1. The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 4 Monooxygenation

2. Monooxygenation
Table 4.1. Typical reactions catalyzed by monooxygenases

3. Reaction catalyzed by lactate oxidase from Mycobacteria
Internal Monooxygenase
Flavin-dependent Hydroxylases
Reaction catalyzed by lactate oxidase from Mycobacteria
Scheme 4.1
No external reducing agent required

4. The lactate oxidase reaction under anaerobic conditions
One Turnover Experiment (enzyme concentration in excess over substrate)
The lactate oxidase reaction under anaerobic conditions
Acting like an oxidase
Scheme 4.2

5. Reaction of Reduced Lactate Oxidase with Pyruvate and Oxygen
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:

6. Possible Mechanisms for Lactate Oxidase
like DAAO
flavin hydroperoxide acts as a nucleophile
electrophilic
substrate
Scheme 4.4

7. NAD(P)H reduction of flavin
External Monooxygenases
NAD(P)H reduction of flavin
O2 activation
Scheme 4.5
Activated O2 is probably in the form of flavin hydroperoxide

8. Mechanism proposed for flavin-dependent hydroxylases
Nucleophilic Substrates
Mechanism proposed for flavin-dependent hydroxylases
stopped-flow spectroscopic evidence for boxed intermediates
flavin hydroperoxide acts as electrophile
electrophilic aromatic substitution
Scheme 4.6

9. Hammett Study p-hydroxybenzoate hydroxylase
log Vmax for hydroxylation vs pKa
linear free energy relationship
 = -0.5
(Electron deficient mechanism)
Consistent with electrophilic aromatic substitution

10. Reaction catalyzed by bacterial luciferase
Electrophilic Substrates
Reaction catalyzed by bacterial luciferase
long-chain aldehydes (electrophilic substrates)
Scheme 4.7

11. Nucleophilic Mechanism for Bacterial Luciferase
on warming
isolated by cryoenzymology
(-30 C in mixed aqueous-organic media)
electrophilic
substrates
Scheme 4.8
detected spectro-photometrically
However, with 8-substituted FMN analogues rate increases with decreasing one electron oxidation potentials of analogues

12. Chemically Initiated Electron Exchange Luminescence (CIEEL) Mechanism for Bacterial Luciferase
SET
Scheme 4.9

13. Dioxirane mechanism for bacterial luciferase
Alternative One-electron Mechanism via a Dioxirane
Dioxirane mechanism for bacterial luciferase
SET
kx/kh vs. p
for 8-substituted flavins
 = -4
(facilitated by e- donation)
Scheme 4.10
Inconsistent with Baeyer-Villiger mechanism ( values +0.2 to 0.6)

14. Baeyer-Villiger Oxidation of Ketones
Scheme 4.11
Migratory aptitude - more e- donating group migrates (in the case above, R)

15. Reaction catalyzed by cyclohexane oxygenase
Ketone Monooxygenases - an Example of a Baeyer-Villiger Oxidation
Reaction catalyzed by cyclohexane oxygenase
Scheme 4.12
C4a-FAD hydroperoxide intermediate detected

16. Other Reactions Catalyzed by Cyclohexanone Oxygenase
same migratory aptitudes as nonenzymatic reaction
Scheme 4.13

17. Cyclohexane Oxygenase Proceeds with Retention of Configuration (like nonenzymatic)
Scheme 4.14

18. Migratory Aptitude of Cyclohexanone Oxygenase-catalyzed Reaction
Same migratory aptitude as nonenzymatic
(3° > 2° > 1° > Me)
Scheme 4.15

19. no loss of D (like nonenzymatic reaction)

20. Baeyer-Villiger-type Mechanism Proposed for Cyclohexanone Oxygenase
electrophilic substrate
Scheme 4.16

21. Reaction of Cyclohexanone Oxygenase with Boranes
same as nonenzymatic reaction
Scheme 4.17

22. Reactions Catalyzed by Ketone Monooxygenase
when R1 = R2 = Me :
R1 = H; R2 = Me :
(same as nonenzymatic reaction)
Scheme 4.18

23. Reactions Catalyzed by the Ketone Monooxygenase from A. calcoaceticus
>95% ee
>95% ee
racemate
Scheme 4.19

24. Reactions Catalyzed by the Ketone Monooxygenase from P. putida
50% ee
>95% ee
>95% ee
racemate
Scheme 4.20

25. Pterin-dependent Monooxygenases aromatic hydroxylation
pteridine ring

26. Tetrahydrobiopterin Fe2+ also required for activity
Only a few enzymes require tetrahydrobiopterin
Important in biosynthesis of dopa, norepinephrine, epinephrine, and serotonin
Reactions similar to flavoenzymes

