Yeast (Saccharomyces cerevisiae) Erv1 (EC number 18.104.22.168), a member of the FAD-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 dithiothreitol (DTT) as an electron donor, our results show that cytochrome c is approximately 7-15-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.
|Journal||The FEBS Journal|
|Early online date||30 Sep 2019|
|Publication status||Published - 2019|
Research Beacons, Institutes and Platforms
- Manchester Institute of Biotechnology