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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...
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Page 1: Israelاسرائيل

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...

Page 2: Israelاسرائيل

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

Page 3: Israelاسرائيل

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

Page 4: Israelاسرائيل

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

Page 5: Israelاسرائيل

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

Page 6: Israelاسرائيل

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

Page 7: Israelاسرائيل

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).

Page 8: Israelاسرائيل

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:

Page 9: Israelاسرائيل

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)

Page 10: Israelاسرائيل

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)

Page 11: Israelاسرائيل

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

Page 12: Israelاسرائيل

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

Page 13: Israelاسرائيل

H-bond interactions within the Cys357 loop

Page 14: Israelاسرائيل

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)

Page 15: Israelاسرائيل

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

Page 16: Israelاسرائيل

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

Page 17: Israelاسرائيل

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

Page 18: Israelاسرائيل

[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

Page 19: Israelاسرائيل

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).

Page 20: Israelاسرائيل

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

Page 21: Israelاسرائيل

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.

Page 22: Israelاسرائيل

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

Page 23: Israelاسرائيل

Possible spin couplings in Compound I

Page 24: Israelاسرائيل

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

Page 25: Israelاسرائيل

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.

Page 26: Israelاسرائيل

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

Page 27: Israelاسرائيل
Page 28: Israelاسرائيل

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

Page 29: Israelاسرائيل

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

Page 30: Israelاسرائيل

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).

Page 31: Israelاسرائيل

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.

Page 32: Israelاسرائيل

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

Page 33: Israelاسرائيل

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).

Page 34: Israelاسرائيل

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

Page 35: Israelاسرائيل

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

Page 36: Israelاسرائيل

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

Page 37: Israelاسرائيل

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).

Page 38: Israelاسرائيل

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)

Page 39: Israelاسرائيل

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]

Page 40: Israelاسرائيل

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

Page 41: Israelاسرائيل

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)

Page 42: Israelاسرائيل

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).

Page 43: Israelاسرائيل

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

Page 44: Israelاسرائيل

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

Page 45: Israelاسرائيل

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)

Page 46: Israelاسرائيل

Transition state of hydrogen abstraction, 2A state, R1pro/B1

TSH

EA = 19.5 / 20.4 kcal/mol

( 2A / 4A )

Page 47: Israelاسرائيل

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

+ +

Page 48: Israelاسرائيل

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

Page 49: Israelاسرائيل

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“

Page 50: Israelاسرائيل

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

Page 51: Israelاسرائيل

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

Page 52: Israelاسرائيل

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

Page 53: Israelاسرائيل

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