Date of Completion


Document Type

Honors College Thesis



Thesis Type

Honors College, College of Arts and Science Honors

First Advisor

Matthew Liptak


chelatases, vitamin B12, coenzyme F430, spectroscopic characterization, mutagenesis


Class II chelatases are enzymes that selectively insert divalent transition metals into tetrapyrroles, which are precursors to a group of biological cofactors that include heme, chlorophyll, vitamin B­12, and coenzyme F430. Although these metalated tetrapyrroles are essential to many of life’s biological processes, the origin of the selective metal insertion in their biosynthesis has yet to be identified. Determining the active site structure for two class II chelatases (Archaeoglobus fulgidus CbiXS and Methanosarcina acetivorans CfbA) will help to determine the origin of selective metal insertion. A combination of site-directed mutagenesis and spectral characterization with electronic absorption spectroscopy and magnetic circular dichroism (MCD) spectroscopy has revealed the essential metal binding residues in the active site of A. fulgidus CbiXS to most likely be His10 and His74. To gain insight into the active site of M. acetivorans CfbA, MCD spectroscopy was used to reveal that cobalt (II) bound to WT CfbA was an octahedral, high spin species, ligated by nitrogen and/or oxygen ligands. These ligands are hypothesized to arise from the histidine residues in the active site of M. acetivorans CfbA (His12 and His78) and an aqueous buffer environment. These data indicate that ligands involved in first sphere interactions with the metal ion are identical between the two proteins. It can be initially hypothesized based on the spectroscopic characterization of the active sites of these two chelatases that the differing metal selectivity does not arise from first sphere interactions with the primary binding residues and is likely to arise from another means.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

Available for download on Wednesday, December 11, 2024