Heme-binding protein CYB5D1 is a radial spoke component required for coordinated ciliary beating.

Coordinated beating is crucial for the function of multiple cilia. However, the molecular mechanism is poorly understood. Here, we characterize a conserved ciliary protein CYB5D1 with a heme-binding domain and a cordon-bleu ubiquitin-like domain. Mutation or knockdown of Cyb5d1 in zebrafish impaired coordinated ciliary beating in the otic vesicle and olfactory epithelium. Similarly, the two flagella of an insertional mutant of the CYB5D1 ortholog in Chlamydomonas (Crcyb5d1) showed an uncoordinated pattern due to a defect in the cis-flagellum. Biochemical analyses revealed that CrCYB5D1 is a radial spoke stalk protein that binds heme only under oxidizing conditions. Lack of CrCYB5D1 resulted in a reductive shift in flagellar redox state and slowing down of the phototactic response. Treatment of Crcyb5d1 with oxidants restored coordinated flagellar beating. Taken together, these data suggest that CrCYB5D1 may integrate environmental and intraciliary signals and regulate the redox state of cilia, which is crucial for the coordinated beating of multiple cilia.

Cytochrome b5 enhances androgen synthesis by rapidly reducing the CYP17A1 oxy-complex in the lyase step.

Cytochrome P450 17A1 (CYP17A1) catalyzes the synthesis of androgens from the steroid precursors pregnenolone and progesterone in a two-step reaction process: allylic hydroxylation and carbo-carbon bond scission. Cytochrome b5 (Cyt-b5 ) is a stimulator of the second lyase reaction, but the chemical mechanism is unclear. We have shown previously that this stimulatory effect requires redox active Cyt-b5 . To investigate the origin of the lyase reaction enhancement by electron transfer from Cyt-b5 , we measured the reduction rates of oxy-ferrous substrate-bound CYP17A1 by Cyt-b5 and by cytochrome P450 reductase (CPR) coincorporated in Nanodiscs using stopped flow spectroscopy. We observed that Cyt-b5 reduces oxy-ferrous CYP17A1 10-fold faster than CPR, with the rate similar to that observed in a ternary complex of all three proteins.

Inactivation of cytochrome P450 (P450) 3A4 but not P450 3A5 by OSI-930, a thiophene-containing anticancer drug.

An investigational anticancer agent that contains a thiophene moiety, 3-[(quinolin-4-ylmethyl)-amino]-N-[4-trifluoromethox)phenyl] thiophene-2-carboxamide (OSI-930), was tested to investigate its ability to modulate the activities of several cytochrome P450 enzymes. Results showed that OSI-930 inactivated purified, recombinant cytochrome P450 (P450) 3A4 in the reconstituted system in a mechanism-based manner. The inactivation was dependent on cytochrome b(5) and required NADPH. Catalase did not protect against the inactivation. No inactivation was observed in studies with human 2B6, 2D6, or 3A5 either in the presence or in the absence of b(5). The inactivation of 3A4 by OSI-930 was time- and concentration-dependent. The inactivation of the 7-benzyloxy-4-(trifluoromethyl)coumarin catalytic activity of 3A4 was characterized by a K(I) of 24 µM and a k(inact) of 0.04 min(-1). This K(I) is significantly greater than the clinical OSI-930 C(max) of 1.7 µM at the maximum tolerated dose, indicating that clinical drug interactions of OSI-930 via this pathway are not likely. Spectral analysis of the inactivated protein indicated that the decrease in the reduced CO spectrum at 450 nm was comparable to the amount of inactivation, thereby suggesting that the inactivation was primarily due to modification of the heme. High-pressure liquid chromatography (HPLC) analysis with detection at 400 nm showed a loss of heme comparable to the activity loss, but a modified heme was not detected. This result suggests either that the heme must have been modified enough so as not to be observed in a HPLC chromatograph or, possibly, that it was destroyed. The partition ratio for the inactivation of P450 3A4 was approximately 23, suggesting that this P450 3A4-mediated pathway occurs with approximately 4% frequency during the metabolism of OSI-930. Modeling studies on the binding of OSI-930 to the active site of the P450 3A4 indicated that OSI-930 would be oriented properly in the active site for oxidation of the thiophene sulfur to give the sulfoxide, which has previously been shown to be a significant metabolite of OSI-930. Because OSI-930 is an inactivator of P450 3A4 but does not exhibit any effect on P450 3A5 activity under the same conditions, it may be an appropriate probe for exploring unique aspects of these two very similar P450s.

