Graduation Semester and Year




Document Type


Degree Name

Doctor of Philosophy in Chemistry


Chemistry and Biochemistry

First Advisor

Brad Pierce


MiaE is a non-heme diiron enzyme which catalyzes the O2-dependent hydroxylation of selected tRNA-nucleotides as part of a multienzyme posttranscriptional hypermodification pathway. This tRNA-modifying enzyme is postulated to signal O2-availability within the pathogenic bacteria, Salmonella typhimurium. Recombinant MiaE was cloned from Salmonella typhimurium genomic DNA, purified to homogeneity, and characterized by steady-state kinetics and spectroscopic techniques (UV-visible, Circular Dichroism, dual mode electron paramagnetic resonance (EPR), and Mössbauer) for comparison to other non-heme diiron enzymes. Remarkably, regardless of the substrate used in peroxide-shunt assays, hydroxylation of the terminal isopentenyl-C4-position was observed with > 97% E-stereoselectivity. The role of tRNA-protein macromolecular interactions on enzymatic reactivity and diiron site conformation was investigated studied using 17 nucleotide RNA oligomer corresponding to the anticodon stem and loop (ACSL) portion of substrate tRNAs. Steady-state reactions utilizing ACSL-substrates were investigated using a recombinant electron transfer chain. A variety of spectroscopic methods were employed to complement kinetic assays and observed structural perturbations within the protein-RNA secondary structure and diiron active site geometry. The tRNA-induced spectroscopic perturbations are reminiscent to be observed in the hydroxylase component of other monooxygenase enzymes upon binding their corresponding effector-protein. Thus, substrate-enzyme interactions may play a regulatory role in tRNA-hydroxylation for MiaE. Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the O2-dependent oxidation of L-cysteine (L-Cys) to produce cysteine sulfinic acid (CSA). In contrast to mammalian CDO, all known bacterial CDO enzymes lack the Cys-Tyr post-translational modification. The relatively uncharacterized ‘Gln-type’ bacterial CDO enzymes offer a unique point of comparison to better understand the role of outer-sphere interactions in thiol dioxygense chemistry. In this work, the ‘Gln-type’ CDO enzyme was cloned from the soil bacteria Azotobacter vinelandii, purified to homogeneity, and characterized kinetically and spectroscopically for comparison to the Mm CDO enzyme. Remarkably, in steady-state assays using 3-mercaptopropionic acid (3-mpa), L-cysteine (cys), and cyteamine (ca), Av CDO exhibits nearly identical maximal velocity (kcat = v0/[E]) for each substrate (0.2 < kcat < 1.0 s-1). However, Av CDO exhibits a specificity (kcat/KM = 18,000 M-1s-1) for 3-mpa over two orders of magnitude higher than observed for either cys (120 M-1s-1) and ca (20 M-1s-1). This observations suggests that the ‘Gln-type’ Av CDO enzyme has been misannotated as a cysteine dioxygenase and is, in fact, a 3-mercaptopropionic acid dioxygenase (MPDO). Complementary X-band EPR experiments were performed on Av CDO using nitric oxide as a surrogate for O2-binding. As with most non-heme mononuclear iron oxidase/oxygenase enzymes, Av CDO exhibits an obligate ordered addition of substrate (3-mpa, cys, and ca) prior to NO. Previously published results demonstrate that the electronic structure of the substrate-bound iron-nitrosyl produced in Mm CDO (Mm ES-NO) exhibits an unusual {FeNO}7 (S = 1/2) electronic ground state. In stark contrast to this, the Av ES-NO exhibits a {FeNO}7 (S = 3/2) species with near axial magnetic symmetry (E/D = 0.009).


Chemistry | Physical Sciences and Mathematics


Degree granted by The University of Texas at Arlington

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