A CONTINUOUS-WAVE AND PULSED EPR CHARACTERIZATION OF THE MN2+ BINDING-SITE IN RHODOBACTER-SPHAEROIDES CYTOCHROME-C-OXIDASE

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 less than or equal to 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 less than or equal to 0.2 Angstrom in ligand bond lengths and less than or equal to 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 alpha and heme alpha(3)-Cu-B [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 alpha and heme alpha(3)-Cu-B 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 Mn2+-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.