Monitoring the role of oxalate in manganese peroxidase

The water proton relaxation rate measurements between 0.01 and 50 MHz on water solutions containing the cyanide adduct of the manganese-depleted manganese peroxidase (MnP-CN^-) and increasing amounts of Mn²⁺ have been determined. The proton relaxivity curves have shown evidence of the formation of the protein/Mn²⁺ complex and have been analyzed in order to obtain spin Hamiltonian parameters and correlation times. Oxalate is shown not to alter the above profiles. This suggests that no protein-Mn²⁺-oxalate ternary complex is formed and that oxalate does not remove Mn²⁺ from the protein. On the basis of high-resolution ¹H NMR experiments, we propose that Ce³⁺ and Gd³⁺ bind at the manganese site, and, on the basis of the charge, we propose that they may mimic Mn³⁺. The water proton relaxation rates of water solutions containing manganese-depleted MnP-CN^- and increasing amounts of Gd³⁺ have been measured and analyzed. Oxalate is shown to remove the trivalent metal ions. This suggests that trivalent metal ions bind oxalate and diffuse away from the protein presumably as oxalate complexes. Implications for the enzymatic mechanism are discussed.