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

• Published in: Biochemical and biophysical research communications Vol. 249; no. 3; pp. 773 - 780
• Main Authors: Sari, Nese, Holden, Marcia J., Mayhew, Martin P., Vilker, Vincent L., Coxon, Bruce
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28.08.1998
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Crystallography, X-Ray; Cysteine - chemistry; Drug Stability; Escherichia coli - genetics; Ferredoxins - chemistry; Ferredoxins - genetics; Iron - chemistry; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Nitrogen Isotopes; Oxidation-Reduction; Pseudomonas putida - chemistry; Pseudomonas putida - genetics; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Sulfur - chemistry
• DOI: 10.1006/bbrc.1998.9227
• ISSN: 0006-291X

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.