Exploration of the Structural Environment of the Iron-Sulfur Cluster in Putidaredoxin by Nitrogen-15 NMR Spectroscopy of Selectively Labeled Cysteine Residues

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• 作者：Sari ; Nese ; Holden ; Marcia J. ; Mayhew ; Martin P. ; Vilker ; Vincent L. ; Coxon ; Bruce
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：1998
• 出版时间：August 28, 1998
• 年：1998
• 卷：249
• 期：3
• 页码：773-780
• 全文大小：175 K

文摘

Putidaredoxin is a di-iron protein whose paramagnetic region is not well characterized by¹H detected NMR. We have studied the structure of this region in greater detail by directly observed¹⁵N NMR of oxidized and reduced putidaredoxin preparations in which the six cysteine residues are selectively labeled with¹⁵N. A new method for preparation of a stable form of reduced putidaredoxin has been developed for use in NMR. The¹⁵N NMR spectra of the oxidized and reduced forms are characteristically different, and we have measured and compared¹⁵N chemical shifts, spin-lattice relaxation times (T₁), and chemical shift/temperature dependences for both forms. Evidence for localized valencies of the iron atoms in the reduced form is presented. From the¹⁵NT₁values of the oxidized form, reduced distances of the cysteine backbone¹⁵N nuclei from the center of the Fe₂S₂cluster have been calculated. These distances are consistent with those calculated from X-ray crystal structure data for five ferredoxins, and confirm the structural similarity of the Fe₂S₂clusters in putidaredoxin and in these ferredoxins in the oxidized state.