NADH-Ferricyanide Reductase Activity Assay

The NADH–ferricyanide reductase activity was measured with minor modification, as recently published [31 (link)]. The medium of the cells was exchanged against 1 mL buffer (50 mM Tris-HCl pH 8.0, 500 μM β-NADH) providing 500 μM potassium ferricyanide (III) (K₃Fe(CN)₆) (Riedel-de-Haen—Honeywell Specialty Chemicals Seelze GmbH, Seelze, Germany) per 35 mm dish and the cells were incubated for 60 min at 37 °C and 5% CO₂. Ferricyanide was reduced to ferrocyanide. Subsequently, an aliquot of the buffer was drawn to measure the absorbance at 420 nm with a photometer (UV1202, Shimadzu, Kyoto, Japan). The NADH–ferricyanide reductase activity was expressed as reduced ferricyanide in nmol/min/4 × 10⁵ cells. Per the experiment, each condition was measured in duplicates.