Transcript The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 4 Monooxygenation Monooxygenation Table 4.1. Typical reactions catalyzed by monooxygenases R1 R2 C H R2 C R3 Ar R1 R1 H R3 C OH R3 R2 Ar OH R1 R1 C C R2 R4 R1 N OH N H R2 O O n R R1 R1 S O S R1 O R2 R2 n+1 :OH CH R2 R1 NH2 C NH2 B+ H O R2 R1 R3 C R2 + NH.

The Organic Chemistry of
Enzyme-Catalyzed Reactions
Chapter 4
Monooxygenation
Monooxygenation
Table 4.1. Typical reactions catalyzed by monooxygenases
R1
R2 C H
R2 C
R3
Ar
R1
R1
H
R3
C
OH
R3
R2
Ar OH
R1
R1
C
C
R2
R4
R1
N OH
N H
2
R2
O
O
n
R
R1
R1
S
O
S
R1
O
R2
R2
n+1
:OH
CH
R2
R1
NH2
C
NH2
B+
H
O
R2
R1
R3
C
R2
+ NH 4+
C
O
R4
Internal Monooxygenase
Flavin-dependent Hydroxylases
Reaction catalyzed by lactate oxidase from
Mycobacteria
HO
CH3
C
COOH
+ O2
E•FMN
CH3COOH + CO2 + H2O
H
4.1
Scheme 4.1
No external reducing agent required
One Turnover Experiment
(enzyme concentration in excess
over substrate)
The lactate oxidase reaction under
anaerobic conditions
FMN
HO
CH3
C
FMNH2
COOH
O
CH3C COOH
4.2
H
Acting like an oxidase
Scheme 4.2
Reaction of Reduced Lactate Oxidase
with Pyruvate and Oxygen
E•FMNH2 + H3C
14
C
COO-
+
O2
E•FMN + CH314COO- + H2O + CO2
O
Scheme 4.3
If O2 is added first, then [14C]pyruvate, pyruvate is
unchanged and H2O2 is formed. Therefore, pyruvate
is an intermediate.
Model study:
CH3CCOOH + H2O2
O
CH3COOH
+ CO2 + H2O
Possible Mechanisms for Lactate Oxidase
B:
CH3
FMNH-
FMN
H
C
COOH
CH3
OH
C
CH3
COOH
R
N
COO-
C
NH
O
N
H
like DAAO
O
via a mechanism such as
shown in Scheme 3.33
18O
2
R
N
N
H
B:
NH
O-
O
H
18O
B+
CH3
- H218O
R
N
N
O
a
18O
- CH3C
18OH
H
O
NH
N
H
18O
O
4.5
N
O
- CO2
N
O
+
via a mechanism such as shown
in Schemes 3.43 or 3.44
OH
R
N
H
N
O
B+
CH3 C
O
b
b
electrophilic
substrate
C
O-
COOB:
O
C
NH
N
H
O flavin
18O
4.3
18O
a
H
hydroperoxide
acts as a
nucleophile
4.4
FMN
H
-FMN
B+
a
b
18OH
18O
+
CO2
+
CH3C
O-
CH3C
C
O
O
4.6
Scheme 4.4
O
b
H3C C
O
COO-
18
18
O
O
:
H2
18O
18O
H
Enzyme bound
:B
External Monooxygenases
NAD(P)H reduction of flavin
O2 activation
R
N
N
O
NAD(P)H
NAD(P)+
R
N
H
N
NH
N
O
NH
N
H
O
O2
Scheme 4.5
O
H2O2
Activated O2 is probably in the form of flavin
hydroperoxide
Nucleophilic Substrates
Mechanism proposed for flavin-dependent hydroxylases
Substrate
E
Substrate
NADH
E
FAD
FAD
Substrate
FADHE
NADH
NAD+
stopped-flow spectroscopic evidence for boxed intermediates
flavin
hydroperoxide
acts as
electrophile
NAD+
R
N
N
N
H
O O
OH
H
N
O
H+
NH
N
H
B+
R
N
O
NH
O2
N
H
O
O
O
NH
O
OH
-OOC
-OOC
H
_
N
see Scheme 3.33
O O
OH
electrophilic OOC
aromatic
substitution
R
N
:B
:B
HO
R
N
N
NH
N
H
O
OH
OH
H
O
+
OH
O
-OOC
-OOC
H
B
B:
- H2O
FAD
