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

Published

August 28, 1998

Author(s)

Fahriye N. Sari, Marcia J. Holden, M P. Mayhew, V L. Vilker, B Coxon

Abstract

Putidaredoxin is a di-iron protein whose paramagnetic region is not well characterized by ¹H detected NMR. We have studied the structure of this region in greater detail by directly observed ¹⁵N NMR of oxidized and reduced putidaredoxin preparations in which the six cysteine residues are selectively labeled with ¹⁵N. A new method for preparation of a stable form of reduced putidaredoxin has been developed. The ¹⁵N NMR spectra of the oxidized and reduced forms are characteristically different, and we have measured and compared ¹⁵N 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 the ¹⁵N T₁ values of the oxidized form, reduced distances of the cysteine backbone ¹⁵N nuclei from the center of the Fe₂S₂ cluster 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 Fe₂S₂ clusters in putidaredoxin and in these ferredoxins in the oxidized state.

Citation

Biochemical and Biophysical Research Communications

Volume

249

Issue

3

Pub Type

Journals

Keywords

anti-Curie dependence, Curie dependence, cysteine, ferredoxins, iron-sulfur cluster, nitrogen-15 labeling, NMR, oxidized form, paramagnetic protein

Citation

Sari, F. , Holden, M. , Mayhew, M. , Vilker, V. and Coxon, B. (1998), Exploration of the Structural Environment of the Iron-Sulfur Cluster in Putidaredoxin by Nitrogen-15 NMR Spectroscopy of Selectively Labeled Cysteine Residues, Biochemical and Biophysical Research Communications (Accessed April 16, 2024)