Direct Cysteine Desulfurase Activity Determination by NMR and the Study of the Functional Role of Key Structural Elements of Human NFS1

SEWELL, KARL ELLIOTH; GOLA, GABRIEL F.; PIGNATARO, MARÍA FLORENCIA; HERRERA, MARÍA GEORGINA; NOGUERA, MARTÍN EZEQUIEL; OLMOS, JUSTO; RAMÍREZ, JAVIER A; CAPECE, LUCIANA; ARAN, MARTÍN; SANTOS, JAVIER

ACS CHEMICAL BIOLOGY

AMER CHEMICAL SOC

Lugar: Washington; Año: 2023

1554-8929

The mitochondrial cysteine desulfurase NFS1 is an essential PLP-dependent enzyme involved iniron–sulfur cluster assembly. The enzyme catalyzes the desulfurization of the L-Cys substrate, producing a persulfide and L-Ala as the products. In this study, we set the measurement of theproduct L-Ala by NMR in vitro by means of 1H -NMR spectra acquisition. This methodologyprovided us with the possibility of monitoring the reaction in both fixed-time and real-timeexperiments, with high sensitivity and accuracy. By studying the I452A, W454A, Q456A, andH457A NFS1 variants, we found that the C-terminal stretch (CTS) of the enzyme is critical forfunction. Specifically, mutation of the extremely conserved position W454 resulted in highlydecreased activity. Additionally, we worked on two singular variants: “GGG” and C158A. Inthe former, the catalytic Cys-loop was altered by including two Gly residues to increase theflexibility of this loop. This variant had significantly impaired activity, indicating that the Cysloop motions are fine-tuned in the wild-type enzyme. In turn, for C158A, we found anunanticipated increase in L-Cys desulfurase activity. Furthermore, we carried out moleculardynamics simulations of the supercomplex dedicated to iron–sulfur cluster biosynthesis, whichincludes NFS1, ACP, ISD11, ISCU, and FXN subunits. We identified CTS as a key element thatestablished interactions with ISCU2 and FXN concurrently; we found specific interactions thatare established when FXN is present, reinforcing the idea that FXN not only forms part of theiron–sulfur cluster assembly site, but also modulates the internal motions of ISCU2.