The developmental increase in adrenocortical 17,20-lyase activity (biochemical adrenarche) is driven primarily by increasing cytochrome b5 in neonatal rhesus macaques.

Adrenarche is thought to be experienced only by humans and some Old World primates despite observed regression of an adrenal fetal zone and establishment of a functional zona reticularis (ZR) in other species like rhesus macaques. Adrenal differentiation remains poorly defined biochemically in nonhuman primates. The present studies defined ZR development in the neonatal rhesus by examining androgen synthetic capacity and factors affecting it in rhesus and marmoset adrenals. Western immunoblots examined expression of 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17), cytochrome b5 (b5), and 3beta-hydroxysteroid dehydrogenase (3betaHSD), among other key enzymes. 17,20-lyase activity was quantified in adrenal microsomes, as was the contribution of b5 to 17,20-lyase activity in microsomes and cell transfection experiments with rhesus and marmoset P450c17. Expression of b5 increased from birth to 3 months, and was positively correlated with age and 17,20-lyase activity in the rhesus. Recombinant b5 addition stimulated 17,20-lyase activity to an extent inversely proportional to endogenous levels in adrenal microsomes. Although 3betaHSD expression also increased with age, P450c17, 21-hydroxylase cytochrome P450, and the redox partner, reduced nicotinamide adenine dinucleotide phosphate-cytochrome P450 oxidoreductase, did not; nor did recombinant cytochrome P450 oxidoreductase augment 17,20-lyase activity. Cotransfection with b5 induced a dose-dependent increase in dehydroepiandrosterone synthesis by both nonhuman primate P450c17 enzymes. We conclude that the increase in 17,20-lyase activity characteristic of an adrenarche in rhesus macaques is driven primarily by increased b5 expression, without the need for a decrease in 3betaHSD, as suggested from human studies. The rhesus macaque is a relevant and accessible model for human ZR development and adrenal function.

Steroidogenic enzymes.

The enzymes and pathways of steroidogenesis are familiar to most endocrinologists, but the biochemistry and molecular biology of these processes are still being studied. This chapter outlines current knowledge about each enzyme. The quantitative regulation of steroidogenesis occurs at the first step, the conversion of cholesterol to pregnenolone. Chronic regulation is principally at the level of transcription of the gene for P450 side chain cleave (P450scc), which is the enzymatically rate-limiting step. Acute regulation is mediated by steroidogenic acute regulatory protein, which facilitates the rapid influx of cholesterol into mitochondria, where P450scc resides. Qualitative regulation, determining the class of steroid produced, is principally determined by P450c17. In the absence of P450c17 in the zona glomerulosa, C21 deoxy steroids are produced, leading to the mineralocorticoid aldosterone. In the presence of the 17alpha-hydroxylase but not the 17,20 lyase activity of P450c17 in the zona fasciculata, C21, 17-hydroxy steroids are produced, leading to the glucocorticoid cortisol. When both the 17alpha-hydroxylase and 17,20 lyase activities of P450c17 are present in the zona reticularis, the androgen precursor dehydroepiandrosterone is produced. The discrimination between 17alpha-hydroxylase and 17,20 lyase activities is regulated by two posttranslational events, the serine phosphorylation of P450c17 and the allosteric action of cytochrome b5, both of which act to optimize the interaction of P450c17 with its obligatory electron donor, P450 oxidoreductase.

Ascaris suum cytochrome b5, an adult-specific secretory protein reducing oxygen-avid ferric hemoglobin.

The anaerobic parasitic nematode Ascaris suum has an oxygen-avid hemoglobin in the perienteric fluid, the biological function of which remains elusive. Here, we report that Ascaris cytochrome b5 is expressed specifically in the intestinal parasitic stage and is secreted into the perienteric fluid, thus co-localizing with Ascaris hemoglobin. We also found that cytochrome b5 reduces Ascaris non-functioning ferric methemoglobin more efficiently than mammalian methemoglobin. Furthermore, a computer graphics model of the electron transfer complex between Ascaris cytochrome b5 and Ascaris hemoglobin strongly suggested that these two proteins are physiological redox partners. Nitric oxide has been reported to react easily with oxygen captured in hemoglobin to form nitrate, but not toxic free radicals, which may result in production of methemoglobin for the cytochrome b5 to regenerate functional ferrous hemoglobin. Therefore, our findings suggest that Ascaris cytochrome b5 is a key redox partner of Ascaris hemoglobin, which acts as an antioxidant.

