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-

被引:22
作者
Antonkine, ML
Bentrop, D
Bertini, I [1 ]
Luchinat, C
Shen, GZ
Bryant, DA
Stehlik, D
Golbeck, JH
机构
[1] Penn State Univ, Dept Biochem & Mol Biol, University Pk, PA 16802 USA
[2] Univ Florence, Dept Chem, Magnet Resonance Ctr, I-50019 Florence, Italy
[3] Free Univ Berlin, Inst Phys Expt, D-14195 Berlin, Germany
来源
JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY | 2000年 / 5卷 / 03期
基金
美国国家科学基金会;
关键词
NMR spectroscopy; paramagnetic; iron-sulfur protein; Photosystem I;
D O I
10.1007/PL00010667
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 H-1 NMR spectroscopy. For this, a sequence-specific assignment of these signals is required. The H-1 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 T-1 relaxation times. The usual fast method of assigning the H-1 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 Angstrom 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 PS I complex.
引用
收藏
页码:381 / 392
页数:12
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