Pulsed EPR studies of the binuclear Mn(III)Mn(IV) center in catalase from Thermus thermophilus

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Abstract
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 14N atom that coordinates the Mn dimer, with magnetic parameters e2Qq = 2.34 MHz, eta = 0.51, and Aiso = 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 spectra are related to the properties of the hyperfine coupling tensors of the Mn ions and of the g tensor, which changes from axial symmetry (gparallel - gperpendicular = 0.018) in the untreated catalase to a nearly isotropic symmetry (gparallel - gperpendicular = 0.002) in the modified enzyme. The three-pulse ESEEM spectrum of the catalase is also completely altered after the reductive methylation, 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 hyperfine interaction from the coupled 14N donor is profoundly modified by the methylation treatment, changing from Aiso = 2.45 MHz to a larger value. The spectra are compared with ESEEM data obtained on two polynuclear Mn systems with 14N donors: the Mn cluster of Photosystem II inhibited by 14NH4Cl, and the model compound [Mn2(bipy)4(mu-O)2](ClO4)3. It is found that the ESEEM data measured on the untreated Mn(III)Mn(IV) catalase resemble those on the Photosystem II manganese site, suggesting that the coupled 14N coordinates the Mn dimer in an analogous fashion. By analogy to the mode of binding of ammonia in Photosystem II proposed by Britt et al. [Britt, R. D., Zimmermann, J. L., Sauer, K., & Klein, M. P. (1989) J. Am. Chem. Soc. 111, 3522-3532], it is proposed that a 14N atom bridges the two Mn ions in Mn(III)Mn(IV) catalase. By contrast, comparison of the data obtained on the methylated enzyme with those on the model compound suggests that the 14N couplings are similar in both systems; this is indicative of a terminal 14N ligand in the modified catalase.(ABSTRACT TRUNCATED AT 400 WORDS)

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