The cytochrome P450 2B4-NADPH cytochrome P450 reductase electron transfer complex is not formed by charge-pairing.

A.I Voznesensky,J.B Schenkman

doi:10.1016/s0021-9258(18)42093-5

Abstract

Attempts to covalently link NADPH-cytochrome P450 reductase to cytochrome P450 2B4 using a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylisopropyl)carbodiimide, were unsuccessful, despite the fact that under the same conditions about 30% of P450 2B4 could be covalently linked with cytochrome b5 in a functionally active complex (Tamburini, P. P., and Schenkman, J. B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 11-15). This suggested that the functional electron transfer complex between P450 2B4 and reductase is not stabilized by electrostatic forces. Raising the ionic strength of the medium is disruptive to salt bridges and was used to further test whether P450 2B4 and the reductase form charge-pairing complexes. Instead of inhibiting electron transfer, high ionic strength increased the apparent fast phase rate constant and the fraction of P450 2B4 reduced in the fast phase. The possibility that electron transfer between NADPH-cytochrome P450 reductase and P450 2B4 is diminished by charge repulsion was examined. Consistent with this hypothesis, the Km of P450 2B4 for reductase was decreased 26-fold by increasing the ionic strength from 10 to 100 mM sodium phosphate without affecting the Vmax. The rate of benzphetamine N-demethylation also was increased by elevation of the ionic strength. Electron transfer from the reductase to other charged redox acceptors, e.g. cytochrome c and ferricyanide, was also stimulated by increased ionic strength. However, no similar stimulation was observed with the uncharged acceptor 1,4-benzoquinone. Polylysine, a polypeptide that binds to anionic sites, enhanced electron transfer from NADPH to ferricyanide and the apparent fast phase of reduction of cytochrome P450. The results are consistent with the hypothesis that charges on NADPH-cytochrome P450 reductase and cytochrome P450 decrease the stability of the electron transfer complex.

Highlights

P450 reductase to cytochrome P450 2B4 using a proteases (Gum and Strobel, 1979; Enoch and Strittmatter, water-soluble carbodiimide, l-ethyl-3-(3-dimethyli- 1979)
In our earlier studies (Tamburini et al, 1986; Tamburini and Schenkman, 1986b) we suggested that the interaction between NADPH-cytochrome P450 reductase and cytochrome P450 involved complementary charge-pairing, based upon the effects of modifications of charged amino acid residues on theseproteins
Acetylation of rat cytochrome P450 amino groups, if anything, reduced the K, for reductase in cytochrome P450-mediated substrate oxidation (Nadlerand Strobel, 1988).Neutralization of carboxyl residues caused an increase in the K,for reductase.Modification of the a-aminogroup of P450 2B4 inhibited its reduction by NADPH, indicating interference with interactionwith NADPH-cytochrome P450 reductase (Bernhardt et al, 1983)

Summary

MATERIALS AND METHODS

Purification of Proteins-Liver microsomes were prepared from 3- added a t a 9 mM level to theP450, amidinated reductase, and kg male New Zealand rabbits after daily treatments with 80 mg/kg sonicated DLPC mixture for varying durations from 1 to 10 sodium phenobarbital intraperitoneally for 4 days. Rabbit NADPH-cytochrome P450 reductase waspurified essentially by themethod of Dignamand Strobel (1975) and was 11nmol/mg. Both enzymes were electrophomin a t 22 “C did not form heterodimers, and longer incubations did not alter the electrophoretic pattern from that, truck 2. XDC Cross-linking of Proteins-Prior to EDC treatment, in some experiments the lysyl c-amino residues of NADPH-cytochrome P450 reductase were modified with ethylacetimidate as described (Tamhurini and Schenkman, 198613).When the reductase and cytochrome 1’450 were incubated for cross-linking, theywere mixedwith dilauroyl phosphatidylcholine vesicles, prepared by sonication (Jansson et al., This evidence was used to support data indicating thesetwo proteins, as well as P450 2B4 and cytochrome bn, interact by complementary charge pairing (Tamburini eatl., 1985). P450 2B4 (6.3PM) and NADPH-cytochrome P450 polylysine (average M,3900) were obtained from Sigma, and potas- reductase were treated with EDC in 50 mM sodium phosphate buffer sium ferricyanide was obtained from Fisher. Incorporated into DLPCvesicles prior to EDC treatment as described under “Materials and Methods.” The molar ratio of NADPH-cyto-

RESULTS

DISCUSSION

Findings

Initial rate