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*We study the structure-function of the intracellular iron-sulfur world in aerobic and thermophilic archaea.LinkIcon

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日本医科大学、医学部、生化学・分子生物学(代謝・栄養学)所属の 岩崎 俊雄グループでは、好熱菌等を主材料とした各種金属酵素の構造機能進化や、アミノ酸要求性の新規大腸菌発現宿主株作成につき、国内外の様々な研究機関と共同研究を実施しています。

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ISC-like [2Fe-2S] ferredoxin (FdxB) from Pseudomonas putida JCM 20004

Biological iron-sulfur (Fe-S) clusters, consisting of non-heme iron and inorganic sulfide atoms, are functionally versatile and may be among the most ancient modular prosthetic groups. The electronic structure and the site of iron reduction of these protein-bound cofactors account for the electron transfer function and mechanism. In the present work we have solved the 1.90-Å structure of the ISC-like [2Fe-2S] ferredoxin called FdxB from the non-pathogenic gammaproteobacterium Pseudomonas putida JCM 20004 (formerly Pseudomonas ovalis IAM 1002). This FdxB protein contains an adrenodoxin like, redox-active [2Fe-2S] cluster, which plays an essential but ill-defined role in the de novo iron-sulfur cluster assembly (ISC) system. It is encoded by the fdxB gene as a constituent of the cognate iscR-iscS1-iscU-iscA-hscB-hscA-fdxB gene cluster for the ISC system (DDBJ-EMBL-GenBank code AB109467; Fig. 1).
In P. putida the ISC pathway is apparently the sole system for in vivo Fe-S cluster assembly whereas the SUF pathway is missing in the bacterial genome (unlike in Escherichia coli).

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Figure 1. Schematic organization of the isc gene cluster, the products of which are involved in the Fe–S cluster-biosynthesis (ISC) machinery, in the whole-genomic DNA sequence of P. putida KT2440, a close relative of strain JCM 20004 used in this work (top). The primary sequence of P. putida JCM 20004 FdxB (DDBJ/EMBL/GenBank code AB109467) is completely identical to that of P. putida KT2440 open reading frame PP_0847. Multiple sequence alignment of selected ISC-like [2Fe–2S] ferredoxins (bottom) indicates that the cluster-binding site of FdxB (bottom) lacks the conserved cysteine residue (underlined) near the [2Fe–2S] cluster (indicated by a red arrow; see Fig. 5) that has been proposed to provide a potential ligand for an S atom or an Fe3+ ion during the action of the ISC machinery. Accession numbers: P. putida JCM 20004 (Pp) FdxB, DDBJ/EMBL/GenBank code BAD01054; Escherichia coli (Ec) Fdx, PDB code 1i7h; Rhodobacter capsulatus (Rc) FdVI, PDB codes 1e9m and 1uwm; Saccharomyces cerevisiae (Sc) YAH1, GenBank code NP_015071. The metal-binding motifs are boxed.

Crystallization

Recombinant FdxB was heterologously overproduced in E. coli BL21-CodonPlus(DE3)-RIL strain, purified and crystallized in its oxidized form by the hanging-drop vapor diffusion and streak-seeding methods using 1.6 M trisodium citrate dihydrate pH 6.5 (Fig. 2). The thin needle-shaped crystals diffract to 1.90 Å resolution and belong to the hexagonal space group P6122, with unit-cell parameters a = 87.58, c = 73.14 Å. The asymmetric unit contains one protein molecule.

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Figure 2. Typical crystals of recombinant FdxB obtained (left) before and (right) after streak-seeding with a human hair.

*One can follow the link to read the original article of this work.

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The 1.90-Å structure of the ISC-like [2Fe-2S] ferredoxin, FdxB

The FdxB structure has a βαββαβ fold with the β-grasp/ubiquitin-like fold motif as found in regular eukaryal and bacterial [2Fe-2S] ferredoxins (also called the vertebrate ferredoxin subclass) such as bovine adrenodoxin (Adx), putidaredoxin (Pdx), and other bacterial ISC-like ferredoxins (e.g. 1i7h.pdb, 1cje.pdb, 1e9m.pdb). FdxB is folded into an (α+β) core fold domain and an extended C-terminal tail (Fig. 3). In the lattice FdxB was found to be homo-dimeric, displaying the isologous association of the extended C-terminal tail from each protomer (Figs. 3, 4).

