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Abstract

To fulfill its essential need for iron, the opportunistic pathogen Fusobacterium nucleatum expresses heme uptake/utilization machinery, encoded a single genetic locus (hmu). The operon includes HmuW, or anaerobilin synthase, which catalyzes the oxygen-independent decyclization of heme, a reaction that releases Fe2+ and produces a linearized tetrapyrrole called anaerobilin. Colocalized with hmuW is hmuF, which is annotated as a member of the flavodoxin protein superfamily. Chapter 2 of this thesis focuses on the elucidation of HmuF function. Like a typical flavodoxin, HmuF binds FMN, but unlike most family members, HmuF does not stabilize the FMN semiquinone oxidation state; instead, the cofactor cycles between oxidized and two-electron reduced hydroquinone state. Through anaerobic UV-visible spectrophotometric assays and high-resolution mass spectrometry, HmuF was shown to traffic heme to HmuW for degradation, and then catalyze multiple reductions of anaerobilin through hydride transfer from the reduced FMN hydroquinone. In this capacity, HmuF provides a protected pathway for F. nucleatum to catabolize heme. Chapter 3 investigates the role of the proximal heme iron ligand, H134, in HmuF function through the creation of the following variants (H143A, H134F, H134M, H134Y and H134C). All variants are able to bind and traffic heme to anaerobilin synthase and catalyze subsequent reduction of the linearized tetrapyrrole. These results reveal that direct protein-iron coordination is not essential for HmuF function. Chapter 4 reports on the use of 19F NMR spectroscopy and MD simulations to reveal the dynamic nature of the cap domain, and how the binding of heme restricts its conformational flexibility. Chapter 5 investigated the role of HutX and HutZ present in the analogous heme uptake/utilization (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 in Esherchia coli O157:H7 and F. nucleatum. We show that HutW from A. fischeri produces anaerobilin, but HutX and/or HutZ do not catalyze anaerobilin reduction. Instead, HutZ binds tightly to heme and prevents the cytotoxicity of labile heme and anaerobilin by sequestering the cofactor from HutW. These results suggest that the distinct chu, hmu, and hut operons functionally converged to protect the cell from anaerobilin accumulation and labile heme cytotoxicity.