Recombinant human erythrocyte cytochrome b5.
The gene encoding the human erythrocyte form of cytochrome b5 (97 residues in length) has been prepared by mutagenesis of an expression vector encoding lipase-solubilized bovine liver microsomal cytochrome b5 (93 residues in length) (Funk et al., 1990). Efficient expression of this gene in Escherichia coli has provided the first opportunity to obtain this protein in quantities sufficient for physical and functional characterization. Comparison of the erythrocytic cytochrome with the trypsin-solubilized bovine liver cytochrome b5 by potentiometric titration 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 this 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 pH in a fashion that is consistent with the presence of a single ionizable group that changes pKa 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 1H NMR spectrum of the erythrocytic ferricytochrome indicates 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, 25 degrees C) with delta H degrees = -2.9 +/- 0.3 kcal/mol and delta S degrees = -5.4 +/- 0.9 eu for this equilibrium.
Lloyd
E
E
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Ferrer
J C
JC
Funk
W D
WD
Mauk
M R
MR
Mauk
A G
AG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D015786:Cytochromes b5 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004912:Erythrocytes / Q000737:chemistry*; D005813:Genes, Synthetic; D006801:Humans; D008099:Liver / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008861:Microsomes / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011199:Potentiometry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013053:Spectrophotometry
D011994:Recombinant Proteins; D015786:Cytochromes b5