Fig. 3

Characterization of NascB in vitro and the proposed mechanism. a HPLC analysis of the NascB-catalyzed reaction. (I) Standard NAS-C, (II) a control reaction, the same as NascB reaction but only omitting the P450 NascB, (III) NascB reaction, using E. coli Fdx and FdxR as electron supply system, (IV) NascB reaction, using spinach Fd and FdR as electron supply system. b X-band (9.3810 GHz) CW EPR spectra recorded at 15 K showing the low-spin (LS) ferric heme signal in the absence (the upper panel) and presence (the lower panel) of the substrate (twofold excess of cW_L-P_L). Simulations computed using the principal g-values are shown in red: (the upper panel) two component model with g = (2.407, 2.253, 1.911) and g = (2.440, 2.245, 1.910); (the lower panel) single component model, g = (2.420, 2.251, 1.918). c Proposed mechanisms of the radical-mediated intramolecular cyclization and intermolecular addition reaction by NascB. The NascB mechanism is proposed to involve the active species compound I (Fe^IV = O+•)^24,25,26,34. It abstracts a proton with an electron from the HN¹ of the substrate to form the active species compound II (Fe^IV(-OH)), which is further converted into the ferric species (1) by accepting a hydrogen radical from C6′ position of the substrate to form one water molecule. The water coordination turns NascB into the resting state P450, completing the catalytic cycle^12,35. A significant high Gibbs free energy (△G) calculated by DFT calculation rule out the possibility of N¹⁰ radical (marked by a dashed rectangle with a red cross). The possibility of Friedel–Crafts reaction from pyrroloindoline C³-radical was ruled out by the insensitivity of NascB-catalyzed reaction to the electron-withdrawing group introduced at C⁷-position of the substrates