Electron paramagnetic resonance (E.P.R.) spectra from single crystals of horse heart ferricytochrome c at 4.2 °K were analyzed to obtain the orientation of the principal g values relative to the crystallographic axes. The axis of the largest principal g value (g3 = 3.06) was within 5° of the heme normal direction reported in the X-ray structure of the same crystals (Dickerson et al.: J. Biol. Chem. 246, 1511 (1971)). The other two g axes (g1 = 1.25, g2 = 2.25) lie within 5° of the N–Fe–N directions in the heme ring, in contrast to met-myoglobin azide (Helcké et al.: Proc. R. Soc. B169, 275 (1968)) and cyanide (Blumberg, W. E.: personal communication) where they lie ~ 45° from the N–Fe–N directions. A version of Eisenberger and Pershan's theory (J. Chem. Phys. 47, 327 (1967)) was used to explain the 400–2000 G variation in linewidth on crystal rotation. The results were explained by combining the broadening produced by a distribution of rhombic crystal field potential (r.m.s. deviation 11%) over the molecular population, with that from a variation in the directions of the principal g values caused by misorientation (r.m.s. deviation 1.5°) of the molecules in the crystal.
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