Molecular cloning, overexpression in Escherichia coli, structural and functional characterization of house fly cytochrome b5.
A microsomal cytochrome b5 cDNA from the house fly, Musca domestica, was cloned and sequenced. The deduced amino acid sequence of the full-length house fly cytochrome b5 (134 residues) is 48% identical to that of rat microsomal cytochrome b5. The house fly cytochrome b5 protein was overexpressed in Escherichia coli, purified, and characterized. Absorption and EPR spectroscopy reveal properties very similar to cytochromes b5 from vertebrates. NMR spectra indicate that the orientation of the heme in the protein relative to its alpha,gamma meso axis is about 1:1. A redox potential of -26 mV versus standard hydrogen electrode was measured by cyclic voltammetry on a modified gold electrode in the presence of hexamminechromium(III) chloride. The cytochrome b5 is reduced by house fly cytochrome P450 reductase in a reconstituted system at a high rate (5.5 s-1), and it stimulates heptachlor epoxidation when reconstituted with house fly cytochrome P450 reductase, cytochrome P450 6A1, phospholipid, and detergent. Cytochrome b5 decreases the apparent Km for P450 reductase and increases the Vmax for heptachlor epoxidation at constant cytochrome P450 6A1 concentrations. The results indicate that cytochrome b5 stimulates a step following the first electron transfer during cytochrome P450 6A1 turnover.
Department of Entomology, University of Arizona, Tucson, Arizona 85721, USA.
V M
VM
Guzov
H L
HL
Houston
M B
MB
Murataliev
F A
FA
Walker
R
R
Feyereisen
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D015786:Cytochromes b5 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary; D004175:Diptera / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis
D018076:DNA, Complementary; D011994:Recombinant Proteins; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System