A PDI-catalyzed thiol-disulfide switch regulates the production of hydrogen peroxide by human Ero1.

Abstract Oxidative folding in the endoplasmic reticulum (ER) involves ER oxidoreductin 1 (Ero1)-mediated disulfide formation in protein disulfide isomerase (PDI). In this process, Ero1 consumes oxygen (O 2 ) and releases hydrogen peroxide (H 2 O 2 ), but none of the published Ero1 crystal structures reveal any potential pathway for entry and exit of these reactants. We report that additional mutation of the Cys 208 –Cys 241 disulfide in hyperactive Ero1α (Ero1α-C104A/C131A) potentiates H 2 O 2 production, ER oxidation, and cell toxicity. This disulfide clamps two helices that seal the flavin cofactor where O 2 is reduced to H 2 O 2 . Through its carboxyterminal active site, PDI unlocks this seal by forming a Cys 208 /Cys 241 -dependent mixed-disulfide complex with Ero1α. The H 2 O 2 -detoxifying glutathione peroxidase 8 also binds to the Cys 208 /Cys 241 loop region. Supported by O 2 diffusion simulations, these data describe the first enzymatically controlled O 2 access into a flavoprotein active site, provide molecular-level understanding of Ero1α regulation and H 2 O 2 production/detoxification, and establish the deleterious consequences of constitutive Ero1 activity.