A refined model for the solution structure of oxidized putidaredoxin

A refined model for the solution structure of oxidized putidaredoxin (Pdx(o)), a Cys(4)Fe(2)S(2) ferredoxin, has been determined. A previous structure (Pochapsky et al. (1994) Biochemistry 33, 6424-6432; PDB entry 1PUT) was calculated using the results of homonuclear two-dimensional NMR experiments. New data has made it possible to calculate a refinement of the original Pdx(o) solution structure, First, essentially complete assignments for diamagnetic N-15 and C-13 resonances of Pdx(o) have been made using multidimensional NMR methods, and N-15- and C-13-resolved NOESY experiments have permitted the identification of many new NOE restraints for structural calculations, Stereospecific assignments for leucine and valine CH3 resonances were made using biosynthetically directed fractional C-13 labeling, improving the precision of NOE restraints involving these residues. Backbone dihedral angle restraints have been obtained using a combination of two-dimensional J-modulated N-15,H-1 HSQC and 3D (HN)CO(CO)NH experiments. Second, the solution structure of a diamagnetic form of Pdx, that of the C85S variant of gallium putidaredoxin, in which a nonligand Cys is replaced by Ser, has been determined (Pochapsky et al. (1998) J. Biomol. NMR 12, 407-415), providing information concerning structural features not observable in the native ferredoxin due to paramagnetism. Third, a crystal structure of a closely related ferredoxin, bovine adrenodoxin, has been published (Muller et al. (1998) Structure 6, 269-280). This structure has been used to model the metal binding site structure in Pdx. A family of fourteen structures is presented that exhibits an rmsd of 0.51 Angstrom for backbone heavy atoms and 0.83 Angstrom for all heavy atoms. Exclusion of the modeled metal binding loop region reduces overall the rmsd to 0.30 Angstrom for backbone atoms and 0.71 Angstrom for all heavy atoms.