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. 2010 Jul 23;285(30):23331-41.
doi: 10.1074/jbc.M110.127449. Epub 2010 May 11.

Iron-sulfur (Fe-S) cluster assembly: the SufBCD complex is a new type of Fe-S scaffold with a flavin redox cofactor

Silke Wollers  1 , Gunhild Layer, Ricardo Garcia-Serres, Luca Signor, Martin Clemancey, Jean-Marc Latour, Marc Fontecave, Sandrine Ollagnier de Choudens
Affiliations

Iron-sulfur (Fe-S) cluster assembly: the SufBCD complex is a new type of Fe-S scaffold with a flavin redox cofactor

Silke Wollers et al. J Biol Chem. .

Abstract

Assembly of iron-sulfur (Fe-S) clusters and maturation of Fe-S proteins in vivo require complex machineries. In Escherichia coli, under adverse stress conditions, this process is achieved by the SUF system that contains six proteins as follows: SufA, SufB, SufC, SufD, SufS, and SufE. Here, we provide a detailed characterization of the SufBCD complex whose function was so far unknown. Using biochemical and spectroscopic analyses, we demonstrate the following: (i) the complex as isolated exists mainly in a 1:2:1 (B:C:D) stoichiometry; (ii) the complex can assemble a [4Fe-4S] cluster in vitro and transfer it to target proteins; and (iii) the complex binds one molecule of flavin adenine nucleotide per SufBC(2)D complex, only in its reduced form (FADH(2)), which has the ability to reduce ferric iron. These results suggest that the SufBC(2)D complex functions as a novel type of scaffold protein that assembles an Fe-S cluster through the mobilization of sulfur from the SufSE cysteine desulfurase and the FADH(2)-dependent reductive mobilization of iron.

