Backbone dynamics of human Cu,Zn superoxide dismutase and of its monomeric F50E/G51E/E133Q mutant: the influence of dimerization on mobility and function.
The backbone assignment of reduced human dimeric Cu,Zn superoxide dismutase (SOD) was performed on a sample 100% enriched in (15)N, (13)C and 70% enriched in (2)H. (15)N T(1), T(2), and T(1)(rho) and (15)N-(1)H NOE assignment was performed at 600 MHz proton frequency on both wild-type SOD and the monomeric F50E/G51E/E133Q mutant. This allowed a comparison of the mobility in the subnanosecond and in the millisecond to microsecond time scales of the two systems. Both proteins are rather rigid, although some breathing of the beta sheets is detected in the wild type dimer. The monomer displays large mobility in the loops in the first part of the sequence, in loop IVa where point mutations have been introduced and at the C-terminus. The dimeric wild type is rigidified at loop IVa and at the C-terminus. Only loop VII shows a higher mobility in the dimer (besides some individual NH moieties). Conformational equilibria are displayed in the monomeric form around cysteines 57 and 146, thus explaining the disorder of arginine 143 which is the most important residue in guiding O(2)(-) toward the copper ion. The larger mobility in the wild type form with respect to the monomer in the picosecond to nanosecond time scale of helix alpha1 and loop VIIb, which provides the correct electrostatic driving force for O(2)(-) in the active channel, has been discussed in terms of favoring the activity of SOD.