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Theoretical Studies on Cytochrome P450

cam
Walter Thiel
Max-Planck-Institut fr Kohlenforschung


Symposium of The Lise Meitner - Minerva Center
Haifa, 19 December 2004


Happy birthday Yitzhak...

QM/MM approach: Overview
QM: density functional theory (B3LYP)
MM: force field (CHARMM22)
QM MM interactions:
electronic embedding
Border region:
hydrogen link atoms L
charge shift for q(M
1
)
Codes:
ChemShell, Turbomole, DL-POLY
M
1

M
2

M
2

M
3

Q
1

Q
2

Q
3

Q
2

L
D. Bakowies and W. Thiel, J. Phys. Chem. 100, 10580 (1996).
P. Sherwood at al, J. Mol. Theochem 632, 1 (2003).

|
|
.
|

\
|
+ + =

J , A
AJ
AJ
AJ
AJ
J , i
AJ
A J
J , i
iJ
J O , I
MM QM
R
B
R
A
R
Z q
r
q
H

6 12
ChemShell: A modular QM/MM package
GAUSSIAN98
GAMESS-UK
Chemshell
Tcl scripts
GROMOS96
CHARMm26
MSI
Integrated
routines:
data
management
geometry
optimisation
molecular
dynamics
generic
force fields
QM/MM
coupling
MNDO99
MOPAC
QM codes MM codes


DL_POLY
TURBOMOLE
CHARMM27
academic
GULP
P. Sherwood et al, J. Mol. Struct. Theochem 632, 1-28 (2003).
MOLPRO
Cytochrome P450
Cam
(Pseudomonas Putida)
heme protein, thiolato ligand
completely buried active site
soluble - extensively
characterized by biochemical /
biophysical techniques
X-ray structures for various
intermediates of the catalytic
cycle
natural substrate camphor,
also other compounds
biohydroxylation of non-
activated C-H bonds

O
H
5exo
H
5endo
P450
cam
+ O
2
+ 2 e
-
+ 2H
+
+ H
2
O
O
OH
H
5endo
camphor
CYP450: Catalytic cycle
Mechanistic features:
electrons from NADPH
(2 3, 4 5)
binding of molecular oxygen
(3 4)
active species 6 (Compound I) not
observed experimentally
hydroxylation mechanism 6 8
under dispute (rebound
mechanism assumed)

N
N
N
N
Fe
COO
COO
N
N
N
N
Fe
Fe
III
OH
2
S
N
N
N
N
Cys
Fe
III
S
N
N
N
N
Cys
R-H
Fe
II
S
N
N
N
N
Cys
R-H
Fe
II
S
N
N
N
N
Cys
R-H
O
O
Fe
III
S
N
N
N
N
Cys
R-H
O
O
Fe
IV
S
N
N
N
N
Cys
R-H
O
Fe
III
S
N
N
N
N
Cys
R
OH
Fe
III
ROH
S
N
N
N
N
Cys
R-H
H
2
O
e
O
2
e
2-
2H
+
H
2
O
H
abs
H
2
O
ROH
1
2
3
4
5
6 (I)
7
8
2
3
4
5
6
7
8
1
Resting state
Initial coordinates: based on
PDB structures 1DZ4
[1]
and
1PHC
[2]
[1] I. Schlichting, J. Berendzen, K. Chu, A. M. Stock, S. A. Maves, D. A. Benson, R. M. Sweet, D. Ringe,
G. A. Petsko, S. G. Sligar, Science 287, 1615 (2000).
[2] T. L. Poulos, B. C. Finzel, A. J. Howard, Biochemistry 25, 5314 (1986).
P450
cam
: Iron(III)-aqua complex
Tyr96
Heme
Cys357
Resting state: QM/MM calculations
N
N
N N
Fe
O
S
R
MM
HN
H
N
C
(H)
O
O
(H)
(H)
MM
MM
(H) MM
R1w: R =
R3w: R =
Leu356
Leu358
Cys357
H
H H
QM: UKS-DFT, B3LYP
Basis: LACVP (ECP) + 6-31G (B1);
ligand atoms: 6-31
+
G*, water protons: 6-31
++
G**(B2)
MM part: CHARMM22 force field
QM/MM: - electrostatic embedding scheme,
- hydrogen link atoms and charge shift model
QM regions: R1w (42 atoms), R3w (59 atoms)
J. C. Schneboom and W. Thiel, J. Phys. Chem. B 108, 7468-7478 (2004).
Resting state: Comparison with experiment
B3LYP/CHARMM22 BLYP/CHARMM22 exp.

