylase). Biochem. Biophys. Res.
Commun. 158, 717–722.
4. Conclusion
[12] Nagano, S. and Poulos, T.L. (2005) Crystallographic study on the dioxygen
complex of wild-type and mutant cytochrome P450cam. Implications for the
dioxygen activation mechanism. J. Biol. Chem. 280, 31659–31663.
[13] Vidakovic, M., Sligar, S.G., Li, H. and Poulos, T.L. (1998) Understanding the role
of the essential Asp251 in cytochrome P450cam using site-directed
mutagenesis, crystallography, and kinetic solvent isotope effect.
Biochemistry 37, 9211–9219.
[14] Khatri, Y., Gregory, M.C., Grinkova, Y.V., Denisov, I.G. and Sligar, S.G. (2014)
Active site proton delivery and the lyase activity of human CYP17A1. Biochem.
Biophys. Res. Commun. 443, 179–184.
[15] Korzekwa, K.R., Trager, W.F., Mancewicz, J. and Osawa, Y. (1993) Studies on
the mechanism of aromatase and other cytochrome P450 mediated
deformylation reactions. J. Steroid Biochem. Mol. Biol. 44, 367–373.
[16] Fishman, J. and Raju, M. (1981) Mechanism of estrogen biosynthesis.
Stereochemistry of C-1 hydrogen elimination in the aromatization of 2 beta-
hydroxy-19- oxoandrostenedione. J. Biol. Chem. 256, 4472–4477.
[17] Hackett, J.C., Brueggemeier, R.W. and Hadad, C.M. (2005) The final catalytic
step of cytochrome P450 aromatase: a density functional theory study. J. Am.
Chem. Soc. 127, 5224–5237.
We observe a normal slowing of steps 1 and 2 of CYP19A1 catal-
ysis (kH/kD of 2.7 and 1.2) consistent with the need for two proton-
ation steps in concomitant formation of the hydroperoxo-ferric
complex and Cpd 1 in D2O. These observations are also consistent
with the previous reports of solvent isotope effects in P450cam and
other P450s [10,41–43]. The presence of kinetic solvent isotope
effect of a similar magnitude when 19-oxo-AD, the substrate for
lyase step of CYP19A1, suggests the involvement of the same
intermediate viz. Cpd 1 for the aromatization step of CYP19A1
as well. In addition, CYP19A1 also showed the highest coupling
for the lyase substrate, 19-oxo-AD, compared with the hydroxylase
substrates, AD and 19-OH-AD, in both protiated and deuterated
solvent system.
[18] Akhtar, M., Calder, M.R., Corina, D.L. and Wright, J.N. (1982) Mechanistic
studies C-19 demethylation in oestrogen biosynthesis. Biochem. J. 201, 569.
[19] Gantt, S.L., Denisov, I.G., Grinkova, Y.V. and Sligar, S.G. (2009) The critical iron–
oxygen intermediate in human aromatase. Biochem. Biophys. Res. Commun.
387, 169–173.
[20] Vaz, A.D.N., Pernecky, S.J., Raner, G.M. and Coon, M.J. (1996) Peroxo-iron and
oxenoid-iron species as alternative oxygenating agents in cytochrome P450-
catalyzed reactions: switching by threonine-302 to alanine mutagenesis of
cytochrome P450 2B4. Proc. Natl. Acad. Sci. 93, 4644–4648.
[21] Shyadehi, A.Z., Lamb, D.C., Kelly, S.L., Kelly, D.E., Schunck, W.-H., Wright, J.N.,
Corina, D. and Akhtar, M. (1996) The mechanism of the acyl-carbon bond
cleavage reaction catalyzed by recombinant sterol 14{alpha}-demethylase of
Candida albicans (other names are: Lanosterol 14{alpha}-demethylase, P-
45014DM, and CYP51). J. Biol. Chem. 271, 12445–12450.
[22] Lee-Robichaud, P., Shyadehi, A.Z., Wright, J.N., Akhtar, M. and Akhtar, M.
(1995) Mechanistic kinship between hydroxylation and desaturation
reactions: acyl-carbon bond cleavage promoted by pig and human CYP17
(P-45017.alpha.; 17.alpha.-hydroxylase-17,20-lyase). Biochemistry 34,
14104–14113.
[23] Gregory, M.C., Denisov, I.G., Grinkova, Y.V., Khatri, Y. and Sligar, S.G. (2013)
Kinetic solvent isotope effect in human P450 CYP17A1-mediated androgen
formation: evidence for a reactive peroxoanion intermediate. J. Am. Chem.
Soc. 135, 16245–16247.
[24] Woodward, J.J., Chang, M.M., Martin, N.I. and Marletta, M.A. (2009) The second
step of the nitric oxide synthase reaction: evidence for ferric-peroxo as the
active oxidant. J. Am. Chem. Soc. 131, 297–305.
[25] Mak, P.J., Luthra, A., Sligar, S.G. and Kincaid, J.R. (2014) Resonance Raman
spectroscopy of the oxygenated intermediates of human CYP19A1 implicates
a compound i intermediate in the final lyase step. J. Am. Chem. Soc. 136,
4825–4828.
Taken together, our results suggest that the involvement of the
same high-valent iron-oxo species, Cpd 1, as the reactive interme-
diate during the conversion of AD, 19-OH-AD and 19-oxo-AD. This
is based on the significant KSIE observed during the first two steps
of hydroxylation as well as the last step of lyase reaction. Our con-
clusions are also in line with the suggestion, albeit indirect, of Cpd1
mediated C–C scission by CYP19A1 provided by the rR, character-
ization of the oxy-complexes of AD- and 19-oxo-AD bound
CYP19A1 in Nanodiscs. This suggestion of involvement of Cpd 1
during C10–C19 lyase reaction by CYP19A1 is in contrast to recent
observation of an inverse KSIE in human CYP17A1 involved in
androgen formation where the reactive peroxoanion intermediate
is involved in the catalytic step [23].
