Extended Data Fig. 7: NFS1 can generate a D-Cys-ketimine intermediate but is unable to mobilise sulfur.

a, Cartoon of the reaction cycle of the cysteine desulfurase NFS1 with L-Cys. The resting state of the enzyme comprises an internal (that is enzyme-bound) aldimine, by which the pyridoxal phosphate (PLP) cofactor is covalently bound to Lys258 as a Schiff base (1). Cys381 and His156 are positioned close to the PLP. Binding of free L-Cys creates an external (that is non-enzyme-bound) L-Cys-aldimine (2) which tautomerizes (catalysed by His156) to an external L-Cys-ketimine (3). The sulfur atom (yellow ball) can be released from L-Cys ketimine and transferred to Cys381, thereby creating an external L-Ala-ketimine and a persulfide (-SSH) on Cys381 (4). The resting state 1 of the enzyme with its internal aldimine is regenerated by rapid release of free L-Ala. Residue numbering is for human NFS1. The reaction scheme was adopted from refs. ^40,41. b, c, Time courses of the generation of the L- and D-Cys-ketimine intermediate (absorption at 340 nm, blue) from the internal aldimine (416 nm, red) by the indicated (NIA)₂ complexes. Data were taken from the spectra shown in Fig. 4b, c. d, Cartoon of the NFS1 reaction with D-Cys. The enzyme can efficiently and rapidly form the D-Cys-aldimine (2) and -ketimine intermediates (3), but for steric reasons cannot transfer the sulfur to Cys381, unlike with L-Cys (a). For 3D inspection of the models in parts a,d, a Chimera X session has been added as Supplementary File 1 (L_D-Cys_Cycle_final.X.py), using the following 3D structures: PDB ID: 7E6D, 6O13, 6O11, 7XEJ. refs. ^40,41.