Probing axial ligands in ferric haemoproteins: An ESR study of myoglobin and horseradish peroxidase in H₂ ¹⁷O

Substitution of H₂ ¹⁶O by H₂ ¹⁷O induces a substantial broadening of the high-field line in the electron-spin resonance spectrum of ferric myoglobin due to the presence of H₂ ¹⁷O at the axial ligand-site. Computer simulations of the experimental spectra yielded the values of the reciprocal relaxation time T₂ ^-1 = 7.8 G and the ¹⁷O-hyperfine coupling constant A = 18 ± 1 G. Under identical experimental conditions no effect of H₂ ¹⁷O was observed in horseradish peroxidase. The latter finding excludes the possibility that a water molecule is liganded to the peroxidase haem-iron and supports either the idea that both axial ligands are amino acid residues or that the haem in ferric horseradish peroxidase is pentacoordinate.