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-valenc

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• 作者：Mikhail L. Antonkine ; Detlef Bentrop ; I. Bertini ; Claudio Luchinat ; Gaozhong Shen ; Donald A. Bryant ; D. Stehlik and J. H. Golbeck
• 关键词：NMR spectroscopy ; Paramagnetic ; Iron ; sulfur protein ; Photosystem I
• 刊名：Journal of Biological Inorganic Chemistry
• 出版年：2000
• 出版时间：June 2000
• 年：2000
• 卷：5
• 期：3
• 页码：381-392
• 全文大小：235 KB

文摘

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 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 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.