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

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...

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Bibliographic Details
Published in:Biochemistry (Easton) 1994-05, Vol.33 (21), p.6424-6432
Main Authors: Pochapsky, Thomas C, Ye, Xiao Mei, Ratnaswamy, Gayathri, Lyons, Teresa A
Format: Article
Language:English
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
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Summary: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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00187a006