Biological Photochemistry:

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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
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Electronic excited states in Biology

• Chemiluminescence
• Bioluminescence charge transfer? radicals?
• Photoinduced electron transfer
• Photosynthesis
• DNA repair
• Photochemistry
• DNA damage
• photosensors

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DNAa polymer of nucleotides connected by
phosphodiester linkages
Nucleic acid bases A, T, C, G
Voet and Voet, Biochemistry, 2nd Ed. Wiley, New
York, 1995
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B-DNA is double-stranded (ds) DNA, forming the
famous double helix (1954 - Watson, Crick,
Franklin)
Watson-Crick base pairing (complementarity)
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DNA absorbs UV radiation
??? transition
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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
  (10-12 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 ? TltgtT pyrimidine dimerization
• Cancer, apoptosisBAD

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UV light damages DNA Bad photochemistry
22 photo-cycloaddition
lt 320 nm
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If DNA damage is left unrepaired then mutations,
cell death, and cancer can develop
http//toms.gsfc.nasa.gov/ery_uv/euv.html
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Pathways involving energy transfer
DA
D G, A, C, T
A G, A, C, T
h?D
DA
h?A
Triplet Energy Transfer
Förster or Dexter Transfer (singlets)
DA
Fluorescence
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Structural quenching pathways
DA
Bright Dark
DhotA
h?D
Intramolecular vibrational relaxation
Conical Intersection
DA
Fluorescence
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Pathways involving electron transfer
DA
h?D
h?EX?
Photoinduced Electron Transfer (PET)
Exciplex (EX) formation (charge transfer)
DA
Fluorescence
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Enzymatic Repair of CPDs by DNA Photolyase uses
blue light as an energy source (Good
photochemistry)

• Repair of the thymidines is direct
• TltgtT? T-T without modifying the DNA backbone
• Wide spread E. coli, Frogs, Rice,
  KangaroosHumans (no!)

• Possible Applications
• Photosomes (AGI Dermatics)
• transgenic crops (wheat?)

Mees, A., et al (2004) Science 306, 1789-1793.
Sancar, A. Structure and function of DNA
photolyase. Biochemistry 33, 2-9 (1994).
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DNA Photolyase (PL) is a flavoprotein (Vitamin
B2) that binds and repairs CPDs

• PL functions efficiently with only FAD (required
  for repair and binding

• PL binds the CPD with high affinity (no light
  required)
• KA 109 M-1 for dsDNA with CPD

Park, H.-W., Kim, S.-T., Sancar, A., and
Deisenhofer, J. (1995) Science 268, 1866-72.
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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 cell
• Surprisingly, flavins are a major biological
  chromophore (DNA repair, circadian rhythms,
  phototropism, etc.)

Biochemistry 2nd Ed., Voet and Voet, J. Wiley
Sons
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Photolyase functions by Photoinduced Electron
Transfer from the FAD to the CPD
Theres a cavity in the protein
FAD
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What happens to substrate conformation upon
binding to Photolyase?
Minor disruption
AA
Photolyase
Moderate disruption
Base Flipping
TltgtT
Severe disruption
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Fluorescent reporter approach to probing double
helical structure
5-probe approach
?
Base Flipping
3-probe approach
?
Base Flipping
The fluorescence quantum yield of the reporter
decreases when base stackedbut why?
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6MAP is an attractive new fluorescent adenosine
analogue
4-amino-6-methyl-8-(2-deoxy-?-D-ribofuranosyl)-7(8
H)-pteridone
Properties1 ?fl 0.2 ?ex 330 nm (? 8,500
M-1cm-1) ?em 430 nm (large Stokes
shift) 1Hawkins, et al, Synthesis and
Fluorescence Characterization of Pteridine
Adenosine Nucleoside Analogs for DNA
Incorporation. Anal. Biochem.298, 231-240 (2001).
K. Yang, S. Matsika, and R.J. Stanley,
Biochemistry 2007
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Base flipping of the CPD monitored by 6MAP
PL
-PL
5-GCAAGTTGGAG-3 3-CGTTCAFCCTC-5
PL
5-GCAAGTTGGAG-3 3-CGTTCFACCTC-5
-PL
Why is the intensity pattern sequence-dependent?
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These data are consistent with disruption of base
stacking due to base flipping of the CPD by
Photolyase
?
Mees et al, Science v. 306, 1789-1793 (2004)
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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, kq?
What are the redox potentials? Cyclic voltammetry
of 6MAP in aprotic organic solvents
submitted to Biochemistry
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The quenching of 6MAP proceeds through
nucleobase oxidation 6MAPNMP?6MAP?NMP?
FBA NB ?GET?(eV) Eact(eV)
6MAP G -0.63 0.000
6MAP A -0.16 0.003
6MAP C 0.021 0.048
6MAP dT -0.009 0.032
(Scandola-Balzani relation)
submitted to Biochemistry
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Whats 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 and MRCI calculations (Matsika)
Stark absorption and emission spectra of 6-MI
(?), a guanine analog, compared with their
absorption and emission spectra (?).
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Another possibility 6MAP emission overlaps the
absorption of the FAD FRET from 6MAP?FAD?
Yang et al, JPC B (2007)
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Fluorescence Energy Transfer Efficiency
R0 ? the Förster distance where ?ET 0.5
rDA ? the distance between a donor (fluorescent
analogue) and an acceptor (FAD in photolyase)
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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.
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The Overlap Integral

