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

Abstract

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.

Keywords: EPR spectroscopy; Fe-S clusters; Hepatitis B; Mössbauer spectroscopy; UV/VIS.

Figure 1.

Characteristic UV/VIS spectra of [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters. The oxidized [2Fe-2S]²⁺ clusters are shown in green, and the one electron reduced forms in light blue. The spectra of the oxidized [3Fe-4S]¹⁺, [4Fe-4S]²⁺, and [4Fe-4S]³⁺ forms are shown in red; the spectra of their one-electron reduced forms are shown in yellow. The spectra have been adapted from references [–41].

Figure 2.

Characteristic X-Band CW EPR spectra of Fe-S clusters. (Top) The g₁–g₂ versus g_av plot is for the [2Fe-2S]¹⁺ clusters. The ligand sets of CCCC and CC(GSH)₂ are shown in green, CCCH and CC(GSH)H in orange, CCCR in pink, CCHH and C(GSH)HH in blue, and CCHE in purple circles. (Bottom) The ligand sets of CCCC are shown in green, CCCH in orange, CCCD in pink, CCCS in purple, CCC(H₂O) and CCC(citrate) in blue, and CCC(H₂O)-RS and CCC(citrate)-RS in grey circles. The spectra are simulations and compilations of the EPR parameters collected in Table 2 and the associated references with Table 2.

Figure 3.

Characteristic Mössbauer spectra and parameters of [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters in their different oxidized states. The overall fit is shown in black; the individual quadrupole doublets (subcomponents) are shown in the colors corresponding to the formal valence of the specific Fe site or Fe pair. The spectra are simulations of parameters that have been adapted from references [,–,,,,,,–83].

Figure 4.

Mössbauer spectra of MBP-HBx isolated from E. coli cells under different expression conditions. The spectra were recorded at 4.2 K and in the presence of a small external field (78 mT), applied parallel to the γ-beam. The experimental spectra are shown with vertical black bars, the fit for the [2Fe-2S]²⁺ clusters with a red-shaded quadrupole doublet and the fit for the [4Fe-4S]²⁺ clusters with a blue-shaded quadrupole doublet. The data have been adapted by reference [111].

Figure 5.

Whole-cell Mössbauer spectra of the cells expressing the MBP-HBx in T7 express cells in the presence and absence of IPTG. The experimental spectra are shown with vertical black bars, the fit for the [2Fe-2S]²⁺ clusters with a red shaded quadrupole doublet and the fit for the [4Fe-4S]²⁺ clusters with a blue shaded quadrupole doublet. The data have been adapted by reference [111].