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

Redox-controlled backbone dynamics in cytochrome c (Cyt c) were revealed by 2D 15N NMR relaxation experiments. 15N T1 and T2 values and 1H-15N NOEs of uniformly 15N-labeled reduced and oxidized Cyt c were measured, and the generalized order parameters (S2), the effective correlation time for internal motion (taue), the 15N exchange broadening contributions (Rex) for each residue, and the overall correlation time (taum) were estimated by model-free dynamics formalism. These dynamic parameters clearly showed that the backbone dynamics of Cyt c are highly restricted due to the covalently bound heme that functions as the stable hydrophobic core. Upon oxidation of the heme iron in Cyt c, the average S2 value was increased from 0.88+/-0.01 to 0.92+/-0.01, demonstrating that the mobility of the backbone is further restricted in the oxidized form. Such increases in the S2 values were more prominent in the loop regions, including amino acid residues near the thioether bonds to the heme moiety and positively charged region around Lys87. Both of the regions are supposed to form the interaction site for cytochrome c oxidase (CcO) and the electron pathway from Cyt c to CcO. The redox-dependent mobility of the backbone in the interaction site for the electron transfer to CcO suggests an electron transfer mechanism regulated by the backbone dynamics in the Cyt c-CcO system.