1. The Mechanism of the Hydride Transfer between Anabaena Tyr303Ser FNRrd/FNRox and NADP+/H A Combined Pre-Steady-State Kinetic/ Ensemble-Averaged Transition State Theory with Multidimensional Tunneling Study The cyanobacterium Anabaena PCC 7119. FNR:NADP + PDB 2BSA

2. Isaias Lans, José Ramón Peregrina and Milagros Medina Departamento de Bioquímica y Biología Molecular y Celular, and Institute of Biocomputation and Physics of Complex Systems (BIFI), Universidad de Zaragoza, E-50009, Zaragoza, Spain. José M. Lluch, Mireia Garcia-Viloca and Àngels Gonzàlez-Lafont Departament de Química and Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain. Darmstadt September 05 - 09, 2010.

3. The cyanobacterium Anabaena PCC 7119 and its photosynthetic chain 2 Fd rd + NADP + + H + 2 Fd ox + NADPH FNR Fd ox + PSI rd Fd rd + PSI ox CO 2 Fixation N 2 Metabolism PSI PSII ATPase Phycobilisoms Cytb 6 f Cytosol

4. 2e - + H + 1e - Fe 2 S 2 NADP + Electron Transfer Chains in Biological Systems FAD

5. 1 2 3 4 NADP + two Fd rd molecules + H + 2 Fd rd + NADP + + H + 2 Fd ox + NADPH FNR

6. Hydride Transfer Mechanism Reduction of NADP + to NADPH is proposed to take place by hydride transfer from the N 5 position of the flavin ring of FNR to the C 4 position of the nicotinamide ring. Tyr303 seems not to be involved in the hydride transfer process but prevents the direct interaction between the flavin ring of FNR and the nicotinamide ring of NADP +. Tyr303 side-chain must be displaced from its position during hydride transfer. AB FAD Y303 NADP + FAD FNR(1GJR)Y303S FNR(2BSA) N 5 -C 4 N 7.78 Å N 5 -C 4 N 3.4 Å N 10 -N 1 N 4.6 Å Rings 30º

7. OBJECTIVES A better understanding of how FNR enzyme works, that is, of how the hydride transfer process that FNR catalyzes takes place in Tyr303Ser FNR To shed light on the role of C-terminal Tyr303 in WT FNR By means of two complementary studies: Theoretical study based on EA-VTST/MT Experimental stopped-flow pre- steady-state kinetic study Lans, I. et al. J. Phys. Chem. B 2010, 114, 3368 – 3379

8. T=279 K EXPERIMENTAL RESULTS BUT: Tyr303 in the WT must be displaced for the hydride transfer to take place !! Tyr303 substitution by a Ser practically deactivates the capacity of the enzyme to reduce NADP + ’ The equilibrium mixture is displaced towards NADPH production, consistent with the physiological main role of the enzyme. 285 s -1 270 s -1 190 s -1

9. NADP + BUT: The structural disposition in the mutant between the flavin and the nicotinamide rings seems favorable to produce the hydride transfer. Anabaena Tyr303Ser FNR:NADP + complex 2BSA

10. OBJECTIVES of the theoretical approach Provide very subtle details of the mechanism that could be unavailable with experimental techniques. By means of a fully microscopical simulation of the hydride transfer in the complete solvated FNR:NADP+ system. Calculate the macroscopic rate constants and kinetic isotope effects of the hydride transfer in FNR that could be compared with the experimental data. By means of Ensemble-Averaged Transition State Theory with Multidimensional Tunneling.

11. Gas-phase model hydride-transfer reaction: stacking AM1 fails to describe the dispersion energy contribution to stacking favoring instead intermolecular hydrogen-bonding. At the MPWB1K/6-31+G(d,p)level lumiflavin and 1-methylnicotinamide are in a vertical stacked configuration. lumiflavin (FADH - /FAD) 1-methylnicotinamide (NADP + /NADPH) MPWB1K/6-31+G(d,p) accounts better for stacking interactions than B3LYP (6 kcal/mol of energy difference in complexation energies).

12. Model of the biological system Initial Cartesian coordinates PDB file 2BSA. Protonation states: PROPKA. H atoms added with HBUILD in CHARMM. System neutralized with 7 Na + Cubic box of water molecules. Total number of atoms: 41896 atoms (4800 protein atoms) Equilibration of the solvated protein/cofactor/coenzyme system NPT molecular dynamics (MD) simulations with periodic boundary conditions (PBC) at 279 K and 1 atm. Particle Mesh Ewald method. SOFTWARE: CHARMM35 CHARMM22 force field +TIP3P 1-D QM/MM Potential Energy Profile COMPUTATIONAL DETAILS ABNR algorithm Mobile part: all atoms within a sphere of 20 Å. Distinguished coordinate z.

