MOLECULAR-CLONING, OVEREXPRESSION IN ESCHERICHIA-COLI, STRUCTURAL ANDFUNCTIONAL-CHARACTERIZATION OF HOUSE-FLY CYTOCHROME B(5)

A microsomal cytochrome b(5) cDNA from the house fly, Musca domestica, was cloned and sequenced, The deduced amino acid sequence of the full -length house fly cytochrome b(5) (134 residues) is 48% identical to t hat of rat microsomal cytochrome b(5). The house fly cyto chrome b(5) protein was overexpressed in Escherichia coli, purified, and character ized. Absorption and EPR spectroscopy reveal properties very similar t o cytochromes b(5) from vertebrates, NMR spectra indicate that the ori entation of the heme in the protein relative to its alpha,gamma meso a xis is about 1:1. A redox potential of -26 mV versus standard hydrogen electrode was measured by cyclic voltammetry on a modified gold elect rode in the presence of hexamminechromium(III) chloride. The cytochrom e b(5) is reduced by house fly cytochrome P450 reductase in a reconsti tuted system at a high rate (5.5 s(-1)), and it stimulates heptachlor epoxidation when res constituted with house fly cytochrome P450 reduct ase, cytochrome P450 6A1, phospholipid, and detergent. Cytochrome b(5) decreases the apparent K-m for P450 reductase and increases the V-max for heptachlor epoxidation at constant cytochrome P450 6A1 concentrat ions. The results indicate that cytochrome b(5) stimulates a step foll owing the first electron transfer during cytochrome P450 6A1 turnover.