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

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.     

The roles of cytochrome b5 in cytochrome P450 reactions

Cytochrome b(5), a 17-kDa hemeprotein associated primarily with the endoplasmic reticulum of eukaryotic cells, has long been known to augment some cytochrome P450 monooxygenase reactions, but the mechanism of stimulation has remained controversial. Studies in recent years have clarified this issue by delineating three pathways by which cytochrome b(5) augments P450 reactions: direct electron transfer of both required electrons from NADH-cytochrome b(5) reductase to P450, in a pathway separate and independent of NADPH-cytochrome P450 reductase; transfer of the second electron to oxyferrous P450 from either cytochrome b(5) reductase or cytochrome P450 reductase; and allosteric stimulation of P450 without electron transfer. Evidence now indicates that each of these pathways is likely to operate in vivo.     

Thermophilic cytochrome P450 enzymes

Thermophilic cytochrome P450 enzymes are of potential interest from structural, mechanistic, and biotechnological points of view. The structures and properties of two such enzymes, CYP119 and CYP175A1, have been investigated and provide the foundation for future work on thermophilic P450 enzymes.     

Heme oxygenase-1 and NADPH cytochrome P450 reductase expression in experimental allergic encephalomyelitis: an expanded view of the stress response

Oxidative stress is implicated in the pathogenesis of experimental allergic encephalomyelitis (EAE), a model for multiple sclerosis. Heme oxygenase-1 (HO-1) is a heat shock protein induced by oxidative stress. HO-1 metabolizes the pro-oxidant heme to the antioxidant biliverdin and CO. HO-1 requires electrons, donated by NADPH cytochrome P450 reductase (henceforth, reductase), for catalytic activity. EAE was induced with a peptide of proteolipid protein in SJL mice, and the expression of HO-1 and reductase in the hindbrain was analyzed. HO-1 protein levels were significantly increased in EAE animals compared with control mice. HO-1 expression was present in ameboid macrophages, reactive microglia, and astrocytes in white matter tracks. Bergmann glia and ameboid macrophages also were occasionally stained in the molecular layer of the cerebellum. Unlike HO-1, reductase protein levels decreased with disease severity. HO-1 and reductase were associated with each other in endoplasmic reticulum micelles, suggesting that the decrease in reductase does not interfere with its association with HO-1. In cells that express HO-1, the association of reductase with HO-1 should competitively inhibit the interaction of reductase with cytochrome P450 isozymes and thereby limit free radical production as the latter two enzymes act cooperatively to produce superoxide. The increase in HO-1 together with the decrease in reductase may be part of a common defense mechanism attempting to minimize tissue damage in several neurological conditions.     

Mass spectrometric identification of lysine residues of heme oxygenase-1 that are involved in its interaction with NADPH-cytochrome P450 reductase

The lysine residues of rat heme oxygenase-1 (HO-1) were acetylated by acetic anhydride in the absence and presence of NADPH-cytochrome P450 reductase (CPR) or biliverdin reductase (BVR). Nine acetylated peptides were identified by MALDI-TOF mass spectrometry in the tryptic fragments obtained from HO-1 acetylated without the reductases (referred to as the fully acetylated HO-1). The presence of CPR prevented HO-1 from acetylation of lysine residues, Lys-149 and Lys-153, located in the F-helix. The heme degradation activity of the fully acetylated HO-1 in the NADPH/CPR-supported system was significantly reduced, whereas almost no inactivation was detected in HO-1 in the presence of CPR, which prevented acetylation of Lys-149 and Lys-153. On the other hand, the presence of BVR showed no protective effect on the acetylation of HO-1. The interaction of HO-1 with CPR or BVR is discussed based on the acetylation pattern and on molecular modeling.     

Structural characterization of human heme oxygenase-1 in complex with azole-based inhibitors

The development of inhibitors specific for heme oxygenases (HO) aims to provide powerful tools in understanding the HO system. Based on the lead structure (2S, 4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[((4-aminophenyl)thio)methyl]-1,3-dioxolane (azalanstat, QC-1) we have synthesized structural modifications to develop novel and selective HO inhibitors. The structural study of human HO-1 (hHO-1) in complex with a select group of the inhibitors was initiated using X-ray crystallographic techniques. Comparison of the structures of four such compounds each in complex with hHO-1 revealed a common binding mode, despite having different structural fragments. The compounds bind to the distal side of heme through an azole “anchor” which coordinates with the heme iron. An expansion of the distal pocket, mainly due to distal helix flexibility, allows accommodation of the compounds without displacing heme or the critical Asp140 residue. Rather, binding displaces a catalytically critical water molecule and disrupts an ordered hydrogen-bond network involving Asp140. The presence of a triazole “anchor” may provide further stability via a hydrogen bond with the protein. A hydrophobic pocket acts to stabilize the region occupied by the phenyl or adamantanyl moieties of these compounds. Further, a secondary hydrophobic pocket is formed via “induced fit” to accommodate bulky substituents at the 4-position of the dioxolane ring.     

