Extended Data Fig. 5: Binding and stopped flow reactions of NicA2 wildtype and v320.

a, Visible absorbance spectra of select time points for the reaction of reduced NicA2 v320 in the presence of 1 mM NMM with O₂ (see Fig. 3b of main text). b, Stopped-flow absorbance traces for the interaction between oxidized NicA2 v320 and NMM at several NMM concentrations. The inset shows the k_obs values of the reaction traces plotted against NMM concentration. Note that the k_obs value was 0.08 s⁻¹ for the secondary decrease in absorbance observed for the reaction of reduced NicA2 v320 in the presence of 1 mM NMM with O₂ (Fig. 3b of main text), which matches the k_obs value when oxidized v320 interacts with 1 mM NMM. c, Reduced NicA2 wildtype and v320 were titrated with NMM, monitoring absorbance at 510 nm to determine the binding coefficient. The inset shows the data points from the titration fit with the tight binding equation. d, Raw traces for the reactions of NicA2 wildtype and v320 with nicotine, both demonstrating biphasic traces. Note the logarithmic x-axis. Raw traces for reactions at all concentrations of nicotine can be seen in Supplementary Fig. 1j–l. e, k_obs values for the reaction of wildtype NicA2 are plotted against the concentration of nicotine. k_obs values from first phase was fit to a hyperbola, k_obs from the second phase was invariant to nicotine concentration. f, k_obs values for the reaction of NicA2 v320 are plotted against the concentration of nicotine, both were invariant to the nicotine concentration. g, The reaction of reduced NicA2 v320 with oxidized CycN was rapid, finishing in roughly 2 seconds similar to the reaction of wildtype NicA2⁹.

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