Generating disulfides enzymatically: Reaction products and electron acceptors of the endoplasmic reticulum thiol oxidase Ero1p

Abstract

Ero1p is a key enzyme in the disulfide bond formation pathway in eukaryotic cells in both aerobic and anaerobic environments. It was previously demonstrated that Ero1p can transfer electrons from thiol substrates to molecular oxygen. However, the fate of electrons under anaerobic conditions and the final fate of electrons under aerobic conditions remained obscure. To address these fundamental issues in the Ero1p mechanism, we studied the transfer of electrons from recombinant yeast Ero1p to various electron acceptors. Under aerobic conditions, reduction of molecular oxygen by Ero1p yielded stoichiometric hydrogen peroxide. Remarkably, we found that reduced Ero1p can transfer electrons to a variety of small and macromolecular electron acceptors in addition to molecular oxygen. In particular, Ero1p can catalyze reduction of exogenous FAD in solution. Free FAD is not required for the catalysis of dithiol oxidation by Ero1p, but it is sufficient to drive disulfide bond formation under anaerobic conditions. These findings provide insight into mechanisms for regenerating oxidized Ero1p and maintaining disulfide bond formation under anaerobic conditions in the endoplasmic reticulum.electron transfer ͉ Ero1 ͉ flavoenzyme ͉ hydrogen peroxide T he thiol oxidase enzyme Ero1p (1, 2), conserved across eukaryotes, generates disulfide bonds de novo in the endoplasmic reticulum (ER) by catalyzing the transfer of electrons from dithiols to molecular oxygen (3). The Ero1p active site consists of a Cys-Xaa-Xaa-Cys amino acid sequence motif (in which Xaa is a non-Cys amino acid) juxtaposed with the isoalloxazine ring system of a bound FAD cofactor (4) (Fig. 1). An additional redox center, a Cys-Xaa 4 -Cys disulfide, has been proposed to accept electrons from substrate proteins and transfer them to the Cys-Xaa-Xaa-Cys disulfide (4, 5).The arrangement of bound flavin, fixed active-site disulfide, and flexible shuttle disulfide is also found in Erv2p (6), a second yeast ER thiol oxidase with no sequence similarity to Ero1p (7,8). Erv2p is a member of the QSOX͞ALR enzyme family (9). The overall reaction of the QSOX͞ALR enzymes can be divided into two half-reactions. In the reductive half-reaction, the enzyme accepts electrons from reducing substrates, resulting in a reduction of the bound flavin cofactor. In the oxidative halfreaction, the enzyme deposits the electrons on an acceptor, such as molecular oxygen, to restore the bound cofactor to its initial state, as shown in Eqs. 1 and 2.Although this scheme describes the simplest scenario, four-, six-, and even eight-electron-reduced states of these f lavindependent sulfhydryl oxidase enzymes may also be possible, depending on the number of redox-active Cys pairs and the rate of internal equilibration between them (10, 11).It is likely that the basic reaction scheme described above for the QSOX͞ALR family applies to Ero1p as well. First, the similar arrangements of the functional groups in the active sites of Ero1p and Erv2p (4) suggest that the two thiol oxidase families may share feat...

Functional in Vitro Analysis of the ERO1 Protein and Protein-disulfide Isomerase Pathway

“…However, the experimental conditions were not well characterized, which could cause discrepancies in the data obtained (10,11,21,25). We found a physical as well as functional pH dependence of ERO1␣ and PDI, with the apparent maximum catalytic rate and binding affinity observed at pH ϳ7 (Fig.…”

Free Radicals: Properties, Sources, Targets, and Their Implication in Various Diseases

“…Another important thiol oxidase enzyme, Erop1p catalyses the transfer of electrons from dithiols to molecular oxygen results in the formation of H 2 O 2 [69]. The other endogenous sources of ROS include prostaglandin synthesis, auto-oxidation of adrenalin, phagocytic cells, reduced riboflavin, FMNH 2 , FADH 2 , cytochrome P 450, immune cell activation, inflammation, mental stress, excessive exercise, infection, cancer, aging, ischemia etc.…”

Interactions of β-Helical Antifreeze Protein Mutants with Ice

“…Thioredoxin (Trx) was prepared as described. 37 Maltose binding protein (MBP) was obtained from TEV cleavage of MBP-His 6-TEV-TmTHP constructs 33 and purified as follows. After the second step of Ni-NTA purification of TmAFP or mutants, beads were washed thoroughly, and bound MBP was eluted.…”