The Cryptic Nature of Fe-S Clusters: A Case Study of the Hepatitis B HBx Oncoprotein

Fe-S clusters are ubiquitous inorganic cofactors found in proteins across all domains of life, including viruses. Their prevalence stems from their unique redox and structural plasticity that supports functions ranging from electron transfer and catalysis to stabilization of protein structure. Although the ability of Fe-S clusters to exchange electrons is often functionally crucial, it can also act as an Achilles heel when these cofactors are exposed to oxidizing conditions, often leading to their degradation. This O₂ sensitivity has rendered certain Fe-S clusters untraceable, particularly when the nascent proteins are isolated under ambient conditions. As a consequence of this O₂ sensitivity, a growing number of proteins with roles in viral infection have been found to harbor Fe-S clusters rather than the annotated Zn²⁺ cofactor. The enigmatic protein X (HBx) of the Hepatitis B Virus is a multifunctional protein essential for viral replication and development of liver disease. Although HBx has defied biochemical characterization for over forty years, it has been shown to coordinate a redox-active Fe-S cluster that represents a significant feature for establishing its molecular function. The present review narrates the approaches to validate the HBx metallocofactor that can be broadly applied as a guide for uncovering the presence of Fe-S clusters in proteins with non-canonical sequence motifs.