israelاسرائيل
DESCRIPTION
خTRANSCRIPT
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Theoretical Studies on Cytochrome P450cam
Walter Thiel
Max-Planck-Institut für Kohlenforschung
Symposium of The Lise Meitner - Minerva Center
Haifa, 19 December 2004
Happy birthday Yitzhak...
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QM/MM approach: Overview
QMQM: density functional theory (B3LYP)
MMMM: force field (CHARMM22)
QM – MM interactionsQM – MM interactions:
„electronic embedding“
Border regionBorder region:
• hydrogen link atoms L
• charge shift for q(M1)
Codes:
ChemShell, Turbomole, DL-POLY
M1
M2
M2
M3
Q1
Q2
Q3
Q2
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
AJ
J,i iJ
JO,IMMQM R
B
R
A
R
Zq
r
qH
612
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ChemShell: A modular QM/MM package
GAUSSIAN98
GAMESS-UK
Chemshell
Tcl scripts
GROMOS96
CHARMm26MSI
Integratedroutines:
datamanagement
geometryoptimisation
moleculardynamics
genericforce fields
QM/MMcoupling
MNDO99
MOPAC
QM codes MM codes
DL_POLY
TURBOMOLE
CHARMM27academic
GULP
P. Sherwood et al, J. Mol. Struct. Theochem 632, 1-28 (2003).
MOLPRO
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Cytochrome P450Cam (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
H5exo
H5endo
P450cam+ O2 + 2 e- + 2H+ + H2O
O
OH
H5endo
camphor
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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
FeIII
OH2
S
N
N
N
N
Cys
FeIII
S
N
N
N
N
Cys
R-H
FeII
S
N
N
N
N
Cys
R-H
FeII
S
N
N
N
N
Cys
R-HO
O
FeIII
S
N
N
N
N
Cys
R-HO
O
FeIV
S
N
N
N
N
Cys
R-HO
FeIII
S
N
N
N
N
Cys
ROH
FeIII
ROH
S
N
N
N
N
Cys
R-H
H2O
e
O2
e2-
2H+
H2O
Habs
H2O
ROH
1
2
3
4
5
6 (I)
7
8 2
3
4
5
6
7
8
1
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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).
P450cam: Iron(III)-aqua complex
Tyr96
Heme
Cys357
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Resting state: QM/MM calculations
N N
NNFe
O
SR
MM
HNHN
C(H)
O
O
(H)
(H)
MM
MM
(H) MM
R1w: R =
R3w: R =
Leu356
Leu358
Cys357
H
H H
QM: UKS-DFT, B3LYPBasis: 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. Schöneboom and W. Thiel, J. Phys. Chem. B 108, 7468-7478 (2004).
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Resting state: Comparison with experiment
B3LYP/CHARMM22 BLYP/CHARMM22 exp.
2A 4A 6A 2A 4A 6A
E(QM/MM) 0 2.37 3.30 0 8.45 18.27 2A
E(QM) 0 0.12 4.13 0 6.07 18.63 -
rFe–O [Å] 2.141 2.475 2.467 2.191 2.605 2.611 2.28(0.2)
rFe–S [Å] 2.269 2.491 2.418 2.239 2.490 2.421 2.25(0.2)
rFe–N [Å] 2.038 2.037 2.095 2.050 2.051 2.113 2.02(0.2)
rFe–H [Å] 2.633 2.928 2.905 2.655 2.980 2.961 2.62
Large QM region R3w, basis set B2:
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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).
P450cam: Iron(III)-aqua complex B3LYP/CHARMM22
[MHz] Aiso Adx Ad
y Adz
H(1) 2.0 4.7 5.0 9.4
H(2) 0.4 4.6 4.4 9.0
[MHz] Atotx Atot
y Atotz
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] 1.5–2.0 4.2 – 4.5 8.4–9.0
exp. [2] 5.0 – 6.4
H(1) H(2)
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Theoretical approaches to Compound I
Gas phase model calculations
• [FeO(porph)(SMe)] Antony et al. 1997; Green 1999: S character
• [FeO(porph)(SH)]
Harris & Loew 1998; Filatov et al. 1999:
A2u character*(FeO)
dxz dyza2u
p(S)
4A2u
*(FeO)
dxz dyz
a2u
p(S)
4S
N
N
N
N
Fe
mesoO
Fe
S
R
O
Fe
S
R
*(FeO) a2u
p(S)
p(S)
• Doublet and quartet states close in energy (2,4A2u normally below 2,4S)
• Electronic nature sensitive to substituent at sulfur (Ogliaro, Shaik et al. 2000)
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QM region and basis set
N N
NNFe
O
SR
CH2 (H)MM
(H)MM
HN
HN
CH2
(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
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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
sp
in d
en
sit
y
QM/MM QM (optimized)
QM (enzyme conformation)
(S)
gr(porph)
av(Cmeso )
av(N)
R1:SH
B3LYP,
basis B3
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H-bond interactions within the Cys357 loop
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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)
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H-bonds within Cys357 loop: N···S and N···O distances (Å)
• 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(Cys357)
Leu358 Gly359 Gln360
side chain Gln360 amide
N-H ··· O=C(Cys357)
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 40a 3.49 3.33 3.56 2.77
snapshot 50 3.48 3.35 3.78 4.22
a manipulated side chain
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Unpaired spin densities for different snapshots
29 50 40 40, man.snapshot
gr(Porph)
(S)
0.70
0.65
0.25
0.20
RH-X (Å) snap 29 snap 50 snap 40 snap 40 man.
