Measuring the reduction potential of taurine:-ketoglutarate dioxygenase and biochemical analysis of a thermophilic ortholog

ABSTRACTMEASURING THE REDUCTION POTENTIAL OF TAURINE:&alpha-KETOGLUTARATE DIOXYGENASE AND BIOCHEMICAL ANALYSIS OF A THERMOPHILIC ORTHOLOGByCeleste Arlene WarrellThis thesis seeks to determine the reduction potential, more specifically, the midpoint potential, of the archetypal non-heme iron/&alpha-ketoglutarate dioxygenase, TauD, and to characterize a putative TauD ortholog from a moderately thermophilic bacterium. The accomplishment of these aims will expand our understanding of the mechanism of action of TauD and other members of this important enzyme superfamily. Two methods were employed to investigate the midpoint potential of TauD. Thin-layer cyclic voltammetry, an electrochemical method, was unsuccessful in directly generating a value for the midpoint of TauD. Instead, I used ultraviolet-visible spectroscopy to monitor titrations with the redox active dye methylene green to calculate the midpoint potential of Fe(III)/Fe(II)-TauD (Em of 207 &plusmn 27 mV vs. the Ag/AgCl electrode). Binding of the co-substrate &alpha-ketoglutarate (0.5 mM) with and without the substrate taurine (0.5 mM) did not significantly shift this value (204 &plusmn 46 mV and 210 &plusmn 32 mV, respectively). Preliminary activity assays suggested that the recombinant Mycobacterium thermoresistibile TauD-like protein is not a taurine:á-ketoglutarate dioxygenase. Ongoing studies by others are now examining an alternate function of this recombinant M. thermoresistibileprotein as a sulfate-degrading enzyme.