Date of Award

5-2011

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

Johannes P. M. Schelvis

Committee Member

Marc L. Kasner

Committee Member

Saliya de Silva

Abstract

Proton-coupled electron-transfer (PCET) is a mechanism of great importance in protein electron transfer and enzyme catalysis, and the involvement of aromatic amino acids in this process is of much interest. The DNA repair enzyme photolyase provides a natural system that allows for the study of PCET using a neutral radical tryptophan (Trp‘). In Escherichia coli photolyase, photoreduction of the flavin adenine dinucleotide (FAD) cofactor in its neutral radical semiquinone form (FADH') results in the formation of FADH and Trp . Charge recombination between these two intermediates requires O A/T # the uptake of a proton by ~ Trp . The rate constant of charge recombination has been measured as a function of temperature in the pH range from 5.5 to 10.0, and the data are analyzed with both classical Marcus and semi-classical Hopfield electron transfer theory. The reorganization energy associated with the charge recombination process shows a pH dependence ranging from 2.3 eV at pH <7 and 1.2 eV at pH(D) 10.0. These findings indicate that at least two mechanisms are involved in the charge recombination reaction. Global analysis of the data supports the hypothesis that PCET during charge recombination can follow two different mechanisms with an apparent switch around pH 6.5. At lower pH, concerted electron proton transfer (CEPT) is the favorable mechanism with a reorganization energy of 2.1 to 2.3 eV. At higher pH, a sequential mechanism becomes dominant with rate-limiting electron-transfer followed by proton uptake which has a reorganization energy of 1.0 to 1.3 eV. The observed 'inverse' deuterium isotope effect at pH < 8 can be explained by a solvent isotope effect that affects the free energy change of the reaction and masks the normal, mass-related kinetic isotope effect that is expected for a CEPT mechanism. To the best of our knowledge, this is the first time that a switch in PCET mechanism has been observed in a protein.

The charge recombination reaction between FADTT and Trp* is also analyzed in Vibrio cholera cryptochrome 1 (VcCry-1), a flavoprotein similar to E. coli photolyase, but with limited DNA repair ability. Several methods for accumulating FADH’ are investigated due to the initially low radical concentration in VcCry-1. A significant effect of potassium ferricyanide on the rate of the charge recombination reaction in VcCry-1 and E. coli photolyase is observed. It is shown that the reaction rate constant increases significantly with an increase in potassium ferricyanide concentration. Transient absorption measurements reveal that the rate of the charge recombination reaction in VcCry-1 is slightly slower than in E. coli photolyase at pFI 7.0.

Photoreduction of FAD and photodecomposition of 5,10- methenyltetrahydrofolate polyglutamate (MTHF) are examined in E. coli photolyase and VcCry-1 with comparison to the published results on Arabidopsis thaliana cryptochrome- 3. Additional information on the MTHF cofactor in VcCry-1 is obtained. Alterations are made to the way the photoreduction and photodecomposition processes are carried out; such as the exposure time to different wavelengths of light, and chemicals used. The results demonstrate that the photoreduction and photodecomposition are much slower processes in VcCry-1 than in E. coli photolyase. The similarity in the behavior of AtCry3 and VcCry-1 compared to E. coli photolyase indicates similarity between these plant and bacterial members of the cryDASH family. Our study shows that the concentration of reducing agent matter, in the photoreduction and photodecomposition processes, and that FADH" may play a crucial, intermediary role.

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