The Biophysical and Spectroscopic Characterization of Redox-active Metallocofactors in a Viral Fe-S Protein and a Diiron HD-domain Phosphohydrolase
Creators: name: Ueda, Chie
Publisher: name: Brandeis University
Year: 2022
Subjects: Ap4A
diiron
fe-s cluster
HBx
metalloprotein
redox

description: It is estimated that around one-fourth of all proteins require metal ions to function. Metal ions can serve a multitude of functions and act as structural elements, catalysts, and electron donors or acceptors. Fe is the fourth most abundant element in the earth’s crust and is incorporated in proteins in the form of hemes, Fe-S clusters, or non-heme iron mononuclear or polynuclear sites. The ability of Fe to readily gain or lose electrons has made Fe-containing proteins especially important for (non)enzymatic electron transfer and for redox regulatory and/or sensory processes. Fe cofactors have essential functions in core biochemical pathways such as photosynthesis and oxidative phosphorylation, making them a requirement to almost all forms of life. On the other hand, Fe in excess catalyzes formation of reactive oxygen species that can damage cellular components, leading to cell death or carcinogenesis. Thus, the properties of Fe cofactors are often finely tuned to the protein’s cellular environment to disfavor such unwanted reactivity. This dissertation examines two redox-active Fe-containing proteins: the Fe-S cluster protein HBx from the Hepatitis B virus and the non-heme diiron HD-domain YqeK hydrolases from Bacillus halodurans and Clostridium acetobutylicum. Chapter 1 of this dissertation is an introduction to Mössbauer spectroscopy, which is a technique central to the study of the viral Fe-S protein HBx that is presented in Chapter 3. Chronic Hepatitis B virus infection currently affects around 350 million people globally and lacks effective therapeutics. HBx is a key player in liver diseases caused by chronic infection, making it a highly attractive drug target. HBx is a non-structural protein that has defied biochemical characterization for more than forty years. Although there is evidence for metal ions such as Fe and Zn to copurify with HBx, the structure, identity, and significance of these cofactors remain unidentified. In Chapter 3, I present a thorough spectroscopic study of a newfound redox-active Fe-S cluster in HBx. The HBx Fe-S cluster undergoes redox-dependent structural rearrangements that are reminiscent of those occurring in Fe-S cluster scaffold proteins, prompting the hypothesis that HBx may act as a rogue Fe-S cluster donor.
description: Chapter 4 of this dissertation describes the characterization of the HD-domain Ap4A hydrolase YqeK. Ap4A is a ubiquitous stress metabolite and second messenger that is involved in the response to environmental stress stimuli, such as oxidation, heat, or antibiotics. YqeK is a bacterial enzyme that can coordinate a redox-active diiron center with redox potentials that are tailored for the organism’s environment (i.e., aerobic vs anaerobic) or utilize a redox-independent heterodinuclear Fe-Zn cofactor, both of which may be strategies for YqeKs from aerobic bacteria to support activity under more oxidative conditions.