Dissertation Defence: Accessory Proteins Involved in Anaerobic Heme Catabolism

Alexandra McGregor, supervised by Dr. Kirsten Wolthers, will defend their dissertation titled “Accessory Proteins Involved in Anaerobic Heme Catabolism” in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry and Molecular Biology.

An abstract for Alexandra McGregor’s dissertation is included below.

Examinations are open to all members of the campus community as well as the general public. Registration is not required for in-person exams.

---

Abstract

To fulfill its essential need for iron, the opportunistic pathogen Fusobacterium nucleatum expresses heme uptake machinery encoded at a single genetic locus. The heme uptake operon includes HmuW, an enzyme capable of catalyzing the oxygen-independent decyclization of heme to release Fe2+ and a linearized tetrapyrrole called anaerobilin. Colocalized with hmuW is hmuF, which encodes for a member of the flavodoxin protein superfamily. Chapter 2 of this thesis outlines the discovery of the function of HmuF, which was found to bind tightly to a molecule of heme in addition to an FMN cofactor. In contrast to canonical flavodoxins, HmuF does not stabilize the semiquinone oxidation state, and instead cycles directly between FMNox and FMNhq. Analysis of the results of this chapter determines that the function of HmuF is to traffic heme to HmuW for degradation, and then catalyze multiple reductions of anaerobilin through hydride transfer from the reduced FMN hydroquinone, providing a protected pathway for F. nucleatum to catabolize heme. Chapter 3 further investigates the heme binding properties of HmuF by mutation of the proximal iron coordination residue His134. All variants are able to bind and traffic heme to anaerobilin synthase for decyclization and catalyze subsequent reduction of the linearized tetrapyrrole, indicating that direct protein-iron coordination is not essential for HmuF function. Chapter 4 presents evidence for the flexibility of the cap domain of HmuF in solution that allows it to complex with HmuW using MD simulations and 19F NMR. Chapter 5 looks at alternative heme degradation accessory proteins by comparing the hmu operon of F. nucleatum and the chu operon of E. coli 0157:H7 to the hut operon of Vibrionaceae. Members of this family encode for anaerobilin synthase (HutW), but do not contain homologs of the accessory proteins (ChuS, ChuY, and HmuF) that mitigate against the cytotoxicity of labile heme and anaerobilin. The results of this chapter show that the anaerobilin synthase HutW from A. fischeri is able to produce anaerobilin, but that the proteins encoded directly downstream of HutW, HutX and HutZ, do not catalyze anaerobilin reduction. Instead, HutZ binds tightly to heme and prevents the cytotoxicity of labile heme and anaerobilin by sequestering it from HutW. These results suggest that the distinct chu, hmu, and hut operons functionally converged to protect the cell from anaerobilin accumulation and the cytotoxicity of heme.