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Mitochondria, besides energy generation, serve as significant cellular redox hubs, mediate stress response and cellular health. However, the effects of endogenous mitochondrial sulfane sulfur (MSS) remain largely uncharacterized as compared with their cytosolic counterparts, cytosolic sulfane sulfur (CSS). To investigate this, we designed a novel artificial substrate for mitochondrial 3\u2010mercaptopyruvate sulfurtransferase (3\u2010MST), a key enzyme involved in MSS biosynthesis. Using cells expressing a mitochondrion\u2010localized persulfide biosensor, we demonstrate this tool's ability to selectively enhance MSS. While H<jats:sub>2<\/jats:sub>S was previously known to suppress human immunodeficiency virus (HIV\u20101), we found that MSS profoundly affected the HIV\u20101 life cycle, mediating viral reactivation from latency. Additionally, we provide evidence for the role of the host's mitochondrial redox state, membrane potential, apoptosis, and respiration rates in managing HIV\u20101 latency and reactivation. Together, dynamic fluctuations in the MSS pool have a significant and possibly conflicting effect on HIV\u20101 viral latency. The precision tools developed herein allow for orthogonal generation of persulfide within both mitochondria and the cytosol and will be useful in interrogating disease biology.<\/jats:p>","DOI":"10.1002\/anie.202411133","type":"journal-article","created":{"date-parts":[[2024,8,2]],"date-time":"2024-08-02T07:47:03Z","timestamp":1722584823000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Orthogonal Persulfide Generation through Precision Tools Provides Insights into Mitochondrial Sulfane Sulfur"],"prefix":"10.1002","volume":"63","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5193-3837","authenticated-orcid":false,"given":"Suman","family":"Manna","sequence":"first","affiliation":[{"name":"Department of Chemistry Indian Institute of Science Education and Research Pune  Pune, Maharashtra 411008 India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ragini","family":"Agrawal","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Cell Biology, Centre for Infectious Disease Research Indian Institute of Science  Bangalore, Karnataka 560012 India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5484-9785","authenticated-orcid":false,"given":"Tarun","family":"Yadav","sequence":"additional","affiliation":[{"name":"Department of Biology Indian Institute of Science Education and Research Pune  Pune, Maharashtra 411008 India"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8922-5905","authenticated-orcid":false,"given":"T. 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