Erv1 (essential for respiration and viability 1) is an FADdependent thiol oxidase of the Erv/ALR (augmenter of liver regeneration) sub-family. It is an essential component of the mitochondrial import and assembly (MIA) pathway, playing an important role in the oxidative folding of the mitochondrial intermembrane space (IMS) proteins and linking the MIA pathway to the mitochondrial respiratory chain via cytochrome c (cyt c). The importance of the Erv/ALR enzymes was also demonstrated in a recent study where a single mutation in the human ALR (R194H) leads to autosomal recessive myopathy [Di Fonzo, Ronchi, Lodi, Fassone, Tigano, Lamperti, Corti, Bordoni, Fortunato, Nizzardo et al. (2009) Am. J. Hum. Genet. 84, 594-604]. However, the molecular mechanism of the disease is still unclear. In the present study, we use yeast Erv1 as a model to provide clear evidence for a progressive functional defect in the catalytic activity of the corresponding Erv1 R182H mutant. We show that the FAD cofactor was released from Erv1 R182H during its catalytic cycle, which led to the inactivation of the enzyme. We also characterized the effects of the mutation on the folding and stability of Erv1 and tested our in vitro findings in vivo using a yeast genetic approach. The results of the present study allow us to provide a model for the functional defect in Erv1 R182H, which could potentially be extended to human ALR R194H and provides insights into the molecular basis of autosomal recessive myopathy.

Mia40 is a highly conserved mitochondrial protein that plays an essential role in the import and oxidative folding of many proteins of the mitochondrial intermembrane space. Mia40 uses its redox active CPC motif to shuttle disulfides between its client proteins (newly imported proteins) and the thiol oxidase Erv1. As a thiol oxidoreductase, no cofactor was found in Mia40, nor is a cofactor required for this function. In the present study we, for the first time based on both in vitro and in vivo studies, show that yeast Mia40 can exist as an Fe-S (iron-sulfur) protein as well. We show that Mia40 binds a [2Fe-2S] cluster in a dimer form with the cluster co-ordinated by the cysteine residues of the CPC motifs. The biological relevance of the cofactor binding was confirmed in vivo by cysteine redox state and iron uptake analyses, which showed that a significant amount of cellular Mia40 binds iron in vivo. Furthermore, our oxygen consumption results suggested that the Fe-S-containing Mia40 is not an electron donor for Erv1. Thus we conclude that Mia40 is a novel Fe-S protein with a new cluster-binding motif (CPC), and apart from the thiol oxidoreductase activity, Mia40 may have another important, as yet undefined, function in cells.

Correct and timely folding is critical to the function of all proteins. The importance of this is illustrated in the biogenesis of the mitochondrial intermembrane space (IMS) “small Tim” proteins. Biogenesis of the small Tim proteins is regulated by dedicated systems or pathways, beginning with synthesis in the cytosol and ending with assembly of individually folded proteins into functional complexes in the mitochondrial IMS. The process is mostly centered on regulating the redox states of the conserved cysteine residues: oxidative folding is crucial for protein function in the IMS, but oxidized (disulfide bonded) proteins cannot be imported into mitochondria. How the redox-sensitive small Tim precursor proteins are maintained in a reduced, import-competent form in the cytosol is not well understood. Recent studies suggest that zinc and the cytosolic thioredoxin system play a role in the biogenesis of these proteins. In the IMS, the mitochondrial import and assembly (MIA) pathway catalyzes both import into the IMS and oxidative folding of the small Tim proteins. Finally, assembly of the small Tim complexes is a multistep process driven by electrostatic and hydrophobic interactions; however, the chaperone function of the complex might require destabilization of these interactions to accommodate the substrate. Here, we review how folding of the small Tim proteins is regulated during their biogenesis, from maintenance of the unfolded precursors in the cytosol, to their import, oxidative folding, complex assembly and function in the IMS.

Yeast (Saccharomyces cerevisiae) essential for respiration and viability 1 (Erv1; EC number http://www.chem.qmul.ac.uk/iubmb/enzyme/1/8/3/2.html), a member of the flavin adenine dinucleotide‐dependent Erv1/ALR disulphide bond generating enzyme family, works together with Mia40 to catalyse protein import and oxidative folding in the mitochondrial intermembrane space. Erv1/ALR functions either as an oxidase or cytochrome c reductase by passing electrons from a thiol substrate to molecular oxygen (O2) or cytochrome c, respectively. However, the substrate specificity for oxygen and cytochrome c is not fully understood. In this study, the oxidase and cytochrome c reductase kinetics of yeast Erv1 were investigated in detail, under aerobic and anaerobic conditions, using stopped‐flow absorption spectroscopy and oxygen consumption analysis. Using DTT as an electron donor, our results show that cytochrome c is ~ 7‐ to 15‐fold more efficient than O2 as electron acceptors for yeast Erv1, and that O2 is a competitive inhibitor of Erv1 cytochrome c reductase activity. In addition, Mia40, the physiological thiol substrate of Erv1, was used as an electron donor for Erv1 in a detailed enzyme kinetic study. Different enzyme kinetic kcat and Km values were obtained with Mia40 compared to DTT, suggesting that Mia40 modulates Erv1 enzyme kinetics. Taken together, this study shows that Erv1 is a moderately active enzyme with the ability to use both O2 and cytochrome c as the electron acceptors, indicating that Erv1 contributes to mitochondrial hydrogen peroxide production. Our results also suggest that Mia40‐Erv1 system may involve in regulation of the redox state of glutathione in the mitochondrial intermembrane space. Erv1 EC number http://www.chem.qmul.ac.uk/iubmb/enzyme/1/8/3/2.html.

Erv1 (essential for respiration and viability 1) is a FAD-dependent sulphydryl oxidase with a tryptophan-rich catalytic domain. We show that Trp95 and Trp183 are important for stabilizing the folding, FAD-binding, and function of Erv1, whilst other four tryptophan residues are not functionally important.