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Alkaline phosphatase (AP) requires disulfide bonds for activity, and copper activated reduced AP both in vitro and when it was expressed in the periplasm of mutants lacking their native disulfide\u2010generating system. However, AP was not activated when it was expressed in the cytoplasm of copper\u2010overloaded cells. Similarly, this copper stress failed to activate OxyR, a transcription factor that responds to the creation of a disulfide bond. The elimination of cellular disulfide\u2010reducing systems did not change these results. Nevertheless, in these cells, the cytoplasmic copper concentration was high enough to impair growth and completely inactivate enzymes with solvent\u2010exposed [4Fe\u20104S] clusters. Experiments with N\u2010acetylcysteine determined that the efficiency of thiol oxidation is limited by the sluggish pace at which oxygen regenerates copper(II) through oxidation of the thiyl radical\u2013Cu(I) complex. We conclude that this slow step makes copper too inefficient a catalyst to create disulfide stress in the thiol\u2010rich cytoplasm, but it can still impact the few thiol\u2010containing proteins in the periplasm. It also ensures that copper accumulates intracellularly in the Cu(I) valence.<\/jats:p>","DOI":"10.1111\/mmi.15032","type":"journal-article","created":{"date-parts":[[2023,2,9]],"date-time":"2023-02-09T08:37:48Z","timestamp":1675931868000},"page":"423-438","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":22,"title":["Excess copper catalyzes protein disulfide bond formation in the bacterial periplasm but not in the cytoplasm"],"prefix":"10.1111","volume":"119","author":[{"given":"Stefanie S.","family":"Eben","sequence":"first","affiliation":[{"name":"Department of Microbiology University of Illinois  Urbana Illinois USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5266-7403","authenticated-orcid":false,"given":"James A.","family":"Imlay","sequence":"additional","affiliation":[{"name":"Department of Microbiology University of Illinois  Urbana Illinois 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