2
A
4
A
6
A
2
A
4
A
6
A
AE(QM/MM) 0 2.37 3.30 0 8.45 18.27
2
A
AE(QM) 0 0.12 4.13 0 6.07 18.63
-

r
FeO
[] 2.141 2.475 2.467 2.191 2.605 2.611 2.28(0.2)
r
FeS
[] 2.269 2.491 2.418 2.239 2.490 2.421 2.25(0.2)
r
FeN
[] 2.038 2.037 2.095 2.050 2.051 2.113 2.02(0.2)
r
FeH
[] 2.633 2.928 2.905 2.655 2.980 2.961 2.62
Large QM region R3w, basis set B2:
Hyperfine coupling constants: Resting state
[1] D. Goldfarb, M. Bernardo, H. Thomann, P. M. H. Kroneck, V. Ullrich, JACS 118, 2686 (1996).
[2] Y.-C. Fann, N. C. Gerber, P. A. Omulski, L. P. Hager, S. G. Sligar, B. M. Hoffman, JACS 116, 5989 (1994).
P450
cam
: Iron(III)-aqua complex B3LYP/CHARMM22
[MHz] A
iso
A
d
x
A
d
y
A
d
z

H
(1)
2.0 4.7 5.0 9.4
H
(2)
0.4 4.6 4.4 9.0
[MHz] A
tot
x
A
tot
y
A
tot
z

N
(B)
6.8 6.1 5.9
N
(A)
6.8 6.2 6.0
N
(C)
6.7 5.8 5.2
N
(D)
6.2 5.5 4.7
exp.
[1]
1.52.0 4.2 4.5 8.49.0
exp.
[2]
5.0 6.4
H
(1)
H
(2)

Theoretical approaches to Compound I
Gas phase model calculations

[FeO(porph)(SMe)]
Antony et al. 1997;
Green 1999:
H
S
character

[FeO(porph)(SH)]
Harris & Loew 1998;
Filatov et al. 1999:
A
2u
character
t*(FeO)
d
xz
d
yz
a
2u
p
t
(S)
4
A
2u
t*(FeO)
d
xz
d
yz
a
2u
p
t
(S)
4
H
S
N
N
N
N
Fe
o
|
meso
O
Fe
S
R
O
Fe
S
R
t*(FeO) a
2u
p
t
(S)
p
o
(S)
Doublet and quartet states close in energy (
2,4
A
2u
normally below
2,4
H
S
)
Electronic nature sensitive to substituent at sulfur (Ogliaro, Shaik et al. 2000)
QM region and basis set
N N
N N
Fe
O
S
R
CH
2
(H)MM
(H)MM
HN
H
N
C
H
2
(H)MM
O
O
(H)MM
R1 =
R2 =
R3 =
R1
(SH)
R2
(SMe)
R3
(cys)
QM atoms 40 43 57
NAO B1 (LACVP,6-31G) 286 299 383
NAO B2 (LACVP, 6-31G, 6-31G*) 316 329 413
NAO B3 (LACVP, 6-31G, 6-31
+
G*) 340 353 437
QM/MM vs. isolated state (gas phase): Unpaired spin densities
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0.800
s
p
i
n

d
e
n
s
i
t
y
QM/MM
QM
(optimized)
QM (enzyme
conformation)
(S)

gr
(porph)

av
(C
meso
)

av
(N)
R1:
SH
B3LYP,
basis B3
H-bond interactions within the Cys357 loop
X-ray structure and MD snapshots
[1] I. Schlichting et al, Science 287, 1615 (2000).



