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.

Fig. 1.

Structure of the Ero1p active site. A ribbon diagram of the active-site region of yeast Ero1p (4) is shown with the bound FAD in orange and disulfides in a ball-and-stick representation. The Cys sulfurs are numbered according to the position of the residue in the yeast Ero1p sequence (RefSeq accession no. NP_013576). The disulfide between Cys-352 and Cys-355 abuts the flavin. The loop containing Cys-100 and Cys-105 is flexible (only one of the two conformations observed in the crystal structures is shown) and may be involved in shuttling electrons from substrate to the Cys-352-Cys-355 disulfide. The structural or functional role of the Cys-90-Cys-349 disulfide has yet to be determined.

Fig. 2.

Absorbance spectrum of Ero1p-bound FAD. The spectrum of Ero1p was measured (black), CTAB was then added to release the bound FAD, and the spectrum was measured again (red). The original spectrum was corrected for the 1% dilution when adding the CTAB before the two spectra were superposed. The absorbance peak of the bound flavin is red-shifted from ≈448 nm to 454 nm, and a shoulder appears at ≈485 nm. Inset is an expansion of the absorbance scale in the visible region.

Fig. 3.

Ero1p (7.4 μM in buffer A) was titrated anaerobically with Trx_red added from a gas-tight syringe. Inset is a plot of absorbance at 454 nm recorded after each addition. Selected spectra from the titration (curves 1-7, corresponding to 0.0, 0.5, 1.0, 1.5, 2.1, 2.5, and 2.9 eq of Trx_red per mol of flavin) are plotted in the main panel.

Fig. 4.

Oxygen is reduced by Ero1p activity to form hydrogen peroxide. (A) The reaction was initiated at time 0 by injecting the shorter construct of Ero1p to a final concentration of 1 μM into a solution containing 50 μM Trx_red, and oxygen consumption was monitored. Approximately half of the oxygen originally consumed during Trx_red oxidation was restored with the addition of catalase (arrow), indicating that oxygen has been reduced to H₂O₂ in the initial reaction. Similar results were obtained for the longer construct. (B) The reaction was initiated at time 0 by injecting the shorter form of Ero1p to a final concentration of 1 μM into a rapidly stirred solution containing 100 μM Trx_red. Samples were withdrawn and mixed with the PeroXOquant detection reagent. Absorbance values were converted to H₂O₂ concentration according to a standard H₂O₂ calibration curve.

Fig. 5.

Determination of the K_m value for oxygen. Ero1p (shorter construct) was injected into a solution containing 12.5 mM DTT in the oxygen electrode chamber. The graph shows the end of the reaction, when the oxygen has been depleted to <10% of its initial value. Inset is the first derivative of the smoothed data, which was used to identify the point at which the rate was half maximal.

Fig. 6.

Effect of exogenous flavin compounds on the rate of oxygen consumption during Ero1p catalysis of disulfide formation. The experiments shown were performed by using the longer Ero1p construct, but the shorter version gave similar results. At time 0, Ero1p was injected to a final concentration of 1 μM into a solution containing 98 μM Trx_red (A)or10mMDTT(B) to obtain the curves indicated by filled symbols. Traces measured in the absence of enzyme are indicated by open symbols. The black curves show the kinetics of oxygen consumption without added flavin compounds. The red, orange, and gold curves show the kinetics of oxygen consumption under similar conditions, except that the indicated flavin compounds were present in the reaction (100 μM FAD, 100 μM FMN, and 100 μM riboflavin, respectively).

Fig. 7.

Reduction of flavins in the absence of oxygen. The reductant was 50μM Trx_red, and the electron acceptors were 21 μM FAD or 30 μM FMN. When present, the shorter construct of Ero1p was at a concentration of 1 μM. The longer version of Ero1p also reduces exogenous flavins (data not shown).

Fig. 8.

Ero1p-catalyzed reduction of protein metal sites in the absence of oxygen. The reductant was 50 μM Trx_red, and the electron acceptors were cytochrome c (open circles) or azurin (filled circles) at 25 μM. In both cases, the uncatalyzed transfer of electrons from donor to acceptor was negligible.