Iron-Sulfur Cluster Complex Assembly in the Mitochondria of Arabidopsis thaliana

Abstract

In plants, the cysteine desulfurase (AtNFS1) and frataxin (AtFH) are involved in the formation of Fe-S groups in mitochondria, specifically, in Fe and sulfur loading onto scaffold proteins, and the subsequent formation of the mature Fe-S cluster. We found that the small mitochondrial chaperone, AtISD11, and AtFH are positive regulators for AtNFS1 activity in Arabidopsis. Moreover, when the three proteins were incubated together, a stronger attenuation of the Fenton reaction was observed compared to that observed with AtFH alone. Using pull-down assays, we found that these three proteins physically interact, and sequence alignment and docking studies showed that several amino acid residues reported as critical for the interaction of their human homologous are conserved. Our results suggest that AtFH, AtNFS1 and AtISD11 form a multiprotein complex that could be involved in different stages of the iron-sulfur cluster (ISC) pathway in plant mitochondria.

Keywords: Arabidopsis; frataxin; mitochondria.

Figure 1

Kinetic assays of AtNFS1:AtISD11:AtFH. L-Ala production from cysteine catalyzed by AtNFS1 in the presence of AtISD11 (1:1, circles); AtFH (1:1, diamonds); AtISD11 and AtFH (1:1:1, black squares) or AtNFS1 alone (empty squares). One unit of activity is defined as the amount of enzyme catalyzing the production of 1 µmol of L-alanine per min. at 37 °C.

Figure 2

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of recombinant proteins. SDS-PAGE analysis of the pull-down assay of the recombinant proteins AtNFS1 (containing a His₆ sequence), AtISD11 and AtFH. Lane 1, AtISD11 was recovered with AtNFS1; lane 2, AtISD11 and AtFH were recovered together with AtNFS1; lane 3, AtNFS1 recovered from the Ni⁺² resin; lane 4, non-specific binding control of AtISD11 and AtFH. MM shows the molecular markers used. Arrows indicate the position of each protein band.

Figure 3

Homology modeling of iron–sulfur cluster (ISC) complex and docking studies. (A) Surface and (B) ribbon models of the protein complex formed by AtNFS1 (light blue), AtISD11 (red), and AtFH (yellow). The docking model was predicted using HADDOCK 2.4. (C) Amino acid sequence alignment of human (FXN) and Arabidopsis frataxin (AtFH) and (D) human (HsNFS1) and Arabidopsis (AtNFS1) cysteine desulfurases. The amino acids highlighted with color in (C) interact with the amino acid with the same color in (D). The amino acids marked in grey in (C) are part of the negatively charged patch in AtFH. Amino acids marked in grey in D are part of the Arg-rich patch in AtNFS1, and those marked in blue are part of the negatively charged helix region that interacts with the α2-helix from AtISD11. (E) Interaction of the acidic AtFH patch with Arg residues on AtNFS1.

Figure 3

Homology modeling of iron–sulfur cluster (ISC) complex and docking studies. (A) Surface and (B) ribbon models of the protein complex formed by AtNFS1 (light blue), AtISD11 (red), and AtFH (yellow). The docking model was predicted using HADDOCK 2.4. (C) Amino acid sequence alignment of human (FXN) and Arabidopsis frataxin (AtFH) and (D) human (HsNFS1) and Arabidopsis (AtNFS1) cysteine desulfurases. The amino acids highlighted with color in (C) interact with the amino acid with the same color in (D). The amino acids marked in grey in (C) are part of the negatively charged patch in AtFH. Amino acids marked in grey in D are part of the Arg-rich patch in AtNFS1, and those marked in blue are part of the negatively charged helix region that interacts with the α2-helix from AtISD11. (E) Interaction of the acidic AtFH patch with Arg residues on AtNFS1.

Figure 4

Secondary structure, sequence alignment and docking studies of AtISD11. (A) Secondary structure of AtISD11 showing the amino acids which interact with AtNFS1 in the protein complex. (B) Amino acid sequence alignment of human (HsISD11) and Arabidopsis (AtISD11) ISD11. Amino acids in grey are predicted to be critical for HsISD11 function. The region marked in bold, from Ser48 to Leu72 in AtISD11 is the second helix region (α2-helix) which interacts with AtNFS1. (C) Modeled interaction between AtNFS1 (light blue) and AtISD11 (red) showing important residues possibly involved in the interaction.

Figure 5

Oxidative degradation assays. Production of malondialdehyde from dRibose, Fe(II), and H₂O₂ in the absence or presence of the recombinant proteins AtFH, AtNFS1, and AtISD11 or bovine serum albumin (BSA). Letters indicate significant differences (p < 0.05).