In the past budget year, the research that has been supported by the ACS PRF Award has led to several important new findings concerning the electron transfer processes in DNA photolyase.  Although continuing problems with the picosecond laser setup prevented work on some of the project aims, alternative but strongly related research was conducted to advance the knowledge of the photoreduction and charge recombination electron-transfer processes in DNA photolyase. 

The permanent dipole moment of the CPD modifies electron transfer in photolyase.  In this project, the potential role of the substrate in modifying the electron-transfer in DNA photolyase was explored.  Two different substrates were analyzed; one with a thymine cyclobutane dimer (T<>T), and one with a cytidine cyclobutane dimer (C<>C).  The charge recombination process is slowed by a factor of 1.4 in the presence of the T<>T-substrate, while it is accelerated by a factor of 1.4 in the presence of the C<>C-substrate.  Calculations were performed to predict the strength and orientation of the permanent dipole moment of each substrate.  The differences in the permanent dipole moments of the substrate are very likely the cause of the differences in the charge recombination rates by establishing different electric fields along the electron transfer pathway.  This conclusion is supported by the finding that the FADH•/FADH^– reduction potential of the FAD-cofactor for the two enzyme-substrate complexes is the same within the margin of error.  Therefore, we conclude that substrate binding but not the substrate dipole moment modifies the FADH•/FADH^– reduction potential and that the substrate dipole moment is largely responsible for changing the charge recombination rates.  The only unknown is the exact binding geometry of the C<>C substrate, which may be different from that of the T<>T substrate and could potentially affect the strength of the electric dipole field near the FAD cofactor.  This strongly suggests that the substrate electric dipole moment will also affect the forward electron transfer in the photoreduction process as well as in the DNA-repair step.  A manuscript is in preparation for submission to the Journal of Physical Chemistry B that will present these and other findings concerning the effect of the substrate dipole moment on the electron transfer properties of photolyase.

Identification of the flavin vibrational normal modes that have contributions from the C8-methyl group.  In photolyase and in other electron-transfer flavoproteins, the C(8)-methyl group of the flavin cofactor is aligned in such a way that it can be part of the electron-transfer pathway in these proteins.  In order to understand whether there are specific interactions between the C(8)-methyl group and the surrounding protein and/or substrate molecule that would facilitate the electron-transfer, it is important to have a spectroscopic method to monitor the C(8)-methyl group.  Therefore, flavin mononucleotide (FMN) was prepared with its C(8)-methyl group deuterated.  The N5-methyl radical of FMN was synthesized to obtain the same information for this redox state of the molecule.  Resonance Raman spectra were collected for these two molecule with and without their C(8)-methyl group deuterated in H₂O and in D₂O.  The results identified several high-frequency vibrations that are sensitive to the C(8)-methyl group with H/D-isotope shifts of 4 to 8 cm^-1 for both molecules.  In both cases, several low-frequency vibrations were observed that showed large H/D-isotope shifts (15 to 30 cm^-1) for the C(8)-methyl group.  Density functional theory (DFT) calculations were performed on FMN to predict the H/D-isotope shifts.  There was very strong agreement between the experimentally observed and computationally predicted isotope shifts, which provided insight into the specific contribution of the C(8)-methyl group to the isotope-sensitive vibrational normal modes.  The results from this project have provided spectroscopic fingerprints of the C8-methyl group of the flavin molecule that can now be used to look for changes in the frequency of these normal modes in electron-transfer flavoproteins and, potentially, investigate the role of this methyl-group in the electron transfer process.  A manuscript describing these findings is being finalized for submission to the Journal of Physical Chemistry A.

In summary, the research has provided new information about the effect of the substrate electric dipole moment on the electron transfer processes in DNA photolyase and determined fingerprint vibrations for the C8-methyl group of the flavin molecule.  Since preliminary results on the tryptophan radical research sponsored by PRF were very promising, this work is expected to be completed in the near future.  The PRF sponsored research has provided (partial) funding for two graduate students, who are currently preparing their dissertations.  Their dissertations will include their research that was sponsored by PRF.