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Paramagnetic 1H NMR spectroscopy of the reduced, unbound Photosystem I subunit PsaC: sequence-specific assignment of contact-shifted resonances and identification of mixed-and equal-valence Fe-Fe pairs in [4Fe-4S] centers FA − and FB −

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Abstract

The PsaC subunit of Photosystem I (PS I) is a 9.3-kDa protein that binds two important cofactors in photosynthetic electron transfer: the [4Fe-4S] clusters FA and FB. The g-tensor orientation of FA − and FB − is believed to be correlated to the preferential localization of the mixed-valence and equal-valence (ferrous) iron pairs in each [4Fe-4S]+ cluster. The preferential position of the mixed-valence and equal-valence pairs, in turn, can be inferred from the study of the temperature dependence of contactshifted resonances by 1H NMR spectroscopy. For this, a sequence-specific assignment of these signals is required. The 1H NMR spectrum of reduced, unbound PsaC from Synechococcus sp. PCC 7002 at 280.4 K in 99% D2O solution shows 18 hyperfine-shifted resonances. The non-solvent-exchangeable, hyperfineshifted resonances of reduced PsaC are clearly identified as belonging to the cysteines coordinating the clusters FA − and FB − by their downfield chemical shifts, by their temperature dependencies, and by their short T 1 relaxation times. The usual fast method of assigning the 1H NMR spectra of reduced [4Fe-4S] proteins through magnetization transfer from the oxidized to the reduced state was not feasible in the case of reduced PsaC. Therefore, a de novo self-consistent sequence-specific assignment of the hyperfine-shifted resonances was obtained based on dipolar connectivities from 1D NOE difference spectra and on longitudinal relaxation times using the X-ray structure of Clostridium acidi urici 2[4Fe-4S] cluster ferredoxin at 0.94 Å resolution as a model. The results clearly show the same sequence-specific distribution of Curie and anti-Curie cysteines for unbound, reduced PsaC as established for other [4Fe-4S]-containing proteins; therefore, the mixed-valence and equal-valence (ferrous) Fe-Fe pairs in FA − and FB − have the same preferential positions relative to the protein. The analysis reveals that the magnetic properties of the two [4Fe-4S] clusters are essentially indistinguishable in unbound PsaC, in contrast to the PsaC that is bound as a component of the PS I complex.

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Abbreviations

FA and FB :

the two [4Fe-4S] clusters of PsaC in PS I

FX :

interpolypeptide iron-sulfur cluster in PS I

NOE:

nuclear Overhauser effect

PS I:

Photosystem I

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, 16802, USA

    Mikhail L. Antonkine, Gaozhong Shen, Donald A. Bryant & John H. Golbeck

  2. Magnetic Resonance Center, Department of Chemistry, University of Florence, Via L. Sacconi 6, 50019, Sesto Fiorentino (Florence), Italy

    Detlef Bentrop, Ivano Bertini & Claudio Luchinat

  3. Institut für Experimentalphysik, Freie Universität Berlin, Arnimallee 14, 14195, Berlin, Germany

    Dietmar Stehlik

Authors
  1. Mikhail L. Antonkine
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  2. Detlef Bentrop
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  3. Ivano Bertini
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  4. Claudio Luchinat
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  5. Gaozhong Shen
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  6. Donald A. Bryant
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  7. Dietmar Stehlik
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  8. John H. Golbeck
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Antonkine, M.L., Bentrop, D., Bertini, I. et al. Paramagnetic 1H NMR spectroscopy of the reduced, unbound Photosystem I subunit PsaC: sequence-specific assignment of contact-shifted resonances and identification of mixed-and equal-valence Fe-Fe pairs in [4Fe-4S] centers FA − and FB − . JBIC 5, 381–392 (2000). https://doi.org/10.1007/PL00010667

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  • DOI: https://doi.org/10.1007/PL00010667

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