Molecular cloning, overexpression in Escherichia coli, structural and functional 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 that of rat microsomal cytochrome b(5). The house fly cyto chrome b(5) protein was overexpressed in Escherichia coli, purified, and characterized. Absorption and EPR spectroscopy reveal properties very similar to cytochromes b(5) 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 b(5) 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 res constituted with house fly cytochrome P450 reductase, 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 concentrations. The results indicate that cytochrome b(5) stimulates a step following the first electron transfer during cytochrome P450 6A1 turnover.