Effect of distal histidines on hydrogen peroxide activation by manganese reconstituted myoglobin.

Myoglobins provide an opportunity to investigate the effect of the secondary coordination sphere on the functionality and reactivity of non-native metal porphyrins inside well-defined protein scaffolds. In this work, we reconstituted myoglobin by the replacement of natural heme with manganese(iii) protoporphyrin IX and firstly investigated the effect of distal histidine on the reaction of Mn(III) porphyrin with H2O2 and one-electron oxidation of ABTS. We have prepared L29H, F43H, H64F, L29H/H64F, F43H/H64F, L29H/F43H and L29H/F43H/H64F mutants and reconstituted apo-myoglobins with manganese(iii) protoporphyrin IX. Distal histidine at the 64 position plays an essential role in binding H2O2 through hydrogen bond formation, which facilitates the coordination of H2O2 to the Mn center. The second histidine at the 43 position is important in the cleavage of the O-O bond and to form the highly valent Mn(iv)-oxo intermediate. His29 has less efficiency to activate H2O2, because it is too far from the Mn center. The cooperative effect of dual distal histidines at positions 64 and 43 on the activation of H2O2 was observed and the F43H Mn(III)Mb mutant exhibited 5-fold and 10-fold reaction rate increases in the activation of H2O2 and one-electron oxidation of ABTS versus wild-type Mn(III)Mb. This is different from the distal histidine effect on the H2O2 activation by heme in Mb. This work will provide new insights to understand the fundamental chemistry of manganese in oxidation, and further construct biomimetic Mn models for peroxidase, inside or outside of protein scaffolds.