Molecular Insights into Frataxin-Mediated Iron Supply for Heme Biosynthesis in <i>Bacillus subtilis</i>

<div><p>Iron is required as an element to sustain life in all eukaryotes and most bacteria. Although several bacterial iron acquisition strategies have been well explored, little is known about the intracellular trafficking pathways of iron and its entry into the systems for co-factor biogenesis. In this study, we investigated the iron-dependent process of heme maturation in <i>Bacillus subtilis</i> and present, for the first time, structural evidence for the physical interaction of a frataxin homologue (Fra), which is suggested to act as a regulatory component as well as an iron chaperone in different cellular pathways, and a ferrochelatase (HemH), which catalyses the final step of heme <i>b</i> biogenesis. Specific interaction between Fra and HemH was observed upon co-purification from crude cell lysates and, further, by using the recombinant proteins for analytical size-exclusion chromatography. Hydrogen–deuterium exchange experiments identified the landscape of the Fra/HemH interaction interface and revealed Fra as a specific ferrous iron donor for the ferrochelatase HemH. The functional utilisation of the <i>in vitro</i>-generated heme <i>b</i> co-factor upon Fra-mediated iron transfer was confirmed by using the <i>B</i>. <i>subtilis</i> nitric oxide synthase bsNos as a metabolic target enzyme. Complementary mutational analyses confirmed that Fra acts as an essential component for maturation and subsequent targeting of the heme <i>b</i> co-factor, hence representing a key player in the iron-dependent physiology of <i>B</i>. <i>subtilis</i>.</p></div

Influence of Fra on the heme maturation pathway.

<p>(A) Kinetics of the conversion of protoporphyrin IX into heme <i>b</i> by HemH. Varying concentrations of Fe²⁺-charged frataxin as the sole iron source were added in a range of 0.1–10 μM, and conversion of protoporphyrin IX into heme <i>b</i> was followed by fluorescence spectroscopy. Data were fitted according to the Michaelis-Menten model, which resulted in a <i>K</i>_m(obs) of about 2.8 ± 0.5 μM and a <i>k</i>_cat(obs) of 0.925 ± 0.059 s^-1 (error bars represent SEM of three independent experiments). (B) <i>In vitro</i> activities of apo-bsNos (1), holo-bsNos (2) and reconstituted apo-bsNos (3). The reconstitution of apo-bsNos with its heme co-factor was carried out by a coupled transfer assay containing Fe²⁺-charged Fra, HemH and protoporphyrin IX, and led to a partial restoration of bsNos activity (error bars represent SEM of three independent experiments). (C) <i>In vivo</i> bsNos activities in equalized amounts of total cellular protein. Control of the assay with <i>B</i>. <i>subtilis</i> WT crude protein extract assayed without the addition of <i>N</i>^ω-hydroxy-l-arginine (1), with <i>B</i>. <i>subtilis</i> WT crude protein extract (2) and with <i>B</i>. <i>subtilis</i> Δ<i>fra</i> crude protein extract (3). The deletion of <i>fra</i> led to a ~12-fold decrease of bsNos activity in the crude mutant cell extract (error bars represent SEM of three independent experiments). (D) Determination of the relative heme contents in <i>B</i>. <i>subtilis</i> WT (red bar) and Δ<i>fra</i> (blue bar) cells by acidic acetone extraction and fluorescence analysis of the heme <i>b</i> soret band emission at 450 nm upon excitation at 380 nm. The amount of cellular heme was found to be ~2.5-fold reduced in the Δ<i>fra</i> mutant cell (error bars represent SEM of three independent experiments). (E) Determination of the relative protoporphyrin IX concentration in <i>B</i>. <i>subtilis</i> WT (red bar) and Δ<i>fra</i> (blue bar) cells by acidic acetone extraction and fluorescence analysis of the protoporphyrin IX soret band emission at 510 nm upon excitation at 410 nm. The amount of cellular protoporphyrin IX was ~1.2-fold elevated in the Δ<i>fra</i> mutant cell (error bars represent SEM of two independent experiments). (F) Fra mediated heme <i>b</i> (protoheme IX) maturation and its delivery to heme-dependent target proteins.</p

Interaction studies of frataxin (Fra) and ferrochelatase (HemH).

