[9] Detergent-solubilized NADH-cytochrome b5 reductase

doi:10.1016/S0076-6879(78)52011-9

Methods in Enzymology

Volume 52, 1978, Pages 102-108

https://doi.org/10.1016/S0076-6879(78)52011-9 Get rights and content

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This chapter talks about solubilized NADH-cytochrome b₅ reductase protein. NADH-cytochrome b₅ reductase is a flavoprotein that is tightly bound to the membranes of endoplasmic reticulum (microsomes) and outer mitochondrial membrane of liver and many other tissues. It catalyzes the NADH-dependent reduction of cytochrome b₅ and several artificial electron acceptors, such as ferricyanide and 2, 6-dichlorophenolindophenol. Despite the fact that the reductase purified after lysosomal solubilization retains the flavin prosthetic group as well as catalytic activities, the possibility exists that it is proteolytically modified product of the native flavoprotein. This is especially likely in view of the finding that proteolytically solubilized cytochrome b₅ is a heme-containing fragment of the native hemoprotein, which can be purified only after solubilization with detergents. It is clear that the proteases remove a peptide segment that is hydrophobic and is responsible for the membrane binding and reconstitution capacity from the native reductase molecule.

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Cited by (93)

Purified NADH-cytochrome b<inf>5</inf> reductase is a novel superoxide anion source inhibited by apocynin: Sensitivity to nitric oxide and peroxynitrite
2014, Free Radical Biology and Medicine
Citation Excerpt :
To prepare fresh liver microsomes the procedure described by Mihara and Sato was followed [32].
Cytochrome b₅ reductase (Cb₅R) is a pleiotropic flavoprotein that catalyzes multiple one-electron reduction reactions with various redox partners in cells. In earlier work from our laboratory, we have shown its implication in the generation of reactive oxygen species (ROS), primarily a superoxide anion overshoot peak, which plays a major role as a triggering event for the acceleration of apoptosis in cerebellar granule neurons in culture. However, the results obtained in that work did not allow us to exclude the possibility that this superoxide anion production could be derived from Cb₅R acting in concert with other cellular components. In this work, we have purified Cb₅R from pig liver and we have experimentally shown that this enzyme catalyzed NADH-dependent production of superoxide anion, assayed with cytochrome c and nitroblue tetrazolium as detection reagents for this particular ROS. The basic kinetic parameters for this novel NADH-dependent activity of Cb₅R at 37 °C are V_max = 3.0±0.5 μmol/min/mg of purified Cb₅R and K_M(NADH) = 2.8±0.3 μM NADH. In addition, we report that apocynin, a widely used inhibitor of nonmitochondrial ROS production in mammalian cell cultures and tissues, is a potent inhibitor of purified Cb₅R activity at the concentrations used in the experiments done with cell cultures. In the presence of apocynin the K_M(NADH) value of Cb₅R increases, and docking simulations indicate that apocynin can bind to a site near to or partially overlapping the NADH binding site of Cb₅R. Other ROS, such as nitric oxide and peroxynitrite, have inhibitory effects on purified Cb₅R, providing the basis for a feedback cellular protection mechanism through modulation of excessive extramitochondrial superoxide anion production by Cb₅R. Both kinetic assays and docking simulations suggest that nitric oxide-induced nitrosylation (including covalent adduction of nitroso functional groups) of Cb₅R cysteines and peroxynitrite-induced tyrosine nitration and cysteine oxidation modified the conformation of the NADH binding domain leading to a decreased affinity of Cb₅R for NADH.
Biochemical and spectroscopic characterization of the human mitochondrial amidoxime reducing components hmARC-1 and hmARC-2 suggests the existence of a new molybdenum enzyme family in eukaryotes
2010, Journal of Biological Chemistry
Citation Excerpt :
Binding of FAD to cyt b5R was determined at 450 nm, and the FAD-to-protein ratio was calculated using an extinction coefficient of 11.3 mm−1 cm−1 (28). Activity of NADH-dependent cyt b5R was determined using the ferricyanide reduction assay according to (29) with minor modifications. One unit is defined as the amount of cyt b5R required to reduce 1 µmol of ferricyanide/min.
The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b₅, and NADH/FAD-dependent cytochrome b₅ reductase. mARC proteins share a significant degree of homology to the molybdenum cofactor-binding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo- or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes.
Clustering of plasma membrane-bound cytochrome b<inf>5</inf> reductase within 'lipid raft' microdomains of the neuronal plasma membrane
2009, Molecular and Cellular Neuroscience
Plasma membrane redox centres play a major role in neuronal defence against oxidative stress and survival. In cerebellar granule neurons in culture (CGN) a large pool of the flavoproteins are associated with the plasma membrane, and the intensity of CGN green/orange autofluorescence correlated with the levels of expression of cytochrome b₅ reductase. Regionalization of cytochrome b₅ reductase in the plasma membrane of CGN by fluorescence resonance energy transfer points out the close proximity between cytochrome b₅ reductase and the ‘lipid raft’ markers cholera toxin B and caveolin-2. This study unravels that membrane-bound cytochrome b₅ reductase is largely enriched at interneuronal contact sites in the neuronal soma and associated with ‘lipid rafts’ of the CGN plasma membrane. We also show that cytochrome b₅ reductase makes a large contribution to the NADH oxidase activity and to the red-shifted flavine fluorescence of purified rat brain synaptic plasma membranes. In conclusion, membrane-bound cytochrome b₅ reductase forms a large mesh of redox centres associated with the neuronal plasma membrane.
