NADH-细胞色素 b5 还原酶在甲状腺过氧化氢生物合成中的作用

应用高香草酸荧光分析技术及NADH－高铁氰化钾还原酶法,对正常和Graves病甲状腺过氧化氢（H2O2）和NADH－细胞色素b5还原酶（b5R)进行测定,发现Graves病甲状腺b5R活性和H2O2水平均明显高于正常,而H2O2酶活性在Graves病和正常甲状腺间无显著差异。加b5R抑制剂对氯汞苯甲酸抑制b5R活性,Graves病和正常甲状腺b5R活性降低近85％,同时H2O2降低近50％,蛋白结合碘形成减少近52％。b5R活性和H2O2水平两者呈显著正相关关系。以上结果表明,b5R参与甲状腺内H2O2的生物合成,是甲状腺内产生H2O2的重要酶系。     

Heterologous expression of an endogenous rat cytochrome b(5)/cytochrome b(5) reductase fusion protein: identification of histidines 62 and 85 as the heme axial ligands

The gene coding for expression of an endogenous soluble fusion protein comprising a b-type cytochrome-containing domain and a FAD-containing domain has been cloned from rat liver mRNA. The 1461-bp hemoflavoprotein gene corresponded to a protein of 493 residues with the heme- and FAD-containing domains comprising the amino and carboxy termini of the protein, respectively. Sequence analysis indicated the heme and flavin domains were directly analogous to the corresponding domains in microsomal cytochrome b(5) (cb5) and cytochrome b(5) reductase (cb5r), respectively. The full-length fusion protein was purified to homogeneity and demonstrated to contain both heme and FAD prosthetic groups by spectroscopic analyses and MALDI-TOF mass spectrometry. The cb5/cb5r fusion protein was able to utilize both NADPH and NADH as reductants and exhibited both NADPH:ferricyanide (k(cat) = 21.7 s(-1), K(NADPH)(m) = 1 microM. K(FeCN6)(m) = 8 microM) and NADPH:cytochrome c (k(cat) = 8.3 s(-1), K(NADPH)(m) = 1 microM. K(cyt c)(m) = 7 microM) reductase activities with a preference for NADPH as the reduced pyridine nucleotide substrate. NADPH-reduction was stereospecific for transfer of the 4R-proton and involved a hydride transfer mechanism with a kinetic isotope effect of 3.1 for NADPH/NADPD. Site-directed mutagenesis was used to examine the role of two conserved histidine residues, H62 and H85, in the heme domain segment. Substitution of either residue by alanine or methionine resulted in the production of simple flavoproteins that were effectively devoid of both heme and NAD(P)H:cytochrome c reductase activity while retaining NAD(P)H:ferricyanide activity, confirming that the former activity required a functional heme domain. These results have demonstrated that the rat cb5/cb5r fusion protein is homologous to the human variant and has identified the heme and FAD as the sites of interaction with cytochrome c and ferricyanide, respectively. Mutagenesis has confirmed the identity of both axial heme ligands which are equivalent to the corresponding residues in microsomal cytochrome b(5).     

Purification of NADH-cytochromeb 5 reductase from rat liver plasma membranes

Summary An NADH-cytochromeb ₅ reductase was purified from rat liver plasma membranes. Rat liver plasma membranes were prepared by aqueous two-phase partition. Peripheral proteins were removed by EDTA extraction and integral membrane proteins were solubilized with Triton X-100. The NADH-cytochromeb ₅ reductase was purified by hydroxyapatite, anion exchange, and gel filtration chromatographies. The purified preparation was homogeneous and estimated to have an apparent molecular weight of 32 kDa on SDS-polyacrylamide gel electrophoresis. Two tryptic peptides of the purified enzyme had sequence homologies with rat, human, and bovine NADH-cytochromeb ₅ reductases.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - BCA bicinchonicic acid - EDTA ethylenediamine tetraacetate acid disodium salt - FeCN ferricyanide - HPLC high-performance liquid chromatography - NADH nicotinamide adenine dinucleotide reduced form - PMSF -phenylmethylsulfonyl fluoride     

Cytochrome b5 reductase and cytochrome b5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test