Scheme 4.6
Hammett Study
p-hydroxybenzoate hydroxylase
X
R
N
N
NH
N
4.8
O
O
log Vmax for hydroxylation vs pKa
linear free energy relationship
(Electron deficient mechanism)
 = -0.5
Consistent with electrophilic aromatic substitution
Electrophilic Substrates
Reaction catalyzed by bacterial luciferase
RCHO
FMN, O2
RCOOH
+ h
NADH
long-chain aldehydes
(electrophilic
substrates)
Scheme 4.7
Nucleophilic Mechanism for Bacterial Luciferase
R
N
N
O
on
warming
R
N
O O
O
NH
N
H
:B
H
N
B
H
H
OH
O
O
OH
h
R
R
N
-H2O
N
FMN
H
B:
O
NH
N
Scheme 4.8
O
NH
N
H
:B
O O
H
O H B
isolated by
cryoenzymology R electrophilic
substrates
(-30 C in mixed
aqueous-organic media)
*
R
N
O
- RCOOH
NH
N
H
N
O
OH
detected spectrophotometrically
However, with 8-substituted FMN analogues rate increases with
decreasing one electron oxidation potentials of analogues
Chemically Initiated Electron Exchange
Luminescence (CIEEL) Mechanism for
Bacterial Luciferase
R
N
N
N
N
H
O
B
H
O
O
B
OH
H
R
O
O
-H+
R
H
R
OH
C
R
NH
O O
H
O
C
OH
O
R
N
N
N
H
O O
H
-H2O
OH
FMN
O
Scheme 4.9
R
OH
O
NH
:
:
NH
N
H
SET
R
N
O
Alternative One-electron Mechanism via a Dioxirane
Dioxirane mechanism for bacterial luciferase
R
N
N
R
N
O
N
R
N
O
N
R
N
O
rate
NH
N
H
O O
O
NH
N
H
:B
H
SET
O O
B
H
O
H
H
O
N
H O
H O
O
O
determining
step
N
H O
H O
O
O
R
R
NH
R
H
N
O
NH
O
H
:B
R
h
R
N
-H2O
N
FMN
H
B:
kx/kh vs. p
for 8-substituted
flavins
 = -4
(facilitated by
e- donation)
N
O
O
N
H O
H O
O
NH
N
*
R
N
O
NH
OH
R
O
Scheme 4.10
Inconsistent with Baeyer-Villiger mechanism ( values +0.2 to 0.6)
Baeyer-Villiger Oxidation of Ketones
O
O
O
R
R' +
Ar C
O
O
O
R
C
O
Scheme 4.11
R'
RO
O
R'
+ ArCOO-
Ar
O
Migratory aptitude - more e- donating group
migrates (in the case above, R)
Ketone Monooxygenases - an Example of a
Baeyer-Villiger Oxidation
Reaction catalyzed by cyclohexane oxygenase
Scheme 4.12
O
O
18
+ NADPH +
18O
2
enzyme
FAD
O
+ H218O
C4a-FAD hydroperoxide intermediate detected
Other Reactions Catalyzed by
Cyclohexanone Oxygenase
O
Me
Ph
Ph
O
Me
O
RCHO
R = alkyl
Scheme 4.13
RCOOH
same migratory aptitudes
as nonenzymatic reaction
Cyclohexane Oxygenase Proceeds with
Retention of Configuration (like nonenzymatic)
O
O
H
D
O
H
D
Scheme 4.14
Migratory Aptitude of Cyclohexanone
Oxygenase-catalyzed Reaction
O
O
O
O
O
Scheme 4.15
Same migratory aptitude
as nonenzymatic
(3° > 2° > 1° > Me)
O
D
D
D
D
4.9
no loss of D (like nonenzymatic reaction)
Baeyer-Villiger-type Mechanism Proposed
for Cyclohexanone Oxygenase
R
N
N
R
N
O
NH
N
H
O O
O
O
N
O
O
H
H
B
O
OH
B:
O
electrophilic
substrate
Scheme 4.16
O
NH
N
O O
:B
N
-H2O
NH
N
H
H
R
N
O
O
FAD
Reaction of Cyclohexanone
Oxygenase with Boranes
