Date of Award
2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Matthew D. Liptak
Abstract
Heme proteins take advantage of an iron atom bound in a conjugated porphyrin scaffold to complete a vast array of chemistry including oxygen transport and catalysis. Human heme oxygenase (hHO) mitigates heme cytotoxicity via degradation to biliverdin through meso-hydroxyheme and verdoheme intermediates. In contrast to hHO, the non-canonical heme oxygenases (ncHOs) IsdI and IsdG are used by Staphylococcus aureus to degrade heme to staphylobilin through an alternative mechanism involving meso-hydroxyheme and formyloxobilin intermediates. For many years, enzyme induced structural and electronic perturbations have been implicated in novel ncHO reactivity. This difference in reactivity between hHO and ncHOs, along with the necessity of HO activity for bacterial proliferation, make ncHOs appealing antibiotic targets. The work discussed herein explores the influence of the IsdI active site on heme reactivity.
Like its homologue MhuD from M. tuberculosis, IsdI was found to host heme in a dynamic equilibrium between ruffled heme with a 2B2g electronic state and planar heme with a 2Eg electronic state. Furthermore, the equilibrium can be predictably shifted toward a 2Eg state through amino acid substitutions. Heme degradation activity was compared across WT, W66Y, and W66F IsdI with absorbance spectroscopy and Mass Spectrometry. Regioselectivity of heme monooxygenation to meso-hydroxyheme was unaffected by changes to ruffling and amino acid redox potential, and thus relies primarily on a conserved Asn residue and heme binding orientation. As opposed to native staphylobilin, W66Y and W66F IsdI variants formed biliverdin and formyloxobilin respectively as major products. This suggests highly hydrophobic WT and W66F IsdI active sites lead to more effective suppression of verdoheme formation. Though W66Y and W66F IsdI induce heme ruffling to roughly the same extent, the Tyr variant oxidized formyloxobilin to staphylobilin more efficiently indicating the presence of an ionizable amino acid in the active site is important for native IsdI oxygenation of meso-hydroxyheme.
Electronic characterization of cytochrome (cyt) P460, a heme protein involved in nitrogen-based metabolism, was also completed. The characteristic lysine cross-link was determined to promote population of a high-spin S = 5/2 configuration. Interestingly, variant P460 was found to host a dynamic heme, which is the first such case outside of ncHO enzymes. On pathway species for the proposed catalytic cycle were also examined. Fe(II) P460 heme was determined to exist as a mixture of S = 0 and S = 2 spin-states while {FeNO}6 P460 heme iron was revealed to have S = 2. This is the first {FeNO}6 complex found to have a high-spin configuration.
Language
en
Number of Pages
168 p.
Recommended Citation
Kocian, Taylor Ann Jeannette, "A Study of the Structural and Electronic Influences of Non-canonical Heme Oxygenase Enzymes on Heme Reactivity" (2025). Graduate College Dissertations and Theses. 2141.
https://scholarworks.uvm.edu/graddis/2141