PH-DEPENDENT PROPERTIES OF SOD STUDIED THROUGH MUTANTS ON LYS-136

The pH dependence of (i) the water H-1 NMR longitudinal relaxation rat es, T1(-1), at 0.03, 0.04, and 0.06 MHz for oxidized copper-zinc super oxide dismutase (Cu2Zn2SOD), (ii) the H-1 NMR shifts of oxidized Cu2Co 2SOD, and (iii) the H-1 NMR shifts of reduced Cu2Zn2SOD have been meas ured for three mutants in which Lys-136 has been substituted with Ala, Gln, and Arg. Lys-136 is sitting in the protein active channel. The p K(a) values of 11.5, on average, obtained for the oxidized form and 10 .8, on average, for the reduced form are virtually the same for the mu tants and the wild type protein. The latter pK(a) is known to be due t o deprotonation of His-63, which is not bridging the metal ions in the reduced species. It appears that the Lys residue cannot be responsibl e for the pK(a) values of the oxidized form nor is capable of affectin g the two pK(a)'s. After the role of Lys-136 was ruled out, the pK(a) of the oxidized species was tentatively assigned to the semicoordinate d water molecule, since all the copper histidine ligands remain coordi nated. The affinity of the mutants for N3- is reduced in the case of G ln, essentially unchanged for Ala, and increased for Arg. SOD activiti es arc similar to those of the wild type, except a slight increase for the Arg mutant. Both results are consistent with a positive charge re latively far from the active site.