O
B(OMe)2
O
B
OMe
OMe
FAD
O
B
OMe
OMe
OH
hydrolysis
same as nonenzymatic reaction
Scheme 4.17
Reactions Catalyzed by Ketone
Monooxygenase
O
R1
R1
O
R2
4.10
O
O
R2
R2
R1
+
4.12
4.11
when R1 = R2 = Me
R1 = H; R2 = Me
O
1
1
:
:
20
1
(same as nonenzymatic reaction)
Scheme 4.18
Reactions Catalyzed by the Ketone
Monooxygenase from A. calcoaceticus
O
R1
O
R2
R1
O
A. calcoaceticus
R2
R2
H
H
1S
4.10
O
O
5R
R1
+
(1S,5R)
R1 = H, R2 = CH3
(1S,5S)
H
1R
5S
4.14
4.13
R1 = R2 = H
H
(1R,5S)
>95% ee
(1R,5S)
>95% ee
racemate
Scheme 4.19
Reactions Catalyzed by the Ketone
Monooxygenase from P. putida
O
R1
O
R2
R2
O
P. putida
R1
O
R1
H
H
1R
4.10
O
5S
R2
+
(1R,5S)
R1 = H, R2 = CH3
(1R,5R)
H
1S
5R
4.16
4.15
R1 = R2 = H
H
50% ee
(1S,5R)
>95% ee
(1S,5R)
>95% ee
racemate
Scheme 4.20
Pterin-dependent Monooxygenases
aromatic hydroxylation
pteridine ring
N
N
N
N
4.17
Tetrahydrobiopterin
H
N
HO
N
NH2
H
NH
N
H
H3C
H
OH
O
4.18
• Fe2+ also required for activity
• Only a few enzymes require tetrahydrobiopterin
• Important in biosynthesis of dopa,
norepinephrine, epinephrine, and serotonin
• Reactions similar to flavoenzymes
Comparison of the Dihydrobiopterin and
Tetrahydrobiopterin with Oxidized Flavin
and Reduced Flavin
R
N
N
R
N
O
NH
N
HO
N
NH
N
OH
NH2
H
N
pteridine
reductase
(NADPH)
R
N
O
reduced flavin
O
4.19
NH2
NH
N
H
N
dihydropteridine
reductase
(NADPH)
R
N
H
O
4.20
H
N
R
Scheme 4.21
O
NH
N
H
O
oxidized flavin
N
H
N
N
H
+
NH2
H
N
NH
O
4.21a
N
NH2
NH
O
4.21b
Reaction Catalyzed by Phenylalanine
Hydroxylase
X
COO-
X
NH3+
+
18O
2
+ H4-pterin
X = 2H, 3H, Cl, Br, alkyl
Phe
hydroxylase
H18O
NIH shift
[1,2] migration
COONH3+
+ H218O + H2-pterin
Scheme 4.22
Similar to flavin hydroxylases except 2H washed
out with flavoenzymes
Possible Intermediate
H
N
R
N
H
N
+
NH2
NH
O O
O
H
4.22
Mechanism of the Reaction Catalyzed by
Tetrahydrobiopterin-dependent Monooxygenases
R
H
N
+
NH2
NH
N
H
H
N
18O
+
NH2
N
H
H2-pterin +
H218O
2
see Scheme 3.33
R
O
B+
N
H
H
18O
+
:
H
N
H18O
NH
R
X
18O
R
X
O
4.23
18O
H
:
R
X
H18O
R
nucleophilic substrate
X
B:
H
X
H18O
Scheme 4.23
R
H18O
NIH shift
X
R
discussed with hemedependent enzymes
Evidence for Arene Oxide Intermediate
Reaction of dihydrophenylalanine with
phenylalanine hydroxylase
COO-
Phe
hydroxylase
NH3+
4.24
Scheme 4.24
COOO
4.25
NH3+
Arene Oxide Mechanism Proposed for
Tetrahydrobiopterin-dependent Monooxygenases
H
2H
COO-
Phe
hydroxylase
NH3+
2H
O
COO-
2H
COO-
O
COO-
HO
2H
NH3+
NH3+
O
H
Scheme 4.25
H
Tyr
NH3+
HO
COO-
2H
2H
COO-
NH3+
NH3+
m-Tyr
Incubation with [4-2H]Phe should favor formation of
m-Tyr (isotope effect), and [3,5-2H2]Phe should favor
Tyr, but they do not.
