| The work described in this thesis focuses on three important flavoproteins: thioredoxin reductase, AidB, and RebC/StaC. These proteins have each been investigated with the goal of understanding the redox properties of the flavin cofactor found in each enzyme.;Chapter 2 focuses on the purification and characterization of a thioredoxin (Trx) and thioredoxin reductase (TrxR) from the archaeon Thermoplasma acidophilum (ta). We establish that while taTrxR does reduce taTrx, it does not do so in a NADPH-dependent manner because the "NADPH binding site" is not conserved. We conclude that taTrxR utilizes a novel reductant in the Trx/TrxR system.;In Chapter 3, we use protein film voltammetry to interrogate the complex redox chemistry of taTrxR in comparison with canonical homolog from E. coli. A reversible electrochemical response is observed that reveals a dynamic behavior dependent upon the temperature of the experiment. The data are interpreted in terms of a model incorporating conformational change where the flavin and disulfide are close in redox potential.;Chapter 4 describes protein film voltammetry of taTrxR modified to lack either the disulfide bond or flavin. We isolated contributions from the disulfide bond and flavin cofactor, and observed redox responses for each cofactor in agreement with data presented in Chapter 3.;In Chapter 5, we examine whether flavin plays a structural role in AidB, an adaptive response protein that currently has unknown function. FAD was removed from AidB, and structural analysis showed flavin binding to AidB induces a structural transformation from the dimer state to tetramer.;Chapter 6 describes the first characterization of AidB redox properties, where we report the two one-electron reduction potentials for flavin. Our results suggest AidB may retain certain characteristics of the acyl-CoA dehydrogenase family of enzymes, and is capable of redox-chemistry within the physiologically relevant window of reduction potential.;Chapter 7 discusses RebC and StaC, analogous flavoenzymes involved in a similar biosynthetic step of two natural products, staurosporine and rebeccamycin. We find that RebC has properties similar to other flavoprotein monooxygenases, and in this group of enzymes, FAD binding affinity is related to the level of substrate oxidation without modulating flavin redox potentials. |