X-ray
+)
snapshot 29 ps snapshot 40 ps



snapshot 40 ps *
)
snapshot 50 ps superimposition

*) side chain of Gln360 manipulated
1)
H-bonds within Cys357 loop: NS and NO distances ()
H-bonds with Leu358 and Gly359 conserved
in all structures
H-bonds involving Gln360 more flexible
Conformation of H-bonds less optimal than
assumed in previous (QM) model studies
backbone N - H S(Cys
357
)
Leu
358
Gly
359
Gln
360

side chain Gln
360
amide
N - H O=C(Cys
357
)
X - ray(1DZ9) 3.51 3.23 3.31 3.00
snapshot 29 3.46 3.32 3.78 4.97
snapshot 40 3.45 3.40 3.57 3.15
snapshot 40
a
3.49 3.33 3.56 2.77
snapshot 50 3.48 3.35 3.78 4.22
a
manipulated side chain
Unpaired spin densities for different snapshots
29 50 40 40, man.
snapshot

gr
(Porph)
(S)
0.70
0.65
0.25
0.20
R
H-X
() snap 29 snap 50 snap 40 snap 40 man.
Gln
360
backbone
N-H S(Cys
357
)
3.81 3.65 3.47 3.43
side chain Gln
360
amide
N-H O=C(Cys
357
)
5.44 4.50 2.86 1.77
N N
N N
Fe
O
S
R
HN
H
N
C
H
2
(H)
O
O
(H)
R =
IV
|
meso
A B
C D
Spectroscopic predictions - P450cam: Compound I
4
A
protein 0.64 0.13 -18 -6 +12
isolated 1.33 0.09 -18 -6 +12
J AE
Q
o (
57
Fe) A
iso
(
57
Fe) A
d
x,y
(
57
Fe) A
d
z
(
57
Fe)
[cm
1
] [mm/s] [mm/s] [MHz] [MHz] [MHz]
2
A
protein -16 0.67 0.13 -29 -14 +23
isolated -27 1.34 0.09 -29 -13 +23
Protein: B3LYP/CHARMM22, isolated: B3LYP, large flexible Fe basis
Optimized geometries
[MHz] A
iso
A
d
x
A
d
y
A
d
z

17
O
oxo

protein

-21.5 -43.9 -40.6 +84.5

isolated

-21.7 -43.5 -41.3 +84.8

14
N
B
pyrr

protein -2.1 +2.1 +1.5 -3.6
isolated -0.3 +1.2 +0.5 -1.6

1
H
meso
protein +3.4 -1.6 -0.1 +1.7
isolated +1.9 -0.9 +0.1 +0.8

1
H
cys-|
protein -7.6 -0.6 0.0 +0.6
isolated -17.0 -1.1 0.0 +1.1
N N
N N
Fe
O
S
R
HN
H
N
C
H
2
(H)
O
O
(H)
R =
IV
|
meso
A B
C D
2
A State
Protein: B3LYP/CHARMM22, isolated: B3LYP, decontracted SVP basis for ligands
Optimized geometries
Spectroscopic predictions - P450cam: Compound I
Doublet-quartet splitting in Compound I : Method
Ab initio MR-CI as implemented in the SORCI procedure [1]:
spectroscopy oriented configuration interaction using the DDCI2 concept
(difference-dedicated CI, Malrieu).
Basis: Fe Wachters [14s11p6d3f]/(8s6p4d2f), O (oxo) aug-cc-p-VDZ, S
and N TZVP, other atoms SV, 504 basis functions.
Technical details for CI: Spin restricted RI-BP86 MOs for quartet, 491
active MOs, CAS (3,3) reference space with 8/1 CSFs for
doublet/quartet, DDCI2 space of 961022/528664 CSFs with variational
treatment of 643279/209828 CSFs for doublet/quartet.
B3LYP: Unrestricted Kohn-Sham calculation, broken symmetry (BS)
solution for doublet, high-spin (HS) solution for quartet, Heisenberg
exchange coupling constant J from Yamaguchi formula [2].
[1] F. Neese, J. Chem. Phys. 119, 9428 (2003).
[2] K Yamaguchi, F. Jensen, A. Dorigo and K. N. Houk, Chem. Phys. Lett. 149, 537 (1988).
Doublet-quartet splitting in Compound I : Results
Ab initio MR - CI :

UDFT (UB3LYP) :

QM/MM calculations with inclusion of protein environment at optimized
UB3LYP/CHARMM geometries:
Ab initio MR - CI : J = -13 cm
-1
, |E| = 39 cm
-1
UDFT (UB3LYP) : J = -16 cm
-1
, |E| = 48 cm
-1
Doublet ground state, antiferromagnetic coupling

