Figure 9. Model of thiol–disulfide exchange reactions in Erv1 catalysis.

This model is focused on the reductive half-reaction. Thiolate anion (S⁻) of the second cysteine (Cys²⁹⁸) of the Mia40 CPC motif nucleophilically attacks Cys³⁰ of Erv1 (stage 1), leading to the formation of Mia40–Erv1 mixed disulfide bond, which is then attacked by S⁻ of the first cysteine (Cys²⁹⁶) of Mia40 for oxidation of the CPC motif and reduction of the Erv1 Cys³⁰–Cys³³ disulfide bond (stage 2). Thiolate of Cys³³ nucleophilically attacks Cys¹³⁰′ of the other subunit of Erv1 (stage 3), leading to the formation of an intermediate Cys³³–Cys¹³⁰′ disulfide bond between two Erv1 subunits and a thiolate to FAD CTC (stage 4). Then, the Cys³³–Cys¹³³′ disulfide is reduced via nucleophilic attack on Cys³³ by Cys³⁰ (step 4), which leads to reoxidation of the Cys³⁰–Cys³³ disulfide bond (stage 5). Subsequently, a pair of electrons from the reduced redox active-site Cys¹³⁰′/Cys¹³³′ is transferred to FAD coupled with the reformation of the Cys¹³⁰′–Cys¹³³′ disulfide bond (stage 6). Finally, the oxidative half-reaction for Erv1 turnover is completed through transfer of a pair of electrons from reduced FAD (FADH₂) to molecular oxygen or cytochrome c. The colour of FAD in stages 4 and 5 correspond to the colour of Erv1C30S and Erv1C130S respectively (the present study and [28]).