Mitochondrial P450 Systems Research Articles

Binding of cytochrome P450 27C1, a retinoid desaturase, to its accessory protein adrenodoxin

Of the 57 human cytochrome P450 (P450) enzymes, seven are mitochondrial: 11A1, 11B1, 11B2, 24A1, 27A1, 27B1, and 27C1. Mitochondrial P450s utilize an electron transport system with adrenodoxin (Adx) and NADPH-adrenodoxin reductase (AdR). AdR reduces Adx, which then transfers electrons to the P450. The interactions between proteins in the mitochondrial P450 system are largely driven by electrostatic interactions, though the specifics vary depending on the P450. Unlike other mitochondrial P450s, the interaction between P450 27C1, a retinoid 3,4-desaturase expressed in the skin, and Adx remains largely uncharacterized. In this work, we utilized an Alexa Fluor 488 C5 maleimide-labeled Adx to measure binding affinities between Adx and P450 27C1 or AdR. Both proteins bound Adx tightly, with Kd values < 100 nM, and binding affinities decreased with increasing ionic strength, supporting the role of electrostatic interactions in mediating these interactions. Cross-linking mass spectrometry and computational modeling were performed to identify interactions between P450 27C1 and Adx. While the residues of Adx identified in interactions were consistent with studies of other mitochondrial P450s, the binding interface of P450 27C1 was quite large and supported multiple Adx binding positions, including ones outside of the canonical Adx binding site. Additionally, Adx did not appear to be an allosteric effector of P450 27C1 substrate binding, in contrast to some other mitochondrial P450s. Overall, we conclude that P450-Adx interactions are P450-specific.

Functional interactions of adrenodoxin with several human mitochondrial cytochrome P450 enzymes

Seven of the 57 human cytochrome P450 (P450) enzymes are mitochondrial and carry out important reactions with steroids and vitamins A and D. These seven P450s utilize an electron transport chain that includes NADPH, NADPH-adrenodoxin reductase (AdR), and adrenodoxin (Adx) instead of the diflavin NADPH-P450 reductase (POR) used by the other P450s in the endoplasmic reticulum. Although numerous studies have been published involving mitochondrial P450 systems, the experimental conditions vary considerably. We compared human Adx and bovine Adx, a commonly used component, and found very similar catalytic activities in reactions catalyzed by human P450s 11B2, 27A1, and 27C1. Binding constants of 6–200 nM were estimated for Adx binding to these P450s using microscale thermophoresis. All P450 catalytic reactions were saturated at 10 μM Adx, and higher concentrations were not inhibitory up to at least 50 μM. Collectively these studies demonstrate the tight binding of Adx (both human and bovine) to AdR and to several mitochondrial P450s and provide guidance for optimization of Adx-dependent P450 reactions.

A tricistronic human adrenodoxin reductase-adrenodoxin–cytochrome P450 27A1 vector system for substrate hydroxylation in Escherichia coli

Cytochrome P450 (P450) 27A1 catalyzes 27-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3, serving as an important component for the maintenance of lipid homeostasis. In eukaryotic cells P450 27A1 is a membrane-bound protein located on the inner mitochondrial membrane and requires two auxiliary reduction partners, adrenodoxin (Adx) and NADPH-adrenodoxin reductase (Adr), for catalysis in the bile acid biosynthesis pathway. A strategy was developed for the functional coexpression of P450 27A1 with Adr and Adx in a tricistronic fashion (single RNA, three proteins) in Escherichia coli, mimicking the mitochondrial P450 system. Intact bacterial cells coexpressing the P450 vector (pTC27A1) efficiently hydroxylated cholesterol at the 27 position as well as vitamin D3 at the 25 position when supplemented with glycerol as a carbon source. Thus, E. coli containing pTC27A1 is able to hydroxylate cholesterol in a self-sufficient fashion and is suitable for further applications of protein interaction, drug discovery, and inhibitor evaluation and for the study of other mitochondrial P450s and oxysterol production in microorganisms without a need for membrane reconstitution, membrane simulation by detergents, or purification of the components.

