Low reduction potential of Ero1α regulatory disulphides ensures tight control of substrate oxidation

Formation of disulphide bonds within the mammalian endoplasmic reticulum (ER) requires the combined activities of Ero1 alpha and protein disulphide isomerase (PDI). As Ero1 alpha produces hydrogen peroxide during oxidation, regulation of its activity is critical in preventing ER-generated oxidative stress. Here, we have expressed and purified recombinant human Ero1 alpha and shown that it has activity towards thioredoxin and PDI. The activity towards PDI required the inclusion of glutathione to ensure sustained oxidation. By carrying out site-directed mutagenesis of cysteine residues, we show that Ero1 alpha is regulated by non-catalytic disulphides. The midpoint reduction potential (E degrees') of the regulatory disulphides was calculated to be approximately -275mV making them stable in the redox conditions prevalent in the ER. The stable regulatory disulphides were only partially reduced by PDI (E degrees' similar to -180 mV), suggesting either that this is a mechanism for preventing excessive Ero1 alpha activity and oxidation of PDI or that additional factors are required for Ero1 alpha activation within the mammalian ER.