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

Abstract

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 ø angular constraints derived from NH-CαH 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 Å for backbone atoms and 1.80 Å for all non-hydrogen atoms. Paramagnetic broadening of resonances within a ca. 8-Å 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. © 1994, American Chemical Society. All rights reserved.