Oxidase uncoupling in heme monooxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs

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Abstract

The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons:1 dioxygen:1 product. However, three alternate unproductive pathways exist where the intermediate iron–oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involves release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent. Superoxide rapidly dismutates and hence both processes produce hydrogen peroxide that can be cytotoxic to the organism. In both cases, the formation of hydrogen peroxide involves the same overall stoichiometry as oxygenases catalysis. The key step in the catalytic cycle of cytochrome P450 involves scission of the oxygen–oxygen bond of atmospheric dioxygen to produce a higher valent iron-oxo state termed “Compound I”. This intermediate initiates a radical reaction in the oxygenase pathway but also can uptake two additional reducing equivalents from reduced pyridine nucleotide (NADPH) and the flavoprotein reductase to produce a second molecule of water. This non-productive decay of Compound I thus yields an overall oxygen to NADPH ratio of 1:2 and does not produce hydrocarbon oxidation. This water uncoupling reaction provides one of a limited means to study the reactivity of the critical Compound I intermediate in P450 catalysis. We measured simultaneously the rates of NADPH and oxygen consumption as a function of substrate concentration during the steady-state hydroxylation of testosterone catalyzed by human P450 CYP3A4 reconstituted in Nanodiscs. We discovered that the “oxidase” uncoupling pathway is also operating in the substrate free form of the enzyme with rate of this pathway substantially increasing with the first substrate binding event. Surprisingly, a large fraction of the reducing equivalents used by the P450 system is wasted in this oxidase pathway. In addition, the overall coupling with testosterone and bromocryptine as substrates is significantly higher in the presence of anionic lipids, which is attributed to the changes in the redox potential of CYP3A4 and reductase.

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). RH+NADPH+O2+2H+ROH+NADP+H2ORH+NADPH+O2+2H+RH+NADP+H2O2RH+2NADPH+O2+4H+RH+2NADP+2H2O

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)

  • E.M. Isin et al.

    Kinetics and thermodynamics of ligand binding by cytochrome P450 3A4

    J. Biol. Chem.

    (2006)
  • I.G. Denisov et al.

    Cooperativity in cytochrome P450 3A4: linkages in substrate binding, spin state, uncoupling, and product formation

    J. Biol. Chem.

    (2007)
  • D.R. Davydov et al.

    Peripheral ligand-binding site in cytochrome P450 3A4 located with fluorescence resonance energy transfer (FRET)

    J. Biol. Chem.

    (2012)
  • I.G. Denisov et al.

    A novel type of allosteric regulation: functional cooperativity in monomeric proteins

    Arch. Biochem. Biophys.

    (2012)
  • W.H. Koppenol

    Oxygen activation by cytochrome P450: a thermodynamic analysis

    J. Am. Chem. Soc.

    (2007)
  • Cited by (0)

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