Identification of the Interactions between Cytochrome P450 2E1 and Cytochrome b5 by Mass Spectrometry and Site-directed Mutagenesis

Abstract

Cytochrome P450s (P450s) 2 are a superfamily of b-type hemoproteins responsible for the metabolism of a wide variety of exogenous compounds such as drugs and carcinogens, and endogenous compounds such as prostaglandins and steroids (1). P450 reactions require input of two electrons (supplemental Fig. S1) (1, 2). The efficiency of electron transfer is one of the key determinants of the reaction kinetics. In microsomal systems, NADPH is the ultimate source of the two electrons, and NADPH-dependent cytochrome P450 oxidoreductase (P450 reductase) together with cytochrome b 5 (b 5 ) facilitates the electron transfer. Knowledge of the interactions between P450s and their redox partners is fundamental to a complete understanding of the mechanisms of P450 reactions.The interactions between P450s and b 5 have drawn much attention because of variable effects b 5 has on different P450 isoforms and P450 reactions. It has been shown that b 5 may stimulate, inhibit or have no effects on P450-catalyzed reactions depending on the particular isoform of P450 and the substrate of the reaction (3-5). However, there is no consensus on whether b 5 transfers electrons to P450, or causes an allosteric effect on P450, or whether both mechanisms are simultaneously operative (6 -10). In addition, the mechanism of substrate-and P450 isoform dependency is unknown (5, 11).CYP2E1 is a P450 isoform whose reactions are highly stimulated in the presence of b 5 . For example, we previously found that b 5 stimulates CYP2E1-catalyzed oxidation of acetaminophen to its toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI), by 25-fold (12). For other CYP2E1-catalyzed reactions, such as aniline p-hydroxylation and 7-ethoxycoumarin O-deethylation, b 5 stimulates the reactions by 270-fold and 67-fold, respectively (5, 11). In contrast to its stimulating effects on CYP3A4, CYP3A5, CYP2C19, CYP2B6, and CYP2C8, apo-b 5 is unable to replace holo-b 5 in stimulating CYP2E1-catalyzed reactions (11,12). The requirement for the heme group increases the probability that b 5 stimulates CYP2E1-catalyzed reactions by facilitating electron transfer rather than by only causing a positive allosteric effect. Because intermolecular complex formation immediately precedes electron trans-* This work was supported by National Institutes of Health Grant GM32165 (tp S. D. N.), the UW NIEHS-sponsored Center for Ecogenetics and Environmental Health Grant P30ES07033, an NCRR high end instrumentation award 1S10RR17262-01 (to D. R. G.), and the WWAMI RCE for Biodefense and Emerging Infectious Diseases 1U54AI57141-01 (to D. R. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Catalytically Relevant Electrostatic Interactions of Cytochrome P450c17 (CYP17A1) and Cytochrome b5

“…All of the five residues of b 5 that interact with CYP2B4 are located on the ␣4-loop-␣5 segment, which are distinct from the residues that interact with CYP17A1. However, CYP2E1 uses Lys-428 and Lys-434 to interact with Glu-58 and Glu-61 of b 5 (35). Even though Lys-434 of CYP2E1 corresponds to Lys-433 in CYP2B4, these residues do not interact with the same residue of b 5 .…”

Correlating Structure and Function of Drug-Metabolizing Enzymes: Progress and Ongoing Challenges

“…In addition, no structures exist to date for membrane P450 enzymes in complex with either b 5 or CPR to guide our understanding of the structural elements of electron transfer and/or allosteric effects. Mutagenesis (Geller et al, 1999;Naffin-Olivos and Auchus, 2006;Im and Waskell, 2011), crosslinking (Bridges et al, 1998;Gao et al, 2006;Zhao et al, 2012;Peng and Auchus, 2013), and protein docking approaches (Bridges et al, 1998;Gao et al, 2006;Im and Waskell, 2011) have provided insights into these protein/protein interactions, supplemented recently by the first structure of the bacterial soluble P450 cam with its redox partner, putidaredoxin (Hiruma et al, 2013;Tripathi et al, 2013), but much is lacking in our current understanding of these aspects of P450 systems. Protein-detected NMR is a highresolution structural technique that has been little applied to P450 systems, but has the potential to provide significant new and orthogonal information on human P450 interactions with both ligands and other proteins in solution, as well as on protein dynamics that can only be inferred from current X-ray structure information.…”

Human Liver Cytochrome P450 3A4 Ubiquitination

“…Indeed, similar intermolecular electrostatic interactions between P450s and their functionally relevant redox partners cytochrome P450 reductase (CPR) and cytochrome b 5 (b 5 ) have been established through site-directed mutagenesis and chemical cross-linking coupled LC-MS/MS analyses (57)(58)(59)(60). Thus, we posited that if such CYP3A4 negatively charged clusters were in fact to interact with positively charged residues of one or more proteins in each of these E2-E3 complexes, then such intermolecular electrostatic interactions would be disrupted in the presence of high salt concentrations in the incubation systems, thereby attenuating if not completely abrogating CYP3A4 ubiquitination.…”