Redox characterisation of Erv1, a key component for protein import and folding in yeast mitochondria.

The mitochondrial import and assembly (MIA) pathway plays a vitally important role in import and oxidative folding of mitochondrial proteins. Erv1, a member of the FAD-dependent Erv1/ALR disulphide bond generating enzyme family, is a key player of the MIA pathway. Although considerable progress has been made, the molecular mechanism of electron transfer within Erv1 is still not fully understood. The reduction potentials of the three redox centres were previously determined to be -320 mV for the shuttle disulphide, -150 mV for the active-site disulphide and -215 mV for FAD cofactor. However, it is unknown why FAD of Erv1 has such a low potential compared with other sulfhydryl oxidases, and why the shuttle disulphide has a potential as low as many of the stable structural disulphides of the substrates of MIA pathway. In this study, the three reduction potentials of Erv1 were reassessed using the wild-type and inactive mutants of Erv1 under anaerobic conditions. Our results show that the standard potentials for the shuttle and active-site disulphides are approximately -250 mV and -215 ~ -260 mV, respectively, and the potential for FAD cofactor is -148 mV. Our results support a model that both disulphide bonds are redox-active, and electron flow in Erv1 is thermodynamically favourable. Furthermore, the redox behaviour of Erv1 was confirmed, for the first time using Mia40, the physiological electron donor of Erv1. Together with previous studies on proteins of MIA pathway, we conclude that electron flow in the MIA pathway is a thermodynamically favourable, smoothly downhill process for all steps. DATABASE: Erv1: EC 1.8.3.2.

Kinetic characterisation of Erv1, a key component for protein import and folding in yeast mitochondria.

Yeast (Saccharomyces cerevisiae) essential for respiration and viability 1 (Erv1; EC number 1.8.3.2), 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 1.8.3.2.

Role of tryptophan residues of Erv1: Trp95 and Trp183 are important for its folding and oxidase function.

Erv1 is an FAD-dependent thiol oxidase of the ERV (essential for respiration and viability)/ALR (augmenter of liver regeneration) sub-family and an essential component of the mitochondrial import and assembly pathway. Erv1 contains six tryptophan residues, which are all located in the highly conserved C-terminal FAD-binding domain. Though important structural roles were predicted for the invariable Trp(95), no experimental study has been reported. In the present study, we investigated the structural and functional roles of individual tryptophan residues of Erv1. Six single tryptophan-to-phenylalanine yeast mutant strains were generated and their effects on cell viability were tested at various temperatures. Then, the mutants were purified from Escherichia coli. Their effects on folding, FAD-binding and Erv1 activity were characterized. Our results showed that Erv1(W95F) has the strongest effect on the stability and function of Erv1 and followed by Erv1(W183F). Erv1(W95F) results in a decrease in the Tm of Erv1 by 23°C, a significant loss of the oxidase activity and thus causing cell growth defects at both 30°C and 37°C. Erv1(W183F) induces changes in the oligomerization state of Erv1, along with a pronounced effect on the stability of Erv1 and its function at 37°C, whereas the other mutants had no clear effect on the function of Erv1 including the highly conserved Trp(157) mutant. Finally, computational analysis indicates that Trp(95) plays a key role in stabilizing the isoalloxazine ring to interact with Cys(133). Taken together, the present study provided important insights into the molecular mechanism of how thiol oxidases use FAD in catalysing disulfide bond formation.

The disease-associated mutation of the mitochondrial thiol oxidase Erv1 impairs cofactor binding during its catalytic reaction.

Erv1 (essential for respiration and viability 1) is an FAD-dependent 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.

Folding and biogenesis of mitochondrial small Tim proteins.

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.

Identification and characterization of mitochondrial Mia40 as an iron-sulfur protein.

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.

Cytosolic thioredoxin system facilitates the import of mitochondrial small Tim proteins.

Thiol-disulphide redox regulation has a key role during the biogenesis of mitochondrial intermembrane space (IMS) proteins. Only the Cys-reduced form of precursor proteins can be imported into mitochondria, which is followed by disulphide bond formation in the mitochondrial IMS. In contrast to the wealth of knowledge on the oxidation process inside mitochondria, little is known about how precursors are maintained in an import-competent form in the cytosol. Here we provide the first evidence that the cytosolic thioredoxin system is required to maintain the IMS small Tim proteins in reduced forms and facilitate their mitochondrial import during respiratory growth.