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 F(A)- and F(B)-

Mikhail L. Antonkine, Detlef Bentrop, Ivano Bertini, Claudio Luchinat, Gaozhong Shen, Donald Ashley Bryant, Dietmar Stehlik, John H. Golbeck

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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 F(A) and F(B). The g-tensor orientation of F(A)- and F(B)- 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 contact-shifted 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, hyperfine-shifted resonances of reduced PsaC are clearly identified as belonging to the cysteines coordinating the clusters F(A)- and F(B)- by their downfield chemical shifts, by their temperature dependencies, and by their short T1 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 A 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 F(A)- and F(B)- 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 PSI complex.

Original languageEnglish (US)
Pages (from-to)381-392
Number of pages12
JournalJournal of Biological Inorganic Chemistry
Volume5
Issue number3
DOIs
StatePublished - Jan 1 2000

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Inorganic Chemistry

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