13. QM H-H- Total number of QM atoms: 58 Frontier GHO atoms: 2 Total number of MM atoms : 41838 Definition of the QM region (atoms in orange) used for the QM/MM calculations

14. FNRrd-NADP + SP FNRox-NADPH 1D-POTENTIAL ENERGY SURFACE: AM1(QM)/CHARMM22-TIP3P(MM) The hydride transfer HT-1 in the complete solvated enzymatic system results in: An endoergic process (7.8 kcal/mol) with a potential energy barrier of 36.7 kcal/mol. The flavin and the nicotinamide rings are set out in a roughly parallel configuration. RDRA.

15. Dual level single-point energy correction,. Low level High Level

16. NPT molecular dynamics (MD) simulations with periodic boundary conditions (PBC) at 279 K and 1 atm. Particle Mesh Ewald method. Potential of mean force (PMF) and classical free energy curve with the umbrella sampling technique along the reaction coordinate z and the weighted histogram analysis method (WHAM).. COMPUTATIONAL DETAILS MPWB1K//AM1(QM)/CHARMM22-TIP3P(MM) CHARMMRATE =CHARMM + POLYRATE

17. The effect of thermal and entropic contributions is small. CLASSICAL POTENTIAL OF MEAN FORCE 34.9 kcal/mol 35.8 kcal/mol The introduction of the MPWB1K energy correction only changes significantly the endergonicity of the reaction. 3.6 kcal/mol 14.9 kcal/mol = 0.04 Ả= -0.07 Ả The location of the transition state moves slightly towards products.

18. It is worth noting that the vibrational contribution lowers the relative free energy of the transition state around 2.5 kcal/mol. QUASICLASSICAL POTENTIAL OF MEAN FORCE 33.3 kcal/mol 14.7 kcal/mol Quantized-vibration correction

19. HT-1 (physiological) 35.0431.87 HT (reverse) 20.0817.02 Quasiclassical activation free energy profile Classical mechanical activation free energy profile Quantal vibrational corrections clearly reduce the free energy barriers. The quassiclassical free energy barrier for the physiological reaction HT-1 is almost twice as big as the corresponding value for the reverse reaction HT. EA-VTST/MT rate constants: activation free energy barriers (kcal/mol)

20. Structural analysis along the reaction coordinate N5 C4N H N10 N1N 3.4 Ả 4.6 Ả 2.75 Ả N 5 – C 4 N N 10 –N 1 N The N 5 – hydride – C 4 N angle has to approach to 180º, and the hydride donor and acceptor atoms have to come closer. Deformation of the contact ion pair FNR rd -NADP + and partial loss of π stacking interaction An energy penalty that increases the free energy cost of the transition state R TS

21. R SP P Z1Z1 Z2Z2 Static-Secondary Zone approximation (Frozen Bath ) Average net transmission coefficient γ For each variational transition state configuration i at z * QC : 1)AM1/CHARMM22 SP location 2)AM1/CHARMM22 MEP 3)Dual-level MEP energy correction (interpolation using ISPE). 4) Quassiclassical transmission factor Γ i 5) Semiclassical transmission coefficient κ i

22.  i (T) = exp{-  G i } Potential of mean force V i (s) for configuration i. Reaction Coordinate z  G i tunneling  I (T) Quassiclasical Transmission Factor Γ

23. Semiclassical transmission coefficient   = = P quantum P classical P cl (E) E 1 V AG V eff V eff,* P qu (E)  = s Action integral along the tunneling path 1/2 P qu (E) s<s< s>s> E

24. Tunneling path MEP Assuming vibrational adiabacity of F-1 modes orthogonal to s s = 0 = 1 Zero-Curvature (ZCT) tunneling E R AB R BC

25. Reaction-path curvature Coupling of vibrations k to the reaction coordinate s “corner cutting” Small-curvature (SCT):

26. HT-1 31.870.998± 0.003 335.70± 142.79 334.82± 142.44 HT 17.020.998± 0.003 335.70± 142.79 334.82± 142.44 EA-VTST/MT rate constants: dynamic effects CHARMMRATE Average net transmission coefficient Quassical transmission factor Semiclassical transmission coefficient: SCT i ≡ configuration of VTSE (kcal/mol)

