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 using19 F NMR spectroscopy. On the basis of the anti-Curie behavior of CF3 signals, we found that the deoxy Mb is in thermal equilibrium between the5 B2 , (dxy )2 (dxz )(dyz )(dz2 )(dx2 -y2 ), and5 E, (dxy )(dxz )2 (dyz )(dz2 )(dx2 -y2 ), states of the heme Fe(II), i.e.,5 B2 ⇆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, the5 E state is slightly dominant over the5 B2 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.