Redox-dependent dynamics of putidaredoxin characterized by amide proton exchange.
Multidimensional NMR methods were used to obtain 1H-15N correlations and 15N 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-450cam (CYP101). A model for the solution structure of oxidized Pdx has been determined recently using NMR methods (Pochapsky TC, Ye XM, Ratnaswamy G, Lyons TA, 1994, Biochemistry 33:6424-6432) and redox-dependent 1H NMR spectral features have been described (Pochapsky TC, Ratnaswamy G, Patera A, 1994, Biochemistry 33:6433-6441). 15N assignments were made with NOESY-(1H/15N) HMQC and TOCSY-(1H/15N) HSQC spectra obtained using samples of Pdx uniformly labeled with 15N. Local dynamics in both oxidation states of Pdx were then characterized by comparison of residue-specific amide proton exchange rates, which were measured by a combination of saturation transfer and H2O/D2O exchange methods at pH 6.4 and 7.4 (uncorrected for isotope effects). In general, where exchange rates for a given site exhibit significant oxidation-state dependence, the oxidized protein exchanges more rapidly than the reduced protein. The largest dependence of exchange rate upon oxidation state is found for 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 between Pdx and CYP101 upon the oxidation state of Pdx.
Lyons
T A
TA
Department of Biology, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
Ratnaswamy
G
G
Pochapsky
T C
TC
D001426:Bacterial Proteins / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D011522:Protons
D001426:Bacterial Proteins; D005288:Ferredoxins; D011522:Protons; C100137:putidaredoxin