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

Abstract

The backbone dynamics of uniformly 15N-labeled reduced and oxidized putidaredoxin (Pdx) have been studied by 2D 15N NMR relaxation measurements. 15N T1 and T2 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 (S2), the effective correlation time for internal motions (tau e), and the 15N exchange broadening contributions (Rex) for each residue, as well as the overall correlation time (tau(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 T1/T2 ratios for residues 33 and 34, giving rise to a large Rex 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.

Redox-controlled backbone dynamics of human cytochrome c revealed by 15N NMR relaxation measurements

“…The backbone fluctuations of these loop regions on the picosecond -nanosecond timescales were suppressed for the oxidized form relative to the reduced form. While the timescale of the backbone dynamics we estimated here are quite fast, previous study also revealed that the amino acid residues in the interaction site for the partner protein show the redox-dependent picosecond -nanosecond backbone dynamics and suggested the electron transfer regulated by the redox-dependent backbone dynamics [20]. Although we cannot exclude the possibility that the binding of CcO might significantly perturb the dynamic properties of Cyt c, the site-specific changes of the backbone dynamics in the interaction site for CcO upon the oxidation strongly suggest that the redox-dependent dynamic property would have some advantages for dissociation of the Cyt c-CcO complex to facilitate the successive electron transfer to CcO.…”

“…As shown in Figure 2 (c), only two amino acid residues (Lys39 and Lys87) exhibited detectable effective internal motion, which is in sharp contrast to the results of putidaredoxin, an example of an iron-sulfur electron transfer [20]. In putidaredoxin, the effective internal motion was detected for 44 and 41 of 106 amide nitrogens in the reduced and oxidized forms, respectively.…”

Backbone Dynamics of Plastocyanin in Both Oxidation States

“…15 N relaxation data (90), R 1 experiments (91), as well as H/D exchange phenomena (92) reveal an increased mobility in the oxidized form also in the case of cytochrome b 5 ,. In the case of putidaredoxin, a [Fe 2 S 2 ] protein, the oxidized form again exhibits higher mobility than the reduced species in the sub-nanosecond and micro-millisecond time scale (93), and this behavior is paralleled by H/D exchange measurements (94). It would appear as if the present cupredoxin, representative of the third large class of electron transfer proteins, constitutes an exception.…”

The Complex Inter-Relationships Between Protein Flexibility and Stability

“…Several other examples exist that utilize a similar approach and reach the same conclusion. [180][181][182] The conformational stability and biological activity of Calmodulin (CaM) before and after methionine oxidation provides another example of the relationships between protein flexibility and oxidation. 183 The rate of methionine oxidation was shown to correlate with solvent exposure based on mass spectrometry measurements and resolution of different forms of oxidized CaM.…”