The double Mn2+ complex of concanavalin A with bound saccharide (SMMPL) was examined by electron paramagnetic resonance (EPR) spectroscopy and magnetic susceptibility measurements. A room temperature X-band (9 GHz) EPR spectrum of SMMPL revealed a relatively weak, broad resonance in contrast to the spectrum with a six-line hyperfine-split pattern observed for the mononuclear, high-spin Mn2+ complex found in Ca2+-Mn2+-concanavalin A with saccharide present (SCMPL). The EPR spectrum of SMMPL at 77 K, however, consisted of a series of overlapping patterns of 11 hyperfine-split lines near g = 2.0 with members of each pattern separated by 47 G, half the value of the hyperfine splitting of SCMPL. These 11-line patterns are preserved at Q-band (35 GHz), indicating that the manganese ions in SMMPL form a spin-coupled, binuclear center. As expected for an exchange-coupled system, the EPR signal of SMMPL at 77 K saturates at a higher microwave power than those for SCMPL or Mn2+ aquoion. There is also a marked loss of EPR signal intensity for SMMPL between 4.2 and 1.4 K, which supports the view that the pair of manganese ions is exchanged-coupled. The temperature dependence of both the magnetic susceptibility and the low-temperature EPR spectral intensity can be explained by a model in which the two high-spin Mn2+ ions of SMMPL are antiferromagnetically-exchanged-coupled with an isotropic coupling constant J = 1.8 cm-1(for the spin Hamiltonian Hex - JS1.S2). Zero-field splitting D’ was estimated to be 375 G from the EPR spectrum. The results provide direct evidence for coupled Mn2+ ions in the SMMPL complex, which is consistent with their binding at the S1 and S2 sites in the protein.
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