The role of frataxin in fission yeast iron metabolism: implications for Friedreich's ataxia.

BACKGROUND: The neurodegenerative disease Friedreich's ataxia is the result of frataxin deficiency. Frataxin is a mitochondrial protein involved in iron-sulfur cluster (Fe-S) cofactor biogenesis, but its functional role in this pathway is debated. This is due to the interconnectivity of iron metabolic and oxidative stress response pathways that make distinguishing primary effects of frataxin deficiency challenging. Since Fe-S cluster assembly is conserved, frataxin overexpression phenotypes in a simple eukaryotic organism will provide additional insight into frataxin function. METHODS: The Schizosaccharomyces pombe frataxin homologue (fxn1) was overexpressed from a plasmid under a thiamine repressible promoter. The S. pombe transformants were characterized at several expression strengths for cellular growth, mitochondrial organization, iron levels, oxidative stress, and activities of Fe-S cluster containing enzymes. RESULTS: Observed phenotypes were dependent on the amount of Fxn1 overexpression. High Fxn1 overexpression severely inhibited S. pombe growth, impaired mitochondrial membrane integrity and cellular respiration, and led to Fxn1 aggregation. Cellular iron accumulation was observed at moderate Fxn1 overexpression but was most pronounced at high levels of Fxn1. All levels of Fxn1 overexpression up-regulated oxidative stress defense and mitochondrial Fe-S cluster containing enzyme activities. CONCLUSIONS: Despite the presence of oxidative stress and accumulated iron, activation of Fe-S cluster enzymes was common to all levels of Fxn1 overexpression; therefore, Fxn1 may regulate the efficiency of Fe-S cluster biogenesis in S. pombe. GENERAL SIGNIFICANCE: We provide evidence that suggests that dysregulated Fe-S cluster biogenesis is a primary effect of both frataxin overexpression and deficiency as in Friedreich's ataxia.

Spectroscopic properties of the metalloregulatory Cd(II) and Pb(II) sites of S. aureus pI258 CadC.

Staphylococcus aureus pI258 CadC is an extrachromosomally encoded metalloregulatory repressor protein from the ArsR superfamily which negatively regulates the expression of the cad operon in a metal-dependent fashion. The metalloregulatory hypothesis holds that direct binding of thiophilic divalent cations including Cd(II), Pb(II), and Zn(II) by CadC allosterically regulates the DNA binding activity of CadC to the cad operator/promoter (O/P). This report presents a detailed characterization of the metal binding and DNA binding properties of wild-type CadC. The results of analytical ultracentrifugation experiments suggest that both apo- and Cd(1)-CadC are stable or weakly dissociable homodimers characterized by a K(dimer) = 3.0 x 10(6) M(-1) (pH 7.0, 0.20 M NaCl, 25.0 degrees C) with little detectable effect of Cd(II) on the dimerization equilibrium. As determined by optical spectroscopy, the stoichiometry of Cd(II) and Pb(II) binding is approximately 0.7-0.8 mol/mol of wild-type CadC monomer. Chelator (EDTA) competition binding isotherms reveal that Cd(II) binds very tightly, with K(Cd) = 4.3 (+/-1.8) x 10(12) M(-1). The results of UV-Vis and X-ray absorption spectroscopy of the Cd(1) complex are consistent with a tetrathiolate (S(4)) complex formed by four cysteine ligands. The (113)Cd NMR spectrum reveals a single resonance of delta = 622 ppm, consistent with an S(3)(N,O) or unusual upfield-shifted S(4) complex. The Pb(II) complex reveals two prominent absorption bands at 350 nm (epsilon = 4000 M(-1) cm(-1)) and 250 nm (epsilon = 41 000 M(-1) cm(-1)), spectral properties consistent with three or four thiolate ligands to the Pb(II) ion. The change in the anisotropy of a fluorescein-labeled oligonucleotide containing the cad O/P upon binding CadC and analyzed using a dissociable CadC dimer binding model reveals that apo-CadC forms a high-affinity complex [K(a) = (1.1 +/- 0.3) x 10(9) M(-1); pH 7.0, 0.40 M NaCl, 25 degrees C], the affinity of which is reduced approximately 300-fold upon the binding of a single molar equivalent of Cd(II) or Pb(II). The implications of these findings on the mechanism of metalloregulation are discussed.