Biological Photochemistry: The fate of electronic excited states in proteins, DNA, and the role of quenching Robert J. Stanley DOE Workshop on Aqueous Scintillators January 19, 2010 emple Chemistry Department Philadelphia, PA

Electronic excited states in Biology Chemiluminescence –Bioluminescence – charge transfer? radicals? Photoinduced electron transfer –Photosynthesis –DNA repair Photochemistry –DNA damage –photosensors

DNA…a polymer of nucleotides connected by phosphodiester linkages 5’ 3’ Nucleic acid bases A, T, C, & G Voet and Voet, Biochemistry, 2nd Ed. Wiley, New York, 1995

B-DNA is double-stranded (ds) DNA, forming the famous double helix ( Watson, Crick, Franklin) Watson-Crick base pairing (complementarity)

DNA absorbs UV radiation  * transition

Quenching of excited states can be desirous or devastating in living systems: DNA UV light absorbed by DNA is rapidly transformed into heat –Conical intersections in the potential surfaces of excited and ground state nucleic acid bases leads to ultrafast degradation of light into heat ( sec.) …GOOD! Excited native DNA bases (Guanine, Adenine, Thymine, Cytosine) can be either excited state donors or acceptors –sequence dependent reaction –*G  8-oxo-G –T-T  T<>T pyrimidine dimerization –Cancer, apoptosis…BAD

UV light damages DNA Bad photochemistry h N NH O T-T O O O HN N CH 3 N HN O O N O O T<>T or CPD CH 3 3 NH C 3 H < 320 nm 2+2 photo-cycloaddition

If DNA damage is left unrepaired then mutations, cell death, and cancer can develop

Förster or Dexter Transfer (singlets) Triplet Energy Transfer Fluorescence D*A h D DA DA * h A Bright Dark Bright or Dark Pathways involving energy transfer D = G*, A*, C*, T* A = G, A, C, T

Conical Intersection Intramolecular vibrational relaxation Fluorescence D*A Bright Dark h D DA “Structural” quenching pathways D hot A

Photoinduced Electron Transfer (PET) Exciplex (EX) formation (charge transfer) Fluorescence D*A h D DA Pathways involving electron transfer Bright Dark Bright or Dark h EX ?

Repair of the thymidines is direct:Repair of the thymidines is direct: T<>T  T-T without modifying the DNA backbone Wide spread: E. coli, Frogs, Rice, Kangaroos…Humans (no!)Wide spread: E. coli, Frogs, Rice, Kangaroos…Humans (no!) Enzymatic Repair of CPDs by DNA Photolyase uses blue light as an energy source (Good photochemistry) Sancar, A. Structure and function of DNA photolyase. Biochemistry 33, 2-9 (1994). Possible Applications: Photosomes® (AGI Dermatics) transgenic crops (wheat?) Mees, A., et al (2004) Science 306,

PL functions efficiently with only FAD (required for repair and bindingPL functions efficiently with only FAD (required for repair and binding PL binds the CPD with high affinity (no light required):PL binds the CPD with high affinity (no light required): K A = 10 9 M -1 for dsDNA with CPD K A = 10 9 M -1 for dsDNA with CPD DNA Photolyase (PL) is a flavoprotein (Vitamin B 2 ) that binds and repairs CPDs Park, H.-W., Kim, S.-T., Sancar, A., and Deisenhofer, J. (1995) Science 268,

Biochemistry 2 nd Ed., Voet and Voet, J. Wiley & Sons Flavin Structure and Oxidation States Flavins can transfer 1 or 2 electrons (unlike nicotinamide) and are used in a large number of redox reactions in the cellFlavins can transfer 1 or 2 electrons (unlike nicotinamide) and are used in a large number of redox reactions in the cell Surprisingly, flavins are a major biological chromophore (DNA repair, circadian rhythms, phototropism, etc.)Surprisingly, flavins are a major biological chromophore (DNA repair, circadian rhythms, phototropism, etc.) FADH — —

