Isonop no electrode apollo 4000

A set of 500-mL flasks was inoculated at an initial OD₆₀₀ of 0.05 in BVMN and incubated at 30 °C and 170 rpm under microoxic conditions for 3 days. Cells were then harvested and washed in a similar way as for the MV⁺-NR and MV⁺-NIR activity assays and resuspended in 1.5 mL of the same buffer. NO consumption rates were determined using a 2-mm ISONOP NO electrode APOLLO 4000^® (World Precision Instruments, Friedberg, Germany), following Cabrera et al. (2016) [69 (link)]. The reaction chamber (≈2.2 mL) contained 1.8 mL phosphate buffer 25 mM (pH 7.41), 100 µL of cell suspension (0.3–0.5 mg protein), 90 µL of 1 M sodium succinate, 100 µL of 320 mM glucose, and 100 µL of an enzyme mix containing 40 units mL⁻¹ of Aspergillus niger glucose oxidase and 250 units mL⁻¹ of bovine liver catalase. Once a steady base line was obtained, 50 µL of a saturated NO solution of 1.91 mM (at 20 °C) [71 (link)] was added to the chamber to start the reaction. Each measurement was stopped when the NO concentration had dropped to zero, meaning that all the NO present had been consumed. NOR activity was expressed as mmol NO consumed·(mg protein·h)⁻¹. Two biological replicates for each Cu condition were used.

In order to investigate the capacity of the different mutants to produce or consume NO, cell cultures at an initial OD600 of about 0.25 were incubated microoxically with KNO₃ as the sole nitrogen source for 24 h, harvested by centrifugation, washed twice with 25 mM Na₂HPO₄/NaH₂PO4 buffer (pH 7.4), and resuspended in 1.5 ml of the same buffer. NO production and consumption activities were determined by using an ISONOP NO electrode APOLLO 4000^®(World Precision Instruments). The reaction chamber (2 ml) was temperature-controlled, magnetically stirred and contained: 1410 μl of 25 mM Na₂HPO₄/NaH₂PO₄ buffer (pH 7.4) and 250 μl of cell suspension (0.4–0.7 mg protein) for NO production or 760 μl of 25 mM Na₂HPO₄/NaH₂PO₄ buffer (pH 7.4) and 900 μl of cell suspension (1.5–2.5 mg protein) for NO consumption. To generate an anoxic atmosphere, 100 μl of an enzymatic mix containing Aspergillus niger glucose oxidase (40 units⋅ml⁻¹), bovine liver catalase (250 units⋅ml⁻¹) (Sigma-Aldrich), 90 μl of 1 M sodium succinate and 100 μl of 320 mM glucose were added to the chamber. Once a steady base line was obtained, 50 μl of 50 mM NaNO₂ (NO production) or 50 μl of 2 mM NO (NO consumption) was added to the chamber to start the reaction.