REDOX-DEPENDENT DYNAMICS OF PUTIDAREDOXIN CHARACTERIZED BY AMIDE PROTON-EXCHANGE

Multidimensional NMR methods were used to obtain H-1-N-15 correlations and N-15 resonance assignments for amide and side-chain nitrogens of oxidized and reduced putidaredoxin (Pdx), the Fe2S2 ferredoxin, which acts as the physiological reductant of cytochrome P-450(cam) (CYP101). A model for the solution structure of oxidized Pdx has been determine d recently using NMR methods (Pochapsky TC, Ye XM, Ratnaswamy G, Lyons TA, 1994, Biochemistry 33:6424-6432) and redox-dependent H-1 NMR spec tral features have been described (Pochapsky TC, Ratnaswamy G, Patera A, 1994, Biochemistry 33:6433-6441). N-15 assignments were made with N OESY-(H-1/N-15) HMQC and TOCSY-(H-1/N-15) HSQC spectra obtained using samples of Pdx uniformly labeled with N-15. Local dynamics in both oxi dation states of Pdx were then characterized by comparison of residue- specific amide proton exchange rates, which were measured by a combina tion of saturation transfer and H2O/D2O exchange methods at pH 6.4 and 7.4 (uncorrected for isotope effects). In general, where exchange rat es for a given site exhibit significant oxidation-state dependence, th e oxidized protein exchanges more rapidly than the reduced protein. Th e largest dependence of exchange rate upon oxidation state is found fo r residues near the metal center and in a region of compact structure that includes the loop-turn Val 74-Ser 82 and the C-terminal residues (Pro 102-Trp 106). The significance of these findings is discussed in light of the considerable dependence of the binding interaction betwee n Pdx and CYP101 upon the oxidation state of Pdx.