EPR polarization studies on Mn catalase from Lactobacillus plantarum.

The binuclear manganese active site of Mn catalase catalyzes redox disproportionation of hydrogen peroxide, forming dioxygen and water. We report here multifrequency EPR and microwave polarization studies of the catalytically active homovalent Mn2+ complex of Lactobacillus plantarum Mn catalase, resolving spectra from each of the thermally accessible multiplet states of the coupled complex by multivariate methods. The experimental spectra have been simulated using computational approaches for the binuclear cluster to predict both intensity and polarization for arbitrary values of the ground state parameters. These two spectroscopic properties define the nature of the ground state wavefunctions and so serve as a sensitive and quantitative measure of the inter-ion interactions in the reduced complex. Interpretation of the spectra in terms of a pair Hamiltonian that includes Heisenberg exchange, dipolar, single site zero field splitting, and Zeeman perturbations leads to the most complete ground state description of the active site metal centers. The results of this spectroscopic analysis support a picture of two high spin ions weakly coupled by exchange interactions (J = 40 cm-1) with relatively small dipole-dipole coupling and single site zero field splittings for the ligand-free reduced enzyme. The coupling between fluoride binding and protonation of the complex has been demonstrated by proton uptake studies. The binding of two fluoride ions in the active site dramatically changes the pair spectra, reflecting a substantially reduced J-coupling (J = 10.5 cm-1) that must be a consequence of perturbation of the bridging ligands. Anion binding to the binuclear Mn complex appears to result in poisoning of the active site by protons, possibly associated with insertion of fluoride into bridging positions of the dimanganese core.