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Although HBx binds either an Fe-S cluster or Zn, its intrinsic disorder and mutational tolerance have hindered its precise characterization. Here, we integrate chemoproteomics with HYSCORE spectroscopy to identify the metal-coordinating ligands in HBx. Histidine coordination is excluded, while C61, C69, C143, and C148 emerge as primary cysteine ligands for the Fe-S cluster, with C137 acting as a conditional ligand. These residues also bind Zn and are associated with HBx transactivation and clinically relevant variants. HBx engages the host cytosolic Fe-S machinery and displays sensitivity to Fe-S-targeting reagents, behavior consistent with Fe-S cluster acquisition and lability. Together, these findings suggest that HBx functionally behaves as an Fe-S cluster-associated protein, highlighting a potentially druggable vulnerability in HBV replication.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>\n                    Fe-S clusters are emerging as key cofactors in viral replication but are often mistaken for Zn due to O\n                    <jats:sub>2<\/jats:sub>\n                    -sensitivity and shared cysteine coordination. The Hepatitis B virus HBx protein, essential for viral replication and hepatocarcinogenesis, has long been mechanistically intractable, with debate over its metallocofactor. Here, we provide evidence that HBx coordinates an Fe-S cluster, placing it within the growing family of viral Fe-S-cluster-containing proteins. Using chemoproteomics, we identify its cysteine ligands, overcoming limitations of mutational analysis in disordered proteins. Although HBx binds both an Fe-S cluster and Zn, its interaction with human Fe-S assembly factors suggests a functional link to Fe-S cluster biology, while its sensitivity to TEMPOL and NO reveals a potentially druggable vulnerabilityin HBx.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.01.20.700672","type":"posted-content","created":{"date-parts":[[2026,1,21]],"date-time":"2026-01-21T20:25:11Z","timestamp":1769027111000},"source":"Crossref","is-referenced-by-count":1,"title":["Promiscuous Metal Site in Hepatitis B Virus X Protein Binds an Fe-S Cluster"],"prefix":"10.64898","author":[{"given":"Jiahua","family":"Chen","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02453, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michelle","family":"Langton","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02453, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrick","family":"Cao","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02453, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Avital","family":"Aaron","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02453, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jackson","family":"Ho","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Boston University, Boston, Massachusetts 02215, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eranthie","family":"Weerapana","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7124-9896","authenticated-orcid":false,"given":"Deborah L.","family":"Perlstein","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Boston University, Boston, Massachusetts 02215, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexey","family":"Silakov","sequence":"additional","affiliation":[{"name":"Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel W.","family":"Bak","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6750-1948","authenticated-orcid":false,"given":"Maria-Eirini","family":"Pandelia","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02453, United States"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026020608160654000_2026.01.20.700672v1.1","doi-asserted-by":"crossref","first-page":"18035","DOI":"10.1038\/nrdp.2018.35","article-title":"Hepatitis B virus infection","volume":"4","year":"2018","journal-title":"Nat Rev Dis Primers"},{"key":"2026020608160654000_2026.01.20.700672v1.2","doi-asserted-by":"publisher","DOI":"10.21037\/jgo.2017.03.14"},{"key":"2026020608160654000_2026.01.20.700672v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.jhep.2011.02.015"},{"key":"2026020608160654000_2026.01.20.700672v1.4","doi-asserted-by":"crossref","unstructured":"E. 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