With CYP2E1 in vitro both the first and the second electron of the catalytic cycle can come from cytochrome b(5) via either NADPH-cytochrome P450 reductase or NADH-cytochrome b(5) reductase, and the presence of cytochrome b(5) stimulates CYP2E1 turnover both in vitro and in vivo. To determine whether electron input via the NADH-dependent pathway was similarly functional in whole cells and necessary for the stimulation by cytochrome b(5), we constructed five plasmids designed to express human CYP2E1 in various combinations with cytochrome b(5) reductase, cytochrome b(5), and cytochrome P450 reductase. CYP2E1 activity in Salmonella typhimurium cells transformed with each plasmid was assessed by mutagenic reversion frequency in the presence of dimethylnitrosamine. A fivefold increase in reversion frequency when cytochrome b(5) was coexpressed with P450 reductase was abolished by disruption of heme-binding in cytochrome b(5) by site-directed mutagenesis (His68Ala), suggesting that electron transfer to cytochrome b(5) was necessary for the stimulation. Addition of cytochrome b(5) reductase to the cytochrome b(5)/P450 reductase coexpression plasmid did not further increase the stimulation by cytochrome b(5), but b(5) reductase could support CYP2E1 activity in the absence of P450 reductase at a level equivalent to that obtained with just CYP2E1 and P450 reductase. Neither cytochrome b(5) reductase nor cytochrome b(5) alone could support CYP2E1 activity. These results demonstrate that the cytochrome b(5) reductase/cytochrome b(5) pathway can support CYP2E1 activity in bacterial cells.     

A mutation in Arabidopsis cytochrome b5 reductase identified by high-throughput screening differentially affects hydroxylation and desaturation

As a model for analyzing the production of novel fatty acids in oilseeds, we used the genetic and molecular techniques available for Arabidopsis to characterize modifying mutations affecting the accumulation of hydroxy fatty acids in the seeds of Arabidopsis plants that express a transgene for the castor bean fatty acid hydroxylase, FAH12. We developed a high-throughput analytical system and used it to identify three complementation classes of mutations with reduced hydroxy fatty acid accumulation from among Arabidopsis M3 seed samples derived from chemical mutagenesis. We identified one of the mutations by positional cloning as a single base pair change in a gene encoding NADH:cytochrome b5 reductase (CBR1, At5g17770). When expressed in yeast, the mutant form of the enzyme was less active and less stable than the wild-type enzyme. Characterization of homozygous mutant lines with and without the FAH12 transgene (FAH12 cbr1-1 and cbr1-1, respectively) indicated that the only detectable consequence of the cbr1-1 mutation was on desaturase and hydroxylase reactions in the developing seed. The leaf and root fatty compositions, as well as the growth, development and seed production of mutant plants were indistinguishable from wild type. Interestingly, while the cbr1-1 mutation reduced the accumulation of hydroxy fatty acids in seeds by 85%, the effects on 18:1 and 18:2 desaturation reactions were much less (<25% and <60%, respectively). These results suggest that there is competition in developing seeds among the several reactions that utilize reduced cytochrome b5.     

Reduction of cytochrome b5 by NADPH-cytochrome P450 reductase

The reduction of mammalian cytochrome b5 (b5) by NADPH-cytochrome P450 (P450) reductase is involved in a number of biological reactions. The kinetics of the process have received limited consideration previously, and a combination of pre-steady-state (stopped-flow) and steady-state approaches was used to investigate the mechanism of b5 reduction. In the absence of detergent or lipid, a reductase-b5 complex is formed and rearranges slowly to an active form. Electron transfer to b5 is rapid within this complex (>30 s(-1) at 23 degrees C), as fast as to cytochrome c. With excess b5 present, a burst of reduction is observed, consistent with rapid electron transfer to one or two b5 molecules per reductase, followed by a subsequent rate-limiting event. In detergent vesicles, the reductase and b5 interact rapidly but electron transfer is slower (approximately 3 s(-1) at 23 degrees C). Experiments with dimyristyl lecithin vesicles yielded results intermediate between the non-vesicle and detergent systems. These steady-state and pre-steady-state kinetics provide views of the different natures of the reduction of b5 by the reductase in the absence and presence of vesicles. Without vesicles, the encounter of the reductase and b5 is rapid, followed by a slow reorganization of the initial complex (approximately 0.07 s(-1)), very fast reduction, and dissociation. In vesicles, encounter is rapid and the slow step (approximately 3 s(-1)) is reduction within a complex less favorable for reduction than in the non-vesicle systems.     

