MIA40 is an oxidoreductase that catalyzes oxidative protein folding in mitochondria

MIA40, found in the mitochondrial intermembrane space, is a central component in the import system that transports certain cysteine motif–containing proteins into the mitochondria. New analyses reveal that MIA40 forms a novel thioredoxin fold. Its redox center catalyzes the formation of the first disulfide bond of a substrate, causing the susbtrate's second disulfide to require only oxygen for its formation. MIA40 has a key role in oxidative protein folding in the mitochondrial intermembrane space. We present the solution structure of human MIA40 and its mechanism as a catalyst of oxidative folding. MIA40 has a 66-residue folded domain made of an α-helical hairpin core stabilized by two structural disulfides and a rigid N-terminal lid, with a characteristic CPC motif that can donate its disulfide bond to substrates. The CPC active site is solvent-accessible and sits adjacent to a hydrophobic cleft. Its second cysteine (Cys55) is essential in vivo and is crucial for mixed disulfide formation with the substrate. The hydrophobic cleft functions as a substrate binding domain, and mutations of this domain are lethal in vivo and abrogate binding in vitro . MIA40 represents a thioredoxin-unrelated, minimal oxidoreductase, with a facile CPC redox active site that ensures its catalytic function in oxidative folding in mitochondria.