Assay of Ndh activity and determination of the reaction products.

Ndh activity was determined as previously described by monitoring the reduction of 2,6-dichlorophenolindophenol (DCIP) with nicotine at 600 nm (ε = 21 mM⁻¹ cm⁻¹) (5). The assay mixture contained 1 mM nicotine, 0.05 mM DCIP, and 50 mM sodium phosphate buffer (pH 7.0). Reduction of 1 μmol of DCIP per min was defined as one unit. The assay was performed using quartz cuvettes (1-cm light path) filled with 1-ml reaction mixture at 30°C using a UV-visible Ultrospec 2100 Pro spectrophotometer (GE Healthcare, USA) and initiated by adding enzyme. In some cases, 0.5 mM phenazine methosulfate (PMS) was added to enhance electron transfer. To identify the reaction products of Ndh, the reaction was performed in 50 mM sodium phosphate buffer (pH 7.0) containing 1 U of Ndh, 2 mM nicotine, and 0.1 mM DCIP for 60 min at 30°C and spectrophotometrically monitored as indicated above. Products were determined by using liquid chromatography-mass spectrometry (LC-MS).