X-ray absorption (XAS), UV-visible, electron paramagnetic resonance (EPR), and electron spin echo envelope modulation (ESEEM) spectroscopies have been used to characterize the interaction of azide and cyanide with the Mn(III)(mu-O)(2)Mn(IV) site in superoxidized Mn catalase. The addition of azide causes no significant change in the X-ray absorption near edge structure (XANES) region and only minor changes in the extended X-ray absorption fine structure (EXAFS) spectra, consistent with only minimal changes in Mn-ligand geometry. In contrast, addition of either azide or cyanide causes an approximately 3-fold increase in the intensity of the visible absorption bands and a small (approximately 4%) decrease in the Mn hyperfine coupling. Anion-binding titrations indicate cooperativity in anion binding. ESEEM experiments on the azide-and cyanide-free enzyme reveal hyperfine (A = 2.88 MHz) and electric quadrupolar couplings (e(2)qQ=2.29 MHz and eta=0.58) for a single N-14 nucleus of the protein. These parameters are slightly altered upon addition of azide or cyanide, but ESEEM studies with N-15-labeled versions of these inhibitors show that the altered modulation is also due to protein-derived N-14. nh, ESEEM experiments show no evidence for coupling of azide-or cyanide-derived nitrogens to the Mn cluster. The outer shell scattering in the EXAFS suggests coordination of histidines to the binuclear Mn cluster, and the quadrupolar couplings observed for the protein-derived ESEEM detectable N-14 nucleus are consistent with those expected for a histidine imidazole coordinated to Mn(TV). Taken together, these results suggest a model in which azide binds to a protein-derived site, rather than binding directly to either Mn. Despite this indirect binding, azide causes minor perturbations in the Mn-2(mu-O)(2) geometry, consistent with a slight flattening of the Mn-2 core.