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ISC-like [2Fe–2S] ferredoxin (FdxB) dimer from Pseudomonas putida JCM 20004: structural and electron–nuclear double resonance characterization

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Abstract

The crystal structure of the ISC-like [2Fe–2S] ferredoxin (FdxB), probably involved in the de novo iron-sulfur cluster biosynthesis (ISC) system of Pseudomonas putida JCM 20004, was determined at 1.90-Å resolution and displayed a novel tail-to-tail dimeric form. P. putida FdxB lacks the consensus free cysteine usually present near the cluster of ISC-like ferredoxins, indicating its primarily electron transfer role in the iron-sulfur cluster. Orientation-selective electron–nuclear double resonance spectroscopic analysis of reduced FdxB in conjunction with the crystal structure has identified the innermost Fe2 site with a high positive spin population as the nonreducible iron retaining the Fe3+ valence and the outermost Fe1 site as the reduced iron with a low negative spin density. The average g max direction is skewed, forming an angle of about 27.3° (±4°) with the normal of the [2Fe–2S] plane, whereas the g int and g min directions are distributed in the cluster plane, presumably tilted by the same angle with respect to this plane. These results are related to those for 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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Abbreviations

Adx:

Adrenodoxin

CW:

Continuous-wave

ENDOR:

Electron-nuclear double resonance

EPR:

Electron paramagnetic resonance

FdVI:

ISC-like [2Fe–2S] ferredoxin from Rhodobacter capsulatus

Fdx:

[2Fe–2S] ferredoxin involved in the ISC pathway

FdxB:

ISC-like [2Fe–2S] ferredoxin from Pseudomonas putida JCM 20004

Fe–S:

Iron–sulfur

Pdx:

Putidaredoxin

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Acknowledgments

We thank Asako Kounosu (Nippon Medical School) for assistance in several sample preparations. This investigation was supported in part by JSPS Grants-in-Aid 18608004 (T.I.), 21659111 (T.I.), and 20500628 (D.O.), a JSPS Bottom-up International Joint Research Program (International Collaborations in Chemistry) grant (T.I.), and grants from the EU and the DFG to R.K. and formerly to Jürgen Hüttermann, whose support over many years is gratefully acknowledged. The diffraction data were collected at the SPring-8 beamline BL41XU with the approval of the Japan Synchrotron Radiation Research Institute (JASRI; proposal no. 2007A1271-NL-np). The coordinates and structural factors have been deposited in the Protein Data Bank (ID 3AH7).

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan

    Toshio Iwasaki

  2. Fachrichtung Biophysik, Universität des Saarlandes, 66421, Homburg/Saar, Germany

    Reinhard Kappl & Gerhard Bracic

  3. Japan Synchrotron Radiation Research Institute (SPring-8/JASRI), Sayo, Hyogo, 679-5198, Japan

    Nobutaka Shimizu & Takashi Kumasaka

  4. Department of Chemistry, Juntendo University, Inba, Chiba, 270-1695, Japan

    Daijiro Ohmori

Authors
  1. Toshio Iwasaki
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  2. Reinhard Kappl
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  3. Gerhard Bracic
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  4. Nobutaka Shimizu
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  5. Daijiro Ohmori
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  6. Takashi Kumasaka
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Corresponding authors

Correspondence to Toshio Iwasaki, Reinhard Kappl or Takashi Kumasaka.

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Iwasaki, T., Kappl, R., Bracic, G. et al. ISC-like [2Fe–2S] ferredoxin (FdxB) dimer from Pseudomonas putida JCM 20004: structural and electron–nuclear double resonance characterization. J Biol Inorg Chem 16, 923–935 (2011). https://doi.org/10.1007/s00775-011-0793-8

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