The High-Level Expression in Escherichia coli of the Membrane-Bound Form of Human and Rat Cytochrome b₅ and Studies on Their Mechanism of Function

Abstract

A T7 expression system is described for the high-level production in Escherichia coli of the membrane-bound form of human and rat cytochrome b₅. The cDNAs of b₅ have been engineered to contain a coding sequence for a four-member histidine domain at the amino-terminus of the recombinant protein permitting the use of a nickel-chelate affinity column for rapid purification of the detergent-solubilized hemoprotein. Results are presented demonstrating the ability of the purified recombinant b₅ proteins to stimulate the rate of oxidation of 17α-hydroxypregnenolone to dehydroepiandrosterone, catalyzed by bovine P450 17A, and to stimulate the 6β-hydroxylation of testosterone, catalyzed by human P450 3A4. These P450-catalyzed reactions have been used to compare the properties of different forms of b₅. Purified b₅ can serve as a "coupling protein" as illustrated by its inhibition of NADPH oxidation, catalyzed by a fusion protein containing the heme domain of P450 3A4 linked to rat NADPH-P450 reductase, and the associated inhibition of hydrogen peroxide formation. Kinetic studies show the formation of a complex of the flavoprotein, NADPH-P450 reductase, with b₅ for the rapid transfer of electrons from NADPH.