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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. FNR:NADP + PDB 2BSA. The cyanobacterium Anabaena PCC 7119. - PowerPoint PPT Presentation
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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
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-LafontDepartament de Química and Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain.
Darmstadt September 05 - 09, 2010.
The cyanobacterium Anabaena PCC 7119 and its photosynthetic chain
2 Fdrd + NADP+ + H+ 2 Fdox + NADPH
FNR
Fdox + PSIrd Fdrd + PSIox
CO2 FixationN2
Metabolism
PSI
PSII
ATPase
Phycobilisoms
Cytb6f
Cytosol
2e- + H+
1e-
Fe2S2
NADP+
Electron Transfer Chains in Biological Systems
FAD
1
2
3
4
NADP+two Fdrd
molecules
+
H+
2 Fdrd + NADP+ + H+ 2 Fdox + NADPH
FNR
Hydride Transfer Mechanism
Reduction of NADP+ to NADPH is proposed to take place by hydride transfer from the N5 position of the flavin ring of FNR to the C4 position of the nicotinamide ring.
Tyr303 seems not to be involved in the hydride transfer process but preventsthe direct interaction between the flavin ring of FNR and the nicotinamide ringof NADP+. Tyr303 side-chain must be displaced from its position during hydride transfer.
A B
FAD
Y303
NADP+
NADP+
FAD
FNR(1GJR) Y303S FNR(2BSA)
N5-C4N7.78 Å
N5-C4N3.4 Å
N10-N1N4.6 Å
Rings30º
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
FNRrd + NADP+ FNRox + NADPH
kB C
[FNRrd-NADP+] [FNRox-NADPH]
Dead time
Dead time
CTC-2 CTC-1
WT FNR Time Dependent Observed Process
kHT
kHT-1
FNRrd + NADP+ FNRox + NADPH
kB C
[FNRrd-NADP+] [FNRox-NADPH]
Dead time
Dead time
CTC-2 CTC-1
Tyr303Ser FNR
kHT
Time Dependent Observed Process
T=279 KEXPERIMENTAL 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
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+ complex2BSA
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 transferin the complete solvated FNR:NADP+ system.
Calculate the macroscopic rate constants and kinetic isotope effectsof the hydride transfer in FNR that could be compared with the
experimental data.
By means of Ensemble-Averaged Transition State Theory with Multidimensional Tunneling.
C7
C8
C9
C9a
C5aC6
N10
C4aN5
N1
C2
N3
C4
O4
O2
C7M
C8M
CH3
C5N
C6N C2N
C3NC4N
CH3
C7N
O7N
N7N
N1N
H H
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 lumiflavinand 1-methylnicotinamide are in a vertical stacked configuration.
lumiflavin (FADH-/FAD)1-methylnicotinamide(NADP+/NADPH)
MPWB1K/6-31+G(d,p) accounts betterfor stacking interactions than B3LYP (6 kcal/mol of energy difference in complexation energies).
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
2)(2
1 oRESDRESD zzkV
ABNR algorithmMobile part: all atoms within a sphere of 20 Å.Distinguished coordinate z.
QM H-
N
N
N-
NH
O
O
C
CHO
C
C
CH2
HO
HO
H
H
H
O
P
O
P
O
O
-O
-O O CH2O
HO OH
N
N
N
N
NH2
N+
NH2
O
O
OHHOPO2
-
O
PO2-
CH2O
HO OPO2H-
N
N
N N
NH2
1 2
34aC
O
4
10a
5
10
5a
9a
6
98
7
1' HH
H H 12
34
5
6
7
5'
H
HN5
FADH- NADP+
Total number of QM atoms: 58Frontier GHO atoms: 2Total number of MM atoms : 41838
Definition of the QM region (atoms in orange) used for the QM/MM calculations
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.
RD RARA RDzRPAM1
C7
C8
C9
C9a
C5aC6
N10
C4aN5
N1
C2
N3
C4
O4
O2
C7M
C8M
CH3
C5N
C6N C2N
C3NC4N
CH3
C7N
O7N
N7N
N1N
H H
RPAM1
AM1cQM
RPAM1
MPWB1KcQM
DLcorr zVzVΔV
Dual level single-point energy correction
,
.
Low level RPAM1
AM1cQM zV
High Level RPAM1
MPWB1KcQM zV
NPT molecular dynamics (MD) simulations with periodic boundaryconditions (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)
)exp( */
RT
zG
h
TkTk
QCQCactBMTVTSTEA
CHARMMRATE =CHARMM + POLYRATE
RPAM
AMcQM
RPAM
KMPWBcQM
CMAMCMDL zVzVzWzW 11
111
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
CMDLz *
= 0.04 Ả CMAMz 1*
= -0.07 Ả
The location of the transition state moves slightly towards products.
RA RDz
It is worth noting that the vibrational contribution lowers the relative freeenergy of the transition state around 2.5 kcal/mol.
QUASICLASSICAL POTENTIAL OF MEAN FORCE
33.3 kcal/mol
14.7 kcal/mol
05.0* QCz
zWzWzW vibCMQC
Quantized-vibration correction
))(max()( * zGzG CMact
CMCMact
HT-1 (physiological) 35.04 31.87
HT (reverse) 20.08 17.02
CMFTRR
CMCMCMact GzWzWzG ,,
RvibCM
FTRRCMQCQC
act WGzWzWzG ,,,
zGzG QCact
QCQCact max*
)exp( */
RT
zG
h
TkTk
QCQCactBMTVTSTEA
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)
Structural analysis along the reaction coordinateN5
C4N
H
N10
N1N
3.4 Ả
4.6 Ả
2.75 ẢN5 – C4N
N10 –N1N
The N5 – hydride – C4N angle hasto approach to 180º, and the hydride donor and acceptor atoms have to come closer.
