Altered heme catabolism by heme oxygenase-1 caused by mutations in human NADPH cytochrome P450 reductase

doi:10.1016/j.bbrc.2010.08.072

Biochemical and Biophysical Research Communications

Volume 400, Issue 3, 24 September 2010, Pages 374-378

https://doi.org/10.1016/j.bbrc.2010.08.072 Get rights and content

Abstract

Human heme oxygenase-1 (HO-1) carries out heme catabolism supported by electrons supplied from the NADPH through NADPH P450 reductase (POR, CPR). Previously we have shown that mutations in human POR cause a rare form of congenital adrenal hyperplasia. In this study, we have evaluated the effects of mutations in POR on HO-1 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified HO-1 to measure heme degradation in a coupled assay using biliverdin reductase. Here we show that mutations in POR found in patients may reduce HO-1 activity, potentially influencing heme catabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had total loss of HO-1 activity, while POR mutations A287P, C569Y and V608F lost 50–70% activity. The POR variants P228L, R316W and G413S, A503V and G504R identified as polymorphs had close to WT activity. Loss of HO-1 activity may result in increased oxidative neurotoxicity, anemia, growth retardation and iron deposition. Further examination of patients affected with POR deficiency will be required to assess the metabolic effects of reduced HO-1 activity in affected individuals.

Research highlights

► Mutations in POR identified from patients lead to reduced HO-1 activities. ► POR mutation Y181D affecting FMN binding results in total loss of HO-1 activity. ► POR mutations A287P, C569Y and V608F, lost 50–70% activity. ► Mutations in FAD binding domain, R457H, Y459H & V492E lost all HO-1 activity. ► POR polymorphisms P228L, R316W, G413S, A503V and G504R have normal activity.

Introduction

Cytochrome P450 oxidoreductase (POR, CPR; EC 1.6.2.4) supplies electron for the metabolic reactions catalyzed by cytochrome P450s in the endoplasmic reticulum [1] as well as heme oxygenase [2], [3] and fatty acid elongase [4]. It also participates in metabolism of several other molecules like mitomycin C [5], doxorubicin [6] cytochrome c, ferricyanide, menadione, dichlorophenolindophenol and nitro blue tetrazolium either directly or as an electron donor to the actual catalyst [7], [8]. The gene (NM_000941) for POR is located on chromosome 7q11.2, and encodes a 82-kDa membrane-bound protein with 680 amino acids (NP_000932). Human POR is a diflavin reductase and contains both the flavin mononucleotide (FMN) and the flavin adenine dinucleotide (FAD) as co-factors and uses NADPH as the preferred electron donor [9], [10], [11]. Electron transfer by POR from NADPH is carried out in several steps starting with binding of NADPH to POR and electrons are then transferred from NADPH first to FAD and then from FAD to FMN [12], [13]. Reduced FMN transfers the electrons one at a time to the redox partners of POR [8]. Heme oxygenase-1 (EC 1.14.99.3) is the initial as well as rate limiting enzyme for microsomal heme degradation and catalyzes heme breakdown to form biliverdin, iron and carbon monoxide and requires electrons from P450 reductase for activity (Fig. 1) [2], [14], [15], [16]. The heme oxygenase itself is not a heme protein and forms a 1:1 complex with heme with absorbance spectral characteristics similar to that of methemoglobin. The conversion of heme into biliverdin by HO is a multistep reaction [3]; the first step is the oxidation of heme to α-hydroxyheme that uses electrons supplied by POR and molecular oxygen (O₂). The second step is the formation of verdoheme with the release of hydroxylated α-meso carbon in the form of carbon monoxide (CO). In the third step α-verdoheme is converted to biliverdin–iron chelate, which also requires O₂ and electrons supplied from POR. The fourth and final step is reduction of the iron of the biliverdin–iron chelate and release of ferrous iron and biliverdin.

We had previously described mutations in POR from patients with disruption of steroid biosynthesis [17], [18] and established that alterations in POR lead to reduced activities of steroid metabolizing enzymes CYP17A1 (17, α-hydroxylase/17,20 lyase) and CYP19A1 (aromatase) [19], [20], [21], [22], [23] and more recently, also of CYP3A4 [24]. In the follow-up studies many POR mutations were reported [25], [26], [27], [28] and a recent large scale sequencing study revealed several polymorphisms in the normal population [29], [30]. In the recent reports from other laboratories several new POR variants have been described [29], [31], [32], [33]. Initially only effects on steroid metabolism were tested with POR variants but more recently we as well as other laboratories have tested drug metabolizing P450s with POR variants [24], [28], [34], [35], [36]. A population genetic variant of POR has been shown to affect CYP3A4 activities but its mechanism remains unclear [37]. Allelic variants of POR are listed on the CYP POR database site (http://www.cypalleles.ki.se/por.htm) [30].