Expression of outer mitochondrial membrane cytochrome b 5 in Escherichia coli. Purification of the recombinant protein and studies of its interaction with electron-transfer partners

In the present work, we report expression in Escherichia coli, purification, and characterization of recombinant full-length cytochrome b(5) from outer mitochondrial membrane. Optimization of expression conditions for cytochrome b(5) from outer mitochondrial membrane allowed reaching expression level up to 10(4) nmol of the hemeprotein per liter of culture. Recombinant cytochrome b(5) from outer mitochondrial membrane was purified from cell lysate by using metal-affinity chromatography. It has physicochemical, spectral, and immunochemical properties similar to those of cytochrome b(5) from rat liver outer mitochondrial membrane. Immobilized recombinant mitochondrial cytochrome b(5) was used as affinity ligand to study its interaction with electron transfer proteins. By using this approach, it is shown that in interaction of NADPH:cytochrome P450 reductase with both forms of cytochrome b(5) an important role is played by hydrophobic interactions between proteins, although the contribution of these interactions in complex formation with NADPH:cytochrome P450 reductase is different for isoforms of cytochrome b(5).     

Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b(5) enzymes

An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.     

Conformational States of the Water-Soluble Fragment of Cytochrome b5. I. pH-Induced Denaturation

Cytochrome b ₅ is a membrane protein that comprises two fragments: one is water-soluble and heme-containing, and the other is hydrophobic and membrane-embedded. The function of electron transfer is performed by the former whose crystal structure is known; however, its conformational states when in the membrane field and interacting with other proteins are still to be studied. Previously, we proposed water–alcohol mixtures for modeling the effect of membrane surface on proteins, and used this approach to study the conformational behavior of positively charged cytochrome c as well as relatively neutral retinol-binding protein also functioning in the field of a negatively charged membrane. The current study describes the conformational behavior of the negatively charged water-soluble fragment of cytochrome b ₅ as dependent on pH. Decreasing pH was shown to transform the fragment state from native to intermediate, similar to the molten globule reported earlier for other proteins in aqueous solutions: at pH 3.0, the fragment preserved a pronounced secondary structure and compactness but lost its rigid tertiary structure. A possible role of this intermediate state in cytochrome b ₅ functioning is discussed.     

Involvement of cytochrome b5 in the cytotoxic response to Escherichia coli Lipopolysaccharide

Cytotoxic lesions, induced by Gram-negative lipopolysaccharides (LPS), occur mainly in liver where the microsomal compartment of hepatocytes is involved in the detoxification mechanisms as well as in the biosynthesis of different active metabolites.The alterations induced by LPS from E. coli 0111: 134 on cytochrome b₅ and its correlation with cytochrome P450, have been studied using an in vivo reversible endotoxic shock model and 24 h non-replicative hepatocyte monolayers.Results show that cytochrome b₅ is directly affected by LPS that induces also a membrane damage with an active release of lactate dehydrogenase (LDH). The increase of cytochrome b₅ levels may enhance the efficiency of the electron transport, thus facilitating the cytochrome P450-associate oxidations and reactions involved in the repair mechanisms of membranes.     

The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b(5)

Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b₅. In both instances, product formation occurs. When the second electron is donated by cytochrome b₅, catalysis (product formation) is ∼10- to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼15% the coupling of NADPH consumption to product formation. Cytochrome b₅ has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b₅ on cytochrome P450 2B4 reactivity can explain how cytochrome b₅ is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b₅ to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochrome b₅ to cytochrome P450 reductase, cytochrome b₅ inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b₅ are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b₅ stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase.     

Human colon tumor cell line LS174T drug metabolizing system

The effects of culture variables on the specific content and activity of various enzymes of the drug mmetabolizing system were assessed in colon tumor cell line LS174T. The NADH reduced cytochrome b₅ (cyt b₅)⁴ spectrum of these cells was similar to rat liver cyt b₅. When released from the membrane by trypsin and concentrated, the cyt b₅ was found to cross react with rabbit antibody to rat liver cyt b₅ and human liver cyt b₅. The enzyme activities were found stable over limited cell passages with control values of 0.03 and 0.13 µol/min/mg protein for NADPH and NADH cytochrome c (cyt c) reducing activity, 0.05 nmol cyt b₅ and 0.013 nmol cytochrome P450 per milligram of microsomal protein. Phenobarbital/hydrocortisone showed a consistent, but not always significant increase in the NADPH and NADH cyt c reduction and benzanthracene an increase in the NADH cyt c reducing activity and cyt b₅ content. Griseofulvin lowered the NADH cyt c reducing activity. Delta-aminolevulinic acid (0.5 mM) caused a significant decrease in the specific activity of all enzymes, as judged by a student's t test, with a p<0.001.Abbreviations cyt b₅ cytochrome b₅ - cyt c cytochrome c - cyt P450 cytochrome P450 - PB Phenobarbital - HC Hydrocortisone - ALA -Aminolevulinic acid - GRIS Griseofulvin - PENT Pentagastrin - PASS Cell Passage - DMH Dimethylhydrazine - BA Benzanth Acene     

