Characterization of Ground State Electron Configurations of High-Spin Quintet Ferrous Heme Iron in Deoxy Myoglobin Reconstituted with Trifluoromethyl Group-Substituted Heme Cofactors

We introduced trifluoromethyl (CF3) group(s) as heme side chain(s) of sperm whale myoglobin (Mb) in order to characterize the electronic nature of heme Fe­(II) in deoxy Mb using 19F NMR spectroscopy. On the basis of the anti-Curie behavior of CF3 signals, we found that the deoxy Mb is in thermal equilibrium between the 5 B 2, (d xy )2(d xz )­(d yz )­(d z 2 )­(d x 2–y 2 ), and 5 E, (d xy )­(d xz )2(d yz )­(d z 2 )­(d x 2–y 2 ), states of the heme Fe­(II), i.e., 5 B 2 ⇆ 5 E. Analysis of the curvature in Curie plots has yielded for the first time ΔH and ΔS values of ∼−20 kJ mol–1 and ∼−60 J K–1 mol–1, respectively, for the thermal equilibrium. Thus, the 5 E state is slightly dominant over the 5 B 2 one at 25 °C. These findings provide not only valuable information about the ground state electronic structure of the high-spin heme Fe­(II) in deoxy native Mb but also an important clue for elucidating the mechanism responsible for acceleration of the spin-forbidden oxygenation of the protein.