RE-EVALUATING AND DELINEATING A PROTEIN FAMILY: BIOCHEMICAL AND BIOPHYSICAL INVESTIGATIONS OF WRBA, FOUNDING MEMBER OF A NAD(P)H:QUINONE OXIDOREDUCTASE FAMILY

Open Access
Author:
Patridge, Eric Vincent
Graduate Program:
Integrative Biosciences
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
March 27, 2009
Committee Members:
  • James Gregory Ferry, Dissertation Advisor
  • James Gregory Ferry, Committee Chair
  • Joseph M Bollinger Jr., Committee Member
  • Ming Tien, Committee Member
  • Squire J Booker, Committee Member
Keywords:
  • WrbA
  • Tryptophan repressor binding protein
  • electrochemistry
  • flavodoxin
  • quinone oxidoreductase
  • flavin
  • electron transfer
  • NADH
  • NADPH
Abstract:
WrbA (tryptophan W repressor binding protein) was discovered in Escherichia coli where it was proposed to play a role in regulation of the tryptophan operon; however, this has been put into question leaving the function unknown. Chapter 3 of this dissertation solidifies and defines the WrbA protein family across all domains of life. Presented with a phylogeny are the first biochemical investigations of WrbA proteins from E. coli and Archaeoglobus fulgidus, a thermophilic species from the Archaea domain. This research is the first to demonstrate that WrbA proteins have NAD(P)H:Quinone Oxidoreductase (NQO) activity. Physiologically relevant kinetic parameters presented here implicate WrbA in two-electron reduction of quinones, protecting against oxidative stress. Throughout the experiments, wild-type WrbA proteins demonstrate monomer-dimer-tetramer equilibria with one FMN cofactor per monomer in the holo-tetramer. Chapter 4 examines multimeric flavodoxin-like NAD(P)H:Quinone Oxidoreductases (NQOs) with many similarities to WrbA proteins, and a phylogeny suggests that WrbA is evolutionarily related to these NQOs. They appear to function in redox-linked processes and protect against environmental stressors, and previous research indicates that reduced dimeric NQO protein is required to protect protein targets from proteasomal degradation. Chapter 5 is the first comprehensive biophysical investigation of any flavodoxin-like NQO protein and it employs E. coli WrbA as the model protein. The study focuses on peptide interactions with the WrbA flavin cofactor, and it incorporates biochemical and biophysical characterizations of wild-type proteins and alanine variants of E. coli WrbA. Fluorescence quenching experiments demonstrate that flavin binding is cooperative and linked to multimerization in E. coli WrbA; monomer WrbA does not bind flavin. Redox potentials were determined with a gold-capillary spectroelectrochemical cell that I constructed and modified from previous investigations. (Construction of the electrochemical cell apparatus is presented in Appendix A.) The experiments show H133 directs electron accumulation to the flavin isoalloxazine ring in WrbA. Specific activities indicate that WrbA proteins retain activity with smaller electron acceptors and that the residues investigated by alanine-scanning are important for enzyme activity. Altogether, this dissertation redefines the WrbA protein family, and it has potential to significantly affect research with other flavodoxin-like NAD(P)H:Quinone Oxidoreductases.