Abstract
Iron, which is found in ~ 8% of enzymes, is at the forefront of almost all physiological and metabolic pathways in both humans and disease-causing pathogens. Fe cofactors are classified into three basic forms: heme, iron-sulfur (Fe-S) clusters, and diiron or mononuclear cofactors, all of which can serve as both structural and functional elements. Fe-containing proteins have central roles in biological redox reactions due to the ability of Fe to interconvert between ferrous (Fe2+) and ferric (Fe3+) forms, and their diverse functions in oxygen transport and utilization, respiration, and energy release are essential for the development and survival of all organisms. This thesis focuses on the characterization of two Fe-containing proteins i) the viral protein, HBx, which binds a novel Fe-S cluster, likely important for Hepatitis B virus (HBV) oncogenesis, and ii) the diiron protein, PhoF, which performs organophosphonate degradation in pathogenic fungi.Despite decades of research, the oncoprotein HBx has evaded biochemical characterization, likely due to the protein’s poor solution behavior, hindering our understanding of its pathogenic mechanisms. Previously, HBx was reported to possess nucleotide hydrolytic activity, providing a possible mechanism by which this protein regulates molecular pathways. The reinvestigation of this activity with new soluble HBx constructs outlined in Chapter 2 demonstrates that this activity stems from the copurifying chaperone, GroEL, rather than HBx, thus revealing that nucleotide hydrolysis is not a mechanism by with HBx contributes to viral pathogenesis.
Instead, HBx binds a previously unidentified redox active Fe-S cluster revealing new possibilities for structural and functional HBx research. Conservation of this cluster in HBx sequences across HBV genotypes, which is presented in Chapter 3, establishes this cofactor as a common feature of HBx and supports its role as a critical factor in HBx modes of action. Identification of the Fe-S ligands has proven technically challenging as the intrinsic disorder of HBx allows for structural rearrangement upon substitutions and heterologous cofactor binding in vitro; however, the findings presented in Chapter 3 identify C61, C69, C143, and C148 as likely Fe-S cluster ligands. The functional relevance of these cysteine residues relates cofactor binding to HBx functionality and provides one example of how pathogenic organisms such as viruses utilize Fe-containing proteins to hijack the host.
Chapter 4 describes the characterization of a second Fe-containing protein, PhoF, which is a member of the HD-domain family of metalloenzymes and coordinates both a mononuclear and dinuclear Fe cofactor. PhoF, which occurs strictly in pathogenic fungi, has herein been characterized as a chimera of the FeII/α-ketoglutarate-dependent hydrolase, PhnY, and the HD-domain oxygenase, PhnZ. This new example of a diiron HD-domain oxygenase represents the first oxidative organophosphonate degradation pathway in fungi and illustrates how domain fusion may be a strategy for these fungi to acquire increased functional versatility.