Mia40 Combines Thiol Oxidase and Disulfide Isomerase Activity to Efficiently Catalyze Oxidative Folding in Mitochondria

• Published in: Journal of molecular biology 2014-12, Vol.426 (24), p.4087-4098
• Main Authors: Koch, Johanna R., Schmid, Franz X.
• Format: Article
• Language: English
• Subjects: Biocatalysis; Cation Transport Proteins - chemistry; Cation Transport Proteins - genetics; Cation Transport Proteins - metabolism; disulfide relay; Disulfides - chemistry; Disulfides - metabolism; Electrophoresis, Polyacrylamide Gel; Isomerism; Kinetics; mitochondrial disulfide formation; mitochondrial import; mitochondrial intermembrane space; Mitochondrial Membrane Transport Proteins - genetics; Mitochondrial Membrane Transport Proteins - metabolism; Models, Molecular; Molecular Chaperones - chemistry; Molecular Chaperones - genetics; Molecular Chaperones - metabolism; Mutation, Missense; Oxidation-Reduction; oxidative protein folding; Oxidoreductases - genetics; Oxidoreductases - metabolism; Protein Disulfide-Isomerases - genetics; Protein Disulfide-Isomerases - metabolism; Protein Folding; Protein Structure, Secondary; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Substrate Specificity; Sulfhydryl Compounds - metabolism
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2014.10.022

Mia40 (a mitochondrial import and assembly protein) catalyzes disulfide bond formation in proteins in the mitochondrial intermembrane space. By using Cox17 (a mitochondrial copper-binding protein) as a natural substrate, we discovered that, in the presence of Mia40, the formation of native disulfides is strongly favored. The catalytic mechanism of Mia40 involves a functional interplay between the chaperone site and the catalytic disulfide. Mia40 forms a specific native disulfide in Cox17 much more rapidly than other disulfides, in particular, non-native ones, which originates from the recently described high affinity for hydrophobic regions near target cysteines and the long lifetime of the mixed disulfide. In addition to its thiol oxidase function, Mia40 is active also as a disulfide reductase and isomerase. We found that species with inadvertently formed incorrect disulfides are rebound by Mia40 and reshuffled, revealing a proofreading mechanism that is steered by the conformational folding of the substrate protein. [Display omitted] •Mitochondrial thiol oxidase Mia40 uses a functional interplay between chaperone site and catalytic disulfide to direct oxidative folding.•Mia40 employs kinetic control to favor formation of native disulfides.•Conformational folding acts as a proofreading mechanism.•Mia40 shows low but significant disulfide isomerase activity.