Single crystal EPR of myoglobin nitroxide. Freezing-induced reversible changes in the molecular orientation of the ligand.

Single crystals of myoglobin nitroxide (MbNO) are examined by the electron paramagnetic resonance spectroscopy at ambient and cryogenic temperatures for both the 14NO and 15NO derivatives. The principal values and the eigenvectors of the g tensor and the hyperfine coupling tensor are determined: g xx = 2.050, g yy = 2.022, and g zz = 1.993; A xi xi = 15.6, A zeta zeta = 21.4, and A eta eta = 26.7 G for the nitrogen in 15NO at ambient temperature. The Fe--N--O bond angle is calculated to be 153 degrees. This result is in good agreement with the x-ray structural result on the six-liganded model compound with the bent Fe--N--O configuration. The principal values and the eigenvectors of the g tensor and the hyperfine coupling tensor are also determined at 77 K for Mb15NO; gxx = 2.076, gyy = 1.979, and gzz = 2.002; A xi xi = 21, A zeta zeta = 24, and A eta eta = 27 G. The Fe--N--O bond angle is calculated to be 109 degrees. The hyperfine splittings attributed to N epsilon atom of proximal histidine are observed in the direction of the gzz at both temperatures. The drastic shift of the EPR spectrum of MbNO single crystal is observed below the freezing point of this crystal. It clearly demonstrates that the conformation of the bonding NO is drastically altered upon freezing. The temperature dependence of the EPR spectra of MbNO below the freezing point cannot be explained only by appropriate combinations of the higher temperature type and the lower temperature type and suggests the contribution from an unpaired electron with the iron dz2 and dyz (or dxz) orbitals. The present EPR results demonstrated that the changes in the molecular orientations are induced by freezing of the biological molecules without disorder of the crystal lattice.