Mitochondrial sulphydryl oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles

Erv1 is an essential component of the mitochondrial import and assembly (MIA) pathway, playing an important role in the oxidative folding of mitochondrial intermembrane space proteins. In the MIA pathway, Mia40, a sulphydryl oxidoreductase with a CPC motif at its active site, oxidises newly-imported substrate proteins. Erv1, a FAD-dependent sulphydryl oxidase, in turn reoxidises Mia40 via its N-terminal Cys30-Cys33 shuttle disulphide. However, it is unclear how the two shuttle cysteines of Erv1 relay electrons from Mia40 CPC to the Erv1 active-site Cys130-Cys133 disulphide. In this study, using yeast genetic approaches we showed that both shuttle cysteine residues of Erv1 are required for cell growth. In organelle and in vitro studies confirmed that both shuttle cysteines were indeed required for import of MIA-pathway substrates and Erv1 enzyme function to oxidise Mia40. Furthermore, our results revealed that the two shuttle cysteines of Erv1 are functionally distinct. While Cys33 is essential for forming the intermediate disulphide Cys33-Cys130’ and transferring electrons to the redox active-site directly, Cys30 plays two important roles: (i) dominantly interacts and receives electrons from Mia40 CPC, and (ii) resolves the Erv1 Cys33-Cys130 intermediate disulphide. Taken together, we conclude that both shuttle cysteines are required for Erv1 function, and play complementary but distinct roles to ensure rapid turnover of active Erv1.