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, 291 (22), 11887-98

The Structure of the Complex Between Yeast Frataxin and Ferrochelatase: CHARACTERIZATION AND PRE-STEADY STATE REACTION OF FERROUS IRON DELIVERY AND HEME SYNTHESIS

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The Structure of the Complex Between Yeast Frataxin and Ferrochelatase: CHARACTERIZATION AND PRE-STEADY STATE REACTION OF FERROUS IRON DELIVERY AND HEME SYNTHESIS

Christopher Söderberg et al. J Biol Chem.

Abstract

Frataxin is a mitochondrial iron-binding protein involved in iron storage, detoxification, and delivery for iron sulfur-cluster assembly and heme biosynthesis. The ability of frataxin from different organisms to populate multiple oligomeric states in the presence of metal ions, e.g. Fe(2+) and Co(2+), led to the suggestion that different oligomers contribute to the functions of frataxin. Here we report on the complex between yeast frataxin and ferrochelatase, the terminal enzyme of heme biosynthesis. Protein-protein docking and cross-linking in combination with mass spectroscopic analysis and single-particle reconstruction from negatively stained electron microscopic images were used to verify the Yfh1-ferrochelatase interactions. The model of the complex indicates that at the 2:1 Fe(2+)-to-protein ratio, when Yfh1 populates a trimeric state, there are two interaction interfaces between frataxin and the ferrochelatase dimer. Each interaction site involves one ferrochelatase monomer and one frataxin trimer, with conserved polar and charged amino acids of the two proteins positioned at hydrogen-bonding distances from each other. One of the subunits of the Yfh1 trimer interacts extensively with one subunit of the ferrochelatase dimer, contributing to the stability of the complex, whereas another trimer subunit is positioned for Fe(2+) delivery. Single-turnover stopped-flow kinetics experiments demonstrate that increased rates of heme production result from monomers, dimers, and trimers, indicating that these forms are most efficient in delivering Fe(2+) to ferrochelatase and sustaining porphyrin metalation. Furthermore, they support the proposal that frataxin-mediated delivery of this potentially toxic substrate overcomes formation of reactive oxygen species.

Keywords: Friedreich ataxia; ataxia; ferrochelatase; frataxin; heme; iron chaperone; iron trafficking; iron-sulfur protein; mitochondria; protoporphyrin.

Figures

FIGURE 1.
FIGURE 1.
Time dependence of heme synthesis at two Fe2+:Yfh1 ratios. Yeast ferrochelatase (20 μm) with bound protoporphyrin IX (15 μm) was mixed with 200 μm ferrous iron (A) in the absence of Yfh1 and in the presence of 100–200 μm Yfh1 (B and C). The Fe2+:Yfh1 molar ratios were 2.0 (B) and 1.3 (C). In all cases, the red, green, yellow, and blue circles represent the kinetic traces at 0, 2, 5, and 10 min, respectively, after the initial shot (i.e. filling the stopped-flow syringes with the reactants).
FIGURE 2.
FIGURE 2.
Yfh1-mediated Fe2+ delivery to ferrochelatase. A, the rate of ferrochelatase-Yfh1 association and ferrochelatase-catalyzed heme synthesis in the presence of Fe2+-bound Yfh1. The decrease in intrinsic protein fluorescence was used to follow the association of Yfh1 with ferrochelatase (green circles); the rate of the ferrochelatase-catalyzed metalation reaction was monitored by following the consumption of the protoporphyrin IX substrate (red circles). Yeast ferrochelatase (10 μm) was mixed with 200 μm Yfh1–200 μm Fe2+ (final concentrations after mixing) in 20 mm MOPS, pH 7.0, containing 0.4 m NaCl and 0.2% (v/v) Tween 80. The observed rate constants were calculated by fitting the decrease in intrinsic protein fluorescence (green circles) or the decrease in protoporphyrin IX fluorescence (red circles) over time to Equation 2 for a single-exponential process. B, effects of Yfh1 and EDTA on the rate of heme synthesis by ferrochelatase. Yeast ferrochelatase (20 μm) incubated with protoporphyrin IX (15 μm) was mixed with 200 μm ferrous iron in the absence (red circles) or presence (80 μm; green circles) of Yfh1. In both cases the addition of EDTA at a concentration of 200 μm inhibited the reaction (orange circles, no Yfh1; blue circles, +Yfh1). Each trace represents the average of 10 experimental measurements. All concentrations given are the final concentrations in the observation chamber after mixing of the reactants.
FIGURE 3.
FIGURE 3.
Size exclusion chromatogram of the frataxin-ferrochelatase complex. A, chromatogram of proteins eluted from the Superdex 200 resin as recorded at A280. Fractions corresponding to the first two of the three peaks were run on SDS-PAGE (numbered 1–13). Fractions from the second peak, primarily fraction no. 10, was used for transmission electron microscopy studies. AU, absorbance units. B, SDS-PAGE gel of fractions 1–13. The first lane contains protein markers. Ferrochelatase (44 kDa) indicated by FC and frataxin (14 kDa) by Yfh1.
FIGURE 4.
FIGURE 4.
EM reconstruction of the complex between yeast ferrochelatase and Yfh1. A, an EM micrograph of the Yfh1-ferrochelatase complex. Particles chosen for the reconstruction are boxed. B, a comparison between projections of the single-particle reconstruction of the complex and the corresponding class averages. C and D, side (B) and top (C) view (looking down to the membrane) single-particle reconstruction of the complex between yeast ferrochelatase and Yfh1.
FIGURE 5.
FIGURE 5.
The Rosetta model of the complex between yeast ferrochelatase and frataxin Yfh1 docked into the density generated from the single particle reconstruction. The orientations in A and B are similar to those shown in Fig. 2, B and C. All structure figures were prepared using PyMOL (70).
FIGURE 6.
FIGURE 6.
Structural details of the complex between yeast ferrochelatase and frataxin Yfh1. A, the cross-links between ferrochelatase (green ribbon model) and frataxin (orange ribbon model), which were analyzed in the MS experiments, are shown as dotted lines, and the corresponding amino acid side chains are shown in sticks representation. Only one ferrochelatase monomer is shown looking down into the porphyrin binding pocket. The cross-link distances between the amino acid residues involved in the cross-linking are shown in the insert. Only distance constrains obtained in the cross-linking experiments where used in the choice of the best model for docking into the EM density and for further analysis. Regions of frataxin helices α1 and α2, shown in NMR experiments (in which monomeric frataxin was used) to be involved in interactions with ferrochelatase, are colored in magenta. For clarity only two monomers of the frataxin trimer are shown on this figure. The third monomer points away from ferrochelatase and is not involved in complex formation. B, the side chains of frataxin and ferrochelatase amino acid residues involved in potential interactions within the complex are shown in sticks representation. A dotted line is drawn from the ferroxidation site of frataxin to Asp-320 of the π-helix of ferrochelatase, and the distance between the two sites is shown. Both regions are colored in violet for clarity. From the model it appears that although the upper frataxin monomer is involved in the stabilization of the complex, the lower monomer would deliver the metal to ferrochelatase.

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