The ligation environment of the tightly bound Mn2+ in cytochrome c oxidase from Rhodobacter sphaeroides has been characterized by electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM). The EPR data show that the Mn2+ is six-coordinate and located in a highly symmetric binding site. Analyses of X- and Q-band EPR spectra show that the zero field splitting parameter D is 115 +/- 25 G (0.0107 +/- 0.0023 cm-1) in the fully oxidized enzyme and 125 +/- 15 G (0.0117 +/- 0.0014 cm-1) in the fully reduced enzyme. For both redox forms of the enzyme the value of E is < or = 25 G (0.0023 cm-1). By comparison with crystal structures of Mn2+ binding proteins, the structural changes at the Mn2+ binding site upon redox state change of the enzyme are estimated to be < or = 0.2 A in ligand bond lengths and < or = 10 degrees in bond angle. This analysis indicates that little modification occurs at the Mn2+ site upon redox change at the other metal centers. Considering the proximity of the Mn2+ site to heme a and heme a3-CuB [Hosler, J. P., Espe, M. P., Zhen, Y., Babcock, G. T., & Ferguson-Miller, S. (1995) Biochemistry 34, 7586-7592], we interpret these results to imply also that there is no large protein conformational change near the heme a and heme a3-CuB sites upon a change in their redox states. Multifrequency 3-pulse ESEEM results provide direct evidence for a nitrogen ligand to the Mn2+, which is assigned to a histidine by comparison with ESEEM studies of Mn(2+)-bound lectins [McCracken, J., Peisach, J., Bhattacharyya, L., & Brewer, F. (1991) Biochemistry 30, 4486-4491] and specifically to His-411 in subunit 1 on the basis of mutagenesis studies (Hosler et al., 1995). From these results a partial model of the Mn2+ binding site has been constructed.