Comparison of Backbone Dynamics of Oxidized and Reduced Putidaredoxin by 15N NMR Relaxation Measurements

• Published in: Biochemistry (Easton) Vol. 38; no. 31; pp. 9862 - 9871
• Main Authors: Sari, Neşe, Holden, Marcia J, Mayhew, Martin P, Vilker, Vincent L, Coxon, Bruce
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 03.08.1999
• Subjects: Amides - chemistry; Bacterial Proteins - chemistry; Entropy; Ferredoxins - chemistry; Models, Molecular; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular - methods; Oxidation-Reduction; Protein Conformation; Pseudomonas putida; Thermodynamics; Tryptophan - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9906423

The backbone dynamics of uniformly 15N-labeled reduced and oxidized putidaredoxin (Pdx) have been studied by 2D 15N NMR relaxation measurements. 15N T 1 and T 2 values and 1H−15N NOEs have been measured for the diamagnetic region of the protein. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameters (S 2), the effective correlation time for internal motions (τ e), and the 15N exchange broadening contributions (R ex) for each residue, as well as the overall correlation time (τ m). Order parameters for the reduced Pdx are generally higher than for the oxidized Pdx, and there is increased mobility on the microsecond to millisecond time scale for the oxidized Pdx, in comparison with the reduced Pdx. These results clearly indicate that the oxidized protein exhibits higher mobility than the reduced one, which is in agreement with the recently published redox-dependent dynamics studied by amide proton exchange. In addition, we observed very high T 1/T 2 ratios for residues 33 and 34, giving rise to a large R ex contribution. Residue 34 is believed to be involved in the binding of Pdx to cytochrome P450cam (CYP101). The differences in the backbone dynamics are discussed in relation to the oxidation states of Pdx, and their impact on electron transfer. The entropy change occurring on oxidation of reduced Pdx has been calculated from the order parameters of the two forms.