Identification of the missing component in the mitochondrial benzamidoxime prodrug-converting system as a novel molybdenum enzyme
2006, Journal of Biological Chemistry
Citation Excerpt :
Thereby, average saturation of HMCS-CT with MPT/Moco, determined as FormA-dephospho, was found to be about 41% (412 ± 96 pmol of FormA/nmol of protein; n = 5). NADH Cyt b5 Reductase Activity—Enzyme activity was determined by modification of the ferricyanide reduction assay (26). Heme Content—Heme content was estimated by recording the sodium dithionite-reduced minus the oxidized spectrum (27).
Amidoximes can be used as prodrugs for amidines and related functional groups to enhance their intestinal absorption. These prodrugs are reduced to their active amidines. Other N-hydroxylated structures are mutagenic or responsible for toxic effects of drugs and are detoxified by reduction. In this study, a N-reductive enzyme system of pig liver mitochondria using benzamidoxime as a model substrate was identified. A protein fraction free from cytochrome b₅ and cytochrome b₅ reductase was purified, enhancing 250-fold the minor benzamidoxime-reductase activity catalyzed by the membrane-bound cytochrome b₅/NADH cytochrome b₅ reductase system. This fraction contained a 35-kDa protein with homologies to the C-terminal domain of the human molybdenum cofactor sulfurase. Here it was demonstrated that this 35-kDa protein contains molybdenum cofactor and forms the hitherto ill defined third component of the N-reductive complex in the outer mitochondrial membrane. Thus, the 35-kDa protein represents a novel group of molybdenum proteins in eukaryotes as it forms the catalytic part of a three-component enzyme complex consisting of separate proteins. Supporting these findings, recombinant C-terminal domain of the human molybdenum cofactor sulfurase exhibited N-reductive activity in vitro, which was strictly dependent on molybdenum cofactor.
Reduction of N<sup>ω</sup>-hydroxy-l-arginine to l-arginine by pig liver microsomes, mitochondria, and human liver microsomes
2006, Biochemical and Biophysical Research Communications
Citation Excerpt :
NADH-cytochrome b5-reductase. NADH-cytochrome b5-reductase was assessed by its NADH-ferricyanide-reductase activity (1 U = 1 μmol of reduced ferricyanide/min) as described by Mihara and Sato [26]. CYP2D enzyme.
N^ω-Hydroxy-l-arginine, the intermediate in nitric oxide formation from l-arginine catalyzed by NO synthase, can be released into the extracellular space. It has been suggested that it can circulate and exert paracrine effects. Since it cannot only be used as substrate by NO synthases, but can also be oxidized by cytochrome P450 and other hemoproteins in a superoxide-dependent manner, it has been proposed that it can serve as NO donor. In the present study, the in vitro reduction of N^ω-hydroxy-l-arginine was examined. Pig and human liver microsomes as well as pig liver mitochondria were capable of reducing N^ω-hydroxy-l-arginine to l-arginine in an oxygen-insensitive enzymatic reaction. These results demonstrate that this metabolic pathway has to be considered when suggesting N^ω-hydroxy-l-arginine as NO-precursor. The reconstituted liver microsomal system of a pig liver CYP2D enzyme, the benzamidoxime reductase, was unable to replace microsomes to produce l-arginine from N^ω-hydroxy-l-arginine.
Oxygen-insensitive enzymatic reduction of oximes to imines
2006, Biochemical Pharmacology
The reduction of oximes to imines under anaerobic and aerobic conditions was studied using (E)- and (Z)-2,4,6-trimethylacetophenone oxime, benzaldoxime and (E)-2,4,6-trimethylbenzaldoxime. Pig and human liver microsomes, pig liver mitochondria and cytosol to a minor extent catalyzed the conversion of both isomeric ketoximes to the corresponding stable imine, the (E)-isomer being the better substrate. All reactions were oxygen-insensitive and required active protein and NADH or NADPH; however, NADH was preferred as cofactor. The reconstituted liver microsomal system of a pig liver CYP2D enzyme (NADH-benzamidoxime reductase), which is known to reduce N-hydroxylated derivatives of strongly basic functional groups, such as amidoximes, is also capable of reducing oximes. As expected, the corresponding imine was detected in relevant amounts when incubating 2,4,6-trimethyl-acetophenone oxime using the reconstituted enzyme system, but reduction rates were significantly lower compared to rates obtained when incubating benzamidoxime. Steric hindrance due to the methyl groups in ortho position to the oxime functionality could be excluded as being responsible for the lower conversion rates according to results obtained in incubations of 2,4,6-trimethylbenzamidoxime. When incubating benzaldoxime, only benzoic acid could be detected as metabolite, since the aldehyde is easily oxidized during incubation procedures, whereas incubations of (E)-2,4,6-trimethylbenzaldoxime also showed the formation of the corresponding aldehyde. These results allow us to postulate that the metabolism of aldoximes like 2,4,6-trimethylbenzaldoxime most likely proceeds through enzymatic reduction of the oxime to yield the intermediate imine, which is subsequently hydrolyzed to the aldehyde and then oxidized to the corresponding benzoic acid.

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[9] Detergent-solubilized NADH-cytochrome b5 reductase

Publisher Summary

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biochem. (Tokyo)

J. Biochem. (Tokyo)

Biochem. J.

[9] Detergent-solubilized NADH-cytochrome b₅ reductase