Given the metabolic importance of heme oxygenase activity and its dependence on POR an examination of the effects of POR mutation on HO-1 activity is crucial. Here we are showing for the first time that mutations in POR may also reduce HO-1 activity. We have tested several POR mutations for their ability to support HO-1 activity and found that POR mutations identified in patients with disordered steroid metabolism also significantly reduced heme oxygenase activity.

Section snippets

Expression and purification of the wild-type and the mutant POR in bacteria

Human POR variants were expressed in bacteria and purified as described previously [18], [19], [24], [38]. In brief; cDNAs for WT or mutant POR variants in a pET22b vector were transformed into E. coli BL21(DE3), single colonies were selected for growth on ampicillin and grown in terrific broth supplemented with 40 mM FeCl₃, 4 mM ZnCl₂, 2 mMCoCl₂, 2 mM Na₂MoO₄, 2 mM CaCl₂, 2 mM CuCl₂, 2 mM H₃BO₃, 10% (v/v) potassium phosphate solution (0.17 M KH₂PO₄, 0.72 M K₂HPO₄ pH 7.4), 0.05 mg/ml riboflavin, 100

Results and discussion

From our initial studies conflicting results for different target proteins were obtained for different mutation in POR. From our studies of POR mutations it seems that mutations that interfere with FAD binding (R457H, Y459H, V492E) are generally lethal and result in an inactive POR molecule (Fig. 2) [17], [18]. Interestingly, mutations in NADPH binding domain often have a milder effect and may affect the activities of partner proteins to lesser extent [17], [18], [19], [29], [35]. An

Acknowledgments

This work was supported by grants to AVP from the Swiss National Science Foundation (3100A3-113719), Novartis Foundation for Biology and Medicine, Roche Research Foundation, Swiss Society for Endocrinology & Diabetology and Bern University Research Foundation.

References (47)

A.Y. Lu et al.
Resolution of the cytochrome P-450-containing ω-hydroxylation system of liver microsomes into three components
J. Biol. Chem.
(1969)
B.A. Schacter et al.
Immunochemical evidence for an association of heme oxygenase with the microsomal electron transport system
J. Biol. Chem.
(1972)
G. Kikuchi et al.
Heme oxygenase and heme degradation
Biochem. Biophys. Res. Commun.
(2005)
Z. Ilan et al.
Evidence for a new physiological role of hepatic NADPH:ferricytochrome (P-450) oxidoreductase. Direct electron input to the microsomal fatty acid chain elongation system
J. Biol. Chem.
(1981)
A. Bartoszek et al.
Enhancement of doxorubicin toxicity following activation by NADPH cytochrome P450 reductase
Biochem. Pharmacol.
(1992)
G.P. Kurzban et al.
Preparation and characterization of FAD-dependent NADPH-cytochrome P-450 reductase
J. Biol. Chem.
(1986)
B.S. Masters
The journey from NADPH-cytochrome P450 oxidoreductase to nitric oxide synthases
Biochem. Biophys. Res. Commun.
(2005)
J.L. Vermilion et al.
Separate roles for FMN and FAD in catalysis by liver microsomal NADPH-cytochrome P-450 reductase
J. Biol. Chem.
(1981)
C.E. Flück et al.
Modeling of human P450 oxidoreductase structure by in silico mutagenesis and Md simulation
Mol. Cell. Endocrinol.
(2009)
T. Yoshida et al.
Purification and properties of heme oxygenase from rat liver microsomes
J. Biol. Chem.
(1979)

R. Tenhunen et al.

Microsomal heme oxygenase characterization of the enzyme

J. Biol. Chem.

(1969)

N. Huang et al.

Diversity and function of mutations in p450 oxidoreductase in patients with Antley–Bixler syndrome and disordered steroidogenesis

Am. J. Hum. Genet.

(2005)

W.L. Miller et al.

P450 oxidoreductase deficiency

Lancet

(2004)

C. Nicolo et al.

Restoration of mutant cytochrome P450 reductase activity by external flavin

Mol. Cell. Endocrinol.

