OXIDATIVE MODIFICATION OF A CARBOXYL-TERMINAL VICINAL METHIONINE IN CALMODULIN BY HYDROGEN-PEROXIDE INHIBITS CALMODULIN-DEPENDENT ACTIVATION OF THE PLASMA-MEMBRANE CA-ATPASE

In order to investigate the possibility that calmodulin (CaM) may be a principal target of reactive oxygen species (ROS) produced undercondi tions of oxidative stress, we have examined wheat germ CaM for the pre sence of highly reactive sites that correlate with the loss of functio n, Using reversed-phase HPLC and FAB mass spectrometry after proteolyt ic digestion, we have identified the sites of modification by hydrogen peroxide, We find that one of the vicinal methionines (i.e., Met(146) or Met(147)) near the C-terminus of CaM is selectively oxidized, The ability of CaM to bind and to activate the plasma membrane (PM) Ca-ATP ase from erythrocytes was measured. There is a 30-fold decrease in the calcium affinity of oxidatively modified CaM. While there is little c hange in the binding constant between the carboxyl-terminal domain of calcium-saturated CaM and a peptide homologous to the autoinhibitory s equence of the PM-Ca-ATPase, we find that there is a 9-fold reduction in the affinity of the aminoterminal domain of CaM with respect to the ability to bind target peptides. The extent of oxidative modification to one of the vicinal methionines near the carboxyl-terminal domain c orrelates with the loss of CaM-dependent activation of the PM-Ca-ATPas e. The presence of oxidatively modified CaM prevents native CaM from a ctivating the PM-Ca-ATPase, indicating that the oxidatively modified C aM binds to the autoinhibitory sequence on the Ca-ATPase in an altered nonproductive conformation. We suggest that the functional sensitivit y of CaM to the oxidation of one of the C-terminal vicinal methionines permits CaM to serve a regulatory role in modulating cellular metabol ism under conditions of oxidative stress. The predominant oxidation of a methionine near the carboxyl terminal of CaM is rationalized in ter ms of the enhanced solvent accessibility of these vicinal methionines.