Structural and thermodynamic encoding in the sequence of rat microsomal cytochrome b(5).

The water-soluble domain of rat microsomal cytochrome b(5) is a convenient protein with which to inspect the connection between amino acid sequence and thermodynamic properties. In the absence of its single heme cofactor, cytochrome b(5) contains a partially folded stretch of 30 residues. This region is recognized as prone to disorder by programs that analyze primary structures for such intrinsic features. The cytochrome was subjected to amino acid replacements in the folded core (I12A), in the portion that refolds only when in contact with the heme group (N57P), and in both (F35H/H39A/L46Y). Despite the difficulties associated with measuring thermodynamic quantities for the heme-bound species, it was possible to rationalize the energetic consequences of both types of replacements and test a simple equation relating apoprotein and holoprotein stability. In addition, a phenomenological relationship between the change in T(m) (the temperature at the midpoint of the thermal transition) and the change in thermodynamic stability determined by chemical denaturation was observed that could be used to extend the interpretation of incomplete holoprotein stability data. Structural information was obtained by nuclear magnetic resonance spectroscopy toward an atomic-level analysis of the effects.

Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4.

The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b(5) (cyt b(5)) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b(5) molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 +/- 2 and 29 +/- 4.5 s(-1), respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of benzphetamine congruent with100-fold (k = 0.15 +/- 0.05 s(-1)) and cyclohexane congruent with10-fold (k = 2.5 +/- 0.35 s(-1)) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b(5), and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b(5) and CPR. Increasing the concentration of cyt b(5) enhanced the amount of product formed by cyt b(5) while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b(5):cyt P450 molar ratios examined, cyt b(5) inhibits the rate of NADPH consumption. Nevertheless, at low cyt b(5):cyt P450 molar ratios

The involvement of lipid radical cycles and the adenine nucleotide translocator in neurodegenerative diseases.

The major cause of neurodegenerative disorders, including mid- to late-life onset Alzheimer's Disease, is permanent oxidative stress in the brain. Polyunsaturated fatty acids (PUFA) and alpha-tocopherol (alpha-TOH) are the most oxygen-sensitive constituents of cells. The presence of alpha-TOH in biological membranes is required but not sufficient to protect them against lipid peroxidation. The data presented in this review consider the role of alpha-TOH and cytochrome b5 which permit operation of lipid-radical cycles and the participation of lipid-radical reactions in key processes occurring in the membrane. Analysis of role of these cycles in membrane bioenergetics led us to a model involving the adenine nucleotide translocator and ATP synthesis in brain mitochondria. This paper summarizes experimental evidence for oxidative and non-oxidative pathways of PUFA metabolism with respective intermediates, which could be relevant to elucidation of new mechanisms of neurodegenerative diseases. Lipid-radical reactions in membranes work as important component of normal cell metabolism. Discussion is focused on the consequences of ineffective electron transfer to peroxyl radicals (LOO.--> LOO-) and excessive oxidative pathway of PUFA metabolism (LOO.-->LOOH) with two reactive secondary products: malondialdehyde and methylglyoxal. Our future aim is to develop a more detailed model supplemented by the formation of lipofuscin and amyloid structures.

Cytochrome P450-dependent monooxygenase system mediated hydrocarbon metabolism and antioxidant enzyme responses in prawn, Macrobrachium malcolmsonii.

We investigated the alteration of cytochrome P450-dependent monooxygenase enzymes and antioxidant enzymes in response to oil effluent in freshwater prawn, Macrobrachium malcolmsonii. The prawns were exposed to two sublethal (10% [0.91 ppt] and 25% [2.3 ppt] of 5-day median lethal concentration) concentrations of oil. After 30 days, treated prawns were transferred into untreated freshwater and depuration was followed for another 30 days. At 7-day intervals, hydrocarbons and detoxifying enzymes were analysed in the hepatopancreas. Accumulation of hydrocarbon in the tissues gradually increased when exposed to sublethal concentration of oil effluent associated with enhanced levels of cytochrome P450, NADPH cytochrome c reductase and cytochrome b(5). During depuration, the levels of accumulated hydrocarbons decreased due to the induction of these detoxifying enzymes. Oil derived hydrocarbon mediated oxyradical production would have occurred in M. malcolmsonii. This was confirmed by elevated levels of superoxide dismutase (SOD) and catalase (CAT). Thus, cytochrome P450-dependent monooxygenase enzymes and antioxidant enzymes in oil-exposed prawns demonstrate a well-established detoxifying mechanism in M. malcolmsonii.

