Iron-binding activity of human iron-sulfur cluster assembly protein hIscA1

Abstract

A human homologue of the iron-sulfur cluster assembly protein IscA (hIscA1) has been cloned and expressed in Escherichia coli cells. The UV-visible absorption and EPR (electron paramagnetic resonance) measurements reveal that hIscA1 purified from E. coli cells contains a mononuclear iron centre and that the iron binding in hIscA1 expressed in E. coli cells can be further modulated by the iron content in the cell growth medium. Additional studies show that purified hIscA1 binds iron with an iron association constant of approx. 2x1019 M-1, and that the iron-bound hIscA1 is able to provide the iron for the iron-sulfur cluster assembly in a proposed scaffold protein, IscU of E. coli, in vitro. The complementation experiments indicate that hIscA1 can partially substitute for IscA in restoring the cell growth of E. coli in the M9 minimal medium under aerobic conditions. The results suggest that hIscA1, like E. coli IscA, is an iron-binding protein that may act as an iron chaperone for biogenesis of iron-sulfur clusters.

Figure 1. UV-visible and EPR spectra of purified human IscA homologue hIscA1

HiscA1 was purified from E. coli cells grown in the LB growth medium. A) UV-visible spectra of hIscA1. Purified hIscA1 (50 µM) (spectrum 2) was incubated with EDTA (10 mM) and L-cysteine (2 mM) at 37°C for 60 min (spectrum 1). The iron-depleted hIscA1 (50 µM) was re-incubated with Fe(NH)₂(SO₄)₂ (50 µM) and dithiothreitol (2 mM) (spectrum 3). Each protein was re-purified by passing through a High-Trap Desalting column after incubation. B) EPR spectra of hIscA1. The protein samples were prepared as described in A), except that the protein concentration was ~450 µM.

Figure 2. Iron binding activity of hIscA1 in vitro

A) Iron binding titration of hIscA1. The iron-depleted hIscA1 (50 µM) was incubated with Fe(NH)₂(SO₄)₂ (0 to 100 µM) in the presence of dithiothreitol (2 mM) at 37°C for 30 min, followed by re-purification of hIscA1. The absorption amplitude at 315 nm of re-purified hIscA1 was plotted as a function of the Fe(NH)₂(SO₄)₂ concentration in the incubation solution. B) Iron binding competition of hIscA1. The iron-saturated hIscA1 (100 µM) was incubated with sodium citrate (0 to 100 mM) in the presence of dithiothreitol (2 mM) at 37°C for 30 min, followed by re-purification of hIscA1. The absorption amplitude at 315 nm of re-purified hIscA1 was plotted as a function of the sodium citrate concentration in the incubation solution. The results are the means ± SD from three independent experiments

Figure 3. Iron binding activity of hIscA1 in E. coli cells

A) The UV-visible absorption spectra of hIscA1 purified from the E. coli cells grown in the M9 minimal medium supplemented with (spectrum 1) or without (spectrum 2) 50 µM ferrous ammonium sulfate. The protein concentration was approx. 50 µM. The insert is a photograph of the SDS/PAGE gel of hIscA1 purified from the E. coli cells grown in the M9 minimal medium supplemented with (lane 1) or without (lane 2) 50 µM ferrous ammonium sulfate. B) The UV-visible absorption spectra of human frataxin purified from the E. coli cells grown in the M9 minimal medium supplemented with (spectrum 1) or without (spectrum 2) 50 µM ferrous ammonium sulfate. The protein concentration was approx. 20 µM. The insert is a photograph of the SDS/PAGE gel of frataxin purified from the E. coli cells grown in the M9 minimal medium supplemented with (lane 1) or without (lane 2) 50 µM ferrous ammonium sulfate. The results are representatives from three independent experiments.

Figure 4. The iron-bound hIscA1 acts as an iron donor for the iron-sulfur cluster assembly in E. coli IscU in vitro

A) Purified E. coli IscU (50 µM) was incubated with E. coli IscS (1 µM), the iron-bound hIscA1 (100 µM), Tris (20 mM, pH 8.0) and NaCl (200 mM) in the presence of dithiothreitol (2 mM) at 37°C for 5 min. L-cysteine (1 mM) was then added to initiate the iron-sulfur cluster assembly reaction. The absorption peak at 456 nm represents formation of the IscU [2Fe-2S] cluster. Spectra were taken every 2 min for 24 min. B) Same as in A) except the iron-bound hIscA1 was replaced with the iron-bound E. coli IscA in the incubation solution.

Figure 5. Complementary role of hIscA1 in E. coli cells

The E. coli iscA⁻¹/sufA⁻¹ double mutant cells containing plasmid expressing E. coli IscA (pBAD/iscA) (A), hIscA1 (pBAD/hiscA1) (B), or the vector only (pBAD) (D), were spotted on the minimal medium plates supplemented with glucose (0.2%) and arabinose (0.002%). The wild-type strain MC4100 cells (C) were also used as control. The plate was incubated at 37°C for 48 hours under aerobic conditions before the photography was taken.