An NMR-Derived Model for the Solution Structure of Oxidized Putidaredoxin, a 2-Fe, 2-S Ferredoxin from Pseudomonas

• Published in: Biochemistry (Easton) Vol. 33; no. 21; pp. 6424 - 6432
• Main Authors: Pochapsky, Thomas C, Ye, Xiao Mei, Ratnaswamy, Gayathri, Lyons, Teresa A
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.05.1994
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Binding Sites; Ferredoxins - chemistry; Ferredoxins - metabolism; Magnetic Resonance Spectroscopy; Metals - metabolism; Molecular Sequence Data; Oxidation-Reduction; Protein Conformation; Protein Structure, Secondary; Pseudomonas; Pseudomonas - chemistry; Solutions
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00187a006

A model for the solution structure of oxidized putidaredoxin (Pdx), a 106-residue globular protein containing a Fe2S2 cluster, has been determined using homonuclear NMR methods. Pdx is the first of the class of Fe2S2Cys4 ferredoxins which act as electron-transfer partners for P-450 monooxygenases to be structurally characterized, and no crystal structure has been determined for Pdx or for any closely homologous protein. Pdx is the physiological redox partner of cytochrome P-450cam. A total of 878 NOE distance constraints, 66 phi angular constraints derived from NH-C alpha H coupling constants, and five paramagnetic broadening constraints were used in simulated annealing structural refinements to obtain a family of structures with pairwise rms deviations of 1.14 A for backbone atoms and 1.80 A for all non-hydrogen atoms. Paramagnetic broadening of resonances within a ca. 8-A radius of the metal cluster prevents the use of NMR-derived constraints in this region of the protein; structural constraints used to model the environment of the metal cluster were obtained from site-directed mutagenesis and model compounds and by comparison with known ferredoxin structures. Pdx retains a similar folding topology to other structurally characterized Fe2S2Cys4 ferredoxins but differs from the other ferredoxins in containing a significantly more compact structure in the C-terminal half of the protein.