Therefore, not an arene oxide intermediate
Cationic Mechanism Proposed for
Tetrahydrobiopterin-dependent Monooxygenases
Fe
COO-
HO
Y
COO-
X
NH3+
O
Phe
COO-
hydroxylase
NH3+
as X is
larger
X
X
NH3+
HO
COONH3+
Y
O
H
X
HO
COO-
COO-
X
NH3+
m-Tyr
Scheme 4.26
NH3+
The larger the size of X, the more m-Tyr product
R
COOH
NH2
 = -5
(cation-like TS‡)
Alternative Species
H
N
R
N
N
H O
O
H
N
NH2+
NH
R
O
N
H
N
NH2+
NH
OH O
O
Fe4+
Fe2+
4.25b
4.25a
These species could account for alkyl
hydroxylation products (heme chemistry),
e.g. with H3C
COOH
NH2
hydroxylation here
Heme-Dependent Monooxygenases
Heme
N
N
FeIII
N
N
COOH
COOH
4.26
Cytochrome P450s (>500 different isozymes) require
NAD(P)H and O2
Protection from xenobiotics
Reactions Catalyzed by Heme-dependent
Monooxygenases
Subs tr ate
Alkane
Pr oduct
Alcohol
Alkene
Epoxide
Arene
Arenol or arene oxide
R2NH, R 2O, R2S
RNH 2, ROH, RSH + RCHO
R3N, R2S
+ + R3N-O, R2S-O
RCH 2X
RCHO + HX
RCH 2OH
RCHO
RCHO
RCOOH
Molecular Oxygen Activation by Hemedependent Monooxygenases
(requires NADPH)
In P450cam
Thr-252
low-spin state
H
H
FeIII
N
N
N
R-H
N
FeIII
S
FeIII more
readily
accepts e-
N
FMNH
N
N
N
N
FeIII
N
S
FMN
NAD(P)H
:O:
N
N
N
FeV
N
S
S
4.33d
4.33c
B
O
R-H
O
O
FMN
N
FeIII
N
N
N
S
S
4.30
R-O-H
R-H : :
O
N
FeIV
N
N
O2
FeII
FADH
FAD
4.28
N
FMN
S
N
H
O
R-H
R-H
N
N
N
4.27
FMN
R-H
O
N
high-spin state
NAD(P)+
4.31
4.29
cytochrome P450
reductase
B
OH
:O:
N
N
N
FeIV
N
4.33b
calculations favor
this structure
means isolated and characterized
R-H
:O:
N
S
H
N
N
FeIII
N
S
4.33a
N
N
-H2O
N
O
FeIII
N
N
S
4.32
Scheme 4.27
Alkane Hydroxylation
Two-step radical mechanism with oxygen
rebound for alkane oxygenation by hemedependent monooxygenases
3° > 2° > 1°
R
R
H
:O:
N
N
FeIV
C
N
N
OH
R'
R''
N
N
FeIV
N
C
R'
R''
N
N
oxygen
rebound
N
N
N
FeIII
S
S
S
4.33b
4.34
4.28
R
HO
C
R'
R''
retention of
configuration
Scheme 4.28
Intermolecular isotope effect < 2
Intramolecular isotope effect > 11
(suggests C-H cleavage is not the
rate-determining step)
C-H cleavage during catalysis
Products from the Reaction of all Exo2,3,5,6-tetradeuterionorbornane with the
CYP2B4 Isozyme of Cytochrome P450
D
OH
D
D
D
D
D
D
4.36
CYP2B4
4.35
D
Scheme 4.29
D
4.37
D
4.38
OH
D
H
H
D
D
OH
D
D
D
D
D
4.39
Scrambling of stereochemistry
supports 2-step radical mechanism
OH
Radical Clocks - detection of
radical intermediates
Radical clock approach for determination of
reaction rates in radical rearrangement reactions
substrate
H
substrate
kOH
substrate
OH
kr
known
rearranged
substrate
kOH = kr (substrate-OH / rearranged substrate-OH)
rearranged
substrate OH
Scheme 4.30
The rate of hydroxylation can be calculated (lifetime
of radical intermediate)
Example of Radical Clock
Cytochrome P450-catalyzed monooxygenation
of a cyclopropane analogue
kOH
H
:O:
N IV N
Fe
N
N
S
HO
a
OH
kr
N
N
FeIV
N
S
N
Scheme 4.31
OH
b
From kr = 2  109 s-1 and the ratio of a/b,
can calculate kOH = 2.4  1011 s-1
Cytochrome P450-catalyzed Oxidation of
Trans-1-methyl-2-phenylcyclopropane
FeIV O
H
a
HO
FeIV OH
a
kOH
4.40
4.42
4.41
b
3 x 1011 s-1 kr
FeIV OH
OH
4.44
OH
4.43
Scheme 4.32
Perdeuteration (CD3) gives increased pathway b
called metabolic switching