Expression and characterization of a functional canine variant of cytochrome b5 reductase

Cytochrome b5 reductase (cb5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 (m/z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation-reduction potentiometric titrations yielded a standard midpoint potential (Eo') for the FAD/FADH2 couple of -273+/-5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited kcat's of 767 and 600 s(-1) for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities, respectively, with Km's of 7, 8, and 12 microM for NADH, K3Fe(CN)6, and cytochrome b5, respectively. Spectral-binding constants (Ks) determined for a variety of NAD+ analogs indicated the highest and lowest affinities were observed for APAD+ (Ks=71 microM) and PCA+ (Ks=>31 mM), respectively, and indicated the binding contributions of the various portions of the pyridine nucleotide. These results provide the first correct sequence for the full-length, membrane-associated form of C. familiaris cb5r and provide a direct comparison of the enzymes from two phylogenetic sources using identical expression systems that indicate that both enzymes have comparable spectroscopic, kinetic, thermodynamic, and structural properties.     

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.     

Up-regulation of antioxidant enzymes and coenzyme Q10 in a human oral cancer cell line with acquired bleomycin resistance

Abstract

Bleomycin (BLM) is an anti-cancer drug that can induce formation of reactive oxygen species (ROS). To investigate the association between up-regulation of antioxidant enzymes and coenzyme Q₁₀ (CoQ₁₀) in acquired BLM resistance, one BLM-resistant clone, SBLM24 clone, was selected from a human oral cancer cell line, SCC61 clone. The BLM resistance of SBLM24 clone relative to a sub-clone of SCC61b cells was confirmed by analysis of clonogenic ability and cell cycle arrest. CoQ₁₀ levels and levels of Mn superoxide dismutase, glutathione peroxidase 1, catalase and thioredoxin reductase 1 were augmented in SBLM24 clone although there was also a mild increase in the expression of BLM hydrolase. Suppression of CoQ₁₀ levels by 4-aminobenzoate sensitized BLM-induced cytotoxicity. The results of suppression on enhanced ROS production by BLM and the cross-resistance to hydrogen peroxide in SBLM24 clone further demonstrated the development of adaptation to oxidative stress during the formation of acquired BLM resistance.     

Site-Directed Mutagenesis of Cytochrome b5 for Studies of Its Interaction with Cytochrome P450

We have shown earlier that microsomal cytochrome b ₅ can form a specific complex with mitochondrial cytochrome P450 (cytochrome P450scc). The formation of the complex between these two heme proteins was proved spectrophotometrically, by affinity chromatography on immobilized cytochrome b ₅, and by measuring the cholesterol side-chain cleavage activity of cytochrome P450scc in a reconstituted system in the presence of cytochrome b ₅. To further study the mechanism of interaction of these heme proteins and evaluate the role of negatively charged amino acid residues Glu42, Glu48, and Asp65 of cytochrome b ₅, which are located at the site responsible for interaction with electron transfer partners, we used sitedirected mutagenesis to replace residues Glu42 and Glu48 with lysine and residue Asp65 with alanine. The resulting mutant forms of cytochrome b ₅ were expressed in E. coli, and full-length and truncated forms (shortened from the C-terminal sequence due to cleavage of 40 amino acid residues) of these cytochrome b ₅ mutants were purified. Addition of the truncated forms of cytochrome b ₅ (which do not contain the hydrophobic C-terminal sequence responsible for interaction with the membrane) to the reconstituted system containing cytochrome P450scc caused practically no stimulation of catalytic activity, indicating an important role of the hydrophobic fragment of cytochrome b ₅ in its interaction with cytochrome P450scc. However, full-length cytochrome b ₅ and the full-length Glu48Lys and Asp65Ala mutant forms of cytochrome b ₅ stimulated the cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc by 100%, suggesting that residues Glu48 and Asp65 of cytochrome b ₅ are not directly involved in its interaction with cytochrome P450scc. The replacement of Glu42 for lysine, however, made the Glu42Lys mutant form of cytochrome b ₅ about 40% less effective in stimulation of the cholesterol side-chain cleavage activity of cytochrome P450scc, indicating that residue Glu42 of cytochrome b ₅ is involved in electrostatic interactions with cytochrome P450scc. Residues Glu42 and Glu48 of cytochrome b ₅ appear to participate in electrostatic interaction with microsomal type cytochrome P450.     