Photolyase functions by Photoinduced Electron Transfer from the FAD to the CPD A large separation between the FADH - and the CPD (~16 Å) would give a slow electron transfer rate (k eT, from Marcus theory)A large separation between the FADH - and the CPD (~16 Å) would give a slow electron transfer rate (k eT, from Marcus theory) Orbital overlap x Driving force Slow electron transfer would compete poorly with 1 FADH — deactivation (about 5 ns)Slow electron transfer would compete poorly with 1 FADH — deactivation (about 5 ns) but  repair > 0.7! There’s a cavity in the protein FAD

What happens to substrate conformation upon binding to Photolyase? Base Flipping Photolyase Minor disruption Moderate disruption Severe disruption AA T<>T

Fluorescent reporter approach to probing double helical structure Base Flipping 5’ 3’ 5’ 3’ 5’-probe approach: Base Flipping 5’ 3’ 5’ 3’ 3’-probe approach: The fluorescence quantum yield of the reporter decreases when base stacked…but why?  

6MAP is an attractive new fluorescent adenosine analogue Properties: 1  fl = 0.2 ex = 330 nm (  ~ 8,500 M -1 cm -1 ) em = 430 nm (large Stokes shift) 1 Hawkins, et al, “Synthesis and Fluorescence Characterization of Pteridine Adenosine Nucleoside Analogs for DNA Incorporation.” Anal. Biochem.298, (2001). 4-amino-6-methyl-8-(2-deoxy-  - D -ribofuranosyl)-7(8H)-pteridone K. Yang, S. Matsika, and R.J. Stanley, Biochemistry 2007

Base flipping of the CPD monitored by 6MAP 5’-GCAAGTTGGAG-3’ 3’-CGTTCAFCCTC-5’ 5’-GCAAGTTGGAG-3’ 3’-CGTTCFACCTC-5’ Why is the intensity pattern sequence-dependent? -PL +PL -PL +PL

These data are consistent with disruption of base stacking due to base flipping of the CPD by Photolyase Photolyase Mees et al, Science v. 306, (2004) ?

Is the fluorescence quantum yield modulation of 6MAP due to PET? Stern-Volmer quenching of 6MAP by G,A,C, and T: what is the rate of quenching, k q ? What are the redox potentials? Cyclic voltammetry of 6MAP in aprotic organic solvents submitted to Biochemistry

The quenching of 6MAP* proceeds through nucleobase oxidation: 6MAP*:NMP  6MAP  ¯:NMP  + (Scandola-Balzani relation) FBANB  G ET  (eV) E act (eV) 6MAP G A C dT submitted to Biochemistry

What’s the mechanism for base analog quenching? What’s the mechanism for base analog quenching? Pathways for energy transduction in a model FBA oligo Absorption Stark spectra of ssDNA with 2AP (  ), a hexamer with 2AP (  ), and a mix of the individual bases (  ). Stark absorption and emission spectra of 6-MI (  ), a guanine analog, compared with their absorption and emission spectra (  ). Stark and MRCI calculations (Matsika)

Another possibility: 6MAP emission overlaps the absorption of the FAD: FRET from 6MAP*  FAD? Yang et al, JPC B (2007)

R 0  the Förster distance where  ET = 0.5 r DA  the distance between a donor (fluorescent analogue) and an acceptor (FAD in photolyase) Fluorescence Energy Transfer Efficiency

R 0 (Å) = The Förster distance  2 : the orientation factor; n : the refractive index of the medium;  D :the fluorescence quantum yield of the donor; J :the overlap integral.