Evidence against a True Radical Intermediate
Another ultrafast radical clock reaction
catalyzed by cytochrome P450
H
H
H
b
OH
kOH
FeIV O
a
4.45
3  1011 s-1
Scheme 4.33
kr
b
OH
very little
kOH has to be faster than the decomposition of a
TS‡ (6  1012 s-1); therefore propose carbocation
after oxidation step
A Hypersensitive Radical Probe Substrate to
Differentiate a Radical from a Cation
Intermediate Generated by Cytochrome P450
Scheme 4.34
CH2
CH3
O
a
Ph
O
4.46
Ph
O
O
HO
HO
Ph
4.47
Ph
4.48
b
+CH2
Ph
O
4.49
Ph
O
HO-
Ph
-tert-butanol
OH
O
Ph
Ph
H
H
O
4.50
O
4.51
based on nonenzymatic reactions
With CYP2B1 - mostly unrearranged, but small amount of
both 4.48 and 4.51; therefore radical lifetime is 70 fs
A Concerted, but Nonsynchronous,
Mechanism Proposed for Cytochrome P450
H
H
R
R
H
R
70 fs
H
R
H
R
O
O
O
O
O
Fe(IV)
Fe
Fe
Fe
Fe(III)
Scheme 4.35
General conclusion:
More than one oxidizing species involving
more than one pathway with multiple highenergy heme complexes (radical and cation)
Alkene Epoxidation
Two-step radical mechanism with oxygen
rebound for alkene oxygenation by hemedependent monooxygenases
R
R'
R
O
:O:
N
FeIV
N
R'
N
N
S
N
N
FeIV
N
N
N
S
4.52
lifetime?
Scheme 4.36
"oxygen
rebound"
N
N
R
R'
N
FeIII
S
O
Evidence for Short-lived Radical
Cytochrome P450-catalyzed epoxidation of
trans-1-phenyl-2-vinylcyclopropane
O
P450
Ph
Ph
4.55
4.56
only
O
Ph
N
FeIV
N
N
S
Scheme 4.37
N
cyclopropyl/carbinyl
radical rearrangement
not detected
Arene Hydroxylation
Isolation of first arene oxide
Cytochrome P450-catalyzed formation of an
arene oxide
O
P450
4.57
Scheme 4.38
Is it an intermediate
or side product?
Evidence for a Cyclohexadienone Intermediate
A common intermediate in the oxygenation of
naphthalene
2H
O
H
O
2H
H
either
H
OH
2H
(H)
O
H2
Scheme 4.39
4.58
same product and
2H incorporation
from both isomers
Should have observed 1- and 2hydroxynaphthalene because of
an isotope effect
Concerted (pathway a) and Stepwise (pathway b)
Mechanisms for the Potential Conversion of an Arene
Oxide to a Cyclohexadienone
H
O
H
H
H
a
concerted
b
O
O
H
H
H
stepwise
O
H
Scheme 4.40
Evidence against concerted: 1) no deuterium isotope effect
2) Hammett plot shows large -
(carbocation intermediate)
Isotope Effect and Hammett Studies Indicate either Radical or
Cation (or both) Intermediates, but not Arene Oxide
Mechanism proposed for heme-dependent oxygenation of
aromatic compounds
a
D
D
R
a :O:
:O:
N
N
FeIV
N
D
R
N
N
b
FeIV
N
S
R
:O:
reasonable
electron
transfer
N
N
N
N
S
FeIII
N
N
S
unfavorable
a
4.59a
4.59b
NIH
shift
oxygen
b rebound
c favorable
N
O
D
R
D
R
N
N
N
FeIII
H
O
S
Electrophilic addition
when R is o/p directing, get mostly p product
when R is m-directing, get m and p products
HO
R
D
Scheme 4.41
Sulfur Oxygenation
Electron transfer mechanism proposed for
heme-dependent oxygenation of sulfides
N
FeIV
N
:O:
..
..
:O:
:O:
N
S
S
S
N
N
N
S
FeIV
N
S
R'
R
R'
R
R'
R
N
N
oxygen
rebound
N
N
N
FeIII
S
X
Scheme 4.42
S
CH3
Linear free energy relationship: log kcat vs. one-electron
oxidation potential as well as +
N-Dealkylation
Electron transfer mechanism proposed for
heme-dependent oxygenation of tertiary amines
R
R
N
H H
4.60
N
N
..
