Divalent metal ions in plant mitochondria and their role in interactions with proteins and oxidative stress-induced damage to respiratory function

Abstract

Understanding the metal ion content of plant mitochondria and metal ion interactions with the proteome are vital for insights into both normal respiratory function and the process of protein damage during oxidative stress. We have analyzed the metal content of isolated Arabidopsis (Arabidopsis thaliana) mitochondria, revealing a 26:8:6:1 molar ratio for iron:zinc:copper:manganese and trace amounts of cobalt and molybdenum. We show that selective changes occur in mitochondrial copper and iron content following in vivo and in vitro oxidative stresses. Immobilized metal affinity chromatography charged with Cu(2+), Zn(2+), and Co(2+) was used to identify over 100 mitochondrial proteins with metal-binding properties. There were strong correlations between the sets of immobilized metal affinity chromatography-interacting proteins, proteins predicted to contain metal-binding motifs, and protein sets known to be oxidized or degraded during abiotic stress. Mitochondrial respiratory chain pathways and matrix enzymes varied widely in their susceptibility to metal-induced loss of function, showing the selectivity of the process. A detailed study of oxidized residues and predicted metal interaction sites in the tricarboxylic acid cycle enzyme aconitase identified selective oxidation of residues in the active site and showed an approach for broader screening of functionally significant oxidation events in the mitochondrial proteome.

Figure 1.

IMAC affinity purification of proteins from Arabidopsis mitochondria. A, Selective purification of mitochondria protein subsets using different metals to charge IMAC resin and EDTA elution. B, Different strengths of Cu²⁺ affinity to IMAC using NH₄Cl step gradient elution. C and D, Different strengths of Co²⁺ (C) and Zn²⁺ (D) using imidazole gradient elution. E, Venn diagram of proteins identified from each metal-binding set using MS (for details, see Supplemental Tables S1–S4).

Figure 2.

Metal-binding motif analysis of proteins. Combinations of His (H), Cys (C), and Met (M) residues within proximity of 12 amino acid residues were considered putative metal-binding motifs. X represents any amino acid, and n denotes the number of amino acids apart. The frequency of motifs occurring in (A) Cu²⁺-IMAC isolated subset (n = 36 proteins; blue), (B) Co²⁺-IMAC isolated subset (n = 27 proteins; pink), and (C) Zn²⁺-IMAC isolated subset (n = 36 proteins; yellow) was compared with that of the Arabidopsis proteome (n = 26,702 proteins; green) and the Arabidopsis mitochondrial proteome (n = 716 proteins; orange). The top six statistically significant motifs are highlighted in the red boxes.

Figure 3.

Peptides identified for plant mitochondrial aconitase (At2g05710). A, Number of peptides consistently identified by MS common to both mock- and H₂O₂-treated enzymes or unique to each treatment. n = 3 to 4 experiments. Peptides noted appeared in at least two experiments. B, Mapping identified peptides onto the 3D structure of the mammalian aconitase. C, BLAST alignment of the region containing the oxidized peptide in plant mitochondrial aconitases and human IRP1 (2b3y). Oxidized (red) and active site (green) residues are indicated in B and C. Substrate recognition site (yellow) and Fe-S cluster ligation site (orange) residues are shown in B. *, Asp-125 (in C) is a conserved active site residue and was found to be oxidized.