Coexpression of redox partners increases the hydrocortisone (cortisol) production efficiency in CYP11B1 expressing fission yeast Schizosaccharomyces pombe

Cytochromes P450 play a vital role in the steroid biosynthesis pathway of the adrenal gland. An example of an essential P450 cytochrome is the steroid 11β-hydroxylase CYP11B1, which catalyses the conversion of 11-deoxycorticol to hydrocortisone. However, despite its high biotechnological potential, this enzyme has so far been unsuccessfully employed in present-day biotechnology due to a poor expression yield and inherent protein instability. In this study, CYP11B1 was biotransformed into various strains of the yeast Schizosaccharomyces pombe, all of which also expressed the electron transfer proteins adrenodoxin and/or adrenodoxin reductase – central components of the mitochondrial P450 system – in order to maximise hydrocortisone production efficiency in our proposed model system. Site-directed mutagenesis of CYP11B1 at positions 52 and 78 was performed in order to evaluate the impact of altering the amino acids at these sites. It was found that the presence of an isoleucine at position 78 conferred the highest 11β-hydroxylation activity of CYP11B1. Coexpression of adrenodoxin and adrenodoxin reductase appeared to further increase the 11β-hydroxylase activity of the enzyme (3.4 fold). Adrenodoxin mutants which were found to significantly enhance enzyme efficiency in other cytochromes in previous studies were also tested in our system. It was found that, in this case, the wild type adrenodoxin was more efficient. The new fission yeast strain TH75 coexpressing the wild type Adx and AdR displays high hydrocortisone production efficiency at an average of 1 mM hydrocortisone over a period of 72 h, the highest value published to date for this biotransformation. Finally, our research shows that pTH2 is an ideal plasmid for the coexpression of the mitochondrial electron transfer counterparts, adrenodoxin and adrenodoxin reductase, in Schizosaccharomyces pombe, and so could serve as a convenient tool for future biotechnological applications.

Antioxidant Protective Mechanisms against Reactive Oxygen Species (ROS) Generated by Mitochondrial P450 Systems in Steroidogenic Cells

Mitochondrial P450 type enzymes catalyze central steps in steroid biosynthesis, including cholesterol conversion to pregnenolone, 11β and 18 hydroxylation in glucocorticoid and mineralocorticoid synthesis, C-27 hydroxylation of bile acids, and 1α and 24 hydroxylation of 25-OH-vitamin D. These monooxygenase reactions depend on electron transfer from NADPH via FAD adrenodoxin reductase and 2Fe-2S adrenodoxin. These systems can function as a futile NADPH oxidase, oxidizing NADPH in absence of substrate, and leak electrons via adrenodoxin and P450 to O2, producing superoxide and other reactive oxygen species (ROS). The degree of uncoupling depends on the P450 and steroid substrate. Studies with purified proteins and overexpression in cultured cells show consistently that adrenodoxin, but not reductase, is responsible for ROS production that can lead to apoptosis. In the ovary and corpus luteum, antioxidant enzyme activities superoxide dismutase, catalase, and glutathione peroxidase parallel steroidogenesis. Antioxidant β-carotene, α-tocopherol, and ascorbate can protect against oxidative damages of P450 systems. In testis Leydig cells, steroidogenesis is associated with aging of the steroidogenic capacity.

Bacterial (CYP101) and mitochondrial P450 systems—how comparable are they?

The bacterial CYP101 system and mitochondrial P450 systems show high similarity. Both systems contain the same protein components, a FAD containing reductase, a ferredoxin of the [2Fe2S] type, and a cytochrome P450. At a first glance they seem to be comparable but there are considerable differences among both proteins. Thus, the ferredoxin components of the two systems display significant structural homology but cannot substitute for each other in functional assays. Going into more detail, pronounced differences between the two systems that affect their biological functions are found.

The Tertiary Structure of Full-Length Bovine Adrenodoxin Suggests Functional Dimers

The three-dimensional X-ray crystal structure of full-length oxidized bovine adrenodoxin (Adx) has been determined at 2.5 Å resolution by molecular replacement using a structure of a truncated form as a starting model. Crystals of Adx belong to a primitive monoclinic space group P21 with four Adx molecules in an asymmetric unit. The unit cell dimensions are a = 59.44 Å, b = 77.03 Å, c = 59.68 Å, and β = 94.83°. The structure has been refined to an R factor of 23.5%. Structures of the four molecules of full-length Adx (127 amino acids) in the asymmetric unit were compared with each other and also with that of the truncated Adx (4–108). The overall topology of full-length Adx remains the same as described earlier for the truncated protein. Differences that do occur are almost wholly confined to alternate side-chain conformations that reflect differing lattice contacts made by two proteins. Extensive interactions found between molecules 1 and 2 in the full-length Adx asymmetric unit may reflect the ability of Adx to form dimers in vivo and are consistent with hydrodynamic measurements which show that in solution there is an equilibrium between monomeric and dimeric forms of Adx. Dimerization of Adx could explain why the truncated form has greater affinity for the P450 redox partner than the full-length form. From these results it can be considered that the mechanism of electron transfer is not necessarily the same in different mitochondrial P450 systems.

