Quantitative analyses of electrostatic interactions between NADPH-cytochrome P450 reductase and cytochrome P450 enzymes

Voznesensky, A. I.; Schenkman, J. B.

A decline in the ionic interactions in the medium with increasing ionic strength (decrease in the ionic activity coefficients) was accompanied by an increase in the fast phase rate constants of CYP2B4 and CYP1A2 reduction. The stimulations were observed both in reconstituted P450 systems and in microsomes. An increase in the ionic strength from 10 to 100 mM sodium phosphate resulted in a 7-fold decrease in the K-m of CYP1A2 for NADPH-cytochrome P450 reductase, while the V-max was unchanged. When ionic interactions were neutralized without changing the ionic strength by addition of charged oligopeptides (polylysine and polyglutamic acid), stimulations of CYP1A2 and CYP2B4 reduction were observed. Increase in the ionic strength also enhanced the rate of cytochrome P450 reduction in control and phenobarbital-induced rat liver microsomes and in reconstituted systems containing purified rat liver CYP2C6, CYP2C12, CYP2C13, and CYP2E1, and rat reductase. A method was devised for the quantification of the number of charges involved in protein-protein interactions based on the estimation of the ionic activity coefficients. Different numbers of charged residues are involved in the repulsion between different P450 forms and the reductase. The product of the number of charges involved in the interaction between rabbit reductase and CYP2B4 is 10.84 compared with the value of 6.64 for the reductase-CYP1A2 interaction.