Hepatic cytochrome P450 reductase-null mice reveal a second microsomal reductase for squalene monooxygenase.

Squalene monooxygenase is a microsomal enzyme that catalyzes the conversion of squalene to 2,3(s)-oxidosqualene, the immediate precursor to lanosterol in the cholesterol biosynthesis pathway. Unlike other flavoprotein monooxygenases that obtain electrons directly from NAD(P)H, squalene monooxygenase requires a redox partner, and for many years it has been assumed that NADPH-cytochrome P450 reductase is this requisite redox partner. However, our studies with hepatic cytochrome P450-reductase-null mice have revealed a second microsomal reductase for squalene monooxygenase. Inhibition studies with antibody to P450 reductase indicate that this second reductase supports up to 40% of the monooxygenase activity that is obtained with microsomes from normal mice. Studies carried out with hepatocytes from CPR-null mice demonstrate that this second reductase is active in whole cells and leads to the accumulation of 24-dihydrolanosterol; this lanosterol metabolite also accumulates in the livers of CPR-null mice, indicating that cholesterol synthesis is blocked at lanosterol demethylase, a cytochrome P450.

The inactivation of cytochrome P450 3A5 by 17alpha-ethynylestradiol is cytochrome b5-dependent: metabolic activation of the ethynyl moiety leads to the formation of glutathione conjugates, a heme adduct, and covalent binding to the apoprotein.

17Alpha-ethynylestradiol (EE) inactivates cytochrome P450 3A5 (3A5) in the reconstituted system in a mechanism-based manner. The inactivation is dependent on NADPH, and it is irreversible. The inactivation of 3A5 by EE is also dependent on cytochrome b5 (b5). The values for the K(I) and k(inact) of the 7-benzyloxy-4-(trifluoromethyl)coumarin O-debenzylation activity of 3A5 are 26 microM and 0.06 min(-1), respectively. Incubation of 3A5 with EE resulted in a 62% loss of catalytic activity, 60% loss in the reduced CO difference spectrum, and 40% decrease in native heme with the formation of a heme adduct. The partition ratio was approximately 25, and the stoichiometry of binding was approximately 0.3 mol of EE metabolite bound/mol of P450 inactivated. Four major metabolites were formed during the metabolism of EE by 3A5. SDS-polyacrylamide gel electrophoresis analysis demonstrated that [3H]EE was irreversibly bound to 3A5 apoprotein. Liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) revealed that two glutathione (GSH) conjugates with m/z values of 620 were formed only in the presence of b5. These two conjugates are formed from the reaction of GSH with the ethynyl group with the oxygen being inserted into either the internal or terminal carbon. A heme adduct with the ion at m/z 927 and two dipyrrole adducts with ions at m/z 579 were detected by LC-MS/MS analysis. In conclusion, 3A5 can activate EE to a 17alpha-oxirene-related reactive species that can then partition the oxygen between the internal and terminal carbons of the ethynyl group to form heme and apoprotein adducts, resulting in the inactivation of P450 3A5.

The stimulatory role of human cytochrome b5 in the bioactivation activities of human CYP1A2, 2A6 and 2E1: a new cell expression system to study cytochrome P450-mediated biotransformation (a corrigendum report on Duarte et al. (2005) Mutagenesis 20, 93-100).