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

Y459H and V492E mutations of cytochrome P450 reductase (CYPOR) cause Antley-Bixler syndrome due to diminished binding of the FAD cofactor. To address whether these mutations impaired the interaction with drug-metabolizing CYPs, a bacterial model of human liver expression of CYP1A2 and CYPOR was implemented. Four models were generated: POR^null, POR^wt, POR^YH, and POR^VE, for which equivalent CYP1A2 and CYPOR levels were confirmed, except for POR^null, not containing any CYPOR. The mutant CYPORs were unable to catalyze cytochrome c and MTT reduction, and were unable to support EROD and MROD activities. Activity was restored by the addition of FAD, with V492E having a higher apparent FAD affinity than Y459H. The CYP1A2-activated procarcinogens, 2-aminoanthracene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and 2-amino-3-methylimidazo(4,5-f)quinoline, were significantly less mutagenic in POR^YH and POR^VE models than in POR^wt, indicating that CYP1A2, and likely other drug-metabolizing CYPs, are impaired by ABS-related POR mutations as observed in the steroidogenic CYPs.     

Thermal inactivation of the reductase domain of cytochrome P450 BM3

Although the reductase domain of cytochrome P450 BM3 (BMR) catalyzes the reduction of cytochrome c and 2,6-dichlorophenolindophenol, we observed a catalytically independent loss of activity. By varying the incubation time for the enzyme prior to reaction initiation, we measured an inactivation rate of 0.22 min(-1). We hypothesized that either an active BMR dimer dissociates to an inactive monomer or BMR undergoes denaturation. We were not able to trap or destabilize a dimer, and BMR inactivation proved to be irreversible. Addition of excess FMN only slightly decreased the rate of inactivation from 0.22 to 0.13 min(-1), indicating inactivation likely does not reflect loss of flavin. When inactivation rates as a function of temperature were fit to the Arrhenius equation, the energy required to inactivate BMR was 9.9 kcal mol(-1)--equivalent to a few hydrogen bonds. The potential instability of BMR under certain conditions raises concerns for the use of BMR as a model or surrogate P450 reductase in other systems.     

Hydroperoxoferric heme intermediate as a second electrophilic oxidant in cytochrome P450-catalyzed reactions

Experimental evidence supporting the catalytic activity of the peroxoferric and hydroperoxoferric cytochrome P450 intermediates as alternative oxidants to the compound I (ferryl) state in the oxygenation of organic substrates is reviewed. The peroxoferric P450 state is proposed to function as a nucleophile in the lyase step of the P450-aromatase reaction. Several systems are reviewed in which the hydroperoxoferric P450 intermediate likely functions as a second electrophilic oxidant, the two-oxidants model. These include alkene epoxidation, sulfoxidation, and hydroxylation of methyl groups on cyclopropane rings. The key use of the P450 mutants from different sources in which the conserved threonine in the distal substrate binding pocket is replaced with alanine, in order to minimize the formation of the compound I intermediate and unmask the reactivity of the hydroperoxoferric state, is emphasized. These data are discussed in the context of the two-states model, which proposes that the compound I P450 intermediate has both high- and low-spin states with different reactivities. A complicated reaction profile emerges for the wide range of P450 reactions involving up to three reactive intermediates, of which the most reactive, the compound I P450 state, has two spin states with different reactivities.     

Effects of substance P on osteoblastic differentiation and heme oxygenase-1 in human periodontal ligament cells

Although substance P (SP) is associated with osteoclast differentiation and bone resorption, little is known about the osteogenic differentiation-inducing effects of SP in periodontal ligament (PDL) cells. This study investigated whether PDL cells could differentiate into osteoblastic-like cells by SP. The expression of osteoblastic differentiation markers such as osteopontin (OPN), osteonectin (ON), osteocalcin (OCN) and bone sialoprotein (BSP) were evaulated by Western blotting. Additionally, SP-mediated heme oxygenase-1 (HO-1) pathways were further clarified.SP increased HO-1 and osteogenic differentiation in concentration- and time-dependent manners, as determined by OPN, ON, OCN and BSP expression. Furthermore, treatment with inhibitors of p38, ERK MAPK, and NF-κB abolished SP-induced osteogenic differentiation and HO-1 expression. SP-induced translocation of Nrf-2 was also observed. The combined results suggest that SP activates the stress-response enzymes HO-1 and Nrf-2, subsequently leading to upregulation of osteogenic differentiation in human PDL cells.     

