Probing the backbone dynamics of oxidized and reduced rat microsomal cytochrome b5 via 15N rotating frame NMR relaxation measurements: biological implications.
Rotating frame 15N relaxation NMR experiments have been performed to study the local mobility of the oxidized and reduced forms of rat microsomal cytochrome b5, in the microsecond to millisecond time range. Measurements of rotating frame relaxation rates (R1rho) were performed as a function of the effective magnetic field amplitude by using off-resonance radio frequency irradiation. Detailed analysis of the two data sets resulted in the identification of slow motions along the backbone nitrogens for both oxidation states of the protein. The local mobility of reduced and oxidized cytochrome b5 turned out to be significantly different; 28 backbone nitrogens of the oxidized form were shown to participate in a conformational exchange process, while this number dropped to 12 in the reduced form. The correlation time, tauex, for the exchange processes could be determined for 21 and 9 backbone nitrogens for oxidized and reduced cytochrome b5, respectively, with their values ranging between 70 and 280 microseconds. The direct experimental evidence provided in this study for the larger mobility of the oxidized form of the protein is consistent with the different backbone NH solvent exchangeability recently documented for the two oxidation states [Arnesano, F., et al. (1998) Biochemistry 37, 173-184]. Our experimental observations may have significant biological implications. The differential local mobility between the two oxidation states is proposed to be an important factor controlling the molecular recognition processes in which cytochrome b5 is involved.
Department of Chemistry, University of Florence, Italy.
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Banci
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Bertini
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Cavazza
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Felli
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Koulougliotis
D000818:Animals; D015786:Cytochromes b5 / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D004579:Electron Transport; D008862:Microsomes, Liver / Q000201:enzymology*; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D051381:Rats; D013816:Thermodynamics
D009587:Nitrogen Isotopes; D015786:Cytochromes b5