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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

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

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  1. Calculation of Reduction Potential of FAD in MCAD using Combined DFTB/MM Simulations Sudeep Bhattacharyay, Marian Stankovich, and Jiali Gao

  2. Overview • Objective • Methods of calculation and strategies • Results • Future Directions

  3. 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

  4. FAD is reduced FAD is reoxidized Medium chain acyl-CoA Dehydrogenase (MCAD) Acyl-CoA Dehydrogenases (CAD) in Electron Transfer Ghisla, S. et al.Eur J Biochem, 2004. 271, 494-508.

  5. N C All -domain Extends into the other dimer Two orthogonal -sheets All -domain MCAD: Structural Information • 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 MCAD ETF

  6. R256Q transient intermediate wt- T168A N O -proton abstraction by the catalytic baseGlu376 -hydride transfer on to the flavin nitrogen A Tale of Two Quasi-independent Processes • Potentials of mean force computation of MCAD demonstrate a STEPWISEmechanism • Both protonand hydridetransfer CONTRIBUTE TO THE OVERALL CATALYTICRATE inwild-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

  7. red (anionic) semiquinone blue (neutral) semiquinone Reduction of Flavin FAD + 2e + 2H+ FADH2

  8. -98.6 kcal/mol -197.6 kcal/mol -99.3 kcal/mol Mancini-Samuelson et al. (1998) Biochemistry, 37, 14605-14612 Experimental Mid-point Potentials Values MCAD-bound FAD yellow blue (neutral) semiquinone red (anionic) semiquinone Gustafson et al. (1986) J. Biol. Chem. 261, 7733-7741

  9. Methods and Strategies • Hybrid QM/MM methods; calculation of electron and proton affinities • Thermodynamic integration through FEP • Dual topology single coordinate method • Boundary condition

  10. Electron and proton affinities

  11. State A State B Free Energy Perturbation 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 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 Thermodynamic integration method 1 1 0 0

  12. G λ (R;λ) = UA/MM(RQM ,RMM ) + UB/MM(RQM ,RMM ) QM/MM Interactions With same number of atoms in the two chemical states Utot (R) = │ĤQM+ĤelQM/MM│ + UvanQM/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 Li, G. et al. J. Phys. Chem. B (2003) 107, 8643 Only the electrostatic term contributes to the free energy derivative as the two states have same cartesian coordinate

  13. AH.E(aq) Aˉ .E(aq) + H+ (aq) G E G(1) AH/A¯ G solv H+ G E = G(1) + G(2) + Gsolv [A¯ -D].E(aq) AH/A¯ H+ G(2) Aˉ .E(aq) + D(g) Aˉ.E(aq) + H+ (g) G = 0.0 Thermodynamic Schemes FEP for reduction potential calculation ΔGRd/Ox E-FAD (ox) E-FAD(red) ΔGRd/Oxisobtained from a single FEP calculation FEP for pKa calculation Li, G. et al. J. Phys. Chem. B (2003) 107, 14521

  14. Representations of Atoms • 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

  15. Reaction zone upto 24 Å 30 Å Buffer zone 24 - 30 Å Reservoir zone beyond 30 Å 45 Å Reaction center average of the coordinates of atoms treated by QM Stochastic Boundary Conditions • 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 • 30 Å water sphere added around the active site center • Deleting all atoms beyond 45 Å • Reservoir zone provides a static forcefield

  16. e e FAD + 2e + 2H+ FADH2 FADFADˉ• FAD2ˉ H+ H+ e FADH• FADHˉ H+ FADH2 Which Route ?

  17. 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 ΔG1Rd/Ox = -85.1 kcal/mol ΔG1Rd/Ox (FEP) = -69.7 kcal/mol ΔG (Born Correction) =-16.4 kcal/mol ΔG2Rd/Ox = -86.1 kcal/mol

  18. (E-FAD2- /E-FADH-) (E-FADH- /E-FADH2) ΔGtotal = ΔG1Rd/Ox + ΔG2Rd/Ox + G +G Calculated Reduction Potential Enzyme mid-point reduction potential for MCAD-bound FAD Em = -145 mV G = -197.9 kcal/mol -85 -86 -56 -30 EstimatedΔGtotal= -256kcal/mol; Overestimated energy ~ 60 kcal/mol FAD + 2e + 2H+ FADH2 Possible reason for this overestimation • Long-range electrostatic interactions

  19. Test Calculations • Stochastic boundary set up with net charge of the complex set to zero • General solvent boundary condition • Matching with experimental results

  20. 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

  21. Innerzone upto16Å Buffer zone upto18 Å Reaction center Secondary buffer (18-20) Å Protein atoms which have 1-3 connections with reservoir zone are kept fixed Reservoir zone > 20 Å Setup of Zones in GSBP • 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

