Color-Modulation of Firefly Luciferin-Luciferase System Investigated by Theoretical Approach

Color-Modulation of Firefly
   Luciferin-Luciferase System
   Investigated by Theoretical
             Approach

                      Isabelle Navizet
        Ya-Jun Liu, Nicolas Ferré, Roland Lindh

Multi-scale modelization and simulation laboratory MSME, UMR 8208 CNRS,
                East-Paris Marne-la-Vallée University, France.
           College of Chemistry, Beijing Normal University, China.

                          navizet@univ-mlv.fr

                         Lyon ISBC 19-23 April 2010

Lund,              Beijing Normal
                                        Uppsala            University
                  Pr. R. Lindh
                                        Université Paris Est
                                        Marne la Vallée
                                        Université
                                        Aix-Marseille

 Dr. I. Navizet                                          Dr. Y-J Liu, Prof.W-H Fang

                                                         Xiao H.Y.   Chen S.F.
www.chimietheorique.fr   Dr. N. Ferré

System: fireflies

                              Oxyluciferase

                                   AMP

Oxyluciferin

Mechanism of firefly bioluminescence:
chemically initiated electron exchange
       luminescence CIEEL

Luciferin Color Modulation
   Fireflies, click beetles and railroad worms share
      the same substrate luciferin, but naturally emit
      light of different wavelengths.

             • Firefly                              green to yellow
             • Railroad worms                       green and red
             •Click beetles                         orange to green

             • Firefly’s luciferase mutants         red

Pictures by V.
Viviani              Nakatsu, Nature 2006,404,372

  How is the color modulation controlled?

5 Hypothesis
• Keto-enol
• Twist angle of the keto anion form.
• Polarization in the micro
  environment (keto, keto-1)
• Resonance structure (keto-1,keto-1’)
• Pocket size

Keto-enol mechanism
Hypothesis: different emission spectra for enol
 and keto form (White, Bioorg. Chem. 1971,92)

-                          -

              Keto-1                   Enol-1
        red                    yellow-green
Experiment: multicolor luminescence require only
keto (Branchini, JACS 2002 124,2112)
Is tautomerization possible inside the protein ?

Twist angle of the keto anion
            form.
Hypothesis: the emitted color depends of the
 twist angle of the keto anion form. (McCapra et
 al)

              Keto-1                 twisted

NO: Twisted structure is a TS state. (Goddard
et al., Nakatsuji et al.)

Polarization in the micro
       environment (keto, keto-1)
  Tv values decrease as the polarization of the
    microenvironment increase.
TD-B3LYP/6-31+G(d,p) Tv in eV (oscillator strength)

3.32(0.293)          3.27(0.336)        3.19(0.360)          2.54(0.645)

                           polarization
u.v.                        Red shift                            blue
 Liu, De Vico, Lindh, J.Photochem. Photobio. A, 2008, 194, 261
                              See Wednesday presentation O36

Resonance structure
          (keto-1,keto-1’)
Color of emitted light is a function of the degree
  of resonance between two extreme electronic
  configurations of the keto-anion.(Branchini,
  Biochem 2004, 43,7255)

 -                                                 -

            Keto-1                       Keto-1’
        green                      red
Bond C-C length single/double

Pocket size

Japanese genji-botaru (Luciola cruciata) 539 residues

 Nakatsu et al. Nature 2006, 440, 372.

Pocket size
Wild type: open->closed->open
Tight pocket
Hypothesis: No much relaxation of
Oxyluciferin before emitting light

Mutant: open->open->open
Loose pocket
Hypothesis: Relaxation of
Oxyluciferin before emitting light
(∆E smaller)

Theoretical studies
• QM: quantum mechanic (only few atoms)

• MM: molecular mechanic (big systems but no
  bond breaking, no electronic excitation)

• QM/MM: combines the 2 methods.

