Bioluminescence: A challenge to computational chemistry. Luca De Vico, Jesper Wisborg-Krogh, and Roland Lindh

Transcription

1 Bioluminescence: A challenge to computational chemistry Luca De Vico, Jesper Wisborg-Krogh, and Roland Lindh

2 Outline Introduction to bioluminescence Examples of bioluminescence The chemistry of bioluminescence The 1,2-dioxetane model The firefly luciferin model Conclusions

3 Introduction to Bioluminescence Definitions Fluorescence is when energy from an external light is absorbed and immediately released at a longer wavelength Phosphorescence is when energy from an external light is absorbed and released at a longer wavelength sometime later Chemiluminescence is the production of light from a chemical reaction Bioluminescence is chemiluminescence inside a living organism

4 Why bioluminescence? Mating Lure and Prey Protection???

5 Mating

6 The Firefly Beetle

7 Lure and Prey

8 Femme Fatal

9 Cookie-cutter

10

11 Protection

12 ???

13 The Chemistry of Chemiluscence

14 Luciferins Bacterial Dinoflagellate Shrimp Most abundant

15 Firefly luciferin

16 The generic reaction

17 Applications Chemical probe ATP in vivo essay Emergency light

18 The 1,2-dioxetane model Simplest system to exhibit chemiluminescence thermal reaction % triplet formed for substituted specie

19 The school book example

20 Previous Study (Robb et al. '99) 6-31G* basis Small CASSCF active space CASSCF surface MR-MP2

21 Key observations Activation energy 17.0 kcal/mol (exptl kcal/mol) No intersection along reaction path of S0 Intersection is perpendicular to the reaction coordinate High TS2 for triplet exit channel No conical intersection considered Energy surfaces are inconsistent with experimental observations.

22 New study Basis-set ANO-RCC with contraction scheme for carbon and oxygen atoms (4s3p2d1f). Full active space: C-C, C-O and O-O bonds considered, and/or π and lone pairs orbitals, for a total of 12 electrons in 10 orbitals. MS-CASPT2//CASSCF fully optimized structures and minimum energy paths. Some features were optimized also at the MS-CASPT2//MS-CASPT2 level of theory.

23 The active space and included states

24 MS-CASPT2//CASSCF S0 80 S1 T1 S0 70 Energy difference (kcal/mol) Activation energy 23.5 kcal/mol Experimental 22.7 kcal/mol Reaction coordinate (a.u)

25 Stationary Points CASSCF (MS-CASPT2) geometrical parameters

26 MS-CASPT2//CASSCF S Energy difference (kcal/mol) 25 S1 T1 S Flat S0 PES after TS before dissociation channel Small peak as reported also in other articles Reaction coordinate (a.u)

27 MS-CASPT2//CASSCF S0 Spin Orbit Coupling 80 S1 T1 S0 70 Energy difference (kcal/mol) singlets + 4 triplets = 16 SOC states Reaction coordinate (a.u) S0, S1 and T1 are mixed.

28 MS-CASPT2//CASSCF T1 Energy difference (kcal/mol) O C C O angle 0 25 O O H H H 3.00 Reaction coordinate (a.u) T1 PES is characterized by a conformational change around C-C bond H

29 MS-CASPT2//CASSCF T1 Energy difference (kcal/mol) The molecule is dropped on T1 PES here from the S0 transition state region Along both paths symmetry is maintained: same C O distance same C pyramidalization Reaction coordinate (a.u)

30 MS-CASPT2//CASSCF T1 TS 70 Energy difference (kcal/mol) 33 TS 180 O 31 O 29 H H H H For the molecule to dissociate it is necessary to take an asymmetric path, perpendicular to the conformational change one. From the two minima, stretching one C O bond while the other shrinks, the molecule reaches a transition state. There is a total of 4 TS The dissociation is into two OCH2 molecules, of which 3.00 one is in an excited state. Reaction coordinate (a.u) S1 PES behaves in the same way

31 MS-CASPT2//MS-CASPT2 MS-CASPT2 PES has the same features of the CASSCF one. First results show that energetics are different. T1 transition states should be lower in energy, to make them more accessible.

32 The Firefly Luciferase The enzyme luciferase carries out the reaction between luciferin, Mg-ATP and molecular oxygen. Light is emitted during relaxation of excited oxyluciferin to its ground state. The reaction has a high quantum yield ( ).