FD(?) the fluorescence intensity of the donor as
a function of wavelength. eA(?) the molar
extinction coefficient of the acceptor at that
wavelength
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The Orientation Factor
mD
mA
rDA

?T ? mD, mA ?D ? mD , rDA ?A ? mA, rDA
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The transition dipole moment direction 6MAP was
calculated from TD-DFT
Yang et al, JPC B (2007)
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Orientation factors and ?ET between Probes and
FADox
From the crystal structure, lit. and TDDFT calcs
experiment
crystal structure
Yang et al, JPC B (2007)
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FRET efficiency vs. orientation
Yang et al, JPC B (2007)
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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)
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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?

?c
MacFarlane and Stanley (2003) Biochemistry 42,
8558-8568
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Transient absorption measurement layout
BBO
CaF2
Sample
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PET to the CPD substrate quenches the FADH?
excited state in 30 ps
MacFarlane and Stanley (2003) Biochemistry 42,
8558-8568
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Whats are the intermediates?
A unidirectional sequential model

• ?A(?,t) ?ci(t)??i(?) C(E - ?0)
• where Ei(?) True spectra of the intermediates
• ?0(?) Ground state absorption spectrum
• Construct C(t) C0eKt (from the K matrix)
• Calculate Ei (?) C-1?A(?,t)
• Minimize ?A(?,t) C(E- ?0) using K matrix

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Pl-red(TTTltgtTT)
The broadband transient absorption data
Pl-red(TTTTT)
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Spectrotemporal intermediates in the repair
reaction E spectra
1PLred? TltgtT
53 ps
2
PLsq TltgtT ?
3
540 ps
620 ps
PLsq T-T ?
4
2753 ps
PLred ? TltgtT or T-T
1

• Fitting the data does not rule out a sequential
  bond breaking mechanism...
• More complicated kinetics cannot be ruled out!

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In conclusionQuenching is a simple term for
many possible mechanisms to shunt electronic
energy in excited molecules
A battery of approaches need to be used to
explore all possible pathways
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The Charge Separation Investigation Team

• Madhavan Narayanan
• Ultrafast spectroscopy
• Protein Chemistry

• Dr. Zhanjia Hou
• Ultrafast spectroscopy
• Single molecule spectroscopy

• Goutham Kodali
• Stark spectroscopy
• Computational chemistry
• Vector dude

• Dr. Alex MacFarlane IV
• Ultrafast spectroscopy
• Electric field effects

• Salim Siddiqui, M.D., Ph.D.
• Stark spectroscopy
• Computational chemistry

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The Group
Gone, but not forgotten..
Funding NSF Molecular Biosciences, REU Petroleum
Research Fund
Collaborators Prof. Aziz Sancar (UNC) Mary
Hawkins (NIH) Prof. Spiridoula Matsika
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A closer look at the damage 5-GCTTAATTCG-3 3-C
GAATTAAGC-5
5 3
A A
Crystal structure Park et al, PNAS 99,
15965-15970 (2002).
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DNA Photolyase (PL) binds its CPD substrate by
base flipping
CPD
Flavin Adenine Dinucleotide
Mees, A., et al (2004) Science 306, 1789-1793.
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Spectral overlaps of probes and FAD
S0?S2
S0?S1
Does FRET explain the intensity pattern
difference?