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-.
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 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 FA- and FB- 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 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.
Antonkine
M L
ML
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park 16802, USA.
Bentrop
D
D
Bertini
I
I
Luchinat
C
C
Shen
G
G
Bryant
D A
DA
Stehlik
D
D
Golbeck
J H
JH
D000458:Cyanobacteria / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008280:Magnetics; D008565:Membrane Proteins; D008961:Models, Structural; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry*; D045331:Photosystem I Protein Complex; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry*; D013696:Temperature
D007506:Iron-Sulfur Proteins; D008565:Membrane Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D011506:Proteins; D011994:Recombinant Proteins; C089909:photosystem I, psaB subunit; D007501:Iron