27. Comparison of the Dihydrobiopterin and Tetrahydrobiopterin with Oxidized Flavin and Reduced Flavin
Scheme 4.21

28. Reaction Catalyzed by Phenylalanine Hydroxylase
NIH shift
[1,2] migration
Scheme 4.22
Similar to flavin hydroxylases except 2H washed out with flavoenzymes

29. Possible Intermediate

30. Mechanism of the Reaction Catalyzed by Tetrahydrobiopterin-dependent Monooxygenases
nucleophilic substrate
discussed with heme-dependent enzymes
Scheme 4.23

31. Reaction of dihydrophenylalanine with phenylalanine hydroxylase
Evidence for Arene Oxide Intermediate
Reaction of dihydrophenylalanine with phenylalanine hydroxylase
Scheme 4.24

32. Arene Oxide Mechanism Proposed for Tetrahydrobiopterin-dependent Monooxygenases
Tyr
Scheme 4.25
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

33. The larger the size of X, the more m-Tyr product
Cationic Mechanism Proposed for Tetrahydrobiopterin-dependent Monooxygenases Fe
as X is larger
Scheme 4.26
m-Tyr
The larger the size of X, the more m-Tyr product
= -5
(cation-like TS‡)

34. Alternative Species
These species could account for alkyl hydroxylation products (heme chemistry), e.g. with
hydroxylation here

35. Heme-Dependent Monooxygenases
Cytochrome P450s (>500 different isozymes) require NAD(P)H and O2
Protection from xenobiotics

36. Reactions Catalyzed by Heme-dependent Monooxygenases+ - + -

37. Molecular Oxygen Activation by Heme-dependent Monooxygenases
(requires NADPH)
In P450cam Thr-252
low-spin state
high-spin state
FeIII more
readily accepts e-
cytochrome P450 reductase
calculations favor this structure
Scheme 4.27
means isolated and characterized

38. Alkane Hydroxylation
Two-step radical mechanism with oxygen rebound for alkane oxygenation by heme-dependent monooxygenases
3° > 2° > 1°
retention of configuration
Scheme 4.28
(suggests C-H cleavage is not the
rate-determining step)
Intermolecular isotope effect < 2
Intramolecular isotope effect > 11
C-H cleavage during catalysis

39. Products from the Reaction of all Exo-2,3,5,6-tetradeuterionorbornane with the CYP2B4 Isozyme of Cytochrome P450
Scheme 4.29
Scrambling of stereochemistry supports 2-step radical mechanism

40. Radical Clocks - detection of radical intermediates
Radical clock approach for determination of reaction rates in radical rearrangement reactions
known
Scheme 4.30
The rate of hydroxylation can be calculated (lifetime of radical intermediate)

41. Cytochrome P450-catalyzed monooxygenation of a cyclopropane analogue
Example of Radical Clock
Cytochrome P450-catalyzed monooxygenation of a cyclopropane analogue a
Scheme 4.31 b
From kr = 2  109 s-1 and the ratio of a/b, can calculate kOH = 2.4  1011 s-1

42. Cytochrome P450-catalyzed Oxidation of Trans-1-methyl-2-phenylcyclopropane
Scheme 4.32
Perdeuteration (CD3) gives
increased pathway b
called metabolic switching

43. Another ultrafast radical clock reaction catalyzed by cytochrome P450
Evidence against a True Radical Intermediate
Another ultrafast radical clock reaction catalyzed by cytochrome P450
3  1011 s-1
Scheme 4.33
very little
kOH has to be faster than the decomposition of a TS‡ (6  1012 s-1); therefore propose carbocation after oxidation step

44. A Hypersensitive Radical Probe Substrate to Differentiate a Radical from a Cation Intermediate Generated by Cytochrome P450
Scheme 4.34
based on nonenzymatic reactions
With CYP2B1 - mostly unrearranged, but small amount of both 4.48 and 4.51; therefore radical lifetime is 70 fs

45. A Concerted, but Nonsynchronous, Mechanism Proposed for Cytochrome P450
Scheme 4.35
General conclusion:
More than one oxidizing species involving more than one pathway with multiple high-energy heme complexes (radical and cation)

46. Alkene Epoxidation
Two-step radical mechanism with oxygen rebound for alkene oxygenation by heme-dependent monooxygenases
lifetime?
Scheme 4.36

47. Evidence for Short-lived Radical
Cytochrome P450-catalyzed epoxidation of trans-1-phenyl-2-vinylcyclopropane
only
cyclopropyl/carbinyl radical rearrangement not detected
Scheme 4.37

48. Cytochrome P450-catalyzed formation of an arene oxide
Arene Hydroxylation
Isolation of first arene oxide
Cytochrome P450-catalyzed formation of an arene oxide
Scheme 4.38
Is it an intermediate or side product?

