Focusing on the Human Mitochondrial Desulfurase NFS1

SEWELL EK; CASTRO IH; SANTOS J

La Plata, Buenos Aires

Congreso; Reunión Anual de la Sociedad Argentina de Biofísica; 2018

SAB

Focusing on the Human Mitochondrial Desulfurase NFS1Ellioth Sewell1,2, Ignacio Castro1,2, Javier Santos1,21Instituto de Química y Fisicoquímica Biológicas, Dr. Alejandro Paladini (UBA-CONICET). C.A.B.A.2Departamento de Fisiología y Biología Molecular y Celular. Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. C.A.B.A.[Fe-S] cluster biosynthesis is a multifaceted mitochondrial process that requires the multiprotein supercomplex NFS1/ACP-ISD11/ISCU, with the positive allosteric modulation of FXN. It is worth noticing that dimerization of NFS1 is key for the proper activity, and therefore the complete active supercomplex is composed of two symmetrically organized molecules of each one of the four proteins. The involvement of each protein in this supercomplex is thought to have crucial and specific consequences in its functioning. For instance, ACP-ISD11 heterodimer is known to stabilize the formation of the NFS1 dimer preventing aggregation, and desulfurase activity is heavily unpaired in its absence. On the other hand, ISCU has a modulatory effect on the desulfurase activity and it is proposed to work as a scaffold for the assembly of the [Fe-S] cluster itself. FXN, has a strong positive effect on the modulation of the activity and it is proposed to bind the iron required for the reaction. Finally, NFS1 is the one responsible for the desulfurase activity itself. By a reaction that involves NFS1 prosthetic group PLP, the enzyme transforms free cysteine to free alanine and forms a persulfide bond with one of its own cysteine residues. This sulfide acquired by NFS1 will be part of the cluster, later on in the reaction sequence.Focusing on the recombinant human NFS1 protein, it was produced and purified requiring strategies to avoid aggregation. Its activity was assessed in the presence of the other protein components of the supercomplex. Desulfurase activity was assessed by measuring the hydrogen sulfide produced during the reaction. Additionally, the activation capability of different FXN variants was studied. As the activity of the supercomplex heavily depends on the interactions between subunits, a NFS1 double mutant containing a single tryptophan (Trp97) was design to study binding equilibrium. Our goal is to accurately measure the shifts in the fluorescence signals due to interactions of the different subunits, as well as a broad characterization comparing with the wild-type NFS1.