RECOMBINANT HUMAN ERYTHROCYTE CYTOCHROME B(5)

The gene encoding the human erythrocyte form of cytochrome b(5) (97 re sidues in length) has been prepared by mutagenesis of an expression ve ctor encoding lipase-solubilized bovine liver microsomal cytochrome b( 5) (93 residues in length) (Funk et al., 1990). Efficient expression o f this gene in Escherichia coil has provided the first opportunity to obtain this protein in quantities sufficient for physical and function al characterization. Comparison of the erythrocytic cytochrome with th e trypsin-solubilized bovine liver cytochrome b(5) by potentiometric t itration indicates that the principal electrostatic difference between the two proteins results from two additional His residues present in the human erythrocytic protein. The midpoint reduction potential of th is protein determined by direct electrochemistry is -9 +/- 2 mV vs SHE at pH 7.0 (mu = 0.10 M, 25.0 degrees C), and this value varies with p H in a fashion that is consistent with the presence of a single ioniza ble group that changes pK(a), from 6.0 +/- 0.1 in the ferricytochrome to 6.3 +/- 0.1 in the ferrocytochrome with Delta H degrees = -3.2 +/- 0.1 kcal/mol and Delta S degrees = -11.5 +/- 0.3 eu (pH 7.0, mu = 0.10 ). The 1D H-1 NMR spectrum of the erythrocytic ferricytochrome indicat es that 90% of the protein binds heme in the ''major'' orientation and 10% of the protein binds heme in the ''minor'' orientation (pH 7.0, 2 5 degrees C) with Delta H degrees = -2.9 +/- 0.3 kcal/mol and Delta S degrees = -5.4 +/- 0.9 eu for this equilibrium.