REDOX-DEPENDENT H-1-NMR SPECTRAL FEATURES AND TERTIARY STRUCTURAL CONSTRAINTS ON THE C-TERMINAL REGION OF PUTIDAREDOXIN

Putidaredoxin (Pdx) is a 106-residue Fe2S2 ferredoxin which acts as th e physiological reductant and effector of cytochrome P-450(cam). Pdx h as two accessible oxidation states, Fe+(3)-Fe+(3) (oxidized) and Fe+3- Fe+2 (reduced), and exhibits redox-dependent binding affinities for cy tochrome P-450(cam), with reduced Pdx binding over 100-fold more tight ly than oxidized Pdx to the oxidized cytochrome P-450(cam) [Hintz, M. J., Mock, D. M., Peterson, L. L., Tuttle, K., & Peterson, J. A. (1982) J. Biol. Chem. 257, 14324-14332]. The analysis of two-dimensional H-1 NMR experiments has yielded sequential H-1 resonance assignments for the diamagnetic regions of the reduced form of Pdx, which are compared to those of oxidized Pdx, described previously [Ye, X. M., Pochapsky, T. C., & Pochapsky, S. S. (1992) Biochemistry 31, 1961-1968]. Increas ed unpaired electron-spin density on the metal cluster in reduced rela tive to oxidized Pdx increases the number of H-1 resonances which are broadened by the metal cluster, and the pattern of paramagnetic broade ning provides information concerning the placement of the metal cluste r within the protein. Two-dimensional exchange experiments on half-red uced samples of Pdx indicate that electron self-exchange is slow on th e chemical shift time scale, with a second-order rate constant less th an or equal to 66 M(-1) s(-1) at 290 K. Spectral changes unrelated to increases in unpaired electron-spin density are also observed. The lar gest changes of this type are observed for features structurally conti guous with the C-terminal region Pro 102-Trp 106. The C-terminal resid ue Trp 106 has been implicated in binding to cytochrome P-450(cam). NO E constraints on the structure of this region are described, and a mod el for the solution structure of Pdx is used to rationalize the observ ed paramagnetic broadening patterns. A hypothesis is offered to ration alize observed redox-state-dependent binding of Pdx to P-450(cam) and changes in midpoint potential of oxidized Pdx upon binding to oxidized P-450(cam).