Molecular cloning, overexpression in Escherichia coli, structural and functional characterization of house fly cytochrome b sub(5)

A microsomal cytochrome b sub(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 sub(5) (134 residues) is 48% identical to that of rat microsomal cytochrome b sub(5). The house fly cytochrome b sub(5) protein was overexpressed in Escherichia coli, purified, and characterized. Absorption and EPR spectroscopy reveal properties very similar to cytochromes b sub(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 sub(5) is reduced by house fly cytochrome P450 reductase in a reconstituted system at a high rate (5.5 s super(-1)), and it stimulates heptachlor epoxidation when reconstituted with house fly cytochrome P450 reductase, cytochrome P450 6A1, phospholipid, and detergent. Cytochrome b sub(5) decreases the apparent K sub(m) for P450 reductase and increases the V sub(max) for heptachlor epoxidation at constant cytochrome P450 6A1 concentrations. The results indicate that cytochrome b sub(5) stimulates a step following the first electron transfer during cytochrome P450 6A1 turnover.