F D ( ): the fluorescence intensity of the donor as a function of wavelength. ε A ( ): the molar extinction coefficient of the acceptor at that wavelength; The Overlap Integral

θ T :  m D, m A θ D :  m D, r DA θ A :  m A, r DA The Orientation Factor mDmD mAmA r DA

The transition dipole moment direction 6MAP was calculated from TD-DFT Yang et al, JPC B (2007)

Orientation factors and  ET between Probes and FAD ox From the crystal structure, lit. and TDDFT calcs crystal structure experiment Yang et al, JPC B (2007)

FRET efficiency vs. orientation Yang et al, JPC B (2007)

NO FRET! The FAD is quenched 100x in the protein (acceptor is dark) A work-around : time- resolved FRET? Quenching mechanism is different for the two probes photoinduced electron transfer vs. ultrafast internal conversion? Does FAD* undergo PET to tryptophan??? Yang et al, JPC B (2007)

Can we identify the kinetics and mechanism of repair? Two color pump probe femtosecond spectroscopy: What is the electron transfer lifetime (  eT ) ? Does repair proceed by a concerted or sequential mechanism? PL red  : T<>T PL red  : T<>T PL sq : T<>T  PL sq : T|_|T  PL sq : T-T  PL red  + T-T PL red  : T-T 7  eT k rec 11 22 k beT k diss k ic, k rad cc MacFarlane and Stanley (2003) Biochemistry 42,

Transient absorption measurement layout BBO CaF 2 Sample

PET to the CPD substrate quenches the FADH  excited state in ~ 30 ps MacFarlane and Stanley (2003) Biochemistry 42,

What’s are the intermediates?  A(,t) =  c i (t)  i ( ) = C(E -  0 ) where E i ( ) = True spectra of the intermediates  0 ( ) = Ground state absorption spectrum  0 ( ) = Ground state absorption spectrum ConstructC(t) = C 0 e Kt (from the K matrix)ConstructC(t) = C 0 e Kt (from the K matrix) CalculateE i ( ) = C -1  A(,t)CalculateE i ( ) = C -1  A(,t) Minimize{  A(,t) – C(E-  0 )} using K matrixMinimize{  A(,t) – C(E-  0 )} using K matrix PL red  : T<>T or T-T PL red  : T<>T PL sq : T<>T  PL sq : T-T  k eT k rec k repair k rad A unidirectional sequential model:

Pl - red +(TTT<>TT) The broadband transient absorption data: Pl - red +(TTTTT)

Spectrotemporal intermediates in the repair reaction: E spectra Fitting the data does not rule out a sequential bond breaking mechanism... More complicated kinetics cannot be ruled out! More complicated kinetics cannot be ruled out! PL SQ PL red  : T<>T or T-T PL red  : T<>T PL sq : T<>T  PL sq : T-T  53 ps 2753 ps 540 ps 620 ps

In conclusion… Quenching is a simple term for many possible mechanisms to shunt electronic energy in excited molecules Photoinduced Electron Transfer (PET) Fluorescence D*A h DA Bright Dark Bright or Dark A battery of approaches need to be used to explore all possible pathways

The C harge S eparation I nvestigation Team Goutham Kodali Stark spectroscopy Computational chemistry “Vector dude” Salim Siddiqui, M.D., Ph.D. Stark spectroscopy Computational chemistry Dr. Zhanjia Hou Ultrafast spectroscopy Single molecule spectroscopy Madhavan Narayanan Ultrafast spectroscopy Protein Chemistry Dr. Alex MacFarlane IV Ultrafast spectroscopy Electric field effects

The Group Collaborators Prof. Aziz Sancar (UNC) Mary Hawkins (NIH) Prof. Spiridoula Matsika Funding NSF Molecular Biosciences, REU Petroleum Research Fund Gone, but not forgotten..

A closer look at the damage… 5’-GCTTAATTCG-3’ 3’-CGAATTAAGC-5’ Crystal structure: Park et al, PNAS 99, (2002). 5’ 3’ AAAA Base stacking is weakened Watson-Crick base pairing is distorted 2.4Å 1.9Å

DNA Photolyase (PL) binds its CPD substrate by base flipping Mees, A., et al (2004) Science 306, Flavin Adenine Dinucleotide CPD

Spectral overlaps of probes and FAD Does FRET explain the intensity pattern difference? S0S2S0S2 S0S1S0S1