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Figures

FIGURE 1.
FIGURE 1.
UV-visible spectrum of reconstituted SufBC2D-[Fe-S] complex. SufBC2D-[Fe-S] complex (50 μm, 3.8 iron and 5 sulfur/complex) in 50 mm Tris-HCl, pH 8.
FIGURE 2.
FIGURE 2.
Mössbauer spectra of SufB-[57Fe-S] and SufBC2D-[57Fe-S]. a, SufB (125 μm, 3.8 iron and 4.9 sulfur/monomer) and b, SufBC2D complex (210 μm, 3.1 iron and 5 sulfur/complex) in 50 mm Tris-HCl, pH 7.5. Spectra were recorded at 4.2 K in a magnetic field of 50 milliteslas applied perpendicular to the γ beam. The black solid lines are composite quadrupole doublet simulations with the parameters cited in the text. The colored solid lines are contributions from [4Fe-4S]2+ clusters (blue) and nonspecifically bound Fe2+ (green).
FIGURE 3.
FIGURE 3.
Properties of SufBC2D-FADH2. (a) UV-visible spectra of anaerobically purified SufBC2D complex after exposure to air; (b) kinetic oxidation of SufBC2D complex (t = 1 min (bold black line) and t = 3, 5, and 7 min (black line); t = 10, 15, 20, and 30 min (dashed bold black line)) showing formation of the neutral semiquinone species (FADH·) at 590 nm (maximum formation at 7 min (black line)). Inset, enlargement of the 500–700 nm region. c, comparison of the air oxidation kinetic of the flavin within the SufBC2D complex (21 μm flavin) (■) with free reduced flavin in solution (16 μm) (▴).
FIGURE 4.
FIGURE 4.
Binding affinity of FADH2 to the apo-SufBC2D complex. The apo-SufBC2D (20 μm) complex was incubated anaerobically with different concentrations of FADH2 (0–200 μm) obtained by photo-induced reduction of FAD. A molecular weight cutoff concentrator was used to separate unbound FADH2 from protein-bound FADH2, and the flavin content of each was determined outside the glove box after oxidation, heat denaturation, centrifugation, and UV-visible analysis of the supernatant. The concentration of SufBC2D-FADH2 ([FADbound]) and that of apo-SufBC2D was determined according to the calculated concentrations of unbound and total FADH2 and the known amount of total apo-SufBC2D. The protein-bound FADH2 as a function of unbound FADH2 in solution was then plotted. The data have been fitted by a saturation hyperbola according to Equation 1.
FIGURE 5.
FIGURE 5.
Iron-sulfur cluster transfer from SufBC2D to AcnB. Holo-SufBC2D complex (0.3 nmol), [Fe-S] (gray bars), or [Fe-S] + FADH2 (hatched bars), was co-incubated in 10 μl of 50 mm Tris-HCl, pH 7.6, with (a) and without (b) 5 mm DTT with apo-AcnB (0.2 nmol). After 5 and 20 min of incubation, the activity of AcnB was measured by monitoring the absorption at 340 nm. For this, a mixture of 1.2 mm MnCl2, 25 mm citrate, 0.5 unit of isocitrate dehydrogenase, and 0.25 mm NADP+ was added to the protein mixture in a final volume of 100 μl. As a control, apo-AcnB was incubated with a 5 molar excess of iron and sulfide, and the activity was assayed (black bars). c, intact cluster transfer from SufBC2D to AcnB. Apo-AcnB (0.2 nmol) was incubated anaerobically with either [4Fe-4S] SufBC2D complex (0.3 nmol) (▴) or 5-fold molar excess of Fe2+ and S2− (■) in 100 μl of 50 mm Tris-HCl, pH 7.6, 5 mm DTT with increasing amounts of bathophenanthroline, and the AcnB activity was measured after 20 min of incubation.
FIGURE 6.
FIGURE 6.
Ferric reductase activity of the SufBC2D-FADH2 complex. a, time-dependent oxidation (t = 0–65 min) of the flavin in the SufBC2D-FADH2 complex (30 μm) in the presence of ferric citrate (300 μm) in 50 mm Tris-HCl, pH 7.5. The light absorption spectrum of the solution in the 300–800 nm range is recorded at 5, 10, 20, 30, 40, 50, and 65 min, and the amount of oxidized flavin was determined from the absorption at 450 nm. Inset, control experiment showing the time-dependent oxidation of the flavin in the SufBC2D-FADH2 complex (30 μm) in the absence of ferric citrate in 50 mm Tris-HCl, pH 7.5 buffer (t = 0 and 65 min). b, reduction of ferric citrate (100 μm) by the SufBC2D-FADH2 complex (10 μm, 0.74 FADH2/complex) in the presence of the Fe2+-chelator ferrozine (300 μm) in the presence (■) and absence (▴) of ATP. The amount of ferrozine-Fe2+ complex (μm), determined from the absorption at 562 nm, was plotted as a function of time (min). c, reduction of the CyaY-Fe3+ protein (10 μm, 20 iron/monomer) by the SufBC2D-FADH2 complex (10 μm with 9.3 μm of FADH2) in the presence of ferrozine (1 mm). The light absorption spectrum of the solution was recorded every 10 min at 562 nm. The arrow indicates the increased formation of the ferrozine-Fe2+ complex.
FIGURE 7.
FIGURE 7.
Complexes formed upon incubation of SufB, SufC, and SufD proteins (from this study and from data obtained in Refs. 24, 29). Only in vivo a SufBC2D stoichiometry may be obtained presumably through an intermediate SufBD, not observed in vitro.
FIGURE 8.
FIGURE 8.
FAD-binding motifs in SufB from E. coli. These motifs that were originally found in the p-cresol-methylhydroxylase family (41) are depicted in boxes (conserved residues are in boldface).
FIGURE 9.
FIGURE 9.
Current view of the mechanism of Fe-S cluster assembly mediated by the SUF system. In a first step, the SufBC2D complex binds 1 eq of FADH2, obtained via the action of a NAD(P)H:flavin oxidoreductase or flavin reductase. SufS and SuE transiently bind to the SufBC2D-FADH2 complex. The whole complex reacts with cysteine to generate persulfides on SufB, and at the same time, the FADH2 cofactor reduces the iron of a ferric iron source CyaY-Fe3+. The resulting ferrous ions react with persulfides to generate the [4Fe-4S] holo-form of SufB. This holo-form of SufBC2D then transfers its cluster to an apoprotein either directly or via SufA. During this process, the flavin is oxidized and released in solution where it can be reduced again by a flavin reductase.
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