Surface-enhanced Raman spectroscopy (SERS) has exhibited great potential in protein identification and quantification. However, the poor spectral reproducibility, originating from random protein immobilization on SERS substrates, still makes it challenging for SERS to probe protein functions without any extrinsic Raman labels. Here, in our study, spacer molecules between proteins and SERS substrates are optimized for both biocompatible protein immobilization and Raman scattering enhancement. We have accordingly prepared iminodiacetic acid (IDA)-functionalized silver substrates, which are used for capturing His-tagged proteins via nickel−imidazole coordination. The controlled immobilization enables excellent SERS spectral reproducibility as evidenced by 6 polypeptides. Furthermore, the interactions between two model proteins, Erv1C (C-terminal domain of flavine adenine dinucleotide-dependent mitochondrial cytochrome c reductase Erv1) and AFP (alpha-fetoprotein), and their ligands Cyt c (cytochrome c) and ATRA (all-trans-retinoic acid) are examined, respectively. The results indicate that the IDA-functionalized silver substrates enable controlled protein immobilization and allow label-free protein function investigation by SERS. As a proof-of-concept study, the proposed functionalized SERS-active substrates combined with immobilized metal-affinity chromatography will be useful for mechanism studies on protein−ligand interactions, which is crucially important for understanding the structural basis of protein functional versatility and will contribute to the fields of drug design and biotechnology.

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.

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 À320 mV for the shuttle disulphide, À150 mV for the active-site disulphide and À215 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 À250 mV and À215~À260 mV, respectively, and the potential for FAD cofactor is À148 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.Abbreviations ALR, augmenter of liver regeneration; DTT, dithiothreitol; Erv, essential for respiration and viability; FAD, flavin adenine dinucleotide; IMS, intermembrane space; MIA, mitochondrial import and assembly; QSOX, quiescin sulfhydryl oxidase; TCEP, tris(2-carboxyethyl)phosphine.Electron flow mechanism of MIA40 pathway E. Ceh-Pavia et al.

Erv1 (EC number 1.8.3.2) is an essential mitochondrial enzyme catalyzing protein import and oxidative folding in the mitochondrial intermembrane space. Erv1 has both oxidase and cytochrome c reductase activities. While both Erv1 and cytochrome c were reported to be membrane associated in mitochondria, it is unknown how the mitochondrial membrane environment may affect the function of Erv1. Here, in this study, we used liposomes to mimic the mitochondrial membrane and investigated the effect of liposomes and cardiolipin on the folding and function of yeast Erv1. Enzyme kinetics of both the oxidase and cytochrome c reductase activity of Erv1 were studied using oxygen consumption analysis and spectroscopic methods. Our results showed that the presence of liposomes has mild impacts on Erv1 oxidase activity, but significantly inhibited the catalytic efficiency of Erv1 cytochrome c reductase activity in a cardiolipin-dependent manner. Taken together, the results of this study provide important insights into the function of Erv1 in the mitochondria, suggesting that molecular oxygen is a better substrate than cytochrome c for Erv1 in the yeast mitochondria.

No abstract