EPR POLARIZATION STUDIES ON MN CATALASE FROM LACTOBACILLUS-PLANTARUM

The binuclear manganese active site of Mn catalase catalyzes redox dis proportionation of hydrogen peroxide, forming dioxygen and water. We r eport here multifrequency EPR and microwave polarization studies of th e catalytically active homovalent Mn2+ complex of Lactobacillus planta rum Mn catalase, resolving spectra from each of the thermally accessib le multiplet states of the coupled complex by multivariate methods. Th e experimental spectra have been simulated using computational approac hes for the binuclear cluster to predict both intensity and polarizati on for arbitrary values of the ground state parameters, These two spec troscopic properties define the nature of the ground state wavefunctio ns and so serve as a sensitive and quantitative measure of the inter-i on interactions in the reduced complex. Interpretation of the spectra in terms of a pair Hamiltonian that includes Heisenberg exchange, dipo lar, 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 demo nstrated by proton uptake studies. The binding of two fluoride ions in the active site dramatically changes the pair spectra, reflecting a s ubstantially reduced J-coupling (J 10.5 cm(-1)) that must be a consequ ence of perturbation of the bridging ligands. Anion binding to the bin uclear 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.