PULSED EPR STUDIES OF THE BINUCLEAR MN(III)MN(IV) CENTER IN CATALASE FROM THERMUS-THERMOPHILUS

The nature of possible protein ligands to the binuclear metal core in manganese catalase from Thermus thermophilus has been addressed by EPR and ESEEM (pulsed EPR) spectroscopies. The three-pulse ESEEM spectrum of the superoxidized Mn(III)Mn(IV) enzyme obtained at 3429 G shows a frequency pattern with peaks at 0.60, 1.45, 2.06, and 5.03 MHz that is assigned to the magnetic coupling in the exact cancellation regime of one N-14 atom that coordinates the Mn dimer, with magnetic parameters e(2)Qq = 2.34 MHz, eta = 0.51, and Al-iso = 2.45 MHz. When the enzyme is chemically modified by reductive methylation, dramatic effects are detected both in the CW-EPR spectrum and in the ESEEM data. Spectral simulations of the CW-EPR signal suggest that the alterations in the s pectra are related to the properties of the hyperfine coupling tensors of the Mn ions and of the g tenser, which changes from axial symmetry (\g(parallel to) - g(perpendicular to)\ = 0.018) in the untreated cat alase to a nearly isotropic symmetry (\g(parallel to) - g(perpendicula r to)\ = 0.002) in the modified enzyme. The three-pulse ESEEM spectrum of the catalase is also completely altered after the reductive methyl ation, with a rather different frequency pattern at 1.57, 2.35, 3.88, and 6.00 MHz. These data are interpreted as indicating that the hyperf ine interaction from the coupled N-14 donor is profoundly modified by the methylation treatment, changing from Al-iso = 2.45 MHz to a larger value. The spectra are compared with ESEEM data obtained on two polyn uclear Mn systems with N-14 donors: the Mn cluster of Photosystem II i nhibited by (NH4Cl)-N-14, and the model compound [Mn-2(bipy)(4)(mu-O)( 2)](ClO4)(3). It is found that the ESEEM data measured on the untreate d Mn(III)Mn(IV) catalase resemble those on the Photosystem II manganes e site, suggesting that the coupled N-14 coordinates the Mn dimer in a n analogous fashion. By analogy to the mode of binding of ammonia in P hotosystem II proposed by Britt et al. [Britt, R. D., Zimmermann, J. L ., Sauer, K., and Klein, M. P. (1989) J. Am. Chem. Soc. 111, 3522-3532 ], it is proposed that a N-14 atom bridges the two Mn ions in Mn(III)M n(IV) catalase. By contrast, comparison of the data obtained on the me thylated enzyme with those on the model compound suggests that the N-1 4 couplings are similar in both systems; this is indicative of a termi nal N-14 ligand in the modified catalase. The analysis of the changes in the amino acid composition of the enzyme that is induced by the red uctive methylation indicates that the amino acid side chain that is pr oposed to bridge the two metal ions in Mn(III)Mn(IV) catalase is most probably the epsilon-amino group of a lysine residue.