Effector role reversal during evolution: the case of frataxin in Fe-S cluster biosynthesis

Abstract

Human frataxin (FXN) has been intensively studied since the discovery that the FXN gene is associated with the neurodegenerative disease Friedreich's ataxia. Human FXN is a component of the NFS1-ISD11-ISCU2-FXN (SDUF) core Fe-S assembly complex and activates the cysteine desulfurase and Fe-S cluster biosynthesis reactions. In contrast, the Escherichia coli FXN homologue CyaY inhibits Fe-S cluster biosynthesis. To resolve this discrepancy, enzyme kinetic experiments were performed for the human and E. coli systems in which analogous cysteine desulfurase, Fe-S assembly scaffold, and frataxin components were interchanged. Surprisingly, our results reveal that activation or inhibition by the frataxin homologue is determined by which cysteine desulfurase is present and not by the identity of the frataxin homologue. These data are consistent with a model in which the frataxin-less Fe-S assembly complex exists as a mixture of functional and nonfunctional states, which are stabilized by binding of frataxin homologues. Intriguingly, this appears to be an unusual example in which modifications to an enzyme during evolution inverts or reverses the mode of control imparted by a regulatory molecule.

Figure 1

Determination of protein ratios that favor complex formation. Cysteine desulfurase activities were measured for (A) the human SD complex with increasing amounts of ISCU2 or IscU_ec, and for (B) the SDU and (C) SDU_ec complexes with increasing amounts of FXN or CyaY. (D) The cysteine desulfurase activity was measured for E. coli IscS with increasing amounts of ISCU2 or IscU_ec.

Figure 2

Stimulation of cysteine desulfurase reaction by addition of frataxin homologs. The k_cat for the cysteine desulfurase reaction was plotted for complexes of human SD with the scaffold protein (ISCU2 or IscU_ec) and frataxin (FXN or CyaY).

Figure 3

Iron-dependent perturbation of cysteine desulfurase activity for SD complexes. (A) Cysteine desulfurase activity plotted as a function of added ferrous iron. (B) Plot of k_cat values for the cysteine desulfurase reaction of SD complexes with (black) and without (gray) 10 equiv of ferrous iron.

Figure 4

Stimulation of Fe-S cluster assembly activity by frataxin homologs. (A) The Fe-S assembly activities were measured at 10 °C for the Na₂S control and SDU, SDUC_ec, and SDUF complexes. The inset shows the activities of the SDU, SDUC_ec, and Na₂S control at 25 °C. (B) The Fe-S assembly activities were measured at 10 °C for the Na₂S control and SDU_ec, SDU_ecC_ec, and SDU_ecF complexes.

Figure 5

Cysteine desulfurase activities for IscS complexes with and without iron. (A) The k_cat for the cysteine desulfurase was plotted for complexes of E. coli IscS (S_ec) without (gray or colored) and with (black) 10 equiv of ferrous iron. (B) The cysteine desulfurase activities for IscS complexes were measured with 100 μM L-cysteine and increasing amounts of ferrous iron.

Figure 6

Frataxin-based inhibition of Fe-S cluster formation for IscS complexes. The rate of Fe-S cluster formation were measured for the S_ecU_ec complex with increasing amounts of (A) CyaY and (C) FXN, and for the S_ecU complex with increasing amounts of (B) CyaY and (D) FXN.

Figure 7

Working model for frataxin regulation of Fe-S cluster biosynthesis. In eukaryotes, a pre-equilibrium model is proposed in the absence of FXN in which a cysteine desulfurase and Fe-S cluster assembly deficient form is favored over a functional form of the SDU complex. FXN binding stabilizes the functional form and promotes sulfur transfer from NFS1 to ISCU2 and Fe-S cluster synthesis activities. In contrast, the prokaryotic S_ecU_ec complex that lacks CyaY exhibits cysteine desulfurase and Fe-S assembly activities. CyaY binding may induce a conformational change in the S_ecU_ec complex that does not significantly affect the cysteine desulfurase activity, but abolishes Fe-S cluster synthesis. The “ON” and “OFF” labels indicate the Fe-S assembly activity of the respective complexes, the blue arrows from the PLP to the mobile Cys loop indicate the presence of cysteine desulfurase activity, and the blue arrows from the Cys loop to the scaffold protein (shown in green) indicate interprotein sulfur transfer. The displayed model represents half of the expected dimeric Fe-S assembly complexes.