Redox-controlled Backbone Dynamics of Human Cytochrome c Revealed by N NMR Relaxation Measurements

Redox-controlled backbone dynamics in cytochrome c (Cyt c) were revealed by 2D N NMR relaxation experiments. N T1 and T2 values and H-N NOEs of uniformly N-labeled reduced and oxidized Cyt c were measured, and the generalized order parameters (S), the effective correlation time for internal motion (τe), the N exchalnge broadening contributions (Rex) for each residue, and the overal correlation time (τm) 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 the oxidation of the heme iron in Cyt c, the average S 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 S 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.