33 Different colours Fireflies, click beetles and railroad worms share the same substrate luciferin, but naturally emit light of different wavelengths. Firefly yellow to green light Click beetles green to orange Railroad worms green to red Firefly s luciferase mutants can emit red light!

34 What s the origin of the variation of the bioluminescence colour? Various hypothesis have been proposed to date. Hypothesis 1: Luciferin could exist in its excited state both as keto or enol form. The equilibrium between the two forms is responsible for the emitted colour. However, experiments showed that only the keto form is responsible for the emition of light. Hypothesis 1 can be excluded.

35 What s the origin of the variation of the bioluminescence colour? Hypothesis 2: The keto form, in its excited singlet state, shows a non planar structure. The emitted colour should then depend on the degree of twist. O S N N O S Previous calculations at different levels of theory disagree whether Hypothesis 2 is plausible or not.

36 What s the origin of the variation of the bioluminescence colour? Our fully optimised structure of the keto(-1) at B3LYP/ G(d,p) is planar, and shows no imaginary frequencies. To our knowledge, this is the best level of theory possible for this kind of analysis. It is unlikely that, inside the luciferase pocket, oxyluciferin has enough space to twist enough in order to change drastically the emitted colour. Hypothesis 2 is excluded!

37 What s the origin of the variation of the bioluminescence colour? Hypothesis 3: The emitted colour could depend on the polarization of the microenvironment of the complex enzyme-oxyluciferin. The higher the polarizability, the larger should be the red-shift of the bioluminescence.

38 What s the origin of the variation of the bioluminescence colour? We simulated the practically complicated microenvironment around oxyluciferin with a simple but reasonable model. We considered only the polarization of the phenolic/phenolate group. - + O S N N S O

39 What s the origin of the variation of the bioluminescence colour? Excitation energy keto 3.32 ev CH2Cl2 + keto 3.27 ev H2O + keto 3.19 ev 2.54 ev keto(-1) Red shift Polarization Structure

40 What s the origin of the variation of the bioluminescence colour? Hypothesis 4: Different resonance based keto forms of the oxyluciferin could exist. Again, the difference in colour should depend on the equilibrium between the two different forms. O S N N S O O S N N S O

41 What s the origin of the variation of the bioluminescence colour? Work is still in progress, but first results indicate that different structures located with a lower quality method (loose convergence criteria, not big enough active space) converge to only one structure when employing the best method feasible, CASSCF with an active space of 18 electrons in 15 orbitals.

42 What s the origin of the variation of the bioluminescence colour? Recently a paper published on Nature (vol. 440, ) dealt with the same problem. An analogue of oxyluciferin was used to freeze luciferase and a red-emitting mutant as they should be before emitting light, and their x-ray structures were resolved. It suggested another possible way of controlling the emitted light wavelength, hypothesis 5.

43 What s the origin of the variation of the bioluminescence colour? The oxyluciferin analogue showed no twisting in all structures, confirming our calculations. Hypothesis 2 is also not to be considered as possible.

44 What s the origin of the variation of the bioluminescence colour? Natural Firefly luciferase OxyLuciferin Mutant

45 What s the origin of the variation of the bioluminescence colour? The tight pocket of the wild-type luciferase should not allow too much structural relaxation of the oxyluciferin before it emits light and decade to its ground state. Most of the chemical energy is then transformed into light with a short wavelength.

46 What s the origin of the variation of the bioluminescence colour? The loose pocket of the mutant, on the other hand, should let the oxyluciferin structure to relax a bit before the decay on the ground state. Part of the chemical energy is wasted, and the emitted light wavelength is longer.

47 What s the origin of the variation of the bioluminescence colour? We are actually working to simulate with our model the amount of energy that can be lost by geometrical relaxation on the S1 surface before decaying on the ground state. S1 hν hν 1 S0 2

48 What s the origin of the variation of the bioluminescence colour? Hypothesis 3 could represent a way of modulating the emitted colour in an artificial environment. Hypothesis 4 could be not possible, but still no final data is available. We are working on elucidating hypothesis 5, which seems to be very appealing.

49 Conclusions Required level of theory MS-CASPT2ANO! 1,2-dioxetane dissociation: MS-CASPT2 gives quantitative results. luciferin: a) micro environment manipulation is possible, b) pocket strain on relaxation before emission is important. MM/QM calculations to come. other important systems, e.g. luminol, etc.