Gln360 backbone
N-H ··· S(Cys357)
3.81 3.65 3.47 3.43
side chain Gln360 amide
N-H ··· O=C(Cys357)
5.44 4.50 2.86 1.77
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N N
NNFe
O
SR
HNHN
CH2
(H)
O
O(H)
R =IV
meso
AB
C D
Spectroscopic predictions - P450cam: Compound I
4Aprotein 0.64 0.13 -18 -6 +12isolated 1.33 0.09 -18 -6 +12
JEQ (57Fe) Aiso (57Fe) Adx,y (57Fe) Ad
z (57Fe)) [cm –1] [mm/s] [mm/s] [MHz] [MHz] [MHz]
2Aprotein -16 0.67 0.13 -29 -14 +23isolated -27 1.34 0.09 -29 -13 +23
Protein: B3LYP/CHARMM22, isolated: B3LYP, large flexible Fe basisOptimized geometries
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[MHz] Aiso Adx Ad
y Adz
17Ooxo protein -21.5 -43.9 -40.6 +84.5
isolated -21.7 -43.5 -41.3 +84.8
14NBpyrr protein -2.1 +2.1 +1.5 -3.6
isolated -0.3 +1.2 +0.5 -1.6
1Hmeso protein +3.4 -1.6 -0.1 +1.7
isolated +1.9 -0.9 +0.1 +0.8
1Hcys- protein -7.6 -0.6 0.0 +0.6
isolated -17.0 -1.1 0.0 +1.1
N N
NNFe
O
SR
HNHN
CH2
(H)
O
O(H)
R =
IV
meso
AB
C D
2A StateProtein: B3LYP/CHARMM22, isolated: B3LYP, decontracted SVP basis for ligandsOptimized geometries
Spectroscopic predictions - P450cam: Compound I
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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).
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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, |ΔE| = 39 cm-1
UDFT (UB3LYP) : J = -16 cm-1, |ΔE| = 48 cm-1
Doublet ground state, antiferromagnetic coupling
J. C. Schöneboom, F. Neese and W. Thiel, submitted
3
AEAEJ
24 )()(
BS
2
HS
2 SS
BSEHSEJ
)()(
J
J
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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.
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Spin Hamiltonian and eigenstates of Compound I
Computed parameters:J = -13 cm-1 (CI); DFe0 = 25 cm-1, EFe0 = 8 cm-1 (BP86).
Eigenstates (cm-1) : 0, 36, 58 <S2> 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.
2FeOy
2FeOxFeO3
22FeOzFeOPFeO SSESDSSJ2H ;;;
ˆˆˆˆˆˆ
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Possible spin couplings in Compound I
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Excited states of Compound I
• Lowest doublet/quartet pair from coupling between FeO triplet (SFeO = 1) and
porphyrin doublet (SP = ).
• Excited quartet/sextet pair from coupling between FeO quintet (SFeO = 2) and
porphyrin doublet (SP = ), in the protein 0.52 eV above the ground state with
J = -26 cm-1 (B3LYP).
• Low-lying quintet states in [FeO(NH3)4(H2O)]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
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Summary : Properties of Compound I
• Protein environment tunes electron and spin density destribution ("chameleon").
• Protein environment affects Mössbauer 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.
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Acknowledgement
Ahmet AltunIris AntesDirk BakowiesSalomon BilleterMarco BocolaHai LinNikolaj OtteJan SchöneboomHans Martin SennFrank TerstegenStephan ThielAlexander TurnerJingjing Zheng
Richard Catlow
Shimrit Cohen
Karl-Erich Jaeger
Christian Lennartz
Frank Neese
Manfred Reetz
Ansgar Schäfer
Sason Shaik
Paul Sherwood
Wilfred van Gunsteren
Support from
European Commission (ESPRIT/QUASI)
German-Israeli Foundation for Scientific Research
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Spectroscopic properties: Formulas for J and HFC
)()34()( 1NNz
iso RPSNA
Isotropic Fermi contact coupling constant for nucleus N:
Electron–nucleus magnetic dipole coupling constant for nucleus N:
kllNNNNkklN
d rrrrPNA |)3(|)( 25 ρ
NeNeN ggP
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:
ao
aoaoaoaoao
oi
ioioioiooi
ANN
SO LLLLLLLLgS
NA,
31131,
,3113
1,
)(
4)(
A
jA
Aiij lrL |)(|Im1 ji
ij lL ||Im2 jAA
iij rlL ||Im 33
214 SS
EEJ BSHS
Heisenberg exchange coupling constant
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Spectroscopic properties: Formulas for Mössbauer parameters
zz
yyxx
V
VV
2/12
31
2
1
zzQ VeQE
Field gradient tensor:
Asymmetry parameter:
Quadrupole splitting:
NC ND
NANB
FeIV
O
SCys
Isomer shift: )0()0()(5
400
220
SA
R
RReZZS
ba A )0(0
F. Neese, Inorg. Chim. Acta 337, 181 (2002).
)3(
|)3(|)(
25
25
ANANANNA
ANA
kllNNNNkkl
RRRRZ
rrrrNV
P
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Spectroscopic properties: Model compounds
N N
NNFe
O
Ph
Ph
PhPhIV
L
J EQ (57Fe) Aiso (57Fe) Adx,y (57Fe) Ad
z (57Fe) [cm –1] [mm/s] [mm/s] [MHz] [MHz] [MHz]
B3LYP:L = - +56 2.21 0.08 -18 -6.5 +13.0L = H2O +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).
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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.
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Orbitals of (FeO)2+ motif in upper valence region
Computed for triplet ground state of [FeO(NH3)4(H2O)]2+ and
assigned under approximate C4v symmetry
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Resting state: Electronic structure
d(xz)d(yz)
d(x2-y2)
d(xy)
d(z2)
N NNN
Fe
O
S R
H H
x
z
2A
d(xz)d(yz)
d(x2-y2)
d(xy)
d(z2)
4A
d(xz)d(yz)
d(x2-y2)
d(xy)
d(z2)
6A
*
(kcal/mol) QM/MM gas phase
D(2A) Q(4A) S(6A) D(2A) Q(4A) S(6A)
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).
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Resting state: Relative energies
-4
-2
0
2
4
6
8
0 50 150 175 195 200
Snapshot [ps]
E
[kca
l/mol
]
E(4A-2A) =E(6A-2A) =
protein: QM/MM energyprotein: QM contribution to QM/MM energygas phase: QM energy
small QM region R1w, basis set B1
Different snapshots from MM-MD trajectory, QM/MM optimized
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Resting state: Effect of the protein
Protein Gas phase
(*-dxz) / eV 0.848 1.057
PA(S) / e 16.452 16.202
EQM(4A-2A) / kcal mol-1 -2.41 5.48
Stabilization of the 4A state
- Kohn-Sham orbital energy difference
- Mulliken electron population at sulfur
- QM energy doublet-quartet gap
OH2
Fe
SR
pz(S)
d(xz) d(yz)
d(x2-y2)
d(xy)
d(z2)
Fe
*
*
*
pZ(S)
pZ(S)
gas phaseenzyme
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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(d 22 yxxz
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Mössbauer spectrum of pentacoordinated ferrous complex
Isomer shift (mm/s), quadrupole splitting |EQ| (mm/s), and
asymmetry parameter .
QM/MM using B3LYP and a large uncontracted basis set at Fe
Quintet: Two electromers with double occupancy of dxz
Only the ground-state quintet electromer matches the experimental data
22 yxd
[1] P. M. Champion et al, Biochemistry 14, 4151 (1975).
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X-ray structure 1DZ9Only one monomer from asymmetric unitTRIS buffer deletedPotassium 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 Å thickness (InsightII, MSI)
Equilibrate inner 8 Å of water layer
Energy minimization, constrain backbone (100 kcal mol-1 Å-2) and sidechains (50 kcal mol-1 Å-2), scale down constraints every 60 steps by 0.65. Final GRMS: 0.04 kcal mol-1 Å-1
Molecular dynamics: 15 ps heating dynamics, 200 ps equilibration dynamics, T = 300K, integration step 1 fs, SHAKE
Heme, Cys357 and outer 8 Å of solvent layer fixed:
Minimize several snapshots from equilibration trajectory
Only MM calculations!