<p>(A) Ultraviolet-visible chromatogram of three independent analytical size-exclusion chromatography runs of heterologously expressed and purified Fra-His₆ (gray dotted line), HemH-Strep II (red dotted line) and both proteins together, revealing the formation of a HemH/Fra interaction complex (blue solid line). The interactions were analysed by using a calibrated Superdex 200 10/300 GL gel-filtration column. The inset shows an SDS-PAGE of the Ni²⁺-NTA elution fraction of <i>B</i>. <i>subtilis</i> WT (WT) crude cell extract and <i>B</i>. <i>subtilis</i> AM09 (AM09) crude cell extract with endogenously expressed Fra-His₆, which was co-purified with several proteins, including ferrochelatase HemH from <i>B</i>. <i>subtilis</i> AM09 crude cell extract. Control experiments did not reveal any unspecific interactions. (B) Fra/HemH dimerization was analysed thermophoretically. Unlabelled HemH was titrated to a constant amount of fluorescent-labelled apo-Fra (squares) and holo-Fra (circles). Dimerization caused a significant change in thermophoresis and a <i>K</i>_d of 1.63 μ 0.02 μM was calculated (red line). Error bars represent SEM of three independent experiments.</p

HDX epitope mapping of the Fra/HemH interaction surface.

<p>The acidic ridge of Fra (top) binds directly above the HemH (bottom) iron co-ordination site in its catalytic centre (blue), leading to a reduction in HDX (red). Upon binding, both enzymes undergo a conformational change, which leads either to an increased (green) or a reduced (red) HDX. Black areas indicate no change in HDX. The results were mapped to crystal structures of <i>B</i>. <i>subtilis</i> Fra (Protein Databank ID code 2OC6) and HemH (Protein Databank ID code 2HK6) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122538#pone.0122538.ref039" target="_blank">39</a>].</p

An Enzymatic Pathway for the Biosynthesis of the Formylhydroxyornithine Required for Rhodochelin Iron Coordination

Rhodochelin, a mixed catecholate–hydroxamate type siderophore isolated from <i>Rhodococcus jostii</i> RHA1, holds two l-δ-<i>N</i>-formyl-δ-<i>N</i>-hydroxyornithine (l-fhOrn) moieties essential for proper iron coordination. Previously, bioinformatic and genetic analysis proposed <i>rmo</i> and <i>rft</i> as the genes required for the tailoring of the l-ornithine (l-Orn) precursor [Bosello, M. (2011) <i>J. Am. Chem. Soc.</i> <i>133</i>, 4587–4595]. In order to investigate if both Rmo and Rft constitute a pathway for l-fhOrn biosynthesis, the enzymes were heterologously produced and assayed <i>in vitro</i>. In the presence of molecular oxygen, NADPH and FAD, Rmo monooxygenase was able to convert l-Orn into l-δ-<i>N</i>-hydroxyornithine (l-hOrn). As confirmed in a coupled reaction assay, this hydroxylated intermediate serves as a substrate for the subsequent <i>N</i>¹⁰-formyl-tetrahydrofolate-dependent (<i>N</i>¹⁰-fH₄F) Rtf-catalyzed formylation reaction, establishing a route for the l-fhOrn biosynthesis, prior to its incorporation by the NRPS assembly line. It is of particular interest that a major improvement to this study has been reached with the use of an alternative approach to the chemoenzymatic FolD-dependent <i>N</i>¹⁰-fH₄F conversion, also rescuing the previously inactive CchA, the Rft-homologue in coelichelin assembly line [Buchenau, B. (2004) <i>Arch. Microbiol.</i> <i>182</i>, 313–325; Pohlmann, V. (2008) <i>Org. Biomol. Chem.</i> <i>6</i>, 1843–1848]

Scheme of the proposed iron channelling pathway.

<p>Ferrous iron is bound to the iron chaperone Fra, which transfers it by physical interaction to the ferrochelatase HemH. Upon incorporation of ferrous iron into the protoporphyrin IX scaffold by HemH, heme <i>b</i> is generated and can serve as a co-factor for heme-dependent target enzymes, such as nitric oxide synthase bsNos.</p

<i>Bs</i>SufS alters the H/D exchange of <i>Bs</i>SufU upon binding.

