Theoretical Studies on Cytochrome P450 cam Walter Thiel Max-Planck-Institut für Kohlenforschung Symposium of The Lise Meitner - Minerva Center Haifa, 19 December 2004 Happy birthday Yitzhak...
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...
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
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
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
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
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
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).
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:
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)
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)
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
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
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: 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
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
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
[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
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, |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
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); 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
Possible spin couplings in Compound I
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
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.
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
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
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
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).
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(NH3)4(H2O)]2+ and
assigned under approximate C4v symmetry
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).
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
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
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
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).
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)
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]
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
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)
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).
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
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 (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)
Transition state of hydrogen abstraction, 2A state, R1pro/B1
TSH
EA = 19.5 / 20.4 kcal/mol
( 2A / 4A )
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
+ +
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
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
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
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
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
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