Expression, Purification, and Physical Properties of Recombinant Flavocytochrome Fusion Proteins Containing Rat Cytochrome b5 Linked to NADPH-Cytochrome P450 Reductase by Different Membrane-Binding Segments

Reconstitution of the enzymatic activities using purified microsomal cytochrome P450s (P450) requires the presence of a membrane-binding segment in the mammalian flavoprotein, NADPH–cytochrome P450 reductase (CPR), and the hemeprotein, cytochrome b5 (b5). The mechanism(s) by which the membrane-binding segments of these proteins exert such a critical role in influencing the reconstitution of the NADPH-supported activity of a P450 remains undefined. In the present work we describe the construction, expression, and purification of four different types of recombinant flavocytochromes containing rat b5 and rat CPR linked by various membrane-binding segments. The physical properties of these artificial fusion proteins have been studied to determine their ability to serve as electron transfer agents. These studies are a prelude to the subsequent study (accompanying paper) evaluating the functional roles of the hydrophobic (membrane-binding) sequences of b5 and CPR in the reconstitution of P450 activities. The present study shows that the purified recombinant fusion proteins can serve as active electron transport carriers from NADPH to cytochrome c as well as b5 by intramolecular as well as intermolecular reactions. It is shown here that the electron transport properties of these purified fusion proteins are influenced by high concentrations of KCl, suggesting a role for charged amino acids in protein–protein interactions. The present study illustrates the application of artificial recombinant flavocytochromes as useful proteins for the study of intramolecular electron transport reactions for comparison with intermolecular interactions.