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Volume 271, Number 43, Issue of October 25, 1996 pp. 26637-26645
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Molecular Cloning, Overexpression in Escherichia coli, Structural and Functional Characterization of House Fly Cytochrome b₅

(Received for publication, June 11, 1996, and in revised form, July 16, 1996)

Victor M. Guzov , Heather L. Houston ^§ , Marat B. Murataliev , F. Ann Walker ^§ and René Feyereisen

From the Departments of  Entomology and ^§ Chemistry, University of Arizona, Tucson, Arizona 85721

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 , 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 s¹), and it stimulates heptachlor epoxidation when reconstituted with house fly cytochrome P450 reductase, cytochrome P450 6A1, phospholipid, and detergent. Cytochrome b₅ 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 b₅ stimulates a step following the first electron transfer during cytochrome P450 6A1 turnover.

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