<p><b>(A)</b> Detected peptic peptides of <i>Bs</i>SufU with the relative fractional uptake after 15 s of incubation in deuterated buffer. <b>(B)</b> Changes in the relative fractional deuterium uptake of <i>Bs</i>SufU after incubation with <i>Bs</i>SufS for 15 s in D₂O buffer compared to <i>Bs</i>SufU alone were mapped onto the surface of <i>Bs</i>SufU (PDB ID 2AZH). The heat map represents the differences in deuterium uptake compared to <i>Bs</i>SufU alone. A decrease (blue) in deuterium uptake signals protection (<i>i</i>.<i>e</i>., a binding event), whereas an increase (red) signals a structural rearrangement. Black regions were not detected. Binding of <i>Bs</i>SufU to <b>(C)</b> the α/β-linker and <b>(D)</b> the Cys128-loop of <i>Bs</i>SufS as a function of deuterium uptake over time. Color code: <i>Bs</i>SufU alone (red), <i>Bs</i>SufU + <i>Bs</i>SufS (green), <i>Bs</i>SufU + <i>Bs</i>Fra (blue), and BsSufU + <i>Bs</i>SufS/<i>Bs</i>Fra (violet). N-terminus (NT) and C-terminus (CT).</p

Characterization of the affinity of <i>Bs</i>Fra for <i>Bs</i>SufS, <i>Bs</i>SufU, and <i>Bs</i>SufS/<i>Bs</i>SufU using microscale thermophoresis.

<p>MST binding curve from the interaction of fluorophore-labeled <i>Bs</i>Fra with <b>(A)</b> <i>Bs</i>SufS, <b>(B)</b> <i>Bs</i>SufS/<i>Bs</i>SufU, and <b>(C)</b> <i>Bs</i>SufU. A Hill model was applied for <i>K</i>_d determination. Fra* indicates fluorophore-tagged frataxin.</p

<i>Bs</i>SufU alters the H/D exchange of <i>Bs</i>SufS upon binding.

<p>Changes in relative fractional deuterium uptake of <i>Bs</i>SufS after incubation with <i>Bs</i>SufU for 15 s in D₂O buffer compared to <i>Bs</i>SufS alone were mapped onto the surface of the <i>Bs</i>SufS <b>(A)</b> monomer and <b>(B)</b> homodimer. A decrease (blue) in deuterium uptake signals protection (<i>i</i>.<i>e</i>., a binding event), whereas an increase (red) signals a structural rearrangement. Black regions were not detected. Binding of <i>Bs</i>SufU to <b>(C)</b> the C-terminus and <b>(D)</b> the α-hinge of <i>Bs</i>SufS as a function of deuterium uptake over time. Color code: <i>Bs</i>SufS alone (green), <i>Bs</i>SufS + <i>Bs</i>SufU (red), <i>Bs</i>SufS + <i>Bs</i>Fra (blue), and BsSufS + <i>Bs</i>SufU/<i>Bs</i>Fra (violet). N-terminus (NT) and C-terminus (CT).</p

<i>Bs</i>Fra alters the H/D exchange of <i>Bs</i>SufS and <i>Bs</i>SufU upon binding the <i>Bs</i>SufS/<i>Bs</i>SufU complex.

<p>Changes in the relative fractional deuterium uptake of <i>Bs</i>SufS/<i>Bs</i>SufU after incubation with <i>Bs</i>Fra for 15 s in D₂O buffer compared to <i>Bs</i>SufS and <i>Bs</i>SufU alone were mapped onto the surface of the <i>Bs</i>SufS <b>(A)</b> monomer and <b>(B)</b> homodimer as well as <b>(C)</b> <i>Bs</i>SufU (PBD ID 2AZH). The heat map represents the differences in deuterium uptake compared to the solo incubation. A decrease (blue) in the uptake signals protection (<i>i</i>.<i>e</i>., a binding event), whereas an increase (red) signals a structural rearrangement. Black regions were not detected. Changes in <b>(D)</b> the SufS Cys361-loop and <b>(E)</b> the β-hook as a function of deuterium uptake over time. Color code: <i>Bs</i>SufS alone (green), <i>Bs</i>SufS + <i>Bs</i>Fra (blue), <i>Bs</i>SufS + <i>Bs</i>SufU (red), and BsSufS + <i>Bs</i>SufU/<i>Bs</i>Fra (violet). Changes in <b>(F)</b> the SufU α5-helix and <b>(G)</b> the Cys41-loop as a function of deuterium uptake over time. Color code: <i>Bs</i>SufU alone (red), <i>Bs</i>SufU + <i>Bs</i>Fra (blue), <i>Bs</i>SufU + <i>Bs</i>SufS (green), and BsSufU + <i>Bs</i>SufS/<i>Bs</i>Fra (violet). N-terminus (NT) and C-terminus (CT).</p