:O:
R''
FeIV
N
N
S
N
N
FeIV
N
S
R
R''
R'
N
:O:
R'
R
R'
N
R'
N
R''
R''
4.61
H
H
..
: OH
N
N
N
R
N
FeIV
N
oxygen
rebound
N
R'
H+
R''
S
:
HO:
H
4.62
R'
Scheme 4.43
R"CHO
+
R
N
H
With primary and secondary amines hydrogen atom abstraction mechanism favored
(see next slide)
O-Dealkylation
Hydrogen atom abstraction mechanism proposed
for heme-dependent oxygenation of ethers
R
R
O
H
R'
O
H
..
: OH
:O:
N
N
FeIV
N
N
N
FeIV
N
S
R
H
N
H+
R'
O
oxygen
rebound
H
HO:
:
S
N
R'
N
N
N
N
FeIII
S
Scheme 4.44
R'CHO
+
ROH
Not electron transfer mechanism-- oxidation potential for oxygen is too high
C-C Bond Cleavage
Reaction catalyzed by aromatase
O
O
19
CH3
3 O2
+ HCO2H
3 NADPH
O
HO
4.63
androstenedione
Scheme 4.45
4.64
estrone
Fate of the Atoms during Aromatase-catalyzed
Conversion of Androstenedione to Estrone
also a substrate
O
CH3
C
H
HR
HS
H
O
H
B
O
4.63
NADPH
+H+
-H2
H
O
4.65
H
-H2
2
NADPH
+H+
H H
O
H
D
2
A
O
H
O
O
4.66
4.67
also a substrate
O2 NADPH
+H+
O
Scheme 4.46
H2O
+
HC OH
+
HO
4.64
First two oxygenation steps proceed by
normal heme hydroxylation mechanism
Three Possible Mechanisms for the Last Step in
the Aromatase-catalyzed Oxygenation of
Androstenedione
heme peroxide
Fe+3 O
O
O
O
O
Fe+3 O
OH
O
Fe+3 O
H
OH
Fe+3
Fe+3 OH
- HCOOH
(1)
O
HO
O
O
4.64
like Fl-OO- addition to aldehydes
Fe+4 O
O
Fe+4 OH
H
O
O
(2)
4.64
HO
+
Fe+4 OH
C
Fe3+ + HCOOH
H
HO
O
Fe+4 O
Fe+4 O
O
O
Fe+4 O
O
Fe+4 O
H
H
CH
O
4.64 +
(3)
O
O
Scheme 4.47
O
HO
Fe+4 OH
Fe3+ + HCOOH
CH
Evidence for Heme Peroxide Mechanism
Oxidation of pregnenolone, catalyzed by an
isozyme of cytochrome P450 (P45017)
H3 C
21
retained
O
H
H
17
HD
16
HO
OH
HD
17
O2
16
NADPH
HO
+
H3CCOOH
HO
HO
4.68
4.69
Scheme 4.48
FeIV-O• would have abstracted a C21 CH3 hydrogen
or a C16 or C17 H
Hydrogen Atom Abstraction Mechanism, Using a
Heme Iron Oxo Species, for the P45017-catalyzed
Oxygenation of Pregnenolone
H3C
H2C
O
H
FeIV O
FeIV OH
HD
17
H
H
HD
17
16
HD
17
16
HO
HO
O
16
HO
HO
+ CH2=C=O
HO
HO
2H
4.70
FeIV OH
Scheme 4.49
4.71
2O
H2C2HCOO2H
FeIII
retained
H
OH
HD
17
16
HO
HO
In 2H2O, ketene would give H2C2HCOO2H; no 2H
found in CH3 group of acetate, therefore not FeIV-O•
Evidence for a Nucleophilic Mechanism, Using Heme Peroxy
Anion Followed by a Radical Decomposition of the Heme
Peroxide, for the P45017-catalyzed Oxygenation of Pregnenolone
H
H3C
B
O
H3C
OH
O
H
O
17
H3C
OH
O
O FeIII
O-FeIII
O FeIII
O
OH
Scheme 4.50
CH3COOH
O FeIII
FeIII
+H+
Mutation of Thr-302 (T302A) in P450 2B4 (needed for formation of iron
oxo species) decreased hydroxylation activity, but increased deacylation
(nucleophilic) activity
H
FeIII
O
OH
O
302T
FeIII
O
OH2
FeIV
O
