Extended Data Fig. 6: NicA2 and CycN’s interaction.

a, Oxidized NicA2 was incubated with nicotine under aerobic conditions in the presence (black filled circles) and absence (red filled circles) of oxidized CycN, and the amount of H₂O₂ produced by the reaction was monitored using the Amplex Red assay. Also included were conditions of NicA2 without nicotine (black empty circles) and with CycN but without nicotine (red empty circles). Only in the condition where NicA2 was incubated with nicotine in the absence of CycN was a significant amount of H₂O₂ produced. Three independent replicates were obtained and plotted. b, Oxidized CycN and reduced NicA2 were combined in an anaerobic stopped-flow spectrophotometer and observed for change in absorbance at 542 nm. When mixed in equimolar amounts (black points), absorbance rose and was maintained at an increased value indicating transition to the flavin semiquinone state. When mixed with excess CycN (blue points), NicA2 first reaches the semiquinone state (observable as an increase in absorbance) before becoming fully oxidized (observable as a subsequent decrease in absorbance). c, 40 µM CycN alone (black dashed line) or 40 µM CycN with 200 µM NicA2 (blue line) were run over a HiLoad Superdex 75 pg size exclusion column. CycN in the presence of excess NicA2 eluted with the same retention time as CycN alone and was well-resolved from the NicA2 peak. The insets show the absorbance spectrum of the two peaks in the chromatogram. Fractions 26 and 39 have spectra consistent with clean NicA2 and CycN, respectively, indicating that the two proteins do not bind with high affinity.