Biochemical and Biophysical Research Communications
Oxidase uncoupling in heme monooxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs
Highlights
► Substantial reducing equivalents are lost in human P450 CYP3A4 via an oxidase channel. ► Substrate binding has a pronounced effect on uncoupling in cytochrome P450. ► Anionic phospholipids improve the overall coupling in CYP3A4 Nanodiscs.
Introduction
The catalytic cycle of cytochrome P450 includes the seven reactions shown in the Scheme 1, with an additional three unproductive branches, termed “uncoupling pathways”, which are depicted as dashed arrows. Reducing equivalents are provided to the heme protein by pyridine nucleotide (NADPH) through the action of a di-flavin reductase. The typical hydroxylation and oxygen transfer reactions catalyzed by the cytochromes P450 involve the consumption of one NADPH molecule and one dioxygen molecule, with one oxygen atom inserted into the product and another one forming water molecule. This stoichiometry of NADPH:O2 of 1:1 is summarized in Eq. (1). The two uncoupling reactions depicted in Scheme 1 involve release of superoxide from the ferrous dioxygen, [4] → [2], and the release of hydrogen peroxide, [5] → [2], also have an overall NADPH:O2 stoichiometry of 1:1 as is illustrated in Eq. (2). Following the generation of a heme peroxo state, the next step in the cyclic catalytic cycle involves cleavage of the O–O bound to produce a higher valent iron-oxo intermediate termed “Compound I” (Cpd I, [6]) following the historical peroxidase nomenclature. Cpd I is a ferryl-oxo porphyrin cation radical and is thought to initiate substrate hydroxylation through the Groves “oxygen rebound” process [1]. However, there is an additional reactivity of Cpd I. Inasmuch as this intermediate is thought to have an extremely positive redox potential [2] it can accept two additional electrons from NADPH and the flavoprotein reductase to reduce the electron deficient heme bound oxygen atom to water. This [6] → [2] pathway shown in Scheme 1 is thus analogous to the reaction of cytochrome oxidase with an NADPH:O2 ratio of 2:1. This oxidase uncoupling is described by Eq. (3).
While this reactivity of the cytochrome P450s has been recognized for some time, it has often been thought to represent a small fraction of the overall pyridine nucleotide reducing equivalents used by the system. Previous investigations have been hampered by problems related to the study of membrane proteins outside a bilayer environment. Systems using aggregated P450s are known to have many artifacts, including the lack of simple first order autoxidation behavior [3]. We have overcome these difficulties by using the Nanodisc system to form a homogeneous and monodisperse sample of human P450 CYP3A4 where detailed mechanistic investigations are possible. CYP3A4 is the major player in human drug metabolism in the liver, metabolizing almost 50% of the drugs currently on the market [4]. Due to the need for the enzyme to recognize a wide variety of substrate structures, the protein is thought to have a highly “plastic” active site. A downside of this flexibility is the poor utilization of pyridine nucleotide reducing equivalents. Although a deleterious process, the oxidase reactivity of Cpd I provides an important opportunity to study the fleeting iron-oxo intermediate.
The distinct NADPH:O2 stoichiometry of Eq. (3) relative to the other processes allows quantitation of the flux through the oxidase pathway by measuring the difference between the rates of NADPH and oxygen consumption, or between NADPH consumption and the sum of the rates of the product formation and H2O2 production. These measurements directly probe the branch point between hydrogen abstraction and subsequent oxygenase catalysis and the facile reduction of this intermediate by two additional electrons. An NADPH:O2 ratio greater than 1:1 indicates excess water production and provides a measure of the efficiency of Cpd I driven catalytic transformation of a given substrate with a given P450. Thus, more “extra” water is formed in the oxidase uncoupling pathway during metabolism of substrates that offer higher barriers for oxygenation, such as hydroxylation versus epoxidation and as reflected in the presence of deuterated analogs [5].
The oxidase uncoupling pathway has been documented for several mammalian cytochromes P450 in reconstituted systems and in microsomes [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. However, earlier approaches suffered from the lack of a clean monomeric heme protein and were not used to gain information about the reactivity and stability of Cpd I through the direct comparison of the rates of productive and unproductive (oxidase) decompositions. We have systematically studied the steady-state rates of water production and product formation for the metabolism of testosterone (TST) and bromocriptine (BC), as catalyzed by the most abundant human cytochrome P450, CYP3A4, with resultant new information about the properties of the Cpd I intermediate state.
Section snippets
Protein expression and purification
Expression and purification of membrane scaffold protein (MSP), cytochrome P450 CYP3A4 and rat P450 reductase (CPR), as well as preparation of CYP3A4 in POPC Nanodiscs (ND) was as described previously [16], [17], [18]. Incorporation of CPR into preformed and purified CYP3A4-Nanodiscs was made by direct addition of oligomeric CPR at 1:4 CYP3A4/CPR molar ratio, as described [19]. All experiments have been performed at 37 °C using POPC Nanodisc system similar to our earlier detailed mechanistic
Results and discussion
Fig. 1 shows the absolute rates of NADPH and oxygen consumption, and their difference, which corresponds to the water uncoupling rate (see Scheme 1). Most interestingly, the unproductive water channel exists even in the absence of substrate. This means that Cpd I is formed with a rate of ∼8 min−1, or 20% of the total oxygen consumption rate (∼42 min−1) even in the absence of substrate. The formation of Cpd I requires facile protonation of the hydroperoxo-ferric intermediate [5B] (Scheme 1) with a
Acknowledgments
This work was supported by NIH grants GM33775 and GM31756 to S.G. Sligar.
References (30)
High-valent iron in chemical and biological oxidations
J. Inorg. Biochem.
(2006)Uncoupling of oxygen activation from hydroxylation in the steroid C-21 hydroxylase of bovine adrenocortical microsomes
Arch. Biochem. Biophys.
(1971)- et al.
On the stoichiometry of the oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P-450. Products of oxygen reduction
J. Biol. Chem.
(1984) - et al.
Kinetic analysis of oxidation of coumarins by human cytochrome P450 2A6
J. Biol. Chem.
(2005) - et al.
Heteroactivator effects on the coupling and spin state equilibrium of CYP2C9
Arch. Biochem. Biophys.
(2006) - et al.
The ferrous-dioxygen intermediate in human cytochrome P450 3A4: substrate dependence of formation of decay kinetics
J. Biol. Chem.
(2006) - et al.
The one-electron autoxidation of human cytochrome P450 3A4
J. Biol. Chem.
(2007) - et al.
Functional reconstitution of monomeric CYP3A4 with multiple cytochrome P450 reductase molecules in Nanodiscs
Biochem. Biophys. Res. Commun.
(2010) - et al.
Oxygen solubilities of media used in electrochemical respiration measurements
Anal. Biochem.
(2003) - et al.
Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment
Arch. Biochem. Biophys.
(2004)