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Figure 3. (Top) Wall-eye stereoview of the backbone tracing of the P. putida FdxB protomer (colors changing gradually from the amino terminus (blue) to the carboxyl terminus (red)) together with the observed 2Fo-Fc map contoured at 1.5 σ (gray). The refined model includes the FdxB protomer from Pro1 to Ala108 plus one extra residue at the amino terminus (Met) derived from the expression vector and a [2Fe-2S] cluster (Fe as dark brown and S as green spheres), as well as 152 water (not shown in the figure), one sodium (yellow sphere) and one chlorine (cyan sphere) molecules from the mother liquor. A total of four amino acids at the C-terminus were disordered and not resolved in the electron density. (Middle) Stereoview of the tail-to-tail dimer organization of FdxB with protomers colored gray and red. (Bottom) Structural alignment of FdxB with related [2Fe-2S] proteins, E. coli Fdx (1I7H.pdb), R. capsulatus FdVI (1E9M.pdb), adrenodoxin (Adx) (1AYF.pdb) and putidaredoxin (Pdx) (1OQQ.pdb), drawn by using the program STRAP. Keys: green arrows, residues located in the dimer interface of the FdxB structure; blue squares, cysteine ligand residues; red circles, residues discussed in text.

The contact surface includes not only the residues in the C-terminal tail (Pro100-Lys101-Tyr102-Ser103-Leu104-Asn105-His106-Ala107-Ala108) but also those in the (α+β) core fold domain (Lys10, Glu36-Met37-Glu38, Cys47 (a [2Fe-2S] cluster ligand), Thr49, Lys74-Ala75-Trp76), plus two internal water molecules and one sodium ion in each protomer (Fig. 4). A possible physiological relevance of this dimeric structure is under investigation.

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Figure 4. Stereoview of the FdxB dimer in a space-fill representation (with each protomer depicted in different colors), viewed from the "C-terminal-tail" face of the protein (top). Close-up stereoviews of the FdxB dimer in the tail-to-tail dimer interface region, depicted in the same view as in Fig. 3, where contact atoms (residues and solvent molecules) are colored in blue and red, except for sodium (yellow sphere) and chloride (cyan sphere) atoms (bottom).

Each FdxB protomer binds a [2Fe-2S] cluster that is coordinated by four terminal cysteine sulfur atoms, where the outermost iron (Fe1) near the protein surface is coordinated by Cys41Sγ and Cys47Sγ and the innermost iron (Fe2) by Cys50Sγ and Cys86Sγ (Fig. 5). In the dimeric structure, two [2Fe-2S] clusters are separated at the closest iron-to-iron (Fe1-Fe1) distance of 25 Å (Fig. 3), suggesting that a rapid interprotomer electron transfer between them would be unlikely to occur. In the place of the consensus free cysteine usually present near the [2Fe-2S] cluster of ISC-like ferredoxins, FdxB has the Lys45 side chain which forms a salt-bridge interaction with Asp65Oδ2 (Fig. 5). Thus, the overall FdxB structural features argue for its primarily electron transfer role in the cognate ISC system, rather than the direct catalytic function.

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Figure 5. The [2Fe-2S] cluster binding site of FdxB. An expanded view of one [2Fe-2S] cluster showing the cluster and iron ligands and the corresponding observed 2Fo-Fc map contoured at 1.5 σ (gray) and 6 σ (red) (left). Locations of Lys45, Asp65 and Met 71 (yellow sticks), together with the corresponding observed 2Fo-Fc map contoured at 1.5 σ (gray) and 6 σ (red), relative to the [2Fe-2S] cluster (right).

*The coordinates and structural factors have been deposited in the Protein Data Bank (PDB ID code: 3AH7). Interactive 3D images of the FdxB structure can be found in Proteopedia.

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Electron-nuclear double resonance (ENDOR) analysis of FdxB

With the molecular structural frame determined from the FdxB structure (PDB code: 3AH7), our ENDOR analysis in collaboration with Dr. Reinhard Kappl (Universität des Saarlandes, Germany) (Fig. 6) has allowed to determine the average gmax direction of the reduced FdxB, which is skewed, pointing roughly towards Cys50Cα and forming an angle of about 27.3 (±4) degrees with the normal of the [2Fe-2S] plane, while the gint- and gmin-directions are distributed in a plane tilted toward the cluster plane (Fig. 7). The site of reduced iron in the reduced FdxB is the outermost Fe1 site with the low negative spin density, while the innermost Fe2 site with the high positive spin population is the non-reducible iron retaining the Fe3+-valence of a reduced cluster (Figs. 5, 7).

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Figure 6. EPR spectrum of reduced FdxB (56Fe, H2O) (left, top), and some continuous wave-1H-ENDOR spectra measured at field positions indicated, showing the evolution of the ENDOR pattern (left, bottom). Outer ENDOR lines are marked A-D for the assignment to specific protons. The gray bars, as examples, give the frequency range of the line width used as input for the automatic calculations. Comparison of ENDOR spectra at the given field positions for samples in aqueous and deuterated buffer (in gray) (right). The gray bars indicate regions with a loss in signal intensity for D2O specimen. The spectra were normalized in intensity by adjusting the resonances at the highest frequency (marked by asterisks). The lower two spectra in the right panel are simulations for all exchangeable NH-protons near the cluster, indicating their frequency range, and for eight β-protons of four cysteine ligands to the [2Fe-2S] cluster (right bottom spectrum). The latter spectrum is reduced in intensity. This figure was prepared by Dr. Reinhard Kappl (Universität des Saarlandes, Germany).

