Comparison of backbone dynamics of oxidized and reduced putidaredoxin by N-15 NMR relaxation measurements

The backbone dynamics of uniformly N-15-labeled reduced and oxidized putida redoxin (Pdx) have been studied by 2D N-15 NMR relaxation measurements. N-1 5 T-1 and T-2 values and H-1-N-15 NOEs have been measured for the diamagnet ic region of the protein. These data were analyzed by using a model-free dy namics formalism to determine the generalized order parameters (S-2), the e ffective correlation time for internal motions (tau(e)), and the N-15 excha nge broadening contributions (R-ex) for each residue, as well as the overal l correlation time (tau(m)). Order parameters for the reduced Pdx are gener ally higher than for the oxidized Pdx, and there is increased mobility on t he microsecond to millisecond time scale for the oxidized Pdx, in compariso n with the reduced Pdx. These results clearly indicate that the oxidized pr otein exhibits higher mobility than the reduced one, which is in agreement with the recently published redox-dependent dynamics studied by amide proto n 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 d ifferences in the backbone dynamics are discussed in relation to the oxidat ion states of Pdx, and their impact on electron transfer. The entropy chang e occurring on oxidation of reduced Pdx has been calculated from the order parameters of the two forms.