J. C. Schneboom, F. Neese and W. Thiel, submitted
3
A E A E
J
2 4
) ( ) (
=
BS
2
HS
2
S S
BS E HS E
J

=
) ( ) (
J
J
Zero-field splitting in the quartet state of Compound I
Contributions from direct electron-electron spin-spin coupling and
spin-orbit coupling (SOC).
SOC terms dominant in Compound I (Fe).
Sum-over-states evaluation of SOC contributions from doublet,
quartet, and sextet configurations at the DFT-BP86 level.
Results in protein environment:
D = 25 cm
-1
, E/D = 0.32
Results in gas phase:
D = 34 cm
-1
, E/D = 0.27
Zero-field splitting D/3 affected by protein environment.




Spin Hamiltonian and eigenstates of Compound I
Computed parameters:
J = -13 cm
-1
(CI); D
Fe0
= 25 cm
-1
, E
Fe0
= 8 cm
-1
(BP86).
Eigenstates (cm
-1)
: 0, 36, 58
<S
2
> values : 1.05, 3.56, 3.63
Population of lowest eigenstate:
100% at 4 K, 54% at 77 K, 38% at 298 K.
Almost equal populations of three lowest eigenstates at 298 K.



( ) ( )
2
FeO y
2
FeO x FeO
3
2
2
FeO z FeO P FeO
S S E S D S S J 2 H
; ; ;

+ + =
Possible spin couplings in Compound I
Excited states of Compound I
Lowest doublet/quartet pair from coupling between FeO triplet (S
FeO
= 1)
and porphyrin doublet (S
P
= ).
Excited quartet/sextet pair from coupling between FeO quintet (S
FeO
= 2)
and porphyrin doublet (S
P
= ), in the protein 0.52 eV above the ground state
with J = -26 cm
-1
(B3LYP).
Low-lying quintet states in [FeO(NH
3
)
4
(H
2
O)]
2+
confirmed by single-point ab
initio calculations at B3LYP optimized triplet geometry which yield the
following relative energies of the quintet state:
MR-CI between -0.11 and +0.07 eV depending on active space and basis,
UCCSD 0.02 eV, UCCSD(T) 0.22 eV,
B3LYP 0.75 eV.

2
1
2
1
Summary : Properties of Compound I
Protein environment tunes electron and spin density destribution
("chameleon").
Protein environment affects Mssbauer parameters (Fe) and hyperfine
coupling constant (ligands).
Ab initio and DFT calculations predict antiferromagnetic doublet ground
state (40 - 50 cm
-1
below quartet).
Spin-orbit coupling causes large zero-field splitting in the quartet (D > 20
cm
-1
) and mixes the lowest doublet and quartet states such that the
ground state has no well-defined spin multiplicity.
There are low lying excited states which may give rise to multi-state
reactivity.
Acknowledgement
Ahmet Altun
Iris Antes
Dirk Bakowies
Salomon Billeter
Marco Bocola
Hai Lin
Nikolaj Otte
Jan Schneboom
Hans Martin Senn
Frank Terstegen
Stephan Thiel
Alexander Turner
Jingjing Zheng

Richard Catlow
Shimrit Cohen
Karl-Erich Jaeger
Christian Lennartz
Frank Neese
Manfred Reetz
Ansgar Schfer
Sason Shaik
Paul Sherwood
Wilfred van Gunsteren
Support from

European Commission (ESPRIT/QUASI)
German-Israeli Foundation for Scientific Research

Spectroscopic properties: Formulas for J and HFC
) ( ) 3 4 ( ) (
1
N N z
iso
R P S N A

t

=
Isotropic Fermi contact coupling constant for nucleus N:
Electronnucleus magnetic dipole coupling constant for nucleus N:

=

kl
l N N N N k kl N
d
r r r r P N A o
v v v
| ) 3 ( | ) (
2 5

N e N e N
g g P | | =
Basis sets with high flexibility in core region required, e.g., standard basis with
decontracted inner s-functions.
Second order spin-orbit contribution (only Fe) to HFC:
( ) ( )
(

+ A

+ A =

a o
ao ao ao ao
a o
o i
io io io io
o i
A
N N
SO
L L L L L L L L g
S
N A
,
3 1 1 3
1
,
,
3 1 1 3
1
,
) (
4
) (
v v v v v
|
o

