Parallel Polarization EPR Characterization of the Mn(III) Center of Oxidized Manganese Superoxide Dismutase

Abstract

Manganese superoxide dismutase (MnSOD) is an enzyme found in mitochondria and chloroplasts of eukaryotes and in the cytoplasm of bacteria.1,2 The redox active Mn ion cofactor catalyzes the one-electron redox cycle by a two-step disproportionation reaction with oxidized Mn3+ in the resting state. In this report, we have used parallel polarization CW-EPR to investigate the paramagnetic Mn3+ ion of the MnSOD enzyme from Escherichia coli overexpressed from pALS1 in HMS174/ DE3. The crystal structure of native MnSOD from E. coli has been determined to 2.1 A resolution.3 Crystal structures have also been solved for MnSOD of Thermus thermophilus4 and human mitochondria.5 These structures show high homology between the bacterial and the eukaryotic MnSOD and confirm that in each case the Mn3+ ion has five ligands (three histidines, one aspartate, and one hydroxide ion) with a distorted trigonal-bipyramidal geometry. In this configuration, the d4 Mn3+ ion is high-spin with an effective total spin S ) 2.6,7 The energy levels of an S ) 2 integer spin system with a positive axial zero-field splitting value, D, are arranged as shown in Figure 1. An EPR signal from the MnSOD Mn3+ ion has not previously been detected with conventional X-band EPR methods. D is predicted to be more than 2 cm-1 based on SQUID saturation magnetization studies of the oxidized form of native MnSOD,7 or between 1-2 cm-1 based on MCD studies.6 Because of the large zero-field splitting values, the conventional perpendicular polarization EPR allowed ∆Ms ) (1 transitions are not possible at normal X-band microwave frequencies (0.3 cm-1) (See Figure 1). However, EPR signals from integer spin systems can be detected when the oscillating magnetic field applied to induce a spin-state transition is oriented parallel to the static magnetic field. The parallel magnetic field orientation allows transitions between the closely spaced Ms ) (2 energy levels (Figure 1 inset), to be observed with signal intensities many orders of magnitude larger than the corresponding perpendicular magnetic field orientation.8 Figure 2a shows the parallel polarization EPR spectrum of the Mn3+ ion of native MnSOD. This signal is centered at an effective g value of 8.17 and consists of six hyperfine lines separated by approximately 100 G. This EPR signal is absent in the conventional perpendicular mode spectrum (data not shown). The spin Hamiltonian describing this system is