Nicotianamine chelates both FeIII and FeII. Implications for metal transport in plants

Abstract

Nicotianamine (NA) occurs in all plants and chelates metal cations, including FeII, but reportedly not FeIII. However, a comparison of the FeII and ZnII affinity constants of NA and various FeIII-chelating aminocarboxylates suggested that NA should chelate FeIII. High-voltage electrophoresis of the FeNA complex formed in the presence of FeIII showed that the complex had a net charge of 0, consistent with the hexadentate chelation of FeIII. Measurement of the affinity constant for FeIII yielded a value of 10(20.6), which is greater than that for the association of NA with FeII (10(12.8)). However, capillary electrophoresis showed that in the presence of FeII and FeIII, NA preferentially chelates FeII, indicating that the FeIINA complex is kinetically stable under aerobic conditions. Furthermore, Fe complexes of NA are relatively poor Fenton reagents, as measured by their ability to mediate H2O2-dependent oxidation of deoxyribose. This suggests that NA will have an important role in scavenging Fe and protecting the cell from oxidative damage. The pH dependence of metal ion chelation by NA and a typical phytosiderophore, 2'-deoxymugineic acid, indicated that although both have the ability to chelate Fe, when both are present, 2'-deoxymugineic acid dominates the chelation process at acidic pH values, whereas NA dominates at alkaline pH values. The consequences for the role of NA in the long-distance transport of metals in the xylem and phloem are discussed.

Figure 1

The structures of the aminocarboxylate ligands used in the study and of DFO.

Figure 2

Computer-generated models of the Fe^III complexes of NA and DMA. The coordinates are based on the Co^III complex of MA (Sugiura et al., 1981) and the ionization state demonstrated by the high-voltage electrophoresis data in Figure 3.

Figure 3

Net charge of Fe^III complexes of NA (•) and MA (○) as determined by high-voltage paper electrophoresis at pH 7.0 under aerobic conditions in the dark.

Figure 4

Spectrophotometric titration against maltol of a 43.5 μm solution of Fe^IIINA complex. Fe^IIINA dissociates with increasing concentrations of maltol, leading to the formation of the orange Fe^IIImaltol complex. The concentrations of maltol used are indicated.

Figure 5

The influence of pH on pFe³⁺ (A) and pFe²⁺ (B) values for hydroxide, NA, DMA, EDTA, and DFO. The total Fe concentration was 1 μm and the total ligand concentration was 10 μm. The higher the pM value, the more effective the ligand.

Figure 6

Migration times of NA and its Fe complexes as measured by CE. A, NA alone; B, Fe^IINA; C, Fe^IIINA.

Figure 7

Computer simulations of the pH dependence of the Fe complexes of NA and DMA. A, Fe^III and NA; B, Fe^III and DMA; C, competition for Fe^III between NA and DMA; D, Fe^II and NA. In all cases, the total Fe concentration is 1 μm, and NA or DMA is present at 10 μm.

Figure 8

Computer simulations of the pH dependence of the Zn and Cu complexes of NA and MA. A, Zn^II and NA; B, Cu^II and NA; C, competition for Zn^II between NA and DMA; D, competition for Cu^II between NA and DMA. The total concentration of Zn^II or Cu^II is 1 μm, and NA or DMA is present at 10 μm.

Figure 9

Computer simulations of the competition between citrate and NA for Fe^III (A) and Fe^II (B). Metal and ligand concentrations are those estimated for the xylem: total Fe = 40 μm, citrate (Cit) = 150 μm, and NA = 20 μm.

Figure 10

Competition for Fe^II between NA and citrate at pH 5.5, as monitored by CE. Citrate was added at 0 min and the amount of Fe^IINA was measured by CE at the times indicated. The inset shows traces from the CE at 2 min (A) and 12 min (B). In each trace, the left peak is NA and the right peak is Fe^IINA.