Cytochrome b5, with a molecular weight of about 1.2 · 104 was highly purified from pig liver. The purified oxidized cytochrome b5 was investigated by the following three methods: 1. (I) Absorption spectrophotometry at 23 °C and 77 °K. 2. (II) Electron Paramagnetic Resonance (EPR) spectroscopy at 20 °K. 3. (III) Kinetic measurements of the reaction with CN- in the temperature range from 22.25 to 46.75 °C. I and IIhhave demonstrated that: 3.1. 1. In the pH region from pH 5.0 to 11.0, oxidized cytochrome b5 is in a purely low-spin state between 23 °C and 20 °K. 3.2. 2. Below pH 4.0, the spin state reversibly changes to high-spin between 23 °C and 20 °K. This high-spin state is found to be due to the hemin released from cytochrome b5. 3.3. 3. Above pH 12.0, the spin state reversibly changes to another type of low-spin state between 23 °C and 20 °K, which is thought to come from a distorted protein structure but not from the liganding of OH-. 3.4. 4. Energy for three t2g orbitals calculated in one hole formalism shows a high symmetry of ligand coordination for the low-spin state at pH 6.2 and a lowering of the symmetry for another type of low-spin state at pH 12.0. III has demonstrated that 3.5. 5. The reaction with CN- is bi-phasic. The fast reaction in the cytochrome b5 monocyanide complex formation, and the slow one is the hemin dicyanide complex formation. 3.6. 6. The activation energy for fast and slow reactions are both 25.1 kcal mole. The values of entropy of activation for fast and slow reactions are 12.1 and 11.5 entropy units, respectively. The protein structure of cytochrome b5 in comparison with that of cytochrome c based on the results above as well as those of X-ray studies by Dickerson, R. E., Takano, T., Eisenberg, D., Kallai, O. B., Samoson, L., Cooper, A. and Margoliash, E. (1971) J. Biol. Chem. 246, 1511-1535 and Mathews, F. S., Levine, M. and Argos, P. (1972) J. Mol. Biol. 64, 449-464 are discussed.
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