Backbone dynamics of human Cu,Zn superoxide dismutase and of its monomericF50E/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 N-15, C-13 and 70% enrich ed in H-2. N-15 T-1, T-2, and T-1 rho and N-15-H-1 NOE assignment was perfo rmed at 600 MHz proton frequency on both wild-type SOD and the monomeric F5 0E/G51E/E133Q mutant. This allowed a comparison of the mobility in the subn anosecond and in the millisecond to microsecond time scales of the two syst ems. Both proteins are rather rigid, although some breathing of the beta sh eets is detected in the wild type dimer. The monomer displays large mobilit y in the loops in the first part of the sequence, in loop IVa where point m utations 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 high er mobility in the dimer (besides some individual NH moieties). Conformatio nal equilibria are displayed in the monomeric form around cysteines 57 and 146, thus explaining the disorder of arginine 143 which is the most importa nt 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 nanosec ond time scale of helix oil and loop VIIb, which provides the correct elect rostatic driving force for O-2(-) in the active channel, has been discussed in terms of favoring the activity of SOD.