This corrigendum report describes the study of the comparison of human cytochrome b(5) (b(5)) with rat b(5) when coupled with human cytochrome P450 CYP1A2, 2A6 or 2E1. Results indicate a role of the N-terminal part of b(5) in the coupling with CYP. Indeed, the plasmid pLCM-b(5)-RED used in our former study on b(5) [Duarte et al. (2005) Mutagenesis, 20(2), 193-100] erroneously contained rat b(5). Plasmid pLCM-b(5)-RED was corrected with human b(5) and subsequently all experimental work was repeated as was described for the rat b(5) plasmid. Although absolute values of contents and activities were lower, all key-findings as found for rat b(5) could be confirmed using human b(5). The physiological relevant co-expression of the members of the cytochrome P450 complex, CYP, NADPH-cytochrome P450 oxidoreductase (RED) and human b(5) could be demonstrated in the different BTC strains, as was found before. The stimulatory effect of human b(5) on the activity of CYP1A2, CYP2A6 and CYP2E1 was in general similar, when compared with rat b(5), though less quantitatively pronounced. This was both the case when using membrane preparations as well as by the bioactivation of procarcinogens using the bacterial mutagenicity assay. Human b(5) stimulated the bioactivation of all compounds as described for rat b(5), except for CYP1A2 mediated bioactivation of 2-aminoanthracene (2AA), which was not stimulated by human b(5). All other main findings of the effect of rat b(5) were confirmed with human b(5), i.e. for CYP2A6: N-nitrosodiethylamine (NNdEA): approximately 14-fold increase ( approximately 23-fold with rat b(5)) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK): approximately 3-fold ( approximately 9-fold with rat b(5)); for CYP2E1: NNdEA: approximately 1.5-fold increase ( approximately 3-fold with rat b(5)); NNK: no mutagenicity with or without human b(5). Both CYP2A6 and CYP2E1 demonstrated total dependence on the presence of human b(5) for N-nitrosodi-n-propylamine (NNdPA) mutagenicity, as was shown before with rat b(5).

Modulation of CYP1A1-mediated oxidation of carcinogenic azo dye Sudan I and its binding to DNA by cytochrome b5.

OBJECTIVES: Modulation of the cytochrome P450 (CYP) 1A1-mediated oxidative activation and detoxication of carcinogenic Sudan I by the heme-protein cytochrome b(5) (b(5)) was investigated. Another aim of the study was to examine the formation of Sudan I-DNA adducts in vivo. METHODS: High performance liquid chromatography (HPLC) with ultraviolet (UV) detection was employed for the separation of Sudan I metabolites formed by human recombinant CYPs and rat CYP1A1. The (32)P-postlabeling technique was utilized to determine Sudan I-DNA adducts. RESULTS: The capabilities of the most efficient CYP enzymes oxidizing Sudan I, human and rat recombinant CYP1A1, as well as of human recombinant CYP1A2, 2A6 and 3A4 were significantly increased by b(5), while reactions catalyzed by human CYP1B1, 2C8, 2C9 and 2E1 were insensitive to this heme protein. Sudan I oxidation catalyzed by CYP2B6, 2C19 and 2D6 was even decreased by b(5). The stimulation of the CYP1A1-mediated Sudan I oxidation was dependent on concentration of b(5). Likewise, the increase in CYP1A1-mediated formation of Sudan I-DNA adducts by b(5) was concentration dependent. Other proteins containing heme such as cytochrome c or myoglobin were without this effect. The major Sudan I-DNA adducts formed in vitro are also generated in vivo, in livers of rats treated with Sudan I. CONCLUSIONS: The data are the first report on the stimulation of CYP1A1-mediated oxidative reactions by b(5). In addition, the results demonstrating covalent binding of Sudan I to rat liver DNA in vivo indicate a genotoxic mechanism of Sudan I carcinogenicity in rats.

Revealing the complementation of ferredoxin by cytochrome b (5) in the Spirulina- (6)-desaturation reaction by N-terminal fusion and co-expression of the fungal-cytochrome b (5) domain and Spirulina- (6)-acyl-lipid desaturase.