=
A
j
A
A i
ij
l r L

| ) ( | Im
1 j i
ij
l L

| | Im
2
=
j A
A
i
ij
r l L

| | Im
3
3

=
2 1
4 S S
E E
J
BS HS

=
Heisenberg exchange coupling constant
Spectroscopic properties: Formulas for Mssbauer parameters
zz
yy xx
V
V V
= q
2 / 1
2
3
1
2
1
|
|
.
|

\
|
+ = A
q
zz Q
V eQ E
Field gradient tensor:
Asymmetry parameter:
Quadrupole splitting:
N
C
N
D
N
A
N
B
Fe
IV
O
S
Cys
Isomer shift: | | ) 0 ( ) 0 ( ) (
5
4
0 0
2 2
0
S A
R
R
R e Z ZS
o
t o
|
.
|

\
|
=
( ) b a
A
= ) 0 (
0
o
F. Neese, Inorg. Chim. Acta 337, 181 (2002).
) 3 (
| ) 3 ( | ) (
2 5
2 5
AN AN AN
N A
AN A
kl
l N N N N k kl
R R R R Z
r r r r N V
v v
v v v
o
o

=
=



P
Spectroscopic properties: Model compounds
N N
N N
Fe
O
Ph
Ph
Ph Ph
IV
L
J AE
Q
o (
57
Fe) A
iso
(
57
Fe) A
d
x,y
(
57
Fe) A
d
z
(
57
Fe)
[cm
1
] [mm/s] [mm/s] [MHz] [MHz] [MHz]
B3LYP:
L = - +56 2.21 0.08 -18 -6.5 +13.0
L = H
2
O +26 1.16 0.11 -18 -5.9 +11.7
exp.
[1]
:
[FeO(TDCPP)]
+
+38 1.48 0.06 -18.3 -8.5 +17.0
[FeO(TMP)]
+
+43

1.62 0.08 -18.3 -9.2 +18.4
[1] D. Mandon, R. Weiss, K. Jayaraj, A. Gold, J. Terner, E. Bill, A. X. Trautwein, Inorg. Chem. 31, 4404 (1992).
Singly occupied orbitals of Compound I
Natural orbitals from spin unrestricted B3LYP calculations on
the quartet state with (A) and without (B) MM point charges.
Orbitals of (FeO)
2+
motif in upper valence region
Computed for triplet ground state of [FeO(NH
3
)
4
(H
2
O)]
2+
and
assigned under approximate C
4v
symmetry
Resting state: Electronic structure
d(xz)
d(yz)
d(x
2
-y
2
)
d(xy)
d(z
2
)
N
N
N N
Fe
O
S
R
H H
x
z
2
A
d(xz)
d(yz)
d(x
2
-y
2
)
d(xy)
d(z
2
)
4
A
d(xz)
d(yz)
d(x
2
-y
2
)
d(xy)
d(z
2
)
6
A
o
t
o*
(kcal/mol) QM/MM gas phase
D(
2
A) Q(
4
A) S(
6
A) D(
2
A) Q(
4
A) S(
6
A)
BLYP 0.00 3.94 19.75 0.00 10.90 21.80
B3LYP 0.00 -2.41 4.50 0.00 5.48 6.92
BH-LYP 0.00 -10.78 -18.05 0.00 -0.84 -13.50
Relative single point energies obtained with different density functionals.
Small QM region R1w, small basis set B1 (LACVP, 6-31G).
Geometries optimized at the B3LYP/B1 level (QM/MM: snapshot 195 ps).
Resting state: Relative energies
-4
-2
0
2
4
6
8
0 50 150 175 195 200
Snapshot [ps]
A
E

[
k
c
a
l
/
m
o
l
]
E(
4
A-
2
A) =
E(
6
A-
2
A) =
protein: QM/MM energy
protein: QM contribution to QM/MM energy
gas phase: QM energy

small QM region R1w, basis set B1
Different snapshots from MM-MD trajectory, QM/MM optimized
Resting state: Effect of the protein
Protein Gas phase
Ac(o*-d
xz
) / eV 0.848 1.057
P
A
(S) / e 16.452 16.202
E
QM
(
4
A-
2
A) / kcal mol
-1
-2.41 5.48
Stabilization of the
4
A state