(2010)

W. Arlt et al.

Congenital adrenal hyperplasia caused by mutant P450 oxidoreductase and human androgen synthesis: analytical study

Lancet

(2004)

M. Kranendonk et al.

Impairment of human CYP1A2-mediated xenobiotic metabolism by Antley–Bixler syndrome variants of cytochrome P450 oxidoreductase

Arch. Biochem. Biophys.

(2008)

A.V. Pandey et al.

Regulation of 17, 20 lyase activity by cytochrome b5 and by serine phosphorylation of P450c17

J. Biol. Chem.

(2005)

S.J. McNally et al.

Optimization of the paired enzyme assay for heme oxygenase activity

Anal. Biochem.

(2004)

A.V. Pandey et al.

A colorimetric assay for heme in biological samples using 96-well plates

Anal. Biochem.

(1999)

J. Wang et al.

The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase

J. Biol. Chem.

(2003)

Y. Higashimoto et al.

Involvement of NADPH in the interaction between heme oxygenase-1 and cytochrome P450 reductase

J. Biol. Chem.

(2005)

W.L. Miller et al.

Genetic variation in human P450 oxidoreductase

Mol. Cell. Endocrinol.

(2009)

H.F. Bligh et al.

Activation of mitomycin C by NADPH:cytochrome P-450 reductase

Cancer Res.

(1990)

Cited by (28)

