Date of Award
2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Matthew D. Liptak
Abstract
For decades, there has been a broad interdisciplinary interest in adapting the metal-tetrapyrrole (MTP) archetype in ways that extend beyond the chemistry of life. Large-scale syntheses of such artificial MTPs have, however, presented challenges in sustainability due to the large volumes of waste metal and solvent generated by traditional synthetic approaches. One alternative that has been proposed to this is to instead leverage the biological origins of these compounds – i.e., strategic modification of the pathways already involved in biosynthesizing MTPs. These pathways are characterized by the particular challenge of inserting a unique metal into the tetrapyrrole center, catalyzed by enzymes known as chelatases; thus, it follows that any biochemical approaches to synthesizing novel MTPs must first conquer the intricacies that dictate substrate scope in chelatases.
The ``primordial'' chelatases that exist today among certain members of the archaea, such as the cobalt chelatase CbiXS and the nickel chelatase CfbA, are of particular interest to this endeavor. Having undergone minimal structural change over the eons, their broad reactivities make them excellent subjects for studying the substrate scopes of chelatases whilst simultaneously serving as prime structural foundations for the future engineering of de novo chelatase enzymes. With this in mind, we have investigated the reactivity of CbiXS across a panel of both metal and tetrapyrrole substrates. Through stoichiometrically controlled assays it was found that although CbiXS experiences product inhibition, the consistency in this inhibition across substrate pairs suggests that it is not a mechanism through which selectivity arises and is reflective of a broad substrate scope.
Further analysis of reaction kinetics revealed that CbiXS is also substrate-inhibited, and this was discovered to be occurring independently through both the metal ion and the tetrapyrrole substrate. Chromatographic studies also found that CbiXS is in fact tetrameric in vitro, providing the necessary context to determine that an allosteric mechanism was likely driving its anomalous kinetic behavior.
In addition to this, the nickel coordination environment of the closely related CfbA was characterized using magnetic circular dichroism. Energies of the observed d-d transitions were consistent with those from an octahedral ligand field, and exhibited temperature-dependent intensities indicating the presence of a spin-degenerate ground state. As such, it was determined that CfbA binds a high-spin, octahedral Ni(II). This in turn indicates that the Ni(II) is kinetically labile toward ligand exchange, providing valuable new mechanistic insights into CfbA as a chelatase.
Language
en
Number of Pages
300 p.
Recommended Citation
Lickey, Bruce, "Spectroscopic Investigations of the Primordial Chelatases CbiXS and CfbA" (2025). Graduate College Dissertations and Theses. 2143.
https://scholarworks.uvm.edu/graddis/2143