Spirulina-acyl-lipid desaturases are integral membrane proteins found in thylakoid and plasma membranes. These enzymes catalyze the fatty acid desaturation process of Spirulina to yield gamma-linolenic acid (GLA) as the final desaturation product. It has been reported that the cyanobacterial desaturases use ferredoxin as an electron donor, whereas the acyl-lipid desaturase in plant cytoplasm and the acyl-CoA desaturase of animals and fungi use cytochrome b (5). The low level of ferredoxin present in Escherichia coli cells leads to an inability to synthesize GLA when the cells are transformed with the Spirulina-(6) desaturase, desD, and grown in the presence of the reaction substrate, linoleic acid. In this study, Spirulina-(6) desaturase, encoded by the desD gene, was N-terminally fused and co-expressed with the cytochrome b (5) domain from Mucor rouxii. The product, GLA, made heterologously in E. coli and Saccharomyces cerevisiae, was then detected and analyzed. The results revealed the production of GLA by Spirulina-(6) desaturase fused or co-expressed with cytochrome b (5) in E. coli cells, in which GLA production by this gene cannot occur in the absence of cytochrome b (5). Moreover, the GLA production ability in the E. coli host cells was lost after the single substitution mutation was introduced to H52 in the HPGG motif of the cytochrome b (5) domain. These results revealed the complementation of the ferredoxin requirement by the fusion or co-expression of the fungal-cytochrome b (5) domain in the desaturation process of Spirulina-(6) desaturase. Furthermore, the free form of cytochrome b (5) domain can also enhance GLA production by the Spirulina-desD gene in yeast cells.

Role of cytochrome b5 in catalysis by cytochrome P450 2B4.

Cytochrome b5 has been shown to stimulate, inhibit or have no effect on catalysis by P450 cytochromes. Its action is known to depend on the isozyme of cytochrome P450, the substrate, and experimental conditions. Cytochrome P450 2B4 (CYP 2B4) has been used in our laboratory as a model isozyme to study the role of cytochrome b5 in cytochrome P450 catalysis using two substrates, methoxyflurane and benzphetamine. One substrate is the volatile anesthetic, methoxyflurane, whose metabolism is consistently markedly stimulated by cytochrome b5. The other is benzphetamine, whose metabolism is minimally modified by cytochrome b5. Determination of the stoichiometry of the metabolism of both substrates showed that the amount of product formed is the net result of the simultaneous stimulatory and inhibitory actions of cytochrome b5 on catalysis. Site-directed mutagenesis studies revealed that both cytochrome b5 and cytochrome P450 reductase interact with cytochrome P450 on its proximal surface on overlapping but non-identical binding sites. Comparison of the rate of reduction of oxyferrous CYP 2B4 and the rate of substrate oxidation by cyt b5 and reductase with stopped-flow spectrophotometric and rapid chemical quench experiments has demonstrated that although cytochrome b5 and reductase reduce oxyferrous CYP 2B4 at the same rate, substrate oxidation proceeds more slowly in the presence of the reductase.

Unusual dehydroxylation of antimicrobial amidoxime prodrugs by cytochrome b5 and NADH cytochrome b5 reductase.

Furamidine is an effective antimicrobial agent; however, oral potency of furamidine is poor. A prodrug of furamidine, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), has greatly improved oral potency. DB289 is transformed to furamidine via O-demethylation, and N-dehydroxylation reactions with four intermediate metabolites formed. The O-demethylation reactions have been shown to be catalyzed by cytochrome P450. The enzymes catalyzing the reductive N-dehydroxylation reactions have not been determined. The objective of this study was to identify the enzymes that catalyze N-dehydroxylation of metabolites M1, a monoamidoxime, and M2, a diamidoxime, formed during generation of furamidine. M1 and M2 metabolism was investigated using human liver microsomes and human soluble cytochrome b5 and NAD cytochrome b5 reductase, expressed in Escherichia coli. Kinetics of M1 and M2 reduction by human liver microsomes exhibited high affinity and moderate capacity. Metabolism was significantly inhibited by antibodies to cytochrome b5 and b5 reductase and by chemical inhibitors of b5 reductase. The amidoximes were efficiently metabolized by liver mitochondria, which contain cytochrome b5/b5 reductase, but not by liver cytosol, which contains minimal amounts of these proteins. Expressed cytochrome b5/b5 reductase, in the absence of any other proteins, efficiently catalyzed reduction of both amidoximes. K(m) values were similar to those for microsomes, and V(max) values were 33- to 36-fold higher in the recombinant system compared with microsomes. Minimal activity was seen with cytochrome b5 or b5 reductase alone or with cytochrome P450 reductase alone or with cytochrome b5. These results indicate that cytochrome b5 and b5 reductase play a direct role in metabolic activation of DB289 to furamidine.

Reduction of sulfamethoxazole and dapsone hydroxylamines by a microsomal enzyme system purified from pig liver and pig and human liver microsomes.