P450 oxidoreductase deficiency
2023, Genetic Steroid Disorders: Second Edition
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 the metabolism of steroid hormones, drugs, and xenobiotics. Mutations in POR cause a complex set of disorders that often resemble defects in steroid metabolizing enzymes CYP17A1, CYP21A2, and CYP19A1. Since the initial reports of POR mutations in 2004, more than 300 different mutations and polymorphisms in the POR gene have been identified and tested for their effect on the activities of several steroid and drug-metabolizing cytochromes P450. Mutations in POR may have variable effects on different cytochrome P450 partners depending on the location of the mutation. The POR mutations that disrupt the binding of cofactors harm the function of all partner proteins, while mutations causing subtle structural changes may lead to altered interaction with partner proteins and the overall effect may be different for each partner.
Differential effects of variations in human P450 oxidoreductase on the aromatase activity of CYP19A1 polymorphisms R264C and R264H
2020, Journal of Steroid Biochemistry and Molecular Biology
Citation Excerpt :
The WT POR and variants were produced in bacteria using the plasmid constructs described in earlier publications [68,74–76]. The protocol for the expression of POR variants and membrane as well as liposome preparation is reported in our earlier publications [64–66,68,74,77]. CYP19A1 activity of proteins was monitored by the tritiated water release assay based on an earlier method described by Lephart and Simpson [78] with slight modifications that have been described previously using an assay system containing lipids, POR and CYP19A1 [64,68].
Aromatase (CYP19A1) converts androgens into estrogens and is required for female sexual development and growth and development in both sexes. CYP19A1 is a member of cytochrome P450 family of heme-thiolate monooxygenases located in the endoplasmic reticulum and depends on reducing equivalents from the reduced nicotinamide adenine dinucleotide phosphate via the cytochrome P450 oxidoreductase coded by POR. Both the CYP19A1 and POR genes are highly polymorphic, and mutations in both these genes are linked to disorders of steroid biosynthesis. We have previously shown that R264C and R264H mutations in CYP19A1, as well as mutations in POR, reduce CYP19A1 activity. The R264C is a common polymorphic variant of CYP19A1, with high frequency in Asian and African populations. Polymorphic alleles of POR are found in all populations studied so far and, therefore, may influence activities of CYP19A1 allelic variants. So far, the effects of variations in POR on enzymatic activities of allelic variants of CYP19A1 or any other steroid metabolizing cytochrome P450 proteins have not been studied. Here we are reporting the effects of three POR variants on the aromatase activities of two CYP19A1 variants, R264C, and R264H. We used bacterially expressed and purified preparations of WT and variant forms of CYP19A1 and POR and constructed liposomes with embedded CYP19A1 and POR proteins and assayed the CYP19A1 activities using radiolabeled androstenedione as a substrate. With the WT-POR as a redox partner, the R264C-CYP19A1 showed only 15% of aromatase activity, but the R264H had 87% of aromatase activity compared to WT-CYP19A1. With P284L-POR as a redox partner, R264C-CYP19A1 lost all activity but retained 6.7% of activity when P284T-POR was used as a redox partner. The R264H-CYP19A1 showed low activities with both the POR-P284 L as well as the POR-P284 T. When the POR-Y607C was used as a redox partner, the R264C-CYP19A1 retained approximately 5% of CYP19A1 activity. Remarkably, The R264H-CYP19A1 had more than three-fold higher activity compared to WT-CYP19A1 when the POR-Y607C was used as the redox partner, pointing toward a beneficial effect. The slight increase in activity of R264C-CYP19A1 with the P284T-POR and the three-fold increase in activity of the R264H-CYP19A1 with the Y607C-POR point toward a conformational effect and role of protein-protein interaction governed by the R264C and R264H substitutions in the CYP19A1 as well as P284 L, P284 T and Y607C variants of POR. These studies demonstrate that the allelic variants of P450 when present with a variant form of POR may show different activities, and combined effects of variations in the P450 enzymes as well as in the POR should be considered when genetic data are available. Recent trends in the whole-exome and whole-genome sequencing as diagnostic tools will permit combined evaluation of variations in multiple genes that are interdependent and may guide treatment options by adjusting therapeutic interventions based on laboratory analysis.
Variability in human drug metabolizing cytochrome P450 CYP2C9, CYP2C19 and CYP3A5 activities caused by genetic variations in cytochrome P450 oxidoreductase
2019, Biochemical and Biophysical Research Communications
Citation Excerpt :
PORD causes defects of adrenal steroidogenesis in addition to several other symptoms [14–21]. Likewise, many other alterations in enzymes activities from mutations in POR has been observed due to the multiplicity of its redox partners, including heme oxygenase, CYP17A1, CYP19A1, and CYP3A4 [13,22–29]. Sequencing and biochemical studies have revealed many diseases caused by mutations in the POR gene [27,30–32].
A broad spectrum of human diseases are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR). Cytochrome P450 proteins perform several reactions, including the metabolism of steroids, drugs, and other xenobiotics. In 2004 the first human patients with defects in POR were reported, and over 250 variations in POR are known. Information about the effects of POR variants on drug metabolizing enzymes is limited and has not received much attention. By analyzing the POR sequences from genomics databases, we identified potentially disease-causing variations and characterized these by in vitro functional studies using recombinant proteins. Proteins were expressed in bacteria and purified for activity assays. Activities of cytochrome P450 enzymes were tested in vitro using liposomes prepared with lipids into which P450 and P450 reductase proteins were embedded. Here we are reporting the effect of POR variants on drug metabolizing enzymes CYP2C9, CYP2C19, and CYP3A5 which are responsible for the metabolism of many drugs. POR Variants A115V, T142A, A281T, P284L, A287P, and Y607C inhibited activities of all P450 proteins tested. Interestingly, the POR variant Q153R showed a reduction of 20–50% activities with CYP2C9 and CYP2C19 but had a 400% increased activity with CYP3A5. The A287P is most common POR mutation found in patients of European origin, and significantly inhibited drug metabolism activities which has important consequences for monitoring and treatment of patients. In vitro, functional assays using recombinant proteins provide a useful model for establishing the metabolic effect of genetic mutations. Our results indicate that detailed knowledge about POR variants is necessary for correct diagnosis and treatment options for persons with POR deficiency and the role of changes in drug metabolism and toxicology due to variations in POR needs to be addressed.
