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 F-19 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 B-5(2), (d(xy))2(d(xz))(d(yz))(d(z2))(d(x2)-(y2)), and 5E, (d(xy))(d(xz))(2)(d(yz))(d(z2))(d(x2)-(y2)), states of the heme Fe(II), i.e., B-5(2) reversible arrow E-5. Analysis of the curvature in Curie plots has yielded for the first time OH and LS.S values of similar to-20 kJ mol(-1) and,similar to-60 J K-1 mol(-1), respectively, for the thermal equilibrium. Thus, the E-5 state is slightly dominant over the B-5(2) one at 25 degrees 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.