In conclusion, our results highlight the involvement of Cpd 1
during the first two steps of aromatization by CYP19A1, a general
mechanism known to be involved in other P450s. We also impli-
cate the involvement of Cpd 1 as a reactive intermediate during
the controversial C10–C19 lyase reaction by human aromatase.
Acknowledgements
This work was supported by the United States National Insti-
tutes of Health grant GM31756 and GM33775 to S.G.S.
[26] Aikens, J. and Sligar, S.G. (1994) Kinetic solvent isotope effects during oxygen
activation by cytochrome P-450cam. J. Am. Chem. Soc. 116, 1143–1144.
[27] Kellis, J.T.J. and Vickery, L.E. (1987) The active site of aromatase cytochrome P-
450. Differential effects of cyanide provide evidence for proximity of heme-
iron and carbon-19 in the enzyme-substrate complex. J. Biol. Chem. 262,
8840–8844.
[28] Tan, L. and Muto, N. (1986) Purification and reconstitution properties of
human placental aromatase. A cytochrome P-450-type monooxygenase. Eur. J.
Biochem. 156, 243–250.
[29] Hagerman, D.D. (1987) Human placenta estrogen synthetase (aromatase)
purified by affinity chromatography. J. Biol. Chem. 262, 2398–2400.
[30] Amarneh, B. and Simpson, E.R. (1995) Expression of a recombinant derivative
of human aromatase P450 in insect cells utilizing the baculovirus vector
system. Mol. Cell. Endocrinol. 109, R1–R5.
References
[1] Sligar, S.G., Makris, T.M. and Denisov, I.G. (2005) Thirty years of microbial P450
monooxygenase research: peroxo-heme intermediates
station in heme oxygenase catalysis. Biochem. Biophys. Res. Commun. 338,
346–354.
– the central bus
[2] Denisov, I.G., Makris, T.M., Sligar, S.G. and Schlichting, I. (2005) Structure and
chemistry of cytochrome P450. Chem. Rev. 105, 2253–2277.
[3] Akhtar, M. and Skinner, S.J. (1968) The intermediary role of a 19-oxoandrogen
in the biosynthesis of oestrogen. Biochem. J. 109, 318–321.
[4] Akhtar, M., Njar, V.C. and Wright, J.N. (1993) Mechanistic studies on aromatase
and related C–C bond cleaving P-450 enzymes. J. Steroid Biochem. Mol. Biol.
44, 375–387.

3122
Y. Khatri et al. / FEBS Letters 588 (2014) 3117–3122
[31] Gartner, C.A., Thompson, S.J., Rettie, A.E. and Nelson, S.D. (2001) Human
aromatase in high yield and purity by perfusion chromatography and its
characterization by difference spectroscopy and mass spectrometry. Protein
Expr. Purif. 22, 443–454.
[32] Kagawa, N., Cao, Q. and Kusano, K. (2003) Expression of human aromatase
(CYP19) in Escherichia coli by N-terminal replacement and induction of cold
stress response. Steroids 68, 205–209.
[33] Kagawa, N., Hori, H., Waterman, M.R. and Yoshioka, S. (2004) Characterization
of stable human aromatase expressed in E. coli. Steroids 69, 235–243.
[34] Sohl, C.D. and Guengerich, F.P. (2010) Kinetic analysis of the three-step steroid
aromatase reaction of human cytochrome P450 19A1. J. Biol. Chem. 285,
17734–17743.
Shepard, P.R.O. and De Montellano, P.R.O., Eds.), pp. 115–127, Humana Press,
New York.
[38] Mulrooney, S.B. and Waskell, L. (2000) High-level expression in Escherichia coli
and purification of the membrane-bound form of cytochrome b(5). Protein
Expr. Purif. 19, 173–178.
[39] Grinkova, Y.V., Denisov, I.G. and Sligar, S.G. (2010) Functional reconstitution of
monomeric CYP3A4 with multiple cytochrome P450 reductase molecules in
nanodiscs. Biochem. Biophys. Res. Commun. 398, 194–198.
[40] Liu, Y. and Ortiz de Montellano, P.R. (2000) Reaction intermediates and single
turnover rate constants for the oxidation of heme by human heme oxygenase-
1. J. Biol. Chem. 275, 5297–5307.
[41] Batabyal, D., Li, H. and Poulos, T.L. (2013) Synergistic effects of mutations in
cytochrome P450cam designed to mimic CYP101D1. Biochemistry 52, 5396–
5402.
[35] Denisov, I.G. and Sligar, S.G. (2011) Cytochromes P450 in nanodiscs. Biochim.
Biophys. Acta 1814, 223–229.
[36] Schuler, M.A., Denisov, I.G. and Sligar, S.G. (2013) Nanodiscs as a new tool to
examine lipid–protein interactions. Methods Mol. Biol. 974, 415–433.
[37] Luthra, A., Gregory, M., Grinkova, Y.V., Denisov, I.G. and Sligar, S.G. (2013)
Nanodiscs in the studies of membrane-bound cytochrome P450 enzymes in:
Cytochrome P450 Protocols: Methods in Molecular Biology (Phillips, I.R.,
[42] Groves, J.T. (2006) High-valent iron in chemical and biological oxidations. J.
Inorg. Biochem. 100, 434–447.
[43] Shimada, H., Sligar, S.G., Yeom, H. and Ishimura, Y. (1997) Heme
monooxygenases in: Oxygenases and Model Systems (Funabiki, T., Ed.), pp.
195–221, Kluwer Academic Publishers, Dordrecht.