- Kohn-Sham orbital energy difference
- Mulliken electron population at sulfur
- QM energy doublet-quartet gap
OH
2
Fe
S
R
p
z
(S)
d(xz)
d(yz)
d(x
2
-y
2
)
d(xy)
d(z
2
)
Fe
o*
o*
o*
p
Z
(S)
p
Z
(S)
gas phase enzyme
Pentacoordinated ferric and ferrous complexes
B3LYP relative energies (kcal/mol), R2/B2W, snapshot 93, for enzyme (QM/MM) and
gas phase (QM).
High-spin ground state for both complexes, in agreement with experiment.
Quintet of (3) with double occupancy of in enzyme (gas phase).

d
x
2
-y
2
d
xz
d
yz
d
xz
1
A (S=0)
2
A (S=1/2)
d
xy
d
yz
d
x
2
-y
2
d
x
2
-y
2
d
xy
d
xz
d
z
2
d
yz
d
x
2
-y
2
d
z
2
o*
t
d
z
2
(or d
xy
)
d
yz
3
A (S=1)
5
A (S=2)
o
t
o*
Ferric complex (2)
d
xy
(or d
z
2
)
d
z
2
d
xz
d
yz
d
xy
d
yz
d
x
2
-y
2
(or, d
xz
)
6
A (S=5/2)
d
xy
d
z
2
d
z
2
d
x
2
-y
2
d
xz
4
A (S=3/2)
d
xy
o
Ferrous complex (3)
d
xz
(or, d
x
2
-y
2
)
QM/MM
QM/MM
QM gas
QM gas
2.4
13.2
12.1
0.7
1.1
5.4
4.9
0
0
0
0
quintet: d
x
2
-y
2
doubly occupied
quintet: d
xz


doubly occupied
4.4
** High-spin ground state
Fe
III
RH
+ e
-
Fe
II
RH
(3)
Cys357
Cys357
-
* B3LYP relative energies (kcal/mol), R2/B2W, snapshot 93
(2)
) d ( d
2 2
y x
xz

Mssbauer spectrum of pentacoordinated ferrous complex
- Isomer shift o (mm/s), quadrupole splitting |AE
Q
| (mm/s), and
asymmetry parameter q.
State
o |AE
Q
| q
1
A 0.65 1.83 0.21
3
A 0.69 1.74 0.53
5
A 0.83 (0.88) 2.83 (4.77) 0.61 (0.08)
exp [1] 0.82 2.42 0.8
-
- QM/MM using B3LYP and a large uncontracted basis set at Fe
- Quintet: Two electromers with double occupancy of d
xz

- Only the ground-state quintet electromer matches the experimental data
( )
2 2
y x
d

[1] P. M. Champion et al, Biochemistry 14, 4151 (1975).
X-ray structure 1DZ9
Only one monomer from asymmetric unit
TRIS buffer deleted
Potassium ion included
H-atom positions constructed with CHARMM
1. Optimize crystallographic water H-atoms
2. Optimize all H-atoms
MM Setup
Add water layer of 16 thickness (InsightII, MSI)
Equilibrate inner 8 of water layer
Energy minimization, constrain backbone (100 kcal mol
-1

-2
)
and sidechains (50 kcal mol
-1

-2
), scale down constraints
every 60 steps by 0.65. Final GRMS: 0.04 kcal mol
-1

-1

Molecular dynamics: 15 ps heating dynamics, 200 ps
equilibration dynamics, T = 300K, integration step 1 fs, SHAKE
Heme, Cys357
and outer 8 of
solvent layer
fixed:
Minimize several snapshots from equilibration trajectory
Only MM
calculations!
16965 atoms solvent
24394 atoms total
Setup
(Compound I)
QM/MM vs. isolated state (gas phase): Geometries
J. C. Schneboom, H. Lin, N. Reuter, W. Thiel, S. Cohen, F. Ogliaro, S. Shaik, J. Am. Chem. Soc. 124, 8142 (2002).
R1(SH) / B3 R3(cys) / B3
4
A
2
A
4
A
2
A
protein (QM/MM) = B3LYP/CHARMM22
isolated (QM) = B3LYP
protein 1.627 1.626
isolated 1.624 1.622
1.626 1.625
1.618 1.617
r
FeO
[]
protein 2.560 2.589
isolated 2.566 2.581
2.585 2.609
2.678 2.697
r
FeS
[]
protein 111.5 111.2
isolated 97.5 97.5
112.0 111.8
115.5 115.1
u
FeS C
[
o
]

