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 dete rmined using homonuclear NMR methods. Pdx is the first of the class of Fe(2)S(2)Cys(4) ferredoxins which act as electron-transfer partners f or P-450 monooxygenases to be structurally characterized, and no cryst al structure has been determined for Pdx or for any closely homologous protein. Pdx is the physiological redox partner of cytochrome P-450(c am). A total of 878 NOE distance constraints, 66 phi angular constrain ts derived from NH-CalphaH coupling constants, and five paramagnetic b roadening constraints were used in simulated annealing structural refi nements to obtain a family of structures with pairwise rms deviations of 1.14 Angstrom for backbone atoms and 1.80 Angstrom for all nonhydro gen atoms. Paramagnetic broadening of resonances within a ca. 8-Angstr om radius of the metal duster prevents the use of NMR-derived constrai nts in this region of the protein; structural constraints used to mode l the environment of the metal cluster were obtained from site-directe d mutagenesis and model compounds and by comparison with known ferredo xin structures. Pdx retains a similar folding topology to other struct urally characterized Fe(2)S(2)Cys(4) ferredoxins but differs from the other ferredoxins in containing a significantly more compact structure in the C-terminal half of the protein.