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In Saccharomyces cerevisiae, about 98% of all mitochondrial proteins are synthesised in the cytosol and are translocated into different mitochondrial subcompartments. The biogenesis of many cysteine-containing proteins in the mitochondrial intermembrane space (IMS) is driven by a sulphydryl oxidoreductase system known as the mitochondrial import and assembly (MIA) machinery that catalyses disulphide bond formation. The MIA machinery contains two essential components: the import receptor Mia40 and the FAD-dependent sulphydryl oxidase Erv1. Current model suggests that Erv1 oxidises Mia40, which in turn transfers a disulphide bond to substrate proteins. However, it was unclear how Erv1 oxidises Mia40. In this study, the roles of individual disulphide bonds of Erv1 and the mechanism of Mia40-Erv1 interaction were investigated. Erv1 contains three disulphide bonds arranged in two CXXC motifs (Cys30-Cys33 and Cys130-Cys133) and one CX16C motif (Cys159-Cys176). Three double cysteine mutants with each cysteine pair corresponding to individual disulphide bonds were generated. Systematic analyses using a wide range of in vitro, in organello and in vivo methods showed that both the CXXC disulphides are required for Erv1 enzymatic function, while the CX16C disulphide has a dual role in stabilising protein folding and mitochondrial import of Erv1. This study showed that Cys30-Cys33 functions as a shuttle disulphide that transfers electrons to the active-site disulphide Cys130-Cys133, and both intersubunit and intermolecular electron transfer can occur. The shuttle disulphide of Erv1 receives electrons from the CPC disulphide (Cys296-Cys298) of Mia40 through formation of a mixed disulphide intermediate. Characterisation of single cysteine mutant proteins of Mia40 and Erv1 suggested that Cys298 of Mia40 favours mixed disulphide bond formation with Cys30 of Erv1, although different mixed disulphides can be formed between all other cysteine combinations in vitro. GSH may play an isomerisation-like role in counteracting non-productive Mia40-Erv1 mixed disulphides. In addition, it was found that Erv1 has a strong affinity towards zinc binding, which inhibits its enzymatic function. The inhibitory effects of zinc can be counteracted in the presence of a chelating agent. |
