Unique roles of iron and zinc binding to the yeast Fe-S cluster scaffold assembly protein "Isu1"

Abstract

Mitochondrial Fe-S cluster biosynthesis is accomplished within yeast utilizing the biophysical attributes of the "Isu1" scaffold assembly protein. As a member of a highly homologous protein family, Isu1 has sequence conservation between orthologs and a conserved ability to assemble [2Fe-2S] clusters. Regardless of species, scaffold orthologs have been shown to exist in both "disordered" and "structured" conformations, a structural architecture that is directly related to conformations utilized during Fe-S cluster assembly. During assembly, the scaffold helps direct the delivery and utilization of Fe(ii) and persulfide substrates to produce [2Fe-2S] clusters, however Zn(ii) binding alters the activity of the scaffold while at the same time stabilizes the protein in its structured state. Additional studies confirm Zn binds to the scaffold's Cys rich active site, and has an impact on the protein's ability to make Fe-S clusters. Understanding the interplay between Fe(ii) and Zn(ii) binding to Isu1 in vitro may help clarify metal loading events that occur during Fe-S cluster assembly in vivo. Here we determine the metal : protein stoichiometry for Isu1 Zn and Fe binding to be 1 : 1 and 2 : 1, respectively. As expected, while Zn binding shifts the Isu1 to its structured state, folding is not influenced by Fe(ii) binding. X-ray absorption spectroscopy (XAS) confirms Zn(ii) binds to the scaffold's cysteine rich active site but Fe(ii) binds at a location distinct from the active site. XAS results show Isu1 binding initially of either Fe(ii) or Zn(ii) does not significantly perturb the metal site structure of alternate metal. XAS confirmed that four scaffold orthologs bind iron as high-spin Fe(ii) at a site composed of ca. 6 oxygen and nitrogen nearest neighbor ligands. Finally, in our report Zn binding dramatically reduces the Fe-S cluster assembly activity of Isu1 even in the presence of frataxin. Given the Fe-binding activity we report for Isu1 and its orthologs here, a possible mechanism involving Fe(ii) transport to the scaffold's active site during cluster assembly has been considered.

Figure 1.. Fe and Zn Binding Affinity to Isu1 Measured using Mag-Fura-2 within a Competition Based Assay.

(A) Representative titration spectra of iron into Isu1 and (B) zinc into Isu1. Initial scan for metal titrations is shown in red with subsequent titrations in black. Mag-fura-2 to protein ratio was varied from 1:2 (red), 1:1 (black), 2:1 (blue) for Fe (C) and Zn titrations (D), respectively. Spectra were collected in duplicate using independent samples to ensure spectral reproducibility.

Figure 2.. Circular Dichroism of Apo, Fe- and Zn-Loaded Isu1.

Representative spectra of apo- (solid line) and 2Fe-loaded (dashed line) Isu1 in panel (A). Representative spectra of apo- (solid) and Zn-loaded (dashed) Isu1 in panel (B). An average of 30 scans were collected for each representative spectrum displayed; trials were performed in duplicate with independent samples to ensure spectral reproducibility.

Figure 3.. Normalized XANES for Fe- and Zn-Loaded Isu1.

(A) Representative spectra for Fe XANES for 2Fe-Isu1 (red) and 1Zn/2Fe-Isu1 (black). (B) Expended display of the Fe-XANES pre-edge features for 2Fe-Isu1 (red) and 1Zn/2Fe-Isu1 (black). (C) Representative Zn XANES for 1Zn-Isu1 (blue) and 1Zn/2Fe-Isu1 (black).

Figure 4:. Raw EXAFS, Fourier Transforms of EXAFS and Spectral Simulations for Fe- and Zn-Isu1.

Full Fe EXAFS for 2Fe-Isu1 and 1Zn/2Fe-Isu1 are shown in (A) and (C), respectively. Full Zn EXAFS for 1Zn-Isu1 and 1Zn/2Fe-Isu1 are shown in (E) and (G), respectively. Fourier transforms for the Fe EXAFS 2Fe-Isu1 and 1Zn/2Fe-Isu1 are shown in (B) and (D), respectively. Fourier transforms for the Zn EXAFS for 1Zn-Isu1 and 1Zn/2Fe-Isu1 are shown in (F) and (H), respectively. Raw data is shown in black while the best-fit simulations for each data set are shown in green.

Figure 5.. EXAFS, Fourier Transforms of EXAFS and Spectral Simulations for Fe-Loaded Isu1 orthologs.

Fe-EXAFS spectra and Fourier transforms of the Fe-EXAFS are shown respectively for a single iron atom bound to the following Isu1 orthologs: H. sapiens (panels A and B), D. melanogaster (panels C and D), S. cerevisiae (panels E and F) and T. maritima (panels G and H). Raw data is shown in black while simulated data is shown in green.

Figure 6.. Sequence alignment of Isu1 orthologs.

Mature protein sequences from H. sapiens, D. melanogaster, S. cerevisiae and T. maritima. Identical residues are shown in blue and conserved/semi conserved residues are shown in green. Conserved 3-Cys active site consists of C69, C96, and C139 (magenta) and D71 (yellow) or H138 (orange) according to the consensus ruler. The methionine conserved only in Eukaryotes (M141) is highlighted in red.

Figure 7.. Sequence Alignment of Isu1 Orthologs.

Mature protein sequences from H. sapiens, D. melanogaster, S. cerevisiae and T. maritima. Identical residues are shown in blue and conserved/semi conserved residues are shown in green. Conserved 3-Cys active site consists of C69, C96, and C139 (magenta) and D71 (yellow) or H138 (orange) according to the consensus ruler. The methionine conserved only in Eukaryotes (M141) is highlighted in red.

Figure 8.. Modeled Zinc Binding Site on Zn-Isu1.

Zn ion (green) coordinated by C69, C96, C139 (magenta) and H138 (orange) or D71 (yellow). Modeled using I-Tasser server.^,