Date of Award

1-2009

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Chemistry and Biochemistry

Thesis Sponsor/Dissertation Chair/Project Chair

J. P. M. Schelvis

Committee Member

M. L. Kasner

Committee Member

D. W. Konas

Abstract

Vibrational spectroscopy of tryptophan can be used as a probe for local protein conformation and to study protein electron transfer. For the interpretation of the vibrational spectra, the proper assignment of the vibrational bands to normal modes is essential. We report the experimentally measured infrared and Raman spectra of tryptophan, indole and 3-methylindole as well as of several of their isotope substituted forms; tryptophan indole-d5 and -15N2, and indole 2-13C. The effect of the exchange of the indole NH to ND on the vibrational spectra of these molecules was also determined. The measurements were made by using Raman spectroscopy with laser excitation at 532 nm and FTIR spectroscopy in KBr pellets. The observed experimental vibrational frequencies and intensities are used to test theoretical predictions of specific isotope shifts of the vibrational frequencies of tryptophan as well as of 3-methylindole and indole. The application of this methodology to tryptophan radicals and to protein electron transfer is also discussed. Consequently, we report experimentally measured data from time-resolved absorption spectroscopy to monitor the rate at which electron transfer occurs from the reduced flavin to the tryptophan radical in DNA photolyase. We also investigate the temperature dependence of this electron transfer process to determine its reorganization energy. Determination of the reorganization energy will allow us to classify the electron transfer as a concerted or sequential proton-coupled electron-transfer. We report values of the reorganization energy of X = 2.08 eV and X = 0.34 eV. The former value puts forward a strong case for concerted electron-proton transfer in photolyase. However, the latter value is too small to be in the range of regular protein electron transfer but this possibility cannot be fully excluded.

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