Down-regulation of aldose reductase renders J774A.1 cells more susceptible to acrolein- or hydrogen peroxide-induced cell death

Aldose reductase (AR) is abundantly expressed in a variety of cell lineages and has been implicated in the cellular response against oxidative stress. However, the exact functional role of AR against oxidative stress remains relatively unclear. This study investigated the role of AR in acrolein- or hydrogen peroxide-induced apoptosis using the J774.A.1 macrophage cell line. Ablation of AR with a small interference RNA or inhibition of AR activity significantly enhanced the acrolein- or hydrogen peroxide-induced generation of reactive oxygen species and aldehydes, leading to increased apoptotic cell death. Blockade of AR activity in J774A.1 cells markedly augmented the acrolein- or hydrogen peroxide-induced translocation of Bax to mitochondria along with reduced Bcl-2 and increased release of cytochrome c from the mitochodria. Taken together, these findings indicate that AR plays an important role in the cellular response against oxidative stress, by sequestering the reactive molecules generated in cells exposed to toxic substances.     

The reaction of neuroglobin with potential redox protein partners cytochrome b5 and cytochrome c

Previously identified, potentially neuroprotective reactions of neuroglobin require the existence of yet unknown redox partners. We show here that the reduction of ferric neuroglobin by cytochrome b(5) is relatively slow (k=6 x 10(2)M(-1)s(-1) at pH 7.0) and thus is unlikely to be of physiological significance. In contrast, the reaction between ferrous neuroglobin and ferric cytochrome c is very rapid (k=2 x 10(7)M(-1)s(-1)) with an apparent overall equilibrium constant of 1 microM. Based on this data we propose that ferrous neuroglobin may well play a role in preventing apoptosis.     

Anaerobic expression of nitric oxide reductase from denitrifying Pseudomonas stutzeri

NO reductase synthesis was investigated immunochemically and by activity assays in cells of Pseudomonas stutzeri ZoBell grown in continuous culture at discrete aeration levels, or in O₂-limited batch cultures supplemented with N oxides as respiratory substrate. Under aerobic conditions, NO reductase was not expressed in P. stutzeri. Oxygen limitation in combination with the presence of nitrate or nitrite derepressed NO reductase synthesis. On transition from aerobic to anaerobic conditions in continuous culture, NO reductase was synthesized below 3% air saturation and reached maximum expression under anaerobic conditions. By use of mutant strains defective in nitrate respiration or nitrite respiration, the inducing effect of individual N oxides on NO reductase synthesis could be discriminated. Nitrite caused definite, concentration-dependent induction, while nitrate promoted moderate enzyme synthesis or amplified effects of nitrite. Exogenous nitric oxide (NO) in concentrations 25 M induced trace amounts of NO reductase; in higher concentrations it arrested cell growth. Nitrite reductase or NO reductase were not detected immunochemically under these conditions. NO generated as an intermediate appeared not to induce NO reductase significantly. Antiserum raised against the P. stutzeri NO reductase showed crossreaction with cell extracts from P. stutzeri JM300, but not with several other denitrifying pseudomonads or Paracoccus denitrificans.     

Structure Guided Chemical Modifications of Propylthiouracil Reveal Novel Small Molecule Inhibitors of Cytochrome b5 Reductase 3 That Increase Nitric Oxide Bioavailability

NADH cytochrome b₅ reductase 3 (CYB5R3) is critical for reductive reactions such as fatty acid elongation, cholesterol biosynthesis, drug metabolism, and methemoglobin reduction. Although the physiological and metabolic importance of CYB5R3 has been established in hepatocytes and erythrocytes, emerging investigations suggest that CYB5R3 is critical for nitric oxide signaling and vascular function. However, advancement toward fully understanding CYB5R3 function has been limited due to a lack of potent small molecule inhibitors. Because of this restriction, we modeled the binding mode of propylthiouracil, a weak inhibitor of CYB5R3 (IC₅₀ = ∼275 μm), and used it as a guide to predict thiouracil-biased inhibitors from the set of commercially available compounds in the ZINC database. Using this approach, we validated two new potent derivatives of propylthiouracil, ZINC05626394 (IC₅₀ = 10.81 μm) and ZINC39395747 (IC₅₀ = 9.14 μm), both of which inhibit CYB5R3 activity in cultured cells. Moreover, we found that ZINC39395747 significantly increased NO bioavailability in renal vascular cells, augmented renal blood flow, and decreased systemic blood pressure in response to vasoconstrictors in spontaneously hypertensive rats. These compounds will serve as a new tool to examine the biological functions of CYB5R3 in physiology and disease and also as a platform for new drug development.