Browsing by Subject "ESEEM"
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Item Spectroscopic and Kinetic Investigation of the Catalytic Mechanism of Tyrosine Hydroxylase(2011-02-22) Eser, Bekir EnginTyrosine Hydroxylase (TyrH) is a pterin-dependent mononuclear non-heme iron oxygenase. TyrH catalyzes the hydroxylation reaction of tyrosine to dihydroxyphenylalanine (DOPA). This reaction is the first and the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. The active site iron in TyrH is coordinated by the common facial triad motif, 2-His-1-Glu. A combination of kinetic and spectroscopic techniques was applied in order to obtain insight into the catalytic mechanism of this physiologically important enzyme. Analysis of the TyrH reaction by rapid freeze-quench Mossbauer spectroscopy allowed the first direct characterization of an Fe(IV) intermediate in a mononuclear nonheme enzyme catalyzing aromatic hydroxylation. Further rapid kinetic studies established the kinetic competency of this intermediate to be the long-postulated hydroxylating species, Fe(IV)O. Spectroscopic investigations of wild-type (WT) and mutant TyrH complexes using magnetic circular dichroism (MCD) and X-ray absorption spectroscopy (XAS) showed that the active site iron is 6-coordinate in the resting form of the enzyme and that binding of either tyrosine or 6MPH4 alone does not change the coordination. However, when both tyrosine and 6MPH4 are bound, the active site becomes 5-coordinate, creating an open site for reaction with O2. Investigation of the kinetics of oxygen reactivity of TyrH complexes in the absence and presence of tyrosine and/or 6MPH4 indicated that there is a significant enhancement in reactivity in the 5-coordinate complex in comparison to the 6-coordinate form. Similar investigations with E332A TyrH showed that Glu332 residue plays a role in directing the protonation of the bridged complex that forms prior to the formation of Fe(IV)O. Rapid chemical quench analyses of DOPA formation showed a burst of product formation, suggesting a slow product release step. Steady-state viscosity experiments established a diffusional step as being significantly rate-limiting. Further studies with stopped-flow spectroscopy indicated that the rate of TyrH reaction is determined by a combination of a number of physical and chemical steps. Investigation of the NO complexes of TyrH by means of optical absorption, electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) techniques revealed the relative positions of the substrate and cofactor with respect to NO, an O2 mimic, and provided further insight into how the active site is tuned for catalytic reactivity upon substrate and cofactor binding.Item Spectroscopic investigation of metal-RNA interactions(Texas A&M University, 2005-02-17) Vogt, Matthew JohnMetal-RNA interactions are important to neutralize the negative charge and aid in correctly folding the RNA. Spectroscopically active metal ions, especially Mn2+, have been used to probe the type of interaction the metal has with RNA. In previous studies, the hammerhead ribozyme, an RNA motif that catalyzes a site-specific phosphodiester bond cleavage reaction, was determined by room temperature EPR (electron paramagnetic resonance) studies to have a set of tightly and weakly bound metal ions. Under high salt concentrations, the hammerhead was found to bind a single Mn2+ ion with high affinity and with a characteristic low temperature EPR signal. Using site specific 15N labeling of a guanine residue in conjunction with ESEEM (electron spin echo envelope modulation) spectroscopy, the high affinity Mn2+ ion was conclusively determined coordinated to G10.1 of the proposed A9/G10.1 site with four water molecules coordinated to the Mn2+ ion. EPR power saturation studies determined that under low salt conditions the hammerhead coordinates up to four Mn2+ ions in relatively close proximity compared to an RNA duplex. EXAFS (extended X-ray absorption fine structure) spectroscopy was used to determine that a Cd2+ ion coordinates to both the Rp and Sp sulfur atoms of a phosphorothioate modification at the A9 phosphate of the hammerhead. Previous EXAFS results for the Mn2+ substituted A9 phosphorothioate suggested that the Mn2+ ion coordinates to the oxygen atom for both isomers. Molecular modeling suggested that the A9/G10.1 metal site will twist the phosphate group in order to accommodate this coordination. A Mn-GMP and Mn-phosphate model complexes were prepared and characterized by EXAFS to assign the origin of the features observed for the hammerhead sample. A series of RNA sequences with internal loops containing the sheared G-A metal ion binding motif showed greater thermal stabilization of the RNA structure in the presence of Mn2+ ions compared to sequences without the motif. The EPR binding isotherms also showed a set of moderately tight metal ion interaction while circular dichroism spectroscopy was used to investigate structural differences between the sequences. These results suggest a mostly electrostatic, not structural role, for the Mn2+ ion interactions with these sequences.