  22. Compared Values of Free Energy Changes

  23. FEP for Electron Additions E-FADˉ·  E-FAD2- E-FAD  E-FADˉ· ΔG1Rd/Ox (FEP) = -51.0 kcal/mol ΔG (Born Correction) =-5.46 kcal/mol ΔG1Rd/Ox = -57.46 kcal/mol ΔG2Rd/Ox (FEP) = -36.67 kcal/mol ΔG (Born Correction) =-16.4 kcal/mol ΔG2Rd/Ox = -53.07 kcal/mol

  24. ΔG2(FEP) = -164.1kcal/mol E(H+) a, self interaction energy of H-atom = -141.9 kcal/mol ΔG b( H+ solvation) = 262.4 kcal/mol ΔG (Born Correction) =5.46 kcal/mol ΔG2(total)= -38.15 kcal/mol (E-FADH-- /E-FADH2) ΔG1(FEP) = -184.8 kcal/mol E(H+) a, self interaction energy of H-atom = -141.9 kcal/mol ΔG b ( H+ solvation) = 262.4 kcal/mol ΔG (Born Correction) =16.4 kcal/mol ΔG1(total)= -47.9 kcal/mol (E-FAD2- /E-FADH-) Calculations for Proton Additions E-FADH-+ H+ E-FADH2 E-FAD2- +H+ E-FADHˉ a Zhou, H. et al. Chemical Physics (2002) 277, 91 b Zhan, C. et al. P. Phys. Chem. A (2001) 105, 11534

  25. e e FADFADˉ• FAD2ˉ H+ H+ e √ E-FADH·  E-FADHˉ FADH• FADHˉ H+ ΔG1Rd/Ox (FEP) = -55.47 kcal/mol ΔG (Born Correction) =-5.46 kcal/mol ΔG1Rd/Ox = -60.93 kcal/mol FADH2 FEP Electron Addition √ √ √ ? √

  26. Computed Free Energy Changes

  27. -197.9 kcal/mol -98.6 kcal/mol Summary Gcomput = -196.6 kcal/mol -6.3 kcal/mol Overestimation ~ 6 kcal/mol Gexpt -57.46 kcal/mol -53.07 kcal/mol e e Enz-FAD Enz-FAD¯• Enz-FAD2- Gexpt - 39.97 kcal/mol H+ H+ -47.9 kcal/mol Gcomput = -97.4 kcal/mol -7.0 kcal/mol Overestimation ~ 6 kcal/mol e H+ Enz-FADH2 Enz-FADH• Enz-FADH¯ -60.93 kcal/mol -38.15 kcal/mol

  28. 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• wascalculated 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

  29. Acknowledgements $ NIH Professor Jiali Gao Professor Don G. Truhlar Dr. Kowangho Nam Dr. Alessandro Cembran Dr. Marian Stankovich Dr. Qiang Cui Dr. Haibo Yu Minnesota Supercomputing Institute

  30. (FAD2- /FADH2) Total = S1+ S2 + GS Converting FAD Reduction Potential FAD + 2e + 2H+ FADH2 Em = -(Total)/nF + NHE; n = number of electrons = 2 F= Faraday constant = 23.06 kcal/mol Total- nFNHE = -nFEm , F = 23.06 kcal/(mol.V) nFNHE = -n4.43 F V = -2x 4.43 x 23.06 kcal/mol = -204.31 kcal/mol For FAD-MCAD: E0 = -0.145V Thus Total- nFNHE = -nF x (-0.145) = -2 x 23.06 x (-0.145) kcal/mol = 6.687 kcal/mol Thus Total= (6.687 + nFNHE ) kcal/mol = (6.687 -204.31) kcal/mol = -197.6 kcal/mol For FAD bound MCAD: Mid point potential, E0 = -0.145V Lenn, N. D. Stankovich, M. T. Liu, H. Biochemistry, 1990, 29, 3709

  31. Calculating Absolute Reduction Potential Standard Hydrogen Electrode (Normal Hydrogen Electrode) = Free energy change, EH0 in the reaction H+ (s) + e¯  ½H2 (g) EH0 For a general reduction process: M (s) + e¯ M¯(s) EMM¯ Then combining the above 2 equations ½H2 (g) + M(s)  M¯(s) + H+ (s) E0 => EMM¯ = (E0 + EH0) E0H =-4.43 eV

  32. Rexact ΔRdiel Rinner = fixed atoms Irregular Solvent-Protein Interfaces Im, W. et al. J. Chem. Phys. 2001, 114, 2924

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