MM (AMBER ff)
 Ebonds =      ∑l 0
                k (l
               bonds
                     − l ) 2

Eangles =      ∑θ
                k (θ
              anglesθ
                     − θ 0 ) 2

 E impr = k ω (ω − ω 0 ) 2
                           Vn ,φ
Ediedrals =      ∑
              diedrals φ     2
                                   [1 + cos(nφ + γ )²]

                        qi q j
Eelec = ∑      fijel
        i< j            ε rij
                              r* 12              6
                                              rij*    
ELJ = ∑ fijLJ          eij*   ij  − 2          
       i< j                   rij           rij    
                                                       
         EMM = Ebonds + Eangles + Eimpr + Edied + Eelec + ELJ   parameters

QM
• Resolution of the Schrödinger equation (time-
  indep.):

                      Hψ i = Ei ψ i
• Goal: find Ei and ψi :
   – Density functional theory (DFT)
   – Post-HF: CASSCF, CASPT2….
• Approximation: ψ combination of
  antisymmetrized product of one-electron spin-
  orbitals, use of a finite basis set.
• Basis set : 6-31G*, ANO-L-VTZP….

QM/MM
E =< ψ H ψ >=< ψ H QM + H MM + H QM / MM ψ >
MM subsyst : see charges and vdW of QM
subsyst (Eelect, EvdW)
QM subsyst : HQM take into account charges of
MM. ESPF (Electrostatic Potential Fitted)

E =< ψ H QM + H    elect
                   QM / MM   ψ > +E  nucl
                                     QM     +EonMMnucl
                                              QM / MM    + EMM

MOLCAS + TINKER + Nicolas Ferré patch
   www.teokem.lu.se/molcas
   sites.univ-provence.fr/lcp-ct/ferre/nf_tinker_qmmm.html

Building models
• Protein structure: PDB data bank
Japanese genji-botaru (Luciola cruciata) Luciferase:
  539 residues

Open: PDB 2D1R
     (WT AMP oxyluciferin)                     Keto-1
     “mutant-like, red”
                                               AMP
Closed: PDB 2D1S
     (WT DLSA)
     “wild, yellow-green”
                                              DLSA

Six different structures studied of two types – so
called “open” and “closed” structures.
All deduced from X-ray structures and modeled with
keto-1

Building models
 • Model 0: Initial corrections, standard protonations,
   missing water and atoms, box of water.
 • Model 1: Local MM and QM re-optimization
 • Model 2: Adding more waters to the cavity
 • Model 3: Structures completely relaxed with MD

              AMP
                                              AMP
                                SER286

                    Open
Ser286   Ile288     closed

QMMM calculation
MOLCAS + TINKER + Nicolas Ferré patch

• QM: Oxyluciferin keto-1,
• MM: protein, water and AMP, AMBER FF99
  parameters
• ESPF (Electrostatic Potential Fitted)

• Optimization: CASSCF 16-in-14 (all Π orbitals
  except the one centred on S7), 6-31G(d) basis
  set, no sym, charge -1, first excited state.

• Electronic transition: CASPT2, 6-31G(d) or
  ANO-L-VTZP basis set, Imaginary shift of 0.05
  if intruder state, 16-in-14 or 18-in-15 on the
  structure optimized in it first excited state.

Emission energy (eV) as a functions
        of structure in luciferase
    CASSCF(16-in-14) S1 opt, CASPT2, 6-31G(d)

   Navizet et al., JACS 2010, 132,706.

Oxyluciferin: same structural
Enzyme and water: different              => Color modulation

Molecular orbitals

Stabilization of HOMO : ∆E ↑ , λ ↓ , blue shift

Model-1-open:
                      2.05(2.03)eV

    The
 H-bonding
   water             Model-1-closed:

network: the         2.13(2.02)eV

 polarizing
   agent
                      Model-2-open:
                      2.14(2.04)eV
 Not pocket effect

Keto/enol ?

                         LUMO
                                                          LUMO

                         HOMO
                                                           HOMO

CASSCF (18 in 15) ANO-RCC-VDZP/CASPT2 (oscillator strength)
Keto-1: 2.18 eV (0.81)           enol-1: 2.20 eV (0.91)

In vacuo, TDDFT : tautomerization TS keto-enol : 64 kcal/mol.
In protein ?

Conclusion
• Not a pocket effect
• Effect of number of water in the pocket
• Enol possible ?

Lund,              Beijing Normal
                                        Uppsala            University
                  Pr. R. Lindh
                                        Université Paris Est
                                        Marne la Vallée
                                        Université
                                        Aix-Marseille

                THANKS FOR YOUR
                ATTENTION !
 Dr. I.
 Navizet
                                                         Dr. Y-J Liu, Prof.W-H Fang

                                        Johannesburg

www.chimietheorique.fr   Dr. N. Ferré                      Xiao H.Y.   Chen S.F.