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 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 (O(2)) 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 O(2) as electron acceptors for yeast Erv1, and that O(2) 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 k (cat) and K (m) 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 O(2) 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.