NADPH P450 oxidoreductase: Structure, function, and pathology of diseases

Abstract

Cytochrome P450 oxidoreductase (POR) is an enzyme that is essential for multiple metabolic processes, chiefly among them are reactions catalyzed by cytochrome P450 proteins for metabolism of steroid hormones, drugs and xenobiotics. Mutations in POR cause a complex set of disorders that often resemble defects in steroid metabolizing enzymes 17α-hydroxylase, 21-hydroxylase and aromatase. Since our initial reports of POR mutations in 2004, more than 200 different mutations and polymorphisms in POR gene have been identified. Several missense variations in POR have been tested for their effect on activities of multiple steroid and drug metabolizing P450 proteins. Mutations in POR may have variable effects on different P450 partner proteins depending on the location of the mutation. The POR mutations that disrupt the binding of co-factors have negative impact on all partner proteins, while mutations causing subtle structural changes may lead to altered interaction with specific partner proteins and the overall effect may be different for each partner. This review summarizes the recent discoveries related to mutations and polymorphisms in POR and discusses these mutations in the context of historical developments in the discovery and characterization of POR as an electron transfer protein. The review is focused on the structural, enzymatic and clinical implications of the mutations linked to newly identified disorders in humans, now categorized as POR deficiency.

Introduction

Cytochrome P450 reductase (POR, also referred as CPR, CYPOR, OR, NCPR, P450R) has a major role in metabolism of drugs and steroids (Flück et al., 2007, Riddick et al., 2013) (Fig. 1). POR was originally described by Horecker in 1950 as a cytochrome c reductase (Horecker and Heppel, 1949, Horecker, 1950). Further studies by Williams and Kamin (1962) and Phillips and Langdon (1962) showed that this protein is located in the endoplasmic reticulum (microsomes). POR was shown to be linked to the microsomal cytochromes, including the cytochrome P450 (P450) proteins described by Omura and Sato (1962) and cytochrome b₅ (CYB5, b₅) (Raw and Mahler, 1959, Waterfield et al., 1969, Nebert, 1970; B. S. Cohen & Estabrook, 1971) involved in drug and steroid hydroxylations (Lu et al., 1969). Studies by Lu and Coon (Lu et al., 1969) provided the definitive evidence for the requirement of POR in cytochrome P450 reactions by dissecting the P450 containing mixed function oxidase system into three distinct components: POR, cytochrome P450, and lipids (Fig. 2). All microsomal P450s depend on POR for supply of electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) for their catalytic activities to metabolize drugs, xenobiotics and steroid hormones. POR also supplies electrons to many other proteins and small molecules (Fig. 1) including heme oxygenase, squalene monooxygenase, cytochrome b₅ (Schacter et al., 1972, Ono and Bloch, 1975, Matsubara et al., 1976, Enoch and Strittmatter, 1979, Nagao et al., 1983, Bligh et al., 1990, Nishino and Ishibashi, 2000, Ramji et al., 2003, Guengerich, 2005, Nicolo et al., 2010, Pandey et al., 2010). The catalytic reactions by P450 systems involve hydroxylations of different substrates. P450-based reactions are also referred as ‘monooxygenation’ reactions due to the ability of cytochrome P450 to mediate the transfer of one atom of molecular oxygen (O₂) into the substrate and reduce the other atom into water. The P450 reaction can be summarized as follows:

RH + O₂ + NAD(P)H + H⁺ → ROH + H₂O + NAD(P)⁺.

RH is the substrate that is hydroxylated by the P450 to form the product ROH, and a water molecule is released as a byproduct of this reaction. In each cycle of monooxygenation reaction two electrons are supplied from NADH/NADPH, and POR works as an electron transporter by accepting two electrons from NADPH and transferring them, one at a time, to the cytochrome P450s during catalysis. POR can also mediate degradation of ferrous heme by H₂O₂ or reactive oxygen species generated during P450 reactions (Masters and Schacter, 1976, Guengerich, 1978) This mechanism of heme degradation is different from the heme oxygenase mediated reaction which also requires POR, as biliverdin was not found to be the intermediate metabolite (W. H. Schaefer et al., 1985).

In some P450s, cytochrome b₅ and post-translational modifications are also involved in P450 reactions (Hildebrandt and Estabrook, 1971, Onoda and Hall, 1982; L. H. Zhang et al., 1995, Auchus et al., 1998, Pandey et al., 2003, Yamaori et al., 2003, Pandey and Miller, 2005) and cytochrome b₅ may act either as a redox facilitator (CYP17A1) or as a donator of second electron to P450s (CYP1A2, CYP3A4, etc.) (Porter, 2002, Mokashi et al., 2003, Shimada et al., 2005). Because all microsomal P450s depend on POR for the supply of electrons, disruption of POR may affect all microsomal P450 enzyme activities with disastrous consequences (Flück et al., 2007). POR knockout mice are embryonically lethal (A. L. Shen et al., 2002, Otto et al., 2003) possibly due to lack of electron transport to P450 enzymes involved in biosynthesis (CYP51A1) and metabolism of cholesterol (CYP17A1, CYP19A1 and CYP21A2) since the liver-specific knockout of POR yields phenotypically and reproductively normal mice, yet with remarkably diminished capacity of hepatic drug metabolism and lipid accumulation (Gu et al., 2003, Henderson et al., 2003, Porter et al., 2011). In this review we will primarily discuss the role of POR in humans; studies on POR in mouse model systems, cell models and translational aspects have been recently reviewed by Riddick et al. (2013).