How the fast (femtosecond-picosecond, fs-ps) protein dynamics contribute to enzymatic function has gained popularity in modern enzymology. With multiple experimental and theoretical studies developed, the most challenging part is to assess both the chemical step kinetics and the relevant motions at the transition state (TS) on the fast time scale. Formate dehydrogenase (FDH), which catalyzes a single hydride transfer reaction, is a model system to address this specific issue. I have crystallized and solved the structure of FDH from Candida boidinii (CbFDH) in complex with NAD+ and azide. With the guidance of the structure information, two active site residues were identified, V123 and I175, which could be responsible for the narrow donor-acceptor-distance (DAD) distribution observed in the wild type CbFDH. This thesis describes studies using kinetic isotope effects (KIEs) and their temperature dependence together with two-dimensional infrared spectroscopy on the recombinant CbFDH and its V123 and I175 mutants. Those mutants were designed to systematically reduce the size of their side chain (I175V, I175A, V123A, V123G and double mutant I175V/V123A), leading to broader distribution of DADs. The kinetic experiments identified a correlation between the DAD distribution and the intrinsic KIEs. The contribution of the fs-ps dynamics was examined via two-dimensional infrared spectroscopy (2D IR) by measuring the vibrational relaxation of TS analog inhibitor, aizde, reflecting the TS environmental motions. Our results provide a test of models for the kinetics of the enzyme-catalyzed reaction that invokes motions of the enzyme at the fs-ps time scale to explain the temperature dependence of intrinsic KIEs.

Proton-transfer reactions are one of the most fundamental chemical reactions. However, the chemical dynamics of these processes remain elusive due to the difficulty of modeling these reactions. Establishing an experimental model system and using infrared absorption and two-dimensional infrared (2D IR) spectroscopies as means for detection, the chemical dynamics of the protonation states that are involved in a ground-electronic-state proton-transfer reaction in solution can be determined. In this study, experimental models are established with formic acid and nitrogenous bases in a low dielectric solvent. A hydrogen bond forms between the acid and the base, which will allow for the proton to transfer between the two molecules to form the neutral and the ion-pair protonation states. The carbon-deuterium (C-D) stretch and the carbonyl (C=O) stretch of the formic acid molecule are used as the reporter groups for the 2D IR measurements. The results of the C-D stretch demonstrate that there is a high sensitivity to the deprotonation, vibrational coupling, and vibrational dynamics trends that are linked to the solute-solvent interactions. The results of the C=O stretch demonstrate a sensitivity to the deprotonation and conformational disorder in which the position of the C=O changes the dynamics of the protonation state. Although, a proton-transfer is not detected, the experimental model system provides an understanding of the features that govern the chemical dynamics of proton-transfer reactions.

Electron transfer is one of the fundamental process occurring in many chemical reactions. Electron transfer process has been under intensive study for many applications, for example artificial photosynthesis, where electrons from photo-excited chromophore molecules are harnessed to produce solar fuels in various forms. Transition metal complexes, such as ruthenium and rhenium complexes, play an important role in the continuing development of artificial photosynthetic devices. The electron transfer process in chromophores involving transition metal complexes often occurs on an ultrafast time scale from sub-ps to ns. To resolve such dynamics, ultrafast spectroscopic techniques are required. A variety of ultrafast techniques, such as time-resolved infrared spectroscopy and multi-pulse transient absorption spectroscopy, were used in this study to unravel the excited state electron transfer dynamics in a series of Re(I) complexes. Transition metal complexes often feature excited states that involve only partial electron transfer between the electron donating and accepting ligands, even for ligands with strong electron donating and accepting properties. It is often difficult to design a compact complex feature a full electron transfer excited state. Therefore, part of the work presented in this thesis was dedicated to the study of the electron transfer extent in the excited states of a series of [Re(N,N)(CO)3L]+ compounds, where N,N stands for electron accepting and L stands for electron donating ligands. By carefully designing the structure and redox properties of both the electron donor and acceptor, we demonstrated that essentially a full-electron charge transfer excited state can be prepared, while the designed Re(I) complex is still compact. To further extend the understanding of the electron transfer in transition metal complexes, modulation of the electron transfer rate in a compact Re(I) complex was studied. By perturbing the electron transfer process with a femtosecond mid-IR pulse, we showed that a 28% increase of the electron transfer rate was achieved. This study demonstrated the possibility of using a small energy mid-IR quanta to change the energy conversion process in a chromophore. Vibrational energy transfer in molecules is another important process in nature. Detailed understanding of the vibrational energy transfer on a molecule level is fundamentally important and essential for the development of molecular optical devices. It was recently discovered that the transport of vibrational energy in molecules can be fast and efficient due to its ballistic character. To understand the mechanism of the ballistic energy transport, experiments with several series of oligomers were performed using a relaxation-assisted two-dimensional infrared method. The energy transport speed was found to be dependent on transport initiation method and the transport pathways for different cases of initiation were identified. Detailed analysis on the chain band structure, group velocity and vibrational relaxation dynamics is presented. / acase@tulane.edu