Deformation of the contact ion pair FNRrd-NADP+
and partial loss of π stacking interaction
An energy penalty that increases the free energy cost of the transition state
R
TS
R SP PZ1
Z2
Static-Secondary Zone approximation(Frozen Bath)
Average net transmission coefficient γ
For each variational transition state configuration For each variational transition state configuration i i at at zz**QCQC::
1) AM1/CHARMM22 SP location2) AM1/CHARMM22 MEP3) Dual-level MEP energy correction (interpolation using ISPE).4) Quassiclassical transmission factor Γi
5) Semiclassical transmission coefficient κi
i(T) = exp{-Gi}
Potential of mean force
Vi (s)for configuration i.
Reaction Coordinate z
Gi
tunnelingI(T)
Quassiclasical Transmission Factor Γ
Semiclassical transmission coefficient
= P quantum
P classical
Pcl(E)
E
1
VAG
Veff
Veff,*
Pqu(E) )(2 Ee
0qu dEeP E
=
s
Action integral along the tunneling path
s
s
sEsVE d)(2)( 21eff
1
1/2Pqu(E)
s< s>
0cl dEeP E
E
Tunneling path
• MEP
ZPE)()( MEPG
Aeff sVsVV
s
s
sEsVE d)(2)( 21eff
1
• Assuming vibrational adiabacity of F-1modes orthogonal to s
s
= 0 = 1
• Zero-Curvature (ZCT) tunneling
ERAB
RBC
Reaction-path curvature
Coupling of vibrations k to the reaction coordinate s
v
“corner cutting”
211
1
2
d
d)(
F
kk
TkK
BCurv
s
vLsB
Small-curvature (SCT):
s
s
sEsVE d)(2)(21
eff1
eff ,GAV
)exp( */
RT
zG
h
TkTk
QCQCactBMTVTSTEA
HT-1 31.87 0.998±0.003 335.70±142.79 334.82±142.44
HT 17.02 0.998±0.003 335.70±142.79 334.82±142.44
ii
EA-VTST/MT rate constants: dynamic effectsCHARMMRATE
Average net transmission coefficient
Quassical transmission factor
Semiclassical transmission coefficient: SCT
i ≡ configuration of VTSE
ii ii)( *zG QCQC
act(kcal/mol)
13.8092.8334.82±142.44335.70±142.790.998±0.00317.0220.08HT
28.642.2X10-10334.82±142.44335.70±142.790.998±0.00331.8735.04HT-1
The hydride transfer from Tyr303Ser FNRrd to NADP+ hardly occurs in agreementwith 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 (kHT = 190 s-1).
EA-VTST/MT rate constants: ii)( *zG QCQC
actMTVTSTEAk /
ln*/ RTzGG QCQC
actMTVTSTEA
act
(s-1)
(kcal/mol)
Phenomenological free energy ofactivation
3.44 3.46 3.48 3.50 3.52 3.54 3.56 3.58 3.602.0
2.5
3.0
Ln
(KIE
)103/T
3.44 3.46 3.48 3.50 3.52 3.54 3.56 3.58 3.602
3
4
5
6
Ln
(ko
bs)
A
B
H
D
Hydride and deuteride transfer in the Tyr303Ser FNR:NADP+ reactant complex: Kinetic Isotopic Effect in the non-photosynthetic direction
-2 -1 0 1-5
0
5
10
15
20
25
30
35
PM
F(k
cal/
mo
l)
z(Å)
H/D
TUNNELHYDRIDE TRANSFER
Parameter Theoretical Experimental
kHT (279k) 92.8 s-1 190 s-1
kDT (279 K) 3.5 s-1 16.3 s-1
KIE 26.3 11.6
AH/AD12.5
FAD:NADPH
(C4-H)FADH-:NADP+ (N5-H)
QM/MMLn(kobs) = lnA - Ea/RT
10.73 kcal/mol
10.68 kcal/mol
Vibration-driven tunneling
Conclusions• The physiological hydride transfer from Tyr303Ser FNRrd to NADP+ is not possible. The reverse reaction, the hydride transfer from NADPH to Tyr303Ser
FNRox, does occur.
• The experimental and theoretical reverse reaction rate constants are in very good accordance.
• At the reactant region the N5-C4N distance might be compatible with the hydride transfer, but the N5-hydride-C4N 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 N5-hydride-C4N 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.
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 (Kd = 5.7 μM), is much lower than in the mutant Tyr303Ser FNR (Kd < 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
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, 811l
PSI
NADPH
NADP+
electrons
FNRhq
FNRsq
FNRqnFdrd (Fldhq)
Fdox (Fldsq)
Transient Interactions in the photosynthetic electron transfer from PSI to NADP+
Corrección Vibracional
-4 -2 0 2
-4
-2
0
2
B
A
B Gauss Fit of B
-4 -2 0 2-3
-2
-1
0
1
C
A
C Gauss Fit of C
Corrección vibracional H Corrección vibracional D
Ajuste a una curva gaussiana
z
E(k
cal/m
ol)
z