Figure 3.

AncFMO1 stopped-flow kinetics.A, reductive half-reaction rates measured in technical triplicates under anaerobic conditions with increasing concentrations of NADH (blue points) or NADPH (red points). The binding constants K_d and k_red values were calculated by fitting the reductive half reaction to the Michaelis–Menten equation with the SD reported in brackets. B, reoxidation spectra of the reduced AncFMO1 with oxygenated buffer over time. C, reoxidation rates with increasing concentration of dioxygen, observed at 368 nm (green triangles) and 448 nm (blue points) in technical triplicates. The dotted lines correspond to the atmospheric concentration of dioxygen (260 μM), present during steady-state kinetics. AncFMO, ancestral flavin-containing monooxygenase; FMO, flavin-containing monooxygenase.