Made available in DSpace on 2014-06-11T19:22:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-17Bitstream added on 2014-06-13T20:08:00Z : No. of bitstreams: 1 goncalves_er_me_sjrp_parcial.pdf: 124608 bytes, checksum: 0a4bcc296ede029511807eb42a3bd883 (MD5) Bitstreams deleted on 2014-08-22T14:57:05Z: goncalves_er_me_sjrp_parcial.pdf,Bitstream added on 2014-08-22T15:02:08Z : No. of bitstreams: 1 000607885.pdf: 2203958 bytes, checksum: 2ad68620c13ca42941cc766689b448fd (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Este trabalho apresenta um estudo do processo de desenovelamento da papaína via temperatura. Para tanto, foi utilizada a técnica espectroscópica de fluorescência, em conjunto com a espectroscopia de correlação bidimensional, com as análises sample-sample e variável- variável aplicadas à região do infravermelho médio. Desta forma, foi possível determinar três temperaturas de pré-transição: 34, 54 e 61ºC; sendo que a temperatura de 54ºC foi evidenciada tanto por fluorescência quanto pela análise sample-sample. Já as temperaturas de 34 e 61ºC foram evidenciadas somente na análise sample-sample. Além disso, a análise variável-variável descreveu a dinâmica conformacional durante o processo de desenovelamento. Assim, pode-se relacionar a temperatura e a dinâmica conformacional. / This work presents a study of the unfolding of papain via temperature. In order to do so, the fluorescence spectroscopy technique was used along with two- dimensional correlation spectroscopy by sample-sample and variable-variable analyses applied to the medium infrared region. Thus, it was possible to determine the three papain thermal pre-transition temperatures, 34, 54 and 61º C. The 54º C one was obtained by fluorescence spectroscopy and sample-sample analyses; whereas the 34 and 61ºC, ones, were detected by sample-sample alone. Additionally, through variable-variable analysis it was possible to describe the conformational dynamics during the unfolding process. Therefore, the relationship between temperature and conformational dynamics could be matched.

Orientador: Marinônio Lopes Cornélio / Banca: Marcelo Andres Fossey / Banca: Hamilton Cabral / Resumo: Este trabalho apresenta um estudo do processo de desenovelamento da papaína via temperatura. Para tanto, foi utilizada a técnica espectroscópica de fluorescência, em conjunto com a espectroscopia de correlação bidimensional, com as análises sample-sample e variável- variável aplicadas à região do infravermelho médio. Desta forma, foi possível determinar três temperaturas de pré-transição: 34, 54 e 61ºC; sendo que a temperatura de 54ºC foi evidenciada tanto por fluorescência quanto pela análise sample-sample. Já as temperaturas de 34 e 61ºC foram evidenciadas somente na análise sample-sample. Além disso, a análise variável-variável descreveu a dinâmica conformacional durante o processo de desenovelamento. Assim, pode-se relacionar a temperatura e a dinâmica conformacional. / Abstract: This work presents a study of the unfolding of papain via temperature. In order to do so, the fluorescence spectroscopy technique was used along with two- dimensional correlation spectroscopy by sample-sample and variable-variable analyses applied to the medium infrared region. Thus, it was possible to determine the three papain thermal pre-transition temperatures, 34, 54 and 61º C. The 54º C one was obtained by fluorescence spectroscopy and sample-sample analyses; whereas the 34 and 61ºC, ones, were detected by sample-sample alone. Additionally, through variable-variable analysis it was possible to describe the conformational dynamics during the unfolding process. Therefore, the relationship between temperature and conformational dynamics could be matched. / Mestre