The histidine–aspartate (HD)-domain protein superfamily contains metalloproteins that share common structural features but catalyze vastly different reactions ranging from oxygenation to hydrolysis. This chemical diversion is afforded by (i) their ability to coordinate most biologically relevant transition metals in mono-, di-, and trinuclear configurations, (ii) sequence insertions or the addition of supernumerary ligands to their active sites, (iii) auxiliary substrate specificity residues vicinal to the catalytic site, (iv) additional protein domains that allosterically regulate their activities or have catalytic and sensory roles, and (v) their ability to work with protein partners. More than 500 structures of HD-domain proteins are available to date that lay out unique structural features which may be indicative of function. In this respect, we describe the three known classes of HD-domain proteins (hydrolases, oxygenases, and lyases) and identify their apparent traits with the aim to portray differences in the molecular details responsible for their functional divergence and reconcile existing notions that will help assign functions to yet-to-be characterized proteins. The present review collects data that exemplify how nature tinkers with the HD-domain scaffold to afford different chemistries and provides insight into the factors that can selectively modulate catalysis.

The viral protein HBx is the key regulatory factor of the hepatitis B virus (HBV) and the main etiology for HBV-associated liver diseases, such as cirrhosis and hepatocellular carcinoma. Historically, HBx has defied biochemical and structural characterization, deterring efforts to understand its molecular mechanisms. Here we show that soluble HBx fused to solubility tags copurifies with either a [2Fe-2S] or a [4Fe-4S] cluster, a feature that is shared among five HBV genotypes. We show that the O₂-stable [2Fe-2S] cluster form converts to an O₂-sensitive [4Fe-4S] state when reacted with chemical reductants, a transformation that is best described by a reductive coupling mechanism reminiscent of Fe-S cluster scaffold proteins. In addition, the Fe-S cluster conversions are partially reversible in successive reduction–oxidation cycles, with cluster loss mainly occurring during (re)oxidation. The considerably negative reduction potential of the [4Fe-4S]^2+/1+ couple (−520 mV) suggests that electron transfer may not be likely in the cell. Collectively, our findings identify HBx as an Fe-S protein with striking similarities to Fe-S scaffold proteins both in cluster type and reductive transformation. An Fe-S cluster in HBx offers new insights into its previously unknown molecular properties and sets the stage for deciphering the roles of HBx-associated iron (mis)regulation and reactive oxygen species in the context of liver tumorigenesis.

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.

Fe–S clusters are emerging as important cofactors in viral replication but are frequently misassigned as Zn due to their O2-sensitivity and overlapping cysteine-based coordination chemistry. The Hepatitis B virus regulatory protein HBx, which is essential for viral replication and hepatocarcinogenesis, has long remained mechanistically intractable because of uncertainty surrounding its physiologically relevant metallocofactor. Although HBx can bind either an Fe–S cluster or Zn, its intrinsic disorder and extensive mutational tolerance have hindered precise characterization of its metal-binding environment. Here, we combine chemoproteomics with HYSCORE spectroscopy to define the metal-coordinating ligands in HBx and overcome limitations associated with conventional mutational analysis of disordered proteins. We exclude histidine coordination and identify C61, C69, C143, and C148 as the primary cysteine ligands for Fe–S cluster binding, with C137 functioning as a conditional auxiliary ligand. These residues also support Zn binding and overlap with regions implicated in HBx transactivation and clinically relevant variants. In addition, HBx engages the host cytosolic Fe–S cluster assembly machinery and displays sensitivity to Fe–S-targeting reagents, behavior consistent with Fe–S cluster acquisition and cofactor lability. Together, these findings propose HBx as an Fe–S cluster-associated viral protein and expand the growing class of viral Fe–S proteins that are critical for infection.

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in biological systems. Given their central role in bacterial metabolism and pathogenesis, the biogenesis of Fe-S clusters is tightly controlled. We reveal a feedback regulatory mechanism involving the sulfide producing SufS/SufU complex within the sulfur utilization (SUF) system of Mycobacterium tuberculosis , the bacterium that causes tuberculosis. In this mechanism, [2Fe-2S] clusters compete with zinc ions for binding to the sulfide transfer protein SufU. Cluster binding induces SufU tetramerization, which prevents its interaction with the cysteine desulfurase SufS, thereby inhibiting SufS activation and limiting sulfide supply for Fe-S cluster biogenesis. These findings uncover an unrecognized regulatory mechanism in M. tuberculosis , ensuring strict control of Fe-S cluster production.