QM/MM vs. isolated state (gas phase): QM energies
J. C. Schneboom, H. Lin, N. Reuter, W. Thiel, S. Cohen, F. Ogliaro, and S. Shaik, JACS 124, 8142 (2002).
108.7 109.0 150.6 150.6
R1(SH) / B3 R3(cys) / B3
4
A
2
A
4
A
2
A
0 0
0 0 QM/MM (protein)
QM (gas phase)
QM (protein)
E
[kcal/mol]
AE
vert

AE
ad

QM/MM = B3LYP/CHARMM22, QM = B3LYP
99.5 99.7 129.7 129.6
Energy differences AE = E(
4
A
2u
) - E (
2
A
2u
)
Energy
a
Geometry
b
R3 (cys)
c
QM/MM (p) QM/MM (p) +0.03 (+0.08)
QM (p) QM/MM (p) +0.04
QM (g) QM/MM (p) +0.06
QM (g) QM (g) +0.08
a
p/g = evaluated with / without protein environment
b
p/g = optimized for protein / gas phase
c
Adiabatic (vertical) energy differences in kcal/mol
B3LYP, basis B3
QM/MM vs. isolated state (gas phase)
Proposed mechanisms for CH hydroxylation by Compound I
Contradictory experimental findings:

Product analysis, KIE- measurements:
rebound mechanism

Radical clock experiments
[1]
:
apparent lifetimes of possible
intermediates too short (t = 80 200 fs)
competing reaction channels, e.g. oxene
insertion
[2]
?
high-spin and low-spin states involved
(two-state-reactivity)
[3]
?
influence of protein pocket ?
Fe
IV
S
R
O
R H
Fe
III
S
R
O
R H
oxene insertion
concerted
Fe
III
S
R
OH
R
H-abstraction
rebound step
stepwise
or
effectively concerted, nonsynchronous
[1] M. Newcomb and P. H. Toy, Acc. Chem. Res. 33, 449 (2000).
[2] M. Newcomb, M.-H. Le Tadi-Biadatti, D. L. Chestney, E. S. Roberts, and P. F. Hollenberg, JACS 117, 12085 (1995).
[3] S. Shaik, M. Filatov, D. Schrder, and H. Schwarz, Chem. Eur. J. 4, 193 (1998).
Investigation of the rebound mechanism
QM subsystems Basis States Environment
R1pro B1:
LACVP+ECP (Fe)
6-31G (others)
1.LS (
2
A)
2.HS (
4
A)

1.QM/MM
(enzyme)
2.Gas phase
R3cam B4:
B1 +
6-31+G* on ligand
atoms,
C
5
, H
5exo

1.LS (
2
A)
2.HS (
4
A)

1.QM/MM
(enzyme)
2.Gas phase

N N
N N
Fe
O
S
(H) MM
H
H
(H)
H
(H)
(H)
(H)
H
N N
N N
Fe
O
S
R
HN
H
N
C
H
2
(H)MM
O
O
(H)MM
H
3
C CH
3
CH
3
O
H
5exo
H
5endo
R =
O
1
2
3
4
7
8
9
6
5
10
Fe
O
S-prot
R
Fe
III
O
S-prot
H
Fe
O
S-prot
R
H
R
HYD
TS
R
Fe
IV
O
S-prot
RH
Cpd I
Fe
III
O
S-prot
R H
PROD
TS
H
H
Rebound mechanism: Computational procedure
R1pro/B1:
PES scan
Full geometry optimizations of
minima and saddle points along
the reaction coordinate
Finite-difference Hessian to
characterize saddle points
R3cam/B4:
Full geometry optimizations of
minima and saddle points obtained
from R1pro/B1 calculations
red: QM region R3cam
yellow: optimized MM atoms
green: fixed MM environment
Hydrogen abstraction: PES scan R1pro/B1
LS (
2
A) state

reference point:
reactive complex
HS (
4
A) state

reference point:
reactive complex
-2
3
8
13
18
2.69 2.54 2.39 2.24 2.09 1.94 1.79 1.64 1.49 1.34 1.19 1.04
R(O-H5exo) [A]
E
n
e
r
g
y

