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

Abstract

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.

Effects of Liposome and Cardiolipin on Folding and Function of Mitochondrial Erv1

“…Next, the effects of the liposomes on the Erv1 oxidase activity were investigated using oxygen consumption analysis ( Figure 4 A), as described previously [ 23 , 36 ]. As shown at the beginning of Figure 4 A, there was no oxygen consumption by DTT in the absence of Erv1.…”

“…Erv/ALR enzymes can act as an oxidase by passing electrons to molecular oxygen and reducing oxygen to hydrogen peroxide, or as a cytochrome c reductase by passing electrons to oxidized cytochrome c ( Figure 1 A). Enzyme kinetic studies revealed that different Erv1/ALR enzymes have different oxidase and cytochrome c reductase activities [ 17 , 21 , 22 , 23 ]. While both yeast Erv1 [ 23 ] and human ALR [ 21 ] seem to prefer cytochrome c more than molecular oxygen as an electron acceptor, the ratio of relative cytochrome c reductase to oxidase activity is quite different for these enzymes, with 15 for yeast Erv1 [ 23 ] and 107 for human ALR [ 21 ], as ALR uses oxygen poorly.…”

“…Enzyme kinetic studies revealed that different Erv1/ALR enzymes have different oxidase and cytochrome c reductase activities [ 17 , 21 , 22 , 23 ]. While both yeast Erv1 [ 23 ] and human ALR [ 21 ] seem to prefer cytochrome c more than molecular oxygen as an electron acceptor, the ratio of relative cytochrome c reductase to oxidase activity is quite different for these enzymes, with 15 for yeast Erv1 [ 23 ] and 107 for human ALR [ 21 ], as ALR uses oxygen poorly. On the other hand, yeast Erv2 [ 17 ] and avian QSOX [ 22 ] are good oxidases and readily reduce oxygen to hydrogen peroxide.…”

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

“…Upon mitochondrial protein import, Mia40 acts as an oxidoreductase interacting with the substrate proteins directly and transfers a disulphide bond to the substrates via the formation of intermolecular disulphide linked complexes . The reduced Mia40 is then re‐oxidised by Erv1 (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/8/3/2.html) for regeneration, and reduced Erv1 can be re‐oxidised by molecular oxygen or oxidised cytochrome c .…”

“…The electrons are then transferred to the active‐site (proximal) disulphide via formation of an intermediate disulphide (Fig. C, C33’‐C130), the cofactor FAD, and then in turn the reduced Erv1 can be re‐oxidised by transferring electrons to molecular oxygen (O 2 ) or cytochrome c for regeneration . Thus, there are three redox‐active centres in Erv1, the shuttle disulphide (C30‐C33), active‐site (proximal) disulphide (C130‐C133) and the FAD cofactor.…”