The effect of extra bound cytochrome b-5 on cytochrome P-450-dependent enzyme activities in liver microsomes

Binding of increasing amounts of detergent-purified cytochrome $\text{b}{}_5$ to rabbit liver microsomes produces a progressive inhibition of NADPH-cytochrome P-450 reductase activity which is accompanied by a similar inhibition of NADPH-supported benzphetamine demethylation. In contrast, NADH-cytochrome P-450 reductase activity in the enriched microsomes is markedly enhanced and this stimulation is accompanied by a similar increase in NADH-peroxidase activity, suggesting that cytochrome $\text{b}{}_5$ in these two reactions functions as an intermediate electron carrier to cytochrome P-450.     

Immunochemical study on the participation of cytochrome b5 in drug oxidation reactions of mouse liver microsomes.

Highly purified divalent and monovalent antibodies against cytochrome $\text{b}{}_5$, anti-$\text{b}{}_5$ immunoglobulin G (IG) and anti-$\text{b}{}_5$ Fab', were used in elucidating the role of this cytochrome in the drug-oxidizing enzyme system of mouse liver microsomes. Anti-$\text{b}{}_5$ IG strongly inhibited not only NADH-supported but also NADPH-supported oxidation of 7-ethoxycoumarin and benzo(a)pyrene, but had no inhibitory action on the oxidation of aniline. Anti-$\text{b}{}_5$ Fab' also inhibited NADH-supported and NADPH-supported benzo(a)pyrene hydroxylation. These observations indicate an essential role of cytochrome $\text{b}{}_5$ in the transfer of electrons not only from NADH but also from NADPH to cytochrome P-450 in the microsomal oxidation of some drugs, but not of aniline.     

Cytochrome b5 as electron donor to rabbit liver cytochrome P-450LM2 in reconstituted phospholipid vesicles

When incorporated into phospholipid vesicles containing NADPH-cytochrome P-450 reductase and P-450_LM2, cytochrome b₅ enhanced the rate of NADPH-supported hydroxylation of 7-ethoxycoumarin or $\text{p}$-nitroanisole about 5-fold. Cytochrome b₅ did not affect the rate of NADPH-oxidation, nor the rate of NADPH-supported formation of the ferrous CO-complex of cytochrome P-450. However, the cytochrome b₅-mediated increase in product formation was found to be correlated with concomitant decreases in the production of H₂O₂ or $\text{O}{}_2{}^{\mathrm{?}}$ in the system, thus strongly indicating cytochrome b₅ being a more efficient donor of the second electron to cytochrome P-450 than is NADPH-cytochrome P-450 reductase.     

A gel-electrophoretically homogeneous preparation of cytochrome P-450 from liver microsomes of phenobarbital-pretreated rabbits

Cytochrome P-450 was purified from liver microsomes of phenobarbital-pretreated rabbits to a specific content of 16 to 17 nmoles per mg of protein with a yield of about 10 %. The purified cytochrome yielded only a single protein band on sodium dodecylsulfate-urea-polyacrylamide gel electrophoresis, and an apparent molecular weight of about 45,000 was estimated for the protein. The preparation was free of cytochrome $\text{b}{}_5$, NADH-cytochrome $\text{b}{}_5$ reductase, and NADPH-cytochrome $\text{c}$ reductase activities. Aniline hydroxylase and ethylmorphine N-demethylase activities could be reconstituted upon mixing the purified cytochrome with an NADPH-cytochrome $\text{c}$ reductase preparation (purified by a detergent method) and phosphatidyl choline.     

Spectral intermediates in the reaction of oxygen with purified liver microsomal cytochrome P-450

Stopped flow spectrophotometry has shown the occurrence of two distinct spectral intermediates in the reaction of oxygen with the reduced form of highly purified cytochrome P-450 from liver microsomes. As indicated by difference spectra, Complex I (with maxima at 430 and 450 nm) is rapidly formed and then decays to form Complex II (with a broad maximum at 440 nm), which resembles the intermediate seen in steady state experiments. In the reaction sequence, P-450_LM^red$\text{→}\text{O}{}_2$Complex I→Complex II→P-450_LM^ox the last step is rate-limiting. The rate of that step is inadequate to account for the known turnover number of the enzyme in benzphetamine hydroxylation unless NADPH-cytochrome P-450 reductase or cytochrome $\text{b}{}_5$ is added. The latter protein does not appear to function as an electron carrier in this process.     

Lifetime of microsomal cytochrome P-450 and steroidogenic enzymes in rat testis as influenced by human chorionic gonadotrophin

The lifetime of different microsomal steroidogenic enzymes and the cytochrome components of the NADPH-cytochrome P-450 pathway have been determined in rat testis by measuring their decrease logarithmically after hypophysectomy. Although both cytochrome P-450 and 17α-hydroxylase show biphasic decay curves, the first decay curve contains 89–94% of the cytochrome P-450 and 17α-hