Calculation of Reduction Potential of FAD in MCAD using Combined DFTB/MM Simulations

Calculation of Reduction Potential of FAD in MCAD using Combined DFTB/MM Simulations

Sudeep Bhattacharyay, Marian Stankovich, and Jiali Gao

Overview

Objective

Methods of calculation and strategies

Results

Future Directions

Flavoenzymes

Mediates electron transfer

Flavin ring shuttles between reduced and oxidized states

Protein environment controls the reduction potential of FAD

Coupled electron-proton transfer

pKa from experiment often misleading if that is observed through a observable signature belonging to a particular redox state

Need to predict correctly through simulation

Requires accuracy in a) reduction potential calculation and b) pKa calculation

Acyl-CoA Dehydrogenases (CAD) in Electron Transfer

Ghisla, S. et al. Eur J Biochem, 2004. 271, 494-508.

Medium chain acyl-CoA Dehydrogenase (MCAD)

FAD is reoxidized

FAD is reduced

MCAD: Structural Information

All -domainExtends into the other dimer

N CAll -domainTwo orthogonal

-sheets

Forms homotetrameric structure

Active site is formed at protein-protein interface

One FAD (cofactor) and one acyl-CoA (substrate) bind to the active site

Each active site work independent of the other

Passes electron to electron transfering protein (ETF) when it binds to MCADETF

N

NN

NO

O

H

H

CH3

CH3

H R

CoA

S

C

O

CC

C5H11

H

H

H

H

Glu376

O

O

(-)

Scheme 1

-hydride transfer on to the flavin nitrogen

NN

-proton abstraction by the catalytic base Glu376

OO

Potentials of mean force computation of MCAD demonstrate a STEPWISE mechanism

Both proton and hydride transfer CONTRIBUTE TO THE OVERALL CATALYTIC RATE in wild-type

Effect of PROTEIN ENVIRONMENT on the two steps can be investigated independently

A very attractive enzyme system to work with i.e. to TUNE the two reaction barriers

Need to know the effect of protein environment on the two processes

Bhattacharyya S. et al. (2005) Biochemistry,44,16549

A Tale of Two Quasi-independent Processes

R256Q

T168A wt-transient intermediate

Reduction of Flavin

blue (neutral)semiquinone

red (anionic)semiquinone

FAD + 2e + 2H+ FADH2

Experimental Mid-point Potentials Values

MCAD-bound FAD

blue (neutral)semiquinone

red (anionic)semiquinone

-98.6 kcal/mol

-99.3 kcal/mol

Gustafson et al. (1986) J. Biol. Chem. 261, 7733-7741

-197.6 kcal/mol

Mancini-Samuelson et al. (1998) Biochemistry, 37, 14605-14612

yellow

Methods and Strategies

Hybrid QM/MM methods; calculation of electron and proton affinities

Thermodynamic integration through FEP

Dual topology single coordinate method

Boundary condition

Electron and proton affinities

Model Reactionsa,b B3LYP/6-31+G(d,p)H (kcal/mol)

SCC-DFTB

H (kcal/mol)

AM1(Gaussian)

H (kcal/mol)

FAD + e¯ FAD¯ -44.3 -37.3 -63.5

FAD¯ + e¯ FAD2- +53.4 +58.2 48.3

FAD + 2e¯ FAD2- +9.1 +20.9 -15.1

FAD2- + H+ FADH¯ -436.5 -442.3 -420.0

FAD + 2e¯ + H+ FADH¯ -427.4 -421.4 -435.1

FADH + e¯ FADH¯ -49.4 -50.3 -57.2

FAD¯ + H+ FADH -333.7 -333.7 -314.3

FADH¯+ H+ FADH2 -331.7 -331.4 -320.2

FAD + 2e¯ + 2H+ FADH2 -759.1 -752.8 -755.3

N

N N

N

O

O

H

CH3

CH3

CH3

Free Energy Perturbation

Potential energy of a hybrid system: Uhybrid = (1-λ)UA + λUB

λ is a coupling parameter varied from 0-1 (0.1, 0.2, ….)

Free energy change ΔG = ∫(∂G(λ)/ ∂λ) dλ = ∫∂U(λ)/ ∂λ)dλ

0

1 1

0

Cartesian coordinates of the QM system kept invariant in the two states

Change of chemical state of the system without any major change of the cartesian coordinate

State A State B

Thermodynamic integration method

QM/MM Interactions

With same number of atoms in the two chemical states

Utot (R) = │ĤQM+ĤelQM/MM │ + Uvan

QM/MM (R) + UbondedQM/MM (R) + UMM (R)

electronic energy van der Waals bonded energy of the of the QM system MM atoms

+ the electrostatic interaction energy

Only the electrostatic term contributes to the free energy derivative as the two states have same cartesian coordinate

Gλ

(R;λ) = UA/MM(RQM ,RMM ) + UB/MM(RQM ,RMM )

Li, G. et al. J. Phys. Chem. B (2003) 107, 8643

Thermodynamic SchemesFEP for reduction potential calculation

E-FAD (ox) E-FAD(red)

FEP for pKa calculation

ΔGRd/Ox

ΔGRd/Ox is obtained from a single FEP calculation

AH.E(aq)

[A¯ -D].E(aq)

Aˉ .E(aq) + D(g)

Aˉ .E(aq) + H+ (aq)

Aˉ.E(aq) + H+ (g)

G solvH+

G E

AH/A¯G(1)

G(2)

G = 0.0

G E = G(1) + G(2) + Gsolv

AH/A¯ H+

Li, G. et al. J. Phys. Chem. B (2003) 107, 14521

Representations of Atoms

N3

N1 N10

N5

O4

O2

H

H

CH3

CH3

HB

HOOH

HO

P1

O

OO

P2

O

O

O5*

O5'

N1

N3

N9 N7

NH2

O

HO

HO

H

H

H

H

44a

10a

65a

7

899a

1'2'

3'

4'

5'

(-)

(-)

5*

4*

2*

3*

1*

8

2

4 5

6

2

QM atoms are treated by SCC-DFTB MM atoms by CHARMM forcefield QM/MM boundary treated with

generalized hybrid orbital (GHO) method or link atom method

Stochastic boundary or general solvent boundary

Stochastic Boundary Conditions

Reaction centeraverage of the coordinates of atoms treated by QM

Reaction zoneupto 24 Å

Buffer zone 24 - 30 Å

Reservoir zonebeyond 30 Å

• 30 Å water sphere added around the active site center

• Deleting all atoms beyond 45 Å

• Reaction zone : Newtonian Mechanics

• Buffer zone: Langevin’s equation of motion Friction coefficient and a harmonic restoring force with a gradiant: Scaled to 0 at reaction zone boundary

• Reservoir zone provides a static forcefield

45 Å

30 Å

Which Route ?

FAD + 2e + 2H+ FADH2 FAD FADˉ • FAD2ˉ

FADH• FADHˉ

e e

H+

H+

e

FADH2

H+

Model Reactionsa,b B3LYP/6-31+G(d,p)

H (kcal/mol)

SCC-DFTB

H (kcal/mol)

AM1(Gaussian)

H (kcal/mol)

FAD + e¯ FAD¯ -44.3 -37.3 -63.5

FAD¯ + e¯ FAD2- +53.4 +58.2 48.3

FAD + 2e¯ FAD2- +9.1 +20.9 -15.1

FAD2- + H+ FADH¯ -436.5 -442.3 -420.0

FAD¯ + H+ FADH -333.7 -333.7 -314.3

FADH + e¯ FADH¯ -49.4 -50.4 -57.2

FAD¯ + e¯ + H+ FADH

-383.1 -384.1 -371.7

FADH¯+ H+ FADH2 -331.7 -331.4 -320.2

FAD + 2e¯ + 2H+ FADH2

-759.1 -752.8 -755.3

Calculations using Stochastic Boundary Condition

FADˉ• + e FAD2-FAD + e FADˉ•

ΔG1Rd/Ox (FEP) = -79.64 kcal/mol

ΔG (Born Correction) =-5.46 kcal/mol ΔG1

Rd/Ox = -85.1 kcal/mol




Calculated Reduction Potential

Enzyme mid-point reduction potential for MCAD-bound FAD

Em = -145 mV G = -197.9 kcal/mol

-85 -86 -56 -30

Estimated ΔGtotal = -256 kcal/mol; Overestimated energy ~ 60 kcal/mol

Possible reason for this overestimation

Long-range electrostatic interactions


(E-FAD2- /E-FADH-) (E-FADH- /E-FADH2)ΔGtotal = ΔG1

Rd/Ox + ΔG2Rd/Ox + G + G

Test Calculations

Stochastic boundary set up with net charge of the complex set to zero

General solvent boundary condition

Matching with experimental results

Treating Solvation with General Solvent Boundary Potential (GSBP)

Inner region atoms (ligand + part of enzyme + solvent) treated explicitly

Outer region: Atoms of enzyme are treated explicitly but the solvent is represented as a continuous dielectric medium

Im, W. et al. J. Chem. Phys. 2001, 114, 2924

Setup of Zones in GSBP

Reaction center

Inner region atoms (ligand + part of enzyme + solvent) treated explicitly (< 16 Å)

Water atoms deleted beyond 18 Å Charges of residues beyond 20 Å set to 0 Protein atom fixed beyond 20 Å Outer region: Atoms of enzyme are treated explicitly but the solvent

is represented as a continuous dielectric medium

Inner zone upto 16 Å

Buffer zone upto 18 Å

Secondary buffer (18-20) Å

Reservoir zone > 20 Å

Protein atoms which have 1-3 connections with reservoir zone are kept fixed

Compared Values of Free Energy Changes

Reaction

Stochastic boundary

with neutralized charge

G(kcal/mol)

General solvent

boundarypotential

G(kcal/mol)

E-FAD E-FADˉ· -57.46 -64.1

E-FADˉ· E-FAD2- -53.07 -46.4

E-FAD2- E-FADHˉ -47.9 -53

FEP for Electron Additions

-120

-100

-80

-60

-40

-20

0

0 0.2 0.4 0.6 0.8 1

l

dU

/dl (

kc

al/

mo

l)

-120

-100

-80

-60

-40

-20

0

20

0 0.2 0.4 0.6 0.8 1

l

dU

/dl

(kc

al/

mo

l)

E-FAD E-FADˉ· E-FADˉ· E-FAD2-







Calculations for Proton Additions

100

140

180

220

260

0 0.2 0.4 0.6 0.8 1

l

du/dl

(kca

l/mol)

100

140

180

220

260

0 0.2 0.4 0.6 0.8 1l

dU

/dl

(kc

al/m

ol)

E-FAD2- + H+ E-FADHˉ E-FADH- + H+ E-FADH2

ΔG2 (FEP) = -164.1kcal/molE(H+) a, self interaction energy of H-atom

= -141.9 kcal/molΔG b( H+ solvation) = 262.4 kcal/molΔG (Born Correction) =5.46 kcal/mol ΔG2

(total) = -38.15 kcal/mol

(E-FADH-- /E-FADH2)ΔG1 (FEP) = -184.8 kcal/molE(H+) a, self interaction energy of H-atom

= -141.9 kcal/molΔG b ( H+ solvation) = 262.4 kcal/molΔG (Born Correction) =16.4 kcal/mol ΔG1

(total) = -47.9 kcal/mol

(E-FAD2- /E-FADH-)

a Zhou, H. et al. Chemical Physics (2002) 277, 91 b Zhan, C. et al. P. Phys. Chem. A (2001) 105, 11534

FEP Electron Addition

-120

-100

-80

-60

-40

-20

0

0 0.2 0.4 0.6 0.8 1

l

dU

/dl

(kc

al/m

ol)

FAD FADˉ • FAD2ˉ

FADH• FADHˉ

e e

H+ H+

e

FADH2

H+

√√

√

√? E-FADH· E-FADHˉ




√

Computed Free Energy Changes

Reaction

Stochastic Boundary with 0

chargeG

(kcal/mol)

E-FAD + e E-FAD¯· -57.46

E-FAD¯· + H+ E-FADH· -39.97

E-FAD¯· + e E-FAD2- -53.07

E-FAD2- + H+ E-FADH¯ -47.9

E-FADH¯ + H+ E-FADH2 -38.15

Enz-FADH· E-FADH2 -60.93

Summary

Gexpt

H+

Enz-FAD

Enz-FADH•

Enz-FAD¯• Enz-FAD2-

Enz-FADH¯

H+

e e-57.46 kcal/mol -53.07 kcal/mol

-47.9 kcal/mol

e

-60.93 kcal/mol

-98.6 kcal/mol - 39.97 kcal/mol

H+

Enz-FADH2

-197.9 kcal/mol

-38.15 kcal/mol

Gexpt

Gcomput

= -97.4 kcal/mol -7.0 kcal/mol

Overestimation ~ 6 kcal/mol

Gcomput

= -196.6 kcal/mol -6.3 kcal/mol

Overestimation ~ 6 kcal/mol

Conclusions

The two-electron two-proton reduction potential of FAD in MCAD was calculated Both reduction potential calculations yield results that are consistent to the

experimental values Computed 2e/2H+ reduction potential for FAD bound MCAD is -180 mV, which is

35 mV more negative than the experimental value The first reduction potential of MCAD-bound FAD was calculated to be ~ -103

kcal/mol compared to the experimentally observed value of -98.6 kcal/mol Computed second reduction potential for MCAD-bound FAD was ~ -105

kcal/mol, about 6 kcal/mol more negative than the experimentally observed value of -99.3 kcal/mol

This calculation shows that at neutral pH MCAD-bound FAD will be converted to the hydroquinone form FADH2 through a two electrons/two protons reduction

pKa calculation of E-FADH¯ and E-FADH2 show that both values are quite high: 35 and 27, respectively. The pKa of E-FADH• was calculated to be ~30

Future Directions Using the method to compute the reduction potential

and pKa of FAD and FMN in aqueous solution pka calculation of acyl-CoA substrate bound to MCAD

Acknowledgements

$ NIHProfessor Jiali Gao Professor Don G. Truhlar

Dr. Kowangho Nam Dr. Alessandro Cembran

Dr. Marian Stankovich Dr. Qiang Cui Dr. Haibo YuMinnesota Supercomputing Institute


Total = S1 + S2 + GS

(FAD2- /FADH2)

Em = -(Total)/nF + NHE ; n = number of electrons = 2F= Faraday constant = 23.06 kcal/mol

Total - nFNHE = -nFEm , F = 23.06 kcal/(mol.V)

nFNHE = -n4.43 F V = -2x 4.43 x 23.06 kcal/mol = -204.31 kcal/molFor FAD-MCAD: E0 = -0.145V

Thus Total - nFNHE = -nF x (-0.145) = -2 x 23.06 x (-0.145) kcal/mol

= 6.687 kcal/mol

Thus Total = (6.687 + nFNHE ) kcal/mol = (6.687 -204.31) kcal/mol

= -197.6 kcal/mol

Converting FAD Reduction Potential

For FAD bound MCAD: Mid point potential, E0 = -0.145VLenn, N. D. Stankovich, M. T. Liu, H. Biochemistry, 1990, 29, 3709

Calculating Absolute Reduction Potential

Standard Hydrogen Electrode (Normal Hydrogen Electrode) = Free energy change, EH

0 in the reaction

H+ (s) + e¯ ½H2 (g) EH0

For a general reduction process:

M (s) + e¯ M¯(s) EMM¯

Then combining the above 2 equations

½H2 (g) + M(s) M¯(s) + H+ (s) E0

=> EMM¯ = (E0 + EH0)

E0H = -4.43 eV

Irregular Solvent-Protein Interfaces

RinnerRexact

ΔRdiel= fixed atoms

Im, W. et al. J. Chem. Phys. 2001, 114, 2924

Calculation of Reduction Potential of FAD in MCAD using Combined DFTB/MM Simulations

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