Date of Award

2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Matthew D. Liptak

Second Advisor

Scott W. Morrical

Abstract

Metal tetrapyrroles represent a large class of earth-abundant catalysts but are limited to naturally-occurring combinations. Chelatase enzymes are responsible for the catalyzed metal insertion into a specific tetrapyrrole. CfbA is a class II chelatase from Archaeaglobis

fulgidus that catalyzes the insertion of nickel into sirohydrochlorin to give rise to Coenzyme F430 required for methanogensis. This archaeal chelatase was used to study the substrate scope of divalent metals and tetrapyrroles to probe various metal tetrapyrrole

combinations. A spectroscopic study established that the CfbA is capable of binding to cobalt and nickel, in addition to various tetrapyrroles. Magnetic circular dichroism (MCD) established that the metal binding site of CfbA contains a labile high spin 6-coordinate cobalt species that is ligated to nitrogen- or oxygen- atoms. The two residues involved in metal binding is likely His10 and His74. Tetrapyrrole binding resulted in a shift in energy. Computational studies have shown that resultant red-shift in energy is due to slight ruffling upon binding to CfbA. The enzymatic capabilities of CfbA was probed with various metal and tetrapyrrole combinations. The rate of insertion was significantly impacted by identity of the metal and the position of the propionate and acetate side chains on the rings of the tetrapyrrole as compared to sirohydrochlorin. Modifications to these side chains resulted

in changes in ruffling. An increase in the ruffling resulted in a decrease in the rate of the reaction. These results have shown a significant expansion of the tetrapyrrole substrate scope. Additionally, detailed insights into the proposed chelatase mechanism have been established.

IsdG serves as the primary enzyme involved in iron acquisition from heme in Staphylococcus aureus. The active site contains a tryptophan residue at 67 that is expected to be involved in heme ruffling. Trp67 was substituted with a smaller amino acid, phenylalanine to determine the role it plays in heme ruffling and degradation. The optical spectroscopic characterization of W67F IsdG resulted in changes to the geometric and electronic structure. The absorbance spectrum of W67F blue-shifted in the Q- and Soret bands indicating a change in the heme ruffling. MCD, VTVH and 1H NMR spectroscopy have shown that the electronic ground state is indicative of a 2Eg state, consistent with reduced heme ruffling. The degradation of heme by W67F IsdG resulted in the formation of biliverdin, a product seen in canonical HOs. These data suggest that Trp67 significantly influences heme ruffling and degradation. Additionally, W67F IsdG follows a unique reaction mechanism compared to IsdG. These data provide information on the development of a selective inhibitor of IsdG to prevent pathogenesis.

Language

en

Number of Pages

314 p.

Available for download on Thursday, September 03, 2020

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