Isolation and characterization of a microperoxidase-8 with a modified histidine axial ligand

Microperoxidase-8, Fe(III)MP-8, the heme octapeptide obtained by horse heart cytochrome c digestion, was studied in the presence of H2O2. A modified form of the catalyst was isolated by HPLC and showed a UV/visible spectrum similar to that of Fe(III)MP-8. ESI-MS measurements revealed a 16 Da increase in molecular mass for the modified catalyst when compared to Fe(III)MP-8, suggesting the insertion of an oxygen atom. ESI-MS2 fragmentation measurements point at oxygen incorporation on the His18 residue of the octapeptide of the modified catalyst. Comparison of the 1H NMR chemical shifts of the methyl protons of the porphyrin ring of Fe(III)MP-8 and the modified catalyst shows a large shift for especially the 3-methyl and 5-methyl resonances, whereas the other 1H NMR chemical shifts are almost unaffected. These observations can best be ascribed to a reorientation of the histidine axial ligand. The latter is suggested to be the consequence of an oxygen insertion, possibly on the imidazole ring of His18, thereby corroborating the data obtained by ESI-MS2. 1H NMR NOE difference measurements on Fe(III)MP-8 and on the modified catalyst supported the assignment of the Hδ2 and Hε1 protons of the His18 imidazole ring. The ring amine proton H61 could not be detected in both forms of the catalyst. For Fe(III)MP-8 this absence of the Hδ1 resonance can be ascribed to fast H/D exchange. For the modified catalyst the NMR data are not contradictory, with an oxygen insertion on position δ1 of the His18 imidazole ring with a fast H/D exchanging hydroxyl proton. Together these data converge in suggesting the H2O2 modified catalyst bears a hydroxylated His18 axial ligand. The mechanism that could underlie Fe(III)MP-8 axial histidine hydroxylation is further discussed.