16965 atoms solvent24394 atoms total
Setup (Compound I)
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QM/MM vs. isolated state (gas phase): Geometries
J. C. Schöneboom, H. Lin, N. Reuter, W. Thiel, S. Cohen, F. Ogliaro, S. Shaik, J. Am. Chem. Soc. 124, 8142 (2002).
R1(SH) / B3R1(SH) / B3 R3(cys) / B3R3(cys) / B3
44AA 22AA 44AA 22AA
protein (QM/MM) = B3LYP/CHARMM22isolated (QM) = B3LYP
protein 1.627 1.626isolated 1.624
1.622
1.626 1.6251.618 1.617
rFe–O [Å]
protein 2.560 2.589isolated 2.566
2.581
2.585 2.6092.678 2.697
rFe–S [Å]
protein 111.5 111.2isolated 97.5
97.5
112.0 111.8115.5 115.1
Fe–S – C [o]
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QM/MM vs. isolated state (gas phase): QM energies
J. C. Schöneboom, 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) / B3R1(SH) / B3 R3(cys) / B3R3(cys) / B3
44AA 22AA 44AA 22AA
0 0 0 0QM/MM (protein)
QM (gas phase)
QM (protein)E [kcal/mol]
Evert
Ead
QM/MM = B3LYP/CHARMM22, QM = B3LYP
99.5 99.7 129.7 129.6
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Energy differences E = E(4A2u) - E (2A2u)
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)
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Proposed mechanisms for C–H hydroxylation by Compound I
Contradictory experimental findings:
• Product analysis, KIE- measurements:
rebound mechanism
• Radical clock experiments [1]:
apparent lifetimes of possible intermediates too short ( = 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 ?
FeIV
S
R
O
R H
FeIII
S
R
OR H
oxene insertion
concerted
FeIII
S
R
OHR
H-abstractionrebound 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. Schröder, and H. Schwarz, Chem. Eur. J. 4, 193 (1998).
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Investigation of the rebound mechanism
QM subsystems Basis States Environment
R1pro B1:LACVP+ECP (Fe)
6-31G (others)
1.LS (2A)
2.HS (4A)
1.QM/MM (enzyme)
2.Gas phase
R3cam B4: B1 +
6-31+G* on ligand atoms,
C5, H5exo
1.LS (2A)
2.HS (4A)
1.QM/MM (enzyme)
2.Gas phase
N N
NNFe
O
S(H) MM
H
H
(H)
H(H)
(H)
(H)H
N N
NNFe
O
SR
HNHN
CH2
(H)MM
O
O(H)MM
H3C CH3
CH3O
H5exo
H5endo
R =
O
1
23
4
7
8 9
6
5
10
Fe
O
S-prot
R
FeIII
O
S-prot
H
Fe
O
S-prot
RHR
HYD TSR
FeIV
O
S-prot
RH
Cpd I
FeIII
O
S-prot
R H
PRODTSH
H
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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
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Hydrogen abstraction: PES scan R1pro/B1
LS (2A) state
reference point:reactive complex
HS (4A) 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]
En
erg
y [k
cal/m
ol]
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]
En
erg
y [k
cal/m
ol]
E(QM/MM) E(QM) E(MM)
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Transition state of hydrogen abstraction, 2A state, R1pro/B1
TSH
EA = 19.5 / 20.4 kcal/mol
( 2A / 4A )
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Mechanism of C–H hydroxylation: Energy profile
J. C. Schöneboom, S. Cohen, H. Lin, S. Shaik, and W. Thiel, J. Am. Chem. Soc. 126, 4017-4034 (2004).
QM/MM geometry optimizations, R1pro/B1Two-state reactivity confirmed
O
Fe
R H
IV
L
O
Fe
RH
L
O
Fe
RH
L
O
Fe
R H
L
O
Fe
R H
L
III
Energy
4A2A,
4A
2A
20/ 2114/ 15
14/ 17
-38/ -43
[kcal/mol]
0/ 0
+ +
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C–H hydroxylation: Energy profile in the gas phase
QM geometry optimizations, R1pro/B1
O
Fe
R H
IV
L
O
Fe
RH
L
O
Fe
RH
L
O
Fe
R H
L
O
Fe
R H
L
III
Energy
IV
4A2A,
4A2A
[kcal/mol]
0/ 0
20/ 20
12/ 13
-50/ -45
- / 16
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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“
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Barriers for hydrogen abstraction: Model system
EA (kcal/mol), basis AE1 (508 basis functions)
BLYP BP86 B97 B3LYP PBE025.2 22.4 20.1 19.5 15.1
EA (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
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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).
4A state4PROD
rFe-O = 2.843 Å
Erel(R1/B1) = 0 kcal/mol
2A state2PROD1
rFe-O = 2.262 Å
Erel(R1/B1) = 4.87 kcal/mol
2A state2PROD2
rFe-O = 2.819 Å
Erel(R1/B1) = 5.18 kcal/mol
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Product complex: X-ray vs. QM/MM (2PROD2)
[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), 2PROD2
Cam-OH
Heme
Tyr96
Cys357
Tyr96
Cam-OH
Heme
Cys357
O
O
H
O
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Hydroxylation mechanism: Summary of results
• Two-state reactivity
• Pre-organization of substrate within the pocket (linear arrangement C–H···O) 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 (FeIII/FeIV) multi-state scenario ?
• Three product minima in agreement with EPR observations