Xenobiotic metabolism in brain

Recent hypothesis suggesting a role for environmental toxins in the pathogenesis of neurodegenerative disorders has stimulated interest in research on xenobiotic metabolizing capability of the brain. In addition to possible irreversible loss of neurons through bioactivation in situ in the nervous tissue, the metabolism of psychoactive drugs in the target tissue can lead to local pharmacological modulation at the site of action. The major drug metabolizing enzymes, cytochromes P-450 (P450) and flavin-containing monooxygenase (FMO) have been detected in rodent brain and human brain tissue obtained at autopsy. The brain microsomal and mitochondrial P450 systems are capable of metabolizing a variety of xenobiotics, while the brain FMO efficiently metabolizes a variety of psychoactive drugs to their respective N-oxides. Immunocytochemical studies have revealed the regional heterogeneity in the distribution of multiple forms of P450 in the brain and the co-localization of P450 and FMO predominantly in the neuronal cells. Although the brain P450 and FMO share many common features with similar enzymes present in other tissues such as liver and lung, there are some distinctive differences. It is evident from the studies carried out so far that the brain can metabolize a variety of lipophilic xenobiotics that enter by way of the blood stream.

Electron Leakage from the Adrenal-Cortex Mitochondrial P450Scc and P450C11 Systems: NADPH and Steroid Dependence

In the present study we examined the coupling of NADPH oxidation to substrate hydroxylation and the effects of steroids on this process in reconstituted P450scc and P450c11 systems. To determine the relative rates of substrate hydroxylation vs electron leakage we assayed both the steroid product and H2O2 in the same sample. For both P450 systems the rates of steroid product and superoxide formation increased as NADPH concentration was increased. However, P450c11 was found to be more leaky. The leakage from the P450scc system was not affected by pregnenolone, the product of cholesterol side chain cleavage. In contrast, corticosterone, the product of P450c11, increased the rate of futile NADPH oxidation by the P450c11 system. We also tested a series of steroids to analyze the stereospecificity of their effects. Relative to the control without steroid, both C-19 and C-21 steroids with 11α-hydroxy groups (11α-OH-testosterone and 11α-OH-cortisol) decreased leakage, and those with 11β-OH groups (11β-OH-testosterone and cortisol) stimulated both NADPH oxidation and electron leakage as measured by H2O2 formation. The results revealed a correlation between the effects previously observed in living cells and in our reconstituted systems. These findings provide further evidence that mitochondrial P450 systems indeed function as a significant source of oxygen radicals in steroidogenic cells.

Routes and regulation of NADPH production in steroidogenic mitochondria.

The first and rate-limiting step of steroidogenesis is catalyzed by the mitochondrial cholesterol side chain cleavage system that is dependent on NADPH. The pathways of NADPH generation in steroidogenic mitochondria include three major routes catalyzed by: 1. NADP-linked malic enzyme, 2. NADP-linked isocitrate dehydrogenase, and 3. nicotinamide nucleotide transhydrogenase. The main route may differ among cell types and across species. Generally operation of alternative routes, with different substrates is not excluded. The oxidation of NADPH by the mitochondrial P450 systems is not tightly coupled with substrate metabolism, as these systems can reduce O2 by a single electron to produce harmful superoxide radical. To minimize such futile NADPH oxidation, NADPH generation may be regulated by two types of mechanisms: 1. Feedback mechanisms that maintain the ratio of NADPH/NADP+ at a steady-state level by enhancing the rate of NADPH production to keep up with its rate of oxidation, e.g., allosteric regulation of enzymes involved in NADPH production. 2. Hormonal signals that enhance the level of NADPH production in coordination with steroidogenesis. One major hypothesis with experimental evidence is that stimulation of mitochondrial NAD(P)H synthesis is mediated by Ca++ as a second messenger of tropic factors. Tropic stimulation of cells increases the levels of Ca++ in the cytosol and then in the mitochondrial matrix, and the rise in Ca++ activates enzymes involved in NAD(P)H synthesis. These regulatory mechanisms most probably operate in concert adjusted to the steroidogenic activity of the cell.

