Iron binding activity is essential for the function of IscA in iron-sulphur cluster biogenesis

Abstract

Iron-sulphur cluster biogenesis requires coordinated delivery of iron and sulphur to scaffold proteins, followed by transfer of the assembled clusters from scaffold proteins to target proteins. This complex process is accomplished by a group of dedicated iron-sulphur cluster assembly proteins that are conserved from bacteria to humans. While sulphur in iron-sulphur clusters is provided by L-cysteine via cysteine desulfurase, the iron donor(s) for iron-sulphur cluster assembly remains largely elusive. Here we report that among the primary iron-sulphur cluster assembly proteins, IscA has a unique and strong binding activity for mononuclear iron in vitro and in vivo. Furthermore, the ferric iron centre tightly bound in IscA can be readily extruded by l-cysteine, followed by reduction to ferrous iron for iron-sulphur cluster biogenesis. Substitution of the highly conserved residue tyrosine 40 with phenylalanine (Y40F) in IscA results in a mutant protein that has a diminished iron binding affinity but retains the iron-sulphur cluster binding activity. Genetic complementation studies show that the IscA Y40F mutant is inactive in vivo, suggesting that the iron binding activity is essential for the function of IscA in iron-sulphur cluster biogenesis.

Fig. 1

Iron binding of primary iron-sulphur cluster assembly proteins. Iron-sulphur cluster assembly proteins IscS, IscU, IscA, HscB and HscA were purified from E. coli cells grown in LB media. a), iron content of purified iron-sulphur cluster assembly proteins. Iron content was presented as the ratio of iron atoms per protein dimer. b), iron content of iron-sulphur cluster assembly proteins (50 μM in monomer) after incubation with Fe(NH₄)₂(SO₄)₂ (50 μM), sodium citrate (5 mM) and dithiothreitol (2 mM). Proteins were re-purified from incubation solutions. Iron content was presented as the ratio of iron atoms per protein dimer. The data are averages plus standard deviations from three independent experiments.

Fig. 2

L-cysteine-mediated iron release from IscA. Iron-bound IscA dimer (300 μM) was incubated with 2 mM L-cysteine under aerobic conditions. Aliquots were taken at indicated time and frozen immediately for the liquid helium-temperature EPR measurements.

Fig. 3

Location of Tyr-40 in E. coli IscA dimer. Tyr-40 is located in the interface between two IscA monomers, and is in the vicinity of the putative iron binding site (adapted from ). The conserved iron binding residues (Cys-99 and Cys-101) of IscA are not visible in the electron density map, likely because of the high flexibility.

Fig. 4

IscA mutant Y40F has a diminished iron binding activity. a), purified wild-type IscA dimer (25 μM) was incubated with indicated concentrations of Fe(NH₄)₂(SO₄)₂ in the presence of dithiothreitol (2 mM) at room temperature for 20 min, followed by re-purification of protein. Spectra were calibrated to the same amplitude of the absorption peak at 260 nm of the IscA sample after reconstitution with two-fold excess of iron. b), same as in a), except IscA mutant Y40F dimer (25 μM) was used. Spectra were calibrated to the same amplitude of the absorption peak at 260 nm of the Y40F sample after reconstitution with two-fold excess of iron.

Fig. 5

In vitro iron-sulphur cluster assembly in IscA and IscA mutant Y40F. Purified IscA mutant Y40F (spectrum 1) or wild-type IscA dimer (spectrum 2) (25 μM) was incubated with IscS (0.5 μM), Fe(NH₄)₂(SO₄)₂ (100 μM), dithiothreitol (2 mM) in buffer containing Tris (20 mM, pH 8.0) and NaCl (200 mM) at 37°C under anaerobic conditions. L-cysteine (1 mM) was then added to the incubation solutions to initiate the iron-sulphur cluster assembly reaction. Spectra were taken 20 min after addition of L-cysteine. The absorption peak at 415 nm reflects the iron-sulphur cluster formation in IscA.

Fig. 6

Complementary activity of the IscA mutant Y40F in E. coli mutant with deletion of IscA and its paralog SufA. The E. coli wild-type (MC4100) and the mutant cells with deletion of IscA/SufA (iscA⁻/sufA⁻) containing expression plasmids were grown in M9 minimal media at 37°C under aerobic conditions. Cell growth was monitored at O.D. at 600 nm for 14 hours after inoculation of 1:100 dilutions.