Oxygraph clark type oxygen electrode

Oxygen consumption was measured at 25 °C using an Oxygraph Clark-type oxygen electrode (Hansatech Instruments) as described previously [25] (link). Briefly, reactions were initiated by adding Ero1α protein to a final concentration of 2 μM in oxygen consumption buffer (100 mM Tris-HAc, pH 8.0, 50 mM NaCl, 2 mM EDTA, 20 μM PDI protein) containing 2 mM GSH or 400 μM Hcy.

SC activity was measured by monitoring the oxygen-reduction rate with an Oxygraph Clark-type oxygen electrode (Hansatech) operated at 25 °C. The reaction cell was loaded with Coenzyme Q₁ (100 µM, Sigma) and NDH-2 (11 nM) in GTBS buffer. NADH (100 µM) was added into the cell, and Coenzyme Q₁ was reduced for 2 min before adding SC to a final concentration of 10.4 µg/mL. The total concentration of SC in the reaction cell was calculated by assuming that the ratio of SC to cyt. bc₁c₄c₅ was ~1:2.2, which was estimated from the cryoEM image analysis. For reactions with inhibitors, the reaction cell was preincubated with either KCN (300 µM) or antimycin A (150 µM). To account for background oxygen consumption each reaction was analyzed by subtracting the value of the slope immediately before the addition of SC from the value of the slope after the addition.

Oxygen consumption was measured at 25 °C using an Oxygraph Clark-type oxygen electrode (Hansatech Instruments) as previously described [18 (link)]. Briefly, reactions were initiated by injecting Ero1 proteins into buffer B (100 mM Tris-HAc, 50 mM NaCl, 2 mM EDTA, pH 8.0) containing PDI/GSH or reduced Trx1 with or without FAD.