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 c alculated using the results of homonuclear two-dimensional NMR experiments. New data has made it possible to calculate a refinement of the original Pd x(o) solution structure, First, essentially complete assignments for diamag netic N-15 and C-13 resonances of Pdx(o) have been made using multidimensio nal NMR methods, and N-15- and C-13-resolved NOESY experiments have permitt ed the identification of many new NOE restraints for structural calculation s, Stereospecific assignments for leucine and valine CH3 resonances were ma de using biosynthetically directed fractional C-13 labeling, improving the precision of NOE restraints involving these residues. Backbone dihedral ang le restraints have been obtained using a combination of two-dimensional J-m odulated N-15,H-1 HSQC and 3D (HN)CO(CO)NH experiments. Second, the solutio n structure of a diamagnetic form of Pdx, that of the C85S variant of galli um putidaredoxin, in which a nonligand Cys is replaced by Ser, has been det ermined (Pochapsky et al. (1998) J. Biomol. NMR 12, 407-415), providing inf ormation concerning structural features not observable in the native ferred oxin due to paramagnetism. Third, a crystal structure of a closely related ferredoxin, bovine adrenodoxin, has been published (Muller et al. (1998) St ructure 6, 269-280). This structure has been used to model the metal bindin g 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 Angstro m for all heavy atoms. Exclusion of the modeled metal binding loop region r educes overall the rmsd to 0.30 Angstrom for backbone atoms and 0.71 Angstr om for all heavy atoms.