Exploration of the structural environment of the iron-sulfur cluster in putidaredoxin by nitrogen-15 NMR spectroscopy of selectively labeled cysteine residues.
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 for use in NMR. 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.
Sari
N
N
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA.
Holden
M J
MJ
Mayhew
M P
MP
Vilker
V L
VL
Coxon
B
B
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics; D007501:Iron / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010084:Oxidation-Reduction; D016958:Pseudomonas putida / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013455:Sulfur / Q000737:chemistry
D001426:Bacterial Proteins; D005288:Ferredoxins; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; C100137:putidaredoxin; D013455:Sulfur; D007501:Iron; D003545:Cysteine