A Fluorometric Assay for Hydrogen Peroxide, Suitable for NAD(P)H-Dependent Superoxide Generating Redox Systems

We report a simple and sensitive fluorimetric method for quantitative assay of the production rate of hydrogen peroxide, and indirectly of superoxide, during electron transfer reactions. The assay requires the inclusion of superoxide dismutase, catalase, and 6% methanol in the tested reaction system, to stochiometrically produce formaldehyde per molecule of H2O2 generated. The reaction is terminated by adding 2 vol of Nash reagent and heating at 60°C for 10 min, to convert accumulated formaldehyde to diacetyldihydrolutidine (DDL). The standard curve for formaldehyde, based on the fluorescence of DDL, is highly reproducible and allows measurement of 1 μM amounts in the reaction sample (coefficient of variation <15%). The excitation and emission wavelengths of DDL at 412 and 505 nm are distant from those of NAD(P)H. Thus, the method can be used in NAD(P)H-dependent enzymatic systems to measure both NAD(P)H oxidation and superoxide production in the same sample. We validated the assay in a mitochondrial P450 system determining the fraction of total electron flow that is channeled to oxy-radical formation. The assay should be useful in the study of this and other superoxide/H2O2 generating systems.

Electron Leakage from the Mitochondrial NADPH-Adrenodoxin Reductase-Adrenodoxin-P450scc (Cholesterol Side Chain Cleavage) System

In electron (e −) transfer systems some e − may "leak," reducing O 2 to a superoxide radical. This study examined the sites and kinetics of e − leakage from the mitochondrial P450scc system. Adrenodoxin reductase alone oxidized NADPH, reducing O 2 to a superoxide radical at a very low rate. However, the reductase-adrenodoxin system reduced O 2 at a rapid steady-state rate with Michaelis- Menten dependence on [adrenodoxin]( V max = 3.5 μM e −/min). After depletion of NADPH, reduced adrenodoxin was oxidized (autooxidation) with pseudo first order kinetics and the rate of e − transfer decreased 10-fold. Ca 2+ (<1 mM) stimulated e − leakage in both phases. The reductase-adrenodoxin-P450scc system exhibited the highest rate of leakage ( V max = 7.8 μM e −/min). At low [adrenodoxin] the majority of e − leaked through P450scc and not through adrenodoxin. In the presence of the substrate, cholesterol, leakage drastically decreased to <0.5 μM e −/min. These results indicate that the mitochondrial P450 systems can leak e −, producing O 2 derived free radicals. Reduction of leakage during P450scc conversion of cholesterol to pregnenolone provides a clue to understanding physiological mechanisms that control e − leakage. These may include coregulation of NADPH and cholesterol availability to the P450scc system and a system of antioxidants for quenching the oxygen radicals.

Structure and function of the mitochondrial P450 system electron transfer proteins, adrenodoxin reductase and adrenodoxin

Article Structure and function of the mitochondrial P450 system electron transfer proteins, adrenodoxin reductase and adrenodoxin was published on July 1, 1992 in the journal Journal of Basic and Clinical Physiology and Pharmacology (volume 3, issue Supplement).

Induction and mitochondrial localization of cytochrome P450scc system enzymes in normal and transformed ovarian granulosa cells.

After ovulation of an oocyte, granulosa cells of the ovarian follicle differentiate into luteal cells and become a major factor dedicated to the synthesis of the steroid hormone progesterone. We recently established granulosa cell lines by cotransfection of granulosa cells with SV-40 and Ha-ras oncogene. In these cells progesterone secretion can be induced by cAMP as in normal rat granulosa cells. The induction of progesterone secretion is observed only after approximately 24 h and closely follows the delayed but quantitatively dramatic induction of the mitochondrial cytochrome P450scc which catalyzes the first step in steroid hormone biosynthesis. The mitochondrial P450 system electron transport proteins, adrenodoxin and adrenodoxin reductase, are also induced but adrenodoxin shows a faster induction. Immunofluorescence studies show that the three enzymes are induced in all cells and incorporated into all mitochondria uniformly. Electron microscopic examination using immunogold technique further confirms this and reveals that adrenodoxin is predominantly located on the matrix side of the inner mitochondrial membrane. Thus, adrenodoxin, which is a small highly charged protein, shows a distribution similar to P450scc which is an integral membrane protein. The uniformity of the response of the cells provides further evidence for the homogeneity of the cell line and makes this new granulosa cell line a highly promising system for the study of the molecular mechanisms involved in changes in gene expression during the process of granulosa cell differentiation.