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Probing electronic and structural properties of the reduced [2Fe−2S] cluster by orientation-selective1H ENDOR spectroscopy: Adrenodoxin versus rieske iron-sulfur protein

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Abstract

The1H electron-nuclear double resonance (ENDOR) spectra in frozen buffer solutions of the reduced [2Fe−2S] clusters in adrenodoxin (Adx) and in the “Rieske” iron-sulfur protein (ISP) from the bovine mitochondrial bc1 complex were measured at low temperatures (5–20 K) and analyzed by spectra reconstruction. A single paramagnetic species with iron valence states (II) and (III) connected uniquely to the cluster irons was found in both proteins. For Adx, the experimental spectra from 23 field positions across the nearly axial (g max=2.0241,g int=1.9347, andg min=1.9331) electron paramagnetic resonance (EPR) spectrum were analyzed. Four larger hyperfine couplings were assigned to the cysteine β-protons near the Fe(III) ion. Transfer into the crystal structure showed that the Fe(III) ion was coordinated to the residues Cys55 and Cys92. The spin density was estimated as +1.60 for the Fe(III) and −0.6 for the Fe(II) ion, respectively. Theg-tensor direction with respect to the cluster showed strong similarities with the earlier assignment inArthospira platensis ferredoxin (Canne C., Ebelshauser M., Gay E., Shergill J.K., Cammack R., Kappl R., Hüttermann J.: J. Biol. Inorg. Chem. 5, 514, 2000). An Adx mutant (T54A) exhibiting a change (70 mV) in redox potential showed no significant influence at the [2Fe−2S] cluster. The Rieske ISP was subjected to the same analysis. The ENDOR spectra from 35 field positions across the rhombic (g max=2.028,g int=1.891, andg min=1.757) EPR spectrum were simulated. Three major proton contributions were identified from the orientation behavior. Two were assigned to cysteine β-protons and one to a β-proton of His141. In contrast to Adx, the direction of theg max-component was found to lie roughly in the FeS-core plane and the largest proton coupling occurred alongg int. The spin population was estimated as about +1.6 for the oxidized and −0.55 for the reduced iron.

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Authors and Affiliations

  1. Fachrichtung Biophysik, Medizinische Fakultät, Universität des Saarlandes, Klinikum Gebäude 76, 66421, Homburg/Saar, Germany

    R. Kappl, M. Ebelshäuser & J. Hüttermann

  2. Fachrichtung Biochemie, Naturwissenschaftlich Technische Fakultät III, Universität des Saarlandes, Saarbrücken, Germany

    F. Hannemann & R. Bernhardt

Authors
  1. R. Kappl
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  2. M. Ebelshäuser
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  3. F. Hannemann
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  4. R. Bernhardt
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  5. J. Hüttermann
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Correspondence to R. Kappl.

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Kappl, R., Ebelshäuser, M., Hannemann, F. et al. Probing electronic and structural properties of the reduced [2Fe−2S] cluster by orientation-selective1H ENDOR spectroscopy: Adrenodoxin versus rieske iron-sulfur protein. Appl. Magn. Reson. 30, 427–459 (2006). https://doi.org/10.1007/BF03166211

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