The Active Center of Methemoglobin Hb(H₂O) Investigated by Mössbauer and Susceptibility Experiments

A fast freezing technique using liquid propane was used to obtain frozen solutions of methemoglobin at pH 7. With this method it was possible to eliminate largely the presence of a low spin component which is usually found in slowly frozen solutions but not present in the sample at room temperature. The magnetic susceptibility and Mössbauer spectra of Hb(H₂O) in the temperature range [formula omitted] have been measured. The data of the high spin compound of Hb(H₂O) were evaluated with a Hamiltonian containing the Coulomb repulsion of the five 3d-electrons of the Fe³⁺ ion, a crystal electric field of C_2v symmetry and the spin orbit coupling. The term describing the crystal electric field depends on five energy parameters ε₁, ε₂, ε₃, D, and E which are determined by least squares fits to the experimental data. The most relevant parameters ε₂and ε₃ which equal the energies of the antibonding single 3d-electron orbitals 3d_z^z and 3d_x²-y² with respect to the 3d_xy orbital are compared with earlier results of these energies ε₂and ε₃ of the high spin compound of Mb(H₂O). From this comparison conclusions regarding the different spatial arrangements of Fe³⁺ in Hb(H₂O) and Mb(H₂O) are drawn.