Biotransformation involving nitrogen are of pharmacological and toxicological relevance. In principle, nitrogen containing functional groups can undergo all the known biotransformation processes such as oxidation, reduction, hydrolysis and formation of conjugates. For the N-reduction of benzamidoxime an oxygen-insensitive liver microsomal enzyme system that required cytochrome b5, NADH-cytochrome b5 reductase and a cytochrome P450 isoenzyme of the subfamily 2D has been described. In previous studies it was demonstrated that N-hydroxylated derivates of strongly basic functional groups are easily reduced by this enzyme system. The N-hydroxylation of sulfonamides such sulfamethoxazole (SMX) and dapsone (DDS) to sulfamethoxazole-hydroxylamine (SMX-HA) and dapsone-hydroxylamine (DDS-N-OH), respectively is the first step in the formation of reactive metabolites. Therefore it seemed reasonable to study the potential of cytochrome b5, NADH-cytochrome b5 reductase and CYP2D to detoxify these N-hydroxylated metabolites by N-reduction. Metabolites were analysed by HPLC analysis. SMX-HA and DDS-N-OH are reduced by cytochrome b5, NADH-cytochrome b5 reductase and CYP2D but also only by cytochrome b5 and NADH-cytochrome b5 reductase without addition of CYP2D. The reduction rate for SMX-HA by cytochrome b5, NADH-cytochrome b5 reductase and CYP2D was 0,65 +/- 0,1 nmol SMX/min/mg protein. The reduction rate by b5 and b5 reductase was 0,37 +/- 0,15 nmol SMX/min/mg protein. For DDS-N-OH the reduction rate by cytochrome b5, NADH-cytochrome b5 reductase and CYP2D was 1.79 +/- 0.85 nmol DDS/min/mg protein and by cytochrome b5 and NADH-cytochrome b5 reductase 1.25 +/- 0.15 nmol DDS/min/mg protein. Cytochrome b5, NADH-cytochrome b5 reductase are therefore involved in the detoxification of these reactive hydroxylamines and CYP2D increased the N-reduction.

Mammalian mitochondrial and microsomal cytochromes b(5) exhibit divergent structural and biophysical characteristics.

The only outer mitochondrial membrane cytochrome b(5) examined to date, from rat (rOM b(5)), exhibits greater stability than known mammalian microsomal (Mc) isoforms, as well as a much higher kinetic barrier for hemin dissociation and a more negative reduction potential. A BlastP search of available databases using the protein sequence of rOM b(5) as template revealed entries for analogous proteins from human (hOM b(5)) and mouse (mOM b(5)). We prepared a synthetic gene coding for the heme-binding domain of hOM b(5), and expressed the protein to high levels. The hOM protein exhibits stability, hemin-binding, and redox properties similar to those of rOM b(5), suggesting that they are characteristic of the OM b(5) subfamily. The divergence in properties between the OM and Mc b(5) isoforms in mammals can be attributed, at least in part, to the presence of two extended hydrophobic patches in the former. The biophysical properties characteristic of the OM proteins may be important in facilitating the two functions proposed for them so far, reduction of ascorbate radical and stimulation of androgen synthesis.

Possible participation of outer mitochondrial membrane cytonchrome B5 in steroidogenesis in zona glomerulosa of rat adrenal cortex.

Outer mitochondrial membrane cytochrome b5 (OMb) originally found in rat liver is an isoform of cytochrome b5 (b5) of the endoplasmic reticulum. In contrast to accumulated data on the physiological roles of b5, functions of OMb have not been well characterized except for its involvement in regeneration of ascorbic acid [i.e., in a semidehydroascorbate reductase (SDAR) system]. By using highly specific antibodies against rat OMb, we found immunohistochemically that OMb in the rat adrenal gland was most abundant in the zona glomerulosa (zG) among the three cortical zones, and the expression level was enhanced on angiotensin II-stimulation. SDAR activity was found in zG and inhibited by anti-OMb antibody. Moreover, the increase in plasma aldosterone concentration under Na+ -deficiency was suppressed by limited ascorbic acid (Asc) availability in rat mutants unable to synthesize Asc, while plasma corticosterone concentration was not affected. These data suggest that OMb, present abundantly in zG, participates in aldosterone formation in zG of rat under angiotensin II-stimulation through regeneration of Asc.