Assignment of the heme axial ligand(s) for the ferric myoglobin (H93G) andheme oxygenase (H25A) cavity mutants as oxygen donors using magnetic circular dichroism

UV-visible absorption and magnetic circular dichroism (MCD) data are report ed for the cavity mutants of sperm. whale H93G myoglobin and human H25A hem e oxygenase in their ferric states at 4 OC. Detailed spectral analyses of H 93G myoglobin reveal that its heme coordination structure has a single wate r ligand at pH 5.0, a single hydroxide ligand at pH 10.0, and a mixture of species at pH 7.0 including five-coordinate hydroxide-bound, and six-coordi nate structures. The five-coordinate aquo structure at pH 5 is supported by spectral similarity to acidic horseradish peroxidase (pH 3.1), whose MCD d ata are reported herein for the first time, and acidic myoglobin (pH 3.4), whose structures have been previously assigned by resonance Raman spectrosc opy. The five-coordinate hydroxide structure at pH 10.0 is supported by MCD and resonance Raman data obtained here and by comparison with those of oth er known five-coordinate oxygen donor complexes. In particular, the MCD spe ctrum of alkaline ferric H93G myoglobin is strikingly similar to that of fe rric tyrosinate-ligated human H93Y myoglobin, whose MCD data are reported h erein for the first time, and that of the methoxide adduct of ferric protop orphyrin IX dimethyl eater ((FePPIXDME)-P-III). Analysis of the spectral da ta for ferric H25A heme oxygenase at neutral pH in the context of the spect ra of other five-coordinate ferric heme complexes with proximal oxygen dono r ligands, in particular the p-nitrophenolate and acetate adducts of (FePPI XDME)-P-III, is most consistent with ligation by a carboxylate group of a n earby glutamyl (or aspartic) acid residue.