Further Evidence for Heme Peroxide
Nucleophilic mechanism, using heme peroxy anion followed
by a Baeyer-Villiger rearrangement, for the lanosterol 14methyl demethylase-catalyzed oxygenation of lanosterol
C8H15
C8H15
2 O2
14
2 NADPH
C
HO
HO
CH3
O
C
O H+
H O
H
O
HO
FeIII
FeIII OO
4.72
Baeyer-Villiger
rearrangement
C8H15
C8H15
no O2 /NADPH
- HCO2H
- H2O
HO
Scheme 4.51
H
4.74
H
O
O
HO
4.73
–O
FeIII
H
isolated, also a substrate
Nucleophilic Mechanism, Using Heme Peroxy Anion, Followed
by a Radical Decomposition of the Heme Peroxide, for the
Lanosterol 14-Methyl Demethylase-catalyzed Oxygenation of
Lanosterol Would Not Give the Baeyer-Villiger Product
C8H15
C8H15
2 O2
2 NADPH
14
C
HO
CH3
O H+
C
HO
H
O
H O
HO
FeIII OO
-HCOOH
C
H
HO
4.74
Scheme 4.52
C8H15
C8H15
-FeIII-OH
HO
O
FeIII
4.72
C8H15
H
HO
O
FeIII
H O
O
H
O
FeIII
No formate ester formed
H
O
O
O
O
4.75
Synthesized to test Baeyer-Villiger
mechanism with aromatase - no estrone
Maybe aromatase and P45017 have
different mechanisms from that of
lanosterol 14-methyl demethylase
Model Studies on the Mechanism of
Aromatase
O
OTHP
OTHP
H2O2
gives
aromatase
product
+ HCO2H
TBDMSO
TBDMSO
4.76
O
OTHP
O
H2O2
no estrone
O
O
4.77
Scheme 4.53
OTHP
O
Revised Aromatase Mechanism
Mechanism proposed for aromatase
initiated by dienol formation
O
O
O
O
FeIIIO
O
O
OH
FeIIIO
OH
O
O
FeIIIOO
H
B
B
O
H
HO
HO
HO
4.78
-HCOOH
O
Ha
HO
Scheme 4.54
FeIIIO
-FeIIIOHb
O
Hb
Ha
HO
4.79
Nonheme Iron Oxygenation
Methane monooxygenases
Glu243
O
binuclear
iron cluster
Glu114 COO
O
H2 O
OOC
FeIII
His147
N
Glu209
FeIII
His246
O
O H
O
N
N
N
Glu144
4.80
CH4  CH3OH
Binuclear Ferric Cluster of Methane Monooxygenase
2 NADPH
H
O
FeIII
FeIII
4.81
2 NADP+
H
H
O
FeII
2
FeII
O
FeIII
*
FeIII
H
O
FeIV
FeIV
4.82
4.84a
4.83a
+2H+
-H2
Scheme 4.55
H
O
FeIII
soluble methane monooxygenase
4.83b
H
FeIII
O
FeIV
FeIV
4.84b
XAS and Mössbauer spectroscopy support 4.83a, not 4.83b
Studies with the hypersensitive cyclopropane probe (4.46,
Scheme 4.34) and methylcubane indicate a cation, not radical,
intermediate
Therefore mechanism like P450
Copper-dependent Oxygenation
Reaction catalyzed by dopamine -monooxygenase
from ascorbic acid
+
HO
NH2
HO
O2
H
2e2H+
HO
+
NH2
HO
4.85
OH
4.86
Scheme 4.56
Optimal activity with 2 CuII per subunit
one CuII catalyzes e- transfer from ascorbate
one CuII catalyzes oxygen insertion into substrate
H2O
Mechanism Proposed for Dopamine -Monooxygenase
CuII
CuII
CuII
O
O
O
O
H
O
H
H2O
H
H
Ar
NH3+
Ar
CuII
CuII
CuII
Ar
O
4.88
4.87
OH
NH3+
Ar
NH3+
H
O
O
HO
NH3+
+H+
Ar
O
HO
H
NH3+
Ar
O
NH3+
Scheme 4.57
Hammett plot  = -1.5 fits better to  than +,
suggesting a radical with a
polar TS‡