Coexpression in yeast of Taxus cytochrome P450 reductase with cytochrome P450 oxygenases involved in Taxol biosynthesis

To maximize redox coupling efficiency with recombinant cytochrome P450 hydroxylases from yew (Taxus) species installed in yeast for the production of the anticancer drug Taxol, a cDNA encoding NADPH:cytochrome P450 reductase from T. cuspidata was isolated. This single-copy gene (2,154 bp encoding a protein of 717 amino acids) resembles more closely other reductases from gymnosperms (approximately 90% similarity) than those from angiosperms (<80% similarity). The recombinant reductase was characterized and compared to other reductases by heterologous expression in insect cells and was shown to support reconstituted taxoid 10beta-hydroxylase activity with an efficiency comparable to that of other plant-derived reductases. Coexpression in yeast of the reductase along with T. cuspidata taxoid 10beta-hydroxylase, which catalyzes an early step of taxoid biosynthesis, demonstrated significant enhancement of hydroxylase activity compared to that supported by the endogenous yeast reductase alone. Functional transgenic coupling of the Taxus reductase with a homologous cytochrome P450 taxoid hydroxylase represents an important initial step in reconstructing Taxol biosynthesis in a microbial host.     

Thirty years of microbial P450 monooxygenase research: peroxo-heme intermediates--the central bus station in heme oxygenase catalysis

Oxygen has always been recognized as an essential element of many life forms, initially through its role as a terminal electron acceptor for the energy-generating pathways of oxidative phosphorylation. In 1955, Hayaishi et al. [Mechanism of the pyrocatechase reaction, J. Am. Chem. Soc. 77 (1955) 5450-5451] presented the most important discovery that changed this simplistic view of how Nature uses atmospheric dioxygen. His discovery, the naming and mechanistic understanding of the first "oxygenase" enzyme, has provided a wonderful opportunity and scientific impetus for four decades of researchers. This volume provides an opportunity to recognize the breakthroughs of the "Hayaishi School." Notable have been the prolific contributions of Professor Ishimura et al. [Oxygen and life. Oxygenases, Oxidases and Lipid Mediators, International Congress Series, Elsevier, Amsterdam, 2002], a first-generation Hayaishi product, to characterization of the cytochrome P450 monooxygenases.     

Crystal structure of the FMN-binding domain of human cytochrome P450 reductase at 1.93 A resolution

The crystal structure of the FMN-binding domain of human NADPH-cytochrome P450 reductase (P450R-FMN), a key component in the cytochrome P450 monooxygenase system, has been determined to 1.93 A resolution and shown to be very similar both to the global fold in solution (Barsukov I et al., 1997, J Biomol NMR 10:63-75) and to the corresponding domain in the 2.6 A crystal structure of intact rat P450R (Wang M et al., 1997, Proc Nat Acad Sci USA 94:8411-8416). The crystal structure of P450R-FMN reported here confirms the overall similarity of its alpha-beta-alpha architecture to that of the bacterial flavodoxins, but reveals differences in the position, number, and length of the helices relative to the central beta-sheet. The marked similarity between P450R-FMN and flavodoxins in the interactions between the FMN and the protein, indicate a striking evolutionary conservation of the FMN binding site. The P450R-FMN molecule has an unusual surface charge distribution, leading to a very strong dipole, which may be involved in docking cytochrome P450 into place for electron transfer near the FMN. Several acidic residues near the FMN are identified by mutagenesis experiments to be important for electron transfer to P4502D6 and to cytochrome c, a clear indication of the part of the molecular surface that is likely to be involved in substrate binding. Somewhat different parts are found to be involved in binding cytochrome P450 and cytochrome c.     

细胞色素P450酶的结构、功能与应用研究进展

细胞色素P450 (cytochrome P450,CYP)酶是广泛存在于微生物、动植物及人体中与膜结合的血红蛋白类酶,具有氧化、环氧化、羟化、去甲基化等多种生物催化活性。CYP酶在药物、类固醇、脂溶性维生素和许多其他类型化学物质的代谢中具有重要作用,其在异源物质的解毒、药物相互作用和内分泌功能等领域的研究是热点问题。本综述对CYP的结构、功能、临床应用与开发前景进行了概述,并对其最新的研究现状和发展前景进行探讨。     