49. A common intermediate in the oxygenation of naphthalene
Evidence for a Cyclohexadienone Intermediate
A common intermediate in the oxygenation of naphthalene
either
same product and 2H incorporation from both isomers
Should have observed 1- and 2-
hydroxynaphthalene because of
an isotope effect
Scheme 4.39

50. Concerted (pathway a) and Stepwise (pathway b) Mechanisms for the Potential Conversion of an Arene Oxide to a Cyclohexadienone
concerted
stepwise
Scheme 4.40
Evidence against concerted: 1) no deuterium isotope effect
2) Hammett plot shows large -
(carbocation intermediate)

51. Electrophilic addition
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
reasonable
unfavorable
NIH shift
favorable
Electrophilic addition
when R is o/p directing, get mostly p product
when R is m-directing, get m and p products
Scheme 4.41

52. Sulfur Oxygenation
Electron transfer mechanism proposed for heme-dependent oxygenation of sulfides
Scheme 4.42
Linear free energy relationship: log kcat vs. one-electron oxidation potential as well as +

53. N-Dealkylation
Electron transfer mechanism proposed for heme-dependent oxygenation of tertiary amines
Scheme 4.43
With primary and secondary amines hydrogen atom abstraction mechanism favored (see next slide)

54. O-Dealkylation
Hydrogen atom abstraction mechanism proposed for heme-dependent oxygenation of ethers
Scheme 4.44
Not electron transfer mechanism--
oxidation potential for oxygen is too high

55. Reaction catalyzed by aromatase
C-C Bond Cleavage
Reaction catalyzed by aromatase
androstenedione
estrone
Scheme 4.45

56. Fate of the Atoms during Aromatase-catalyzed Conversion of Androstenedione to Estrone
also a substrate
also a substrate
Scheme 4.46
First two oxygenation steps proceed by normal heme hydroxylation mechanism

57. Three Possible Mechanisms for the Last Step in the Aromatase-catalyzed Oxygenation of Androstenedione
heme peroxide
like Fl-OO- addition to aldehydes
Scheme 4.47

58. Evidence for Heme Peroxide Mechanism
Oxidation of pregnenolone, catalyzed by an isozyme of cytochrome P450 (P45017)
retained
Scheme 4.48
FeIV-O• would have abstracted a C21 CH3 hydrogen or a C16 or C17 H

59. Hydrogen Atom Abstraction Mechanism, Using a Heme Iron Oxo Species, for the P45017-catalyzed Oxygenation of Pregnenolone
Scheme 4.49
retained
In 2H2O, ketene would give H2C2HCOO2H; no 2H found in CH3 group of acetate, therefore not FeIV-O•

60. 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
Scheme 4.50
Mutation of Thr-302 (T302A) in P450 2B4 (needed for formation of iron oxo species) decreased hydroxylation activity, but increased deacylation (nucleophilic) activity

61. 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
Scheme 4.51
isolated, also a substrate

62. 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
No formate ester formed
Scheme 4.52

63. Synthesized to test Baeyer-Villiger mechanism with aromatase - no estrone
Maybe aromatase and P45017 have different mechanisms from that of lanosterol 14-methyl demethylase

64. Model Studies on the Mechanism of Aromatase
gives aromatase product
Scheme 4.53

65. Mechanism proposed for aromatase initiated by dienol formation
Revised Aromatase Mechanism
Mechanism proposed for aromatase initiated by dienol formation
Scheme 4.54

66. Nonheme Iron Oxygenation
Methane monooxygenases
binuclear iron cluster
CH4  CH3OH

67. Binuclear Ferric Cluster of Methane Monooxygenase*
Scheme 4.55
soluble methane monooxygenase
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

68. Reaction catalyzed by dopamine -monooxygenase
Copper-dependent Oxygenation
Reaction catalyzed by dopamine -monooxygenase
from ascorbic acid
Scheme 4.56
Optimal activity with 2 CuII per subunit
one CuII catalyzes e- transfer from ascorbate
one CuII catalyzes oxygen insertion into substrate

69. Mechanism Proposed for Dopamine -Monooxygenase
Scheme 4.57
Hammett plot  = -1.5
fits better to  than +, suggesting a radical with a polar TS‡