We used single-molecule fluorescence spectroscopy to probe the conformation of calmodulin (CaM) bound to oxidatively modified plasma-membrane Ca(2+)-ATPase (PMCAox). We found that oxidative modification altered the coupling between the ATP binding domain and the autoinhibitory domain. Oxidative modification of PMCA is known to result in a loss of activity for the enzyme. Conformations of PMCAox-CaM complexes were probed by single-molecule polarization modulation spectroscopy, which measured the orientational mobility of fluorescently labeled CaM bound to PMCAox. We detected an enhanced population of PMCAox-CaM complexes with a low orientational mobility in the presence of ATP, whereas nonoxidized PMCA-CaM complexes existed almost exclusively in a high-mobility state in the presence of ATP. We have previously attributed such high-mobility states to PMCA-CaM complexes with a dissociated autoinhibitory/CaM binding domain, whereas the lower-mobility state was attributed to autoinhibited PMCA-CaM complexes with a nondissociated autoinhibitory domain [Osborn, K. D., et al. (2004) Biophys. J. 87, 1892-1899]. In the absence of ATP, the orientational mobility distributions are similar for CaM complexed with oxidized PMCA or nonoxidized PMCA. These results suggest that oxidative modification of PMCA reduced the propensity of the autoinhibitory domain to dissociate from binding sites near the catalytic core of the enzyme with bound nucleotide upon CaM stimulation in the presence of Ca(2+). This interpretation was further supported by chymotrypsin proteolysis, which probes the tightness of binding of the autoinhibitory domain to sites near the catalytic core of the enzyme. Enhanced proteolysis was observed for PMCA upon binding CaM or ATP. In contrast, proteolysis was partially blocked for oxidatively modified PMCA, even in the presence of ATP.