Extended Data Fig. 9: CycN stopped-flow data.

a, Absorbance traces for the stopped flow reaction between ligand-free NicA2-Fl_red with variable concentrations of CycN_ox. In this case, the traces were able to capture formation and depletion of NicA2-Fl_SQ that occurred as the reaction proceeded. CycN contributes a substantial amount of absorbance at 542 nm. Accordingly, the traces were adjusted so that they all end at the same absorbance value to facilitate comparison. Note the logarithmic timescale. b, Signal change for the stopped-flow reaction was also monitored at 552 nm, a wavelength suitable for observing reduction of CycN. The trace required two exponentials (red curve) with similar amplitudes to fit properly, as one exponential (blue curve) was insufficient. Signal change occurred at the same time as NicA2 oxidation monitored at 542 nm, indicating that the processes occurred simultaneously. Note the logarithmic timescale. c, Signal change at 542 nm for the reaction of NMM-NicA2-Fl_red with CycN_ox. Curiously, traces at 542 nm, where NicA2-Fl_SQ is detectable, did not show the increase in absorbance that we observed with ligand-free NicA2-Fl_red; traces at this wavelength showed a decrease in absorbance that occurred in two phases, with the first phase contributing 80–90% of the total absorbance. This observation suggests that N-methylmyosmine in the active site inhibits NicA2’s FAD from populating a semiquinone state during the reaction with CycN. The decrease in absorbance at 542 nm may simply be due the small decrease in charge-transfer absorbance of N-methylmyosmine bound NicA2 that occurs when the flavin gets oxidized (Extended Data Fig. 4b). Reaction traces at 552 nm still showed the two kinetic phases with increasing absorbance (Extended Data Fig. 9b). Note the logarithmic timescale. d, Absorbance traces at 552 nm for the stopped flow reaction between NMM-NicA2-Fl_red and variable concentrations of CycN_ox. Traces fit best to two exponentials and the k_obs values are reported in Fig. 4c of the main text. The traces were adjusted so that they all begin at the same absorbance value for comparison. Note the logarithmic timescale.