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 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 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.

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