27. 13.8092.8334.82 ± 142.44 335.70± 142. 79 0.998± 0.003 17.02 20.08HT 28.642.2X10 -10 334.82 ± 142.44 335.70± 142.79 0.998± 0.003 31.87 35.04HT-1 The hydride transfer from Tyr303Ser FNRrd to NADP+ hardly occurs in agreement with our stopped-flow kinetic measurements. Conversely, the reverse reaction, HT, does happen, with a reaction rate constant of 92.8 s -1 in very good agreement with our stopped-flow kinetic measurements (k HT = 190 s -1 ). EA-VTST/MT rate constants: (s -1 ) (kcal/mol) Phenomenological free energy of activation

28. A B H D Hydride and deuteride transfer in the Tyr303Ser FNR:NADP + reactant complex: Kinetic Isotopic Effect in the non-photosynthetic direction H/DH/D TUNNEL HYDRIDE TRANSFER ParameterTheoreticalExperimental k HT (279k)92.8 s -1 190 s -1 k DT (279 K)3.5 s -1 16.3 s -1 KIE26.311.6 A H /A D 12.5 FAD:NADPH (C4-H) FADH - :NADP + (N5-H) QM/MM Ln(k obs ) = lnA - Ea/RT 10.73 kcal/mol 10.68 kcal/mol Vibration-driven tunneling

30. Conclusions The physiological hydride transfer from Tyr303Ser FNR rd to NADP + is not possible. The reverse reaction, the hydride transfer from NADPH to Tyr303Ser FNR ox, does occur. The experimental and theoretical reverse reaction rate constants are in very good accordance. At the reactant region the N 5 -C 4 N distance might be compatible with the hydride transfer, but the N 5 -hydride-C 4 N angle is very far from collinearity, and therefore, the hydride shift is quite inefficient. In going from the reactant region to the transition- state region the N 5 -hydride-C 4 N angle approaches 180º to make the hydride transfer easier, and the hydride donor and acceptor atoms come closer. Since the width of the hydride transfer reaction path is small, the hydride transfer involves an important degree of quantum mechanical tunneling. The H/D KIE is essentially temperature-independent. All those geometric deformations, including a partial loss of the π stacking interaction, involve a large free energy penalty to reach the transition state. The difference between the direct and the reverse reaction rates comes from the important positive reaction free energy (15 kcal/mol). Such a difference must be due to an important stabilization of the close contact ionic pair FADH - :NADP + versus the situation corresponding to the product, where, after the hydride transfer, the two moieties are neutral.

31. Any factor able to distort the formation of that close contact ionic pair would destabilize the reactant, so increasing the rate constant of the direct reaction. Very interestingly, the affinity for the coenzyme in the WT FNR (K d = 5.7 μM), is much lower than in the mutant Tyr303Ser FNR (K d < 0.01 μM), this fact being consistent with the feasibility of the direct hydride transfer corresponding to the physiological main role of the enzyme. Could this lower affinity in the WT FNR be attributed to the presence of Tyr303 distorting in some way the formation of a close contact ionic pair as stable as in the Tyr303Ser mutant, with the corresponding thermodynamic and kinetic consequences? Possible role of Tyr303 in WT FNR? WT FNR Tyr303Ser FNR

32. Chair Persons: Manuel Yáñez Otilia Mó Co-chair: Saulo Vázquez -2011 Contact: watoc2011@uam.es

33. Catalytic mechanism of hydride transfer. Role of transient charge transfer interactions. –charge-transfer complex An electron-donor–electron-acceptor complex, characterized by electronic transition(s) to an excited state in which there is a partial transfer of electronic charge from the donor to the acceptor moiety. –charge-transfer (CT) transition An electronic transition in which a large fraction of an electronic charge is transferred from one region of a molecular entity, the electron donor, to another, the electron acceptor (intramolecular CT) or from one molecular entity to another (intermolecular CT). Mulliken, R. S. (1952), J. Am. Chem. Soc. 74, 811 l

34. PSI NADPH NADP + electrons FNR hq FNR sq FNR qn Fd rd (Fld hq ) Fd ox (Fld sq ) Transient Interactions in the photosynthetic electron transfer from PSI to NADP +

35. Corrección Vibracional Corrección vibracional HCorrección vibracional D Ajuste a una curva gaussiana z E(kcal/mol) z