TY - JOUR
T1 - Paramagnetic 1H NMR spectroscopy of the reduced, unbound photosystem I subunit PsaC
T2 - 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)-
AU - Antonkine, Mikhail L.
AU - Bentrop, Detlef
AU - Bertini, Ivano
AU - Luchinat, Claudio
AU - Shen, Gaozhong
AU - Bryant, Donald A.
AU - Stehlik, Dietmar
AU - Golbeck, John H.
N1 - Funding Information:
Acknowledgements This work was supported by the European Union, Large-Scale Facility grant ERBCHGECT950033 to the Florence laboratory, by U.S. National Science Foundation grants to J.H.G. (MCB-9723661) and to D.A.B (MCB-9723469), and by the DFG under SFB 312, A1 (D.S.). G.S. was supported by an NSF-RTG Grant (DBI9602232) to the Pennsylvania State University. We thank Dr. Andreas Kamlowski for his contribution in the early stages of the project.
PY - 2000/6
Y1 - 2000/6
N2 - 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.
AB - 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.
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U2 - 10.1007/PL00010667
DO - 10.1007/PL00010667
M3 - Article
C2 - 10907749
AN - SCOPUS:0033623966
SN - 0949-8257
VL - 5
SP - 381
EP - 392
JO - Journal of Biological Inorganic Chemistry
JF - Journal of Biological Inorganic Chemistry
IS - 3
ER -