Human P450 oxidoreductase deficiency
2018, Encyclopedia of Endocrine Diseases
P450 oxidoreductase (POR) is the essential electron donor to all microsomal cytochrome P450 proteins including the steroid metabolizing CYP17A1, CYP19A1, and CYP21A2, as well as CYP51A1 and CYP26B1 which are involved in bone formation. Human POR mutations therefore present with a very broad phenotype comprising congenital adrenal hyperplasia, disordered sexual and pubertal development, and skeletal malformations resembling the Antley–Bixler craniosynostosis syndrome. Clinical manifestation may be severe with as early as prenatal, or very mild and adult onset. Diagnosis of POR deficiency may be suspected from the clinical examination, but requires biochemical assessment of combined 21- and 17-hydroxylase deficiencies seen by urinary gas chromatographic mass spectrometric steroid profiling. However, confirmation of PORD requires genetic analysis. So far more than 200 mutations and polymorphisms have been reported in the POR gene. The A287P is the most frequently reported mutation in European patients, while R457H predominates in Japanese patients. Overall, genotype–phenotype correlation is rather poor for POR variants. This is mostly due to variable effects on interacting partner proteins. POR also supports drug metabolizing P450s with proven adverse effects of several POR variants in in vitro and in vivo tests.
P450 Oxidoreductase deficiency: Analysis of mutations and polymorphisms
2017, Journal of Steroid Biochemistry and Molecular Biology
Cytochrome P450 oxidoreductase (POR) is required for metabolic reactions of steroid and drug metabolizing cytochrome P450 proteins located in endoplasmic reticulum. Mutations in POR cause a complex set of disorders resembling combined deficiencies of multiple steroid metabolizing enzymes. The P450 oxidoreductase deficiency (PORD) was first reported in patients with symptoms of defects in steroidogenic cytochrome P450 enzymes and ambiguous genitalia, and bone malformation features resembling Antley-Bixler syndrome. POR is now classified as a separate and rare form of congenital adrenal hyperplasia (CAH), which may cause disorder of sexual development (DSD). Since the initial description of PORD in 2004, a large number of POR mutations and polymorphisms have been described. In this report we have performed computational analysis of mutations and polymorphisms in POR linked to metabolism of steroids and xenobiotics and pathology of PORD from the reported cases. The mutations in POR that were identified in patients with disruption of steroidogenesis also have severe effects on cytochrome P450 proteins involved in metabolism of drugs. Different variations in POR show a range of diverse effects on different partner proteins that are often linked to the location of the particular variants. The variations in POR that cause defective binding of co-factors always have damaging effects on all partner proteins, while the mutations causing subtle structural changes may lead to altered interaction with partner proteins and the overall effect may be different for each individual partner. Computational analysis of available sequencing data and mutation analysis shows that Japanese (R457H), Caucasian (A287P) and Turkish (399–401) populations can be linked to unique founder mutations. Other mutations identified so far were identified as rare alleles or in single isolated reports. The common polymorphism of POR is the variant A503V which can be found in about 27% of alleles in general population but there are remarkable differences among different sub populations.
Impact on CYP19A1 activity by mutations in NADPH cytochrome P450 oxidoreductase
2017, Journal of Steroid Biochemistry and Molecular Biology
Cytochrome P450 aromatase (CYP19A1), in human placenta metabolizes androgens to estrogens and uses reduced nicotinamide adenine dinucleotide phosphate through cytochrome P450 oxidoreductase (POR) for the energy requirements of its metabolic activities. Mutations in the human POR lead to congenital adrenal hyperplasia due to loss of activities of several steroid metabolizing enzymatic reactions conducted by the cytochrome P450 proteins located in the endoplasmic reticulum. Effect of POR mutations on different P450 activities depend on individual partner proteins. In this report we have studied the impact of mutations found in the POR on the enzymatic activity of CYP19A1. We expressed wild type as well mutant human POR proteins in bacteria and purified the recombinant proteins, which were then used in an in vitro reconstitution system in combination with CYP19A1 and lipids for enzymatic analysis. We found that several mutations as well as polymorphisms in human POR can cause reduction of CYP19A1 activity. This would affect metabolism of estrogens in people with variations of POR allele. The POR mutants Y181D and R616X were found to have no activity in supporting CYP19A1 reactions. The POR mutations Y607C and delF646 showed a loss of 60–90% activity and two polymorphic forms of POR, R316W and G413S showed similar to WT activity. One POR variant, Q153R had almost double the activity of WT. Loss of CYP19A1 activity may contribute to disordered steroidogenesis in female patients with POR mutations as well as in mothers with POR variants carrying a male child.

View all citing articles on Scopus

View full text

Altered heme catabolism by heme oxygenase-1 caused by mutations in human NADPH cytochrome P450 reductase

Abstract

Research highlights

Introduction

Section snippets

Expression and purification of the wild-type and the mutant POR in bacteria

Results and discussion

Acknowledgments

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Biochem. Pharmacol.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Mol. Cell. Endocrinol.

J. Biol. Chem.

J. Biol. Chem.

Am. J. Hum. Genet.

Lancet

Mol. Cell. Endocrinol.

Lancet

Arch. Biochem. Biophys.

J. Biol. Chem.

Anal. Biochem.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

Mol. Cell. Endocrinol.

Activation of mitomycin C by NADPH:cytochrome P-450 reductase

Cancer Res.