[
k
c
a
l
/
m
o
l
]
E(QM/MM) E(QM) E(MM)
-2
3
8
13
18
2.68 2.54 2.39 2.23 2.09 1.94 1.78 1.64 1.49 1.34 1.19 1.04
R(O-H5exo) [A]
E
n
e
r
g
y

[
k
c
a
l
/
m
o
l
]
E(QM/MM) E(QM) E(MM)
Transition state of hydrogen abstraction,
2
A state, R1pro/B1
TS
H

E
A
= 19.5 / 20.4 kcal/mol
(
2
A /
4
A )
Mechanism of CH hydroxylation: Energy profile
J. C. Schneboom, S. Cohen, H. Lin, S. Shaik, and W. Thiel, J. Am. Chem. Soc. 126, 4017-4034 (2004).
QM/MM geometry optimizations, R1pro/B1
Two-state reactivity confirmed
O
F e
R H
I V
L
O
F e
R
H
L
O
F e
R
H
L
O
F e
R
H
L
O
F e
R H
L
I I I
E n e r g y
4
A
2
A ,
4
A
2
A
2 0 / 2 1
1 4 / 1 5

14/ 17
- 3 8 / - 4 3
[ k c a l / m o l ]
0 / 0
+ +
CH hydroxylation: Energy profile in the gas phase
QM geometry optimizations, R1pro/B1

O
Fe
R H
IV
L
O
Fe
R
H
L
O
Fe
R
H
L
O
Fe
R
H
L
O
Fe
R H
L
III
Energy
IV
4
A
2
A,
4
A
2
A
[kcal/mol]
0/ 0
20/ 20
12/ 13
-50/ -45
- / 16
Rebound barriers for quartet / doublet: MO diagram
N. Harris, S. Cohen, M. Filatov, F. Ogliaro, and S. Shaik, Angew. Chem. Int. Ed. 39, 2003 (2000).
Electronic situation during rebound step:

a) MO diagram
rebound barrier in HS state due to
occupation of antibonding orbital
b) electron counting diagram
filling of porphyrin hole
Barriers for hydrogen abstraction: Model system
AE
A
(kcal/mol)
,
basis AE1 (508 basis functions)
BLYP BP86 B97 B3LYP PBE0
25.2 22.4 20.1 19.5 15.1
AE
A
(kcal/mol)
,
B3LYP functional: dependence on basis set (no. of basis functions)
SV (122) B1 (123) SVP (211) TZVP (271) B4 (186) AE1 (508) AE2 (892)
25.4 22.7 21.7 21.4 19.5 19.5 19.2
Product release: Three minima
R. Davydov, T. M. Makris, V. Kofman, D. E. Werst, S. G. Sligar, and B. M. Hoffman, JACS 123, 1403 (2001).
4
A state
4
PROD
r
Fe-O
= 2.843

E
rel
(R1/B1) = 0 kcal/mol
2
A state
2
PROD1
r
Fe-O
= 2.262

E
rel
(R1/B1) = 4.87 kcal/mol
2
A state
2
PROD2
r
Fe-O
= 2.819

E
rel
(R1/B1) = 5.18 kcal/mol
Product complex: X-ray vs. QM/MM (
2
PROD2)
[1] H. Li, N. Shakunthala, L. M. Havran, J. D. Winkler, and T. Poulos, JACS 117, 6297 (1995).
white: X-ray structure 1NOO
[1]
; yellow: B3LYP/CHARMM (R1/B1),
2
PROD2
Cam-OH
Heme
Tyr96
Cys357
Tyr96
Cam-OH
Heme
Cys357
O
O
H
O
Hydroxylation mechanism: Summary of results
Two-state reactivity

Pre-organization of substrate within the pocket (linear arrangement CHO)
lower entropic cost compared to gas phase

Protein pocket essential for enantioselectivity in rebound step
orientation of substrate through hydrogen bond to Tyr96

Small influence of protein environment on abstraction barrier

Protein influences relative stability of redox electromers (Fe
III
/Fe
IV
)
multi-state scenario ?

Three product minima in agreement with EPR observations