PROBING THE BACKBONE DYNAMICS OF OXIDIZED AND REDUCED RAT MICROSOMAL CYTOCHROME B(5) VIA N-15 ROTATING-FRAME NMR RELAXATION MEASUREMENTS - BIOLOGICAL IMPLICATIONS

Rotating frame N-15 relaxation NMR experiments have been performed to study the local mobility of the oxidized and reduced forms of rat micr osomal cytochrome b(5), in the microsecond to millisecond time range. Measurements of rotating frame relaxation rates (R-1 rho) were perform ed as a function of the effective magnetic field amplitude by using of f-resonance radio frequency irradiation. Detailed analysis of the two data sets resulted in the identification of slow motions along the bac kbone nitrogens for both oxidation states of the protein. The local mo bility of reduced and oxidized cytochrome b(5) turned out to be signif icantly different; 28 backbone nitrogens of the oxidized form were sho wn to participate in a conformational exchange process, while this num ber dropped to 12 in the reduced form. The correlation time, tau(ex), for the exchange processes could be determined for 21 and 9 backbone n itrogens for oxidized and reduced cytochrome b(5), respectively, with their values ranging between 70 and 280 mu s. The direct experimental evidence provided in this study for the larger mobility of the oxidize d form of the protein is consistent with the different backbone NH sol vent exchangeability recently documented for the two oxidation states [Arnesano, F., et al. (1998) Biochemistry 37, 173-184]. Our experiment al observations may have significant biological implications. The diff erential local mobility between the two oxidation states is proposed t o be an important factor controlling the molecular recognition process es in which cytochrome b(5) is involved.