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 Title: The F(430) cofactor of methyl coenzyme M reductase: Ligand binding to the nickel and chemical modification of the tetrapyrrole substituents Author(s): Hamilton, Cristi Lynn Department / Program: Chemistry Discipline: Chemistry Degree Granting Institution: University of Illinois at Urbana-Champaign Degree: Ph.D. Genre: Dissertation Subject(s): Chemistry, Analytical Chemistry, Inorganic Biophysics, General Abstract: Chemical and spectroscopic studies on the nickel enzyme methyl-CoM reductase from M. thermoautotrophicum (strain $\Delta$H) were undertaken to better characterize the nickel site. The major goals of this work are two-fold: (1) To further characterize the molecular and electronic structure of the methyl-CoM reductase nickel cofactor F$\sb{430}$ as isolated and in the holoenzyme; (2) To probe the possible roles of the F$\sb{430}$ cofactor in the methyl-CoM reductase-catalyzed reduction of CH$\sb3$SCoM to CH$\sb4$. In addition, the electronic and magnetic properties of the nickel, and the iron-sulfur centers in the different redox states of methyl viologen-reducing hydrogenase from the same bacterium were investigated.The electronic and magnetic properties of methyl-CoM reductase, F$\sb{430}$ and various ligated forms at F$\sb{430}$ were measured using variable-temperature magnetic circular dichroism (MCD) spectroscopy. Low-temperature magnetization data allowed the determination of the axial zero-field splitting parameter, D, of the S = 1 ground state of enzyme-bound F$\sb{430}$ as well as bis-ligand complexes of the isolated cofactor. The values of D suggest oxygenic axial ligation to F$\sb{430}$ in the holoenzyme.To evaluate the role of F$\sb{430}$ in substrate binding, the affinity of native F$\sb{430}$, diepimeric F$\sb{430}$, and F$\sb{560}$ for different types of axial ligands and the conformational changes associated with axial ligation were investigated using uv/visible and circular dichroism spectroscopy. Differences in ligand binding affinities among the F$\sb{430}$ isomers can be explained by macrocyclic conformational differences.To solubilize F$\sb{430}$ in nonaqueous solvents, the five peripheral carboxylates have been amidated with a number of alkylamines using a carbodiimide coupling method. These amides should be useful in evaluating the possible role of F$\sb{430}$ as electron transfer agent and preliminary investigations are discussed.MCD and EPR studies on oxidized and H$\sb2$- and dithionite-reduced forms of the methyl viologen-reducing hydrogenase are presented. These studies provide the first direct spectroscopic evidence of the presence of S $>$ ${1\over2}$ (4Fe-4S) $\sp{1+}$ clusters in the reduced enzyme as well as suggest a spin interaction between these clusters and the nickel site. Issue Date: 1990 Type: Text Language: English URI: http://hdl.handle.net/2142/22068 Rights Information: Copyright 1990 Hamilton, Cristi Lynn Date Available in IDEALS: 2011-05-07 Identifier in Online Catalog: AAI9021693 OCLC Identifier: (UMI)AAI9021693
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