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

Heme oxygenase-1 (HO-1) catalyzes the physiological degradation of heme at the expense of molecular oxygen using electrons donated by NADPH-cytochrome P450 reductase (CPR). In this study, we investigated the effect of NADP(H) on the interaction of HO-1 with CPR by surface plasmon resonance. We found that HO-1 associated with CPR more tightly in the presence of NADP(+) (K(D) = 0.5 microm) than in its absence (K(D) = 2.4 microm). The HO-1 mutants, K149A, K149A/K153A, and R185A, showed almost no heme degradation activity with NADPH-CPR, whereas they exhibited activity comparable to that of the wild type when sodium ascorbate was used. R185A showed a 100-fold decreased affinity for CPR compared with wild type, even in the presence of NADP(+) (K(D) = 36.3 microm). The affinities of K149A and K149A/K153A for CPR were decreased 7- and 9-fold (K(D) = 16.8 and 21.8 microm), respectively. In contrast to R185A, the affinities of K149A and K149A/K153A were improved by the addition of NADP(+) (K(D) = 5.2 and 9.6 microm, respectively), as was the case with wild type. Computer modeling of the HO-1/CPR complex showed that the guanidino group of Arg(185) is located within the hydrogen bonding distance of 2'-phosphate of NADPH, suggesting that Arg(185) contributes to the binding to CPR through an electrostatic interaction with the phosphate group. On the other hand, Lys(149) is close to a cluster of acidic amino acids near the FMN binding site of CPR. Thus, Lys(149) and Lys(153) appear to interact with CPR in such a way as to orient the redox partners for optimal electron transfer from FMN of CPR to heme of HO-1.     

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

Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.     

Measurement of immunoreactive cytochrome P450 2E1 in human leucocytes

We describe initial results on a Western blotting method, using a ployclonal antibody and chemiluminescence detection, for the measurement of cytochrome P450 2E1 in human lymphocytes. The method has been used to study the levels of 2E1 in lymphocytes isolated from 5 ml blood samples collected from a small group of well-controlled type 1 diabetics and healthy individuals. The described method offers increased sensitivity compared with a previously published method and does not need in vitro culturing of the lymphocytes prior to 2E1 measurement. The apparent molecular weight of the lymphocyte P450 2E1 was 55 kDa. There was approximately a six-fold difference in expression levels of 2E1 detected by this immunochemical technique across the study population.     

Co-incorporation of heterologously expressed Arabidopsis cytochrome P450 and P450 reductase into soluble nanoscale lipid bilayers

Heterologous expression of CYP73A5, an Arabidopsis cytochrome P450 monooxygenase, in baculovirus-infected insect cells yields correctly configured P450 detectable by reduced CO spectral analysis in microsomes and cell lysates. Co-expression of a housefly NADPH P450 reductase substantially increases the ability of this P450 to hydroxylate trans-cinnamic acid, its natural phenylpropanoid substrate. For development of high-throughput P450 substrate profiling procedures, membrane proteins derived from cells overexpressing CYP73A5 and/or NADPH P450 reductase were incorporated into soluble His(6)-tagged nanoscale lipid bilayers (Nanodiscs) using a simple self-assembly process. Biochemical characterizations of nickel affinity-purified and size-fractionated Nanodiscs indicate that CYP73A5 protein assembled into Nanodiscs in the absence of NADPH P450 reductase maintains its ability to bind its t-cinnamic acid substrate. CYP73A5 protein co-assembled with P450 reductase into Nanodiscs hydroxylates t-cinnamic acid using reduced pyridine nucleotide as an electron source. These data indicate that baculovirus-expressed P450s assembled in Nanodiscs can be used to define the chemical binding profiles and enzymatic activities of these monooxygenases.     

Establishment of a yeast system that stably expresses human cytochrome P450 reductase: application for the study of drug metabolism of cytochrome P450s in vitro

Cytochrome P450s (CYPs) hold a balance in studying pharmacokinetics, toxico-kinetics, drug metabolism, and drug-drug interactions, which require association with cytochrome P450 reductase (CPR) to achieve optimal activity. A novel system of Saccharomyces cerevisiae useful for expression studies of mammalian microsomal CYPs was established. Human CPR (hCPR) was co-expressed with human CYP3A4 (hCYP3A4) in this system, and two expression plasmids pTpLC and pYeplac195-3A4 containing the cDNA of hCPR and hCYP3A4 were constructed, respectively. The two plasmids were applied first and controlled by phosphoglycerate kinase (PGK) promoter. S. cerevisiae BWG1-7alpha transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-hCYP3A4 immunoglobulin (Ig) and anti-hCPR Ig. The activity of hCPR in yeast BWG-CPR was 443.2 nmol reduced cytochrome c/min/mg, which was about three times the CPR activity of the microsome prepared from the parental yeast. The protein amount of hCYP3A4 in BWG-CPR/3A4 was 35.53 pmol/mg, and the 6beta-hydroxylation testosterone formation activity of hCYP3A4 expressed was 7.5 nmol/min/nmol CYP, 30 times higher than the activity of hCYP3A4 expressed in the parental yeast, and almost two times the activity of hCYP3A4 from homologous human liver microsome. Meanwhile, BWG-CPR/3A4 retained 100 generations under nonselective culture conditions, indicating this yeast was a mitotically stable transformant. BWG-CPR was further tested daily by the PCR amplification of hCPR of yeast genome, Western blot analysis, and the activity assay of hCPR of yeast microsome. This special expression host for CYPs was validated to be stable and efficient for the expression of CYPs, applying as an effective selection model for the drug metabolism in vitro.