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Figure 7. Skewed orientations of the gmax-component with respect to the molecular frame of the [2Fe-2S] cluster of FdxB. The possible orientations of the gmax-component (red) in a tilted arrangement with respect to the [2Fe-2S] cluster of FdxB, derived from calculation with the automatic routine including the four lines A-D and an exchangeable NαH proton (see Fig. 6). The intermediary and minimal g-directions are distributed in the perpendicular plane (green, gint; blue gmin). Atoms are colored as H, white; C, gray; N, blue; O, red; S, yellow; Fe, orange spheres. This figure was prepared by Dr. Reinhard Kappl (Universität des Saarlandes, Germany).

From a structural point of view, the larger number of polarized (or polarizable) bonds (NH, OH) and the extended hydrogen bonding network around Fe1 in FdxB may be the crucial factor favoring the accommodation of the reducing electron at the outermost Fe1 site (Fig. 8). These results are related with other [2Fe-2S] proteins in different electron transport chains (e.g., adrenodoxin) and suggest a significant distortion of the electronic structure of the reduced [2Fe-2S] cluster under the influence of the protein environment around each iron site in general.

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Figure 8. Stereoview of the hydrogen bond network around the [2Fe-2S] cluster binding site (in ball-and-stick representation). Residue numbers are indicated in the figure.

*One can follow the links to read the original article of this work and interactive 3D complement in Proteopedia.

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2D electron spin-echo envelope modulation (ESEEM/HYSCORE) analysis of FdxB

The 1.90-Å structure of P. putida FdxB (PDB ID code: 3AH7) shows an extensive (N/O)–H…S hydrogen bond network around the [2Fe–2S] cluster, with nearly the same pattern as found in other closely related homologs such as adrenodoxin and putidaredoxin, including a conserved N–H…S hydrogen bond between the Nε of Gln87 and the Cys86 Sγ ligand at the Nε–Sγ distance of 3.4 Å in FdxB (Fig. 9a). The extensive (N/O)–H…S hydrogen bond network around the biological iron–sulfur clusters is one of the most important themes in modulating their redox properties and potential electron transfer pathways in biological electron transfer conduits. Paramagnetic relaxation due to the reduced (antiferromagnetically coupled Fe3+ – Fe2+) cluster severely broadens signals from nuclei in the vicinity (8 Å) of the paramagnetic centers, making it difficult to fully assign hyperfine interactions with the paramagnetic metal center by multidimensional heteronuclear NMR. Pulsed EPR (2D ESEEM, also called hyperfine sublevel correlation, HYSCORE) can also be used to characterize the nuclear spin interactions with the paramagnetic center. Uniformly 15N-labeled FdxB (denoted as U-FdxB) was prepared, but the 2D ESEEM spectra did not resolve individual nuclei, showing only two broad (aggregate) 15N cross-features with apparent hyperfine splittings of 1.2 and 0.2 MHz, respectively (Fig. 9b).

Selective labeling of amino acids with stable isotopes, applied in conjunction with a number of spectroscopic techniques such as NMR, EPR, resonance Raman, and infrared (IR) spectroscopies, is undoubtedly a powerful method in deciphering the structures and mechanisms of proteins. Therefore, FdxB was prepared with glutamine specifically 15N-labeled at the Nε position (denoted as Gln-FdxB). The HYSCORE spectra collected from Gln-FdxB clearly resolve, for the first time, the sharp cross-peaks assigned to a single N–H…S hydrogen bond interaction between the 15Nε of Gln87 and the terminal Cys86 Sγ ligand of the reduced cluster in Gln-FdxB, with the isotropic hyperfine splitting of 0.9 MHz (Fig. 9c). This is usually impossible even with the use of uniformly 15N-labeled proteins, presumably because of the significant spectral overlap of many weakly coupled 15N nuclei from the peptide backbone near the cluster.

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Figure 9. The hydrogen bond network around the [2Fe-2S](Cys)4 cluster binding site in the 1.90-Å structure of P. putida FdxB (PDB code: 3AH7) (a) and the 2D ESEEM (HYSCORE) characterization of weakly coupled 15N nuclei in uniformly 15N-labeled FdxB (U-FdxB) (b) and selectively Gln15Nε-labeled FdxB (Gln-FdxB) (c) in the reduced state. The HYSCORE spectra in 3D stacked presentation (b,c) were measured at the low-field edge of the EPR line near g// (342.5 mT, left) and the intermediate position at g (357 mT, right). The cross-peaks for 15Nε of Gln87, which is involved in the N-H…S hydrogen bond with Cys86 Sγ (a), were clearly resolved with the hyperfine splitting of ~0.9 MHz ((2.01, 0.92) MHz and 15A=1.09 MHz near g// (left); (1.95, 1.13) MHz and 15A=0.82 MHz at g (right)) in the Gln-FdxB spectra (c).

*One can follow the link to read the original article of this work.

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