The F(430) cofactor of methyl coenzyme M reductase: Ligand binding to the nickel and chemical modification of the tetrapyrrole substituents
Hamilton, Cristi Lynn
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Permalink
https://hdl.handle.net/2142/22068
Description
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
Issue Date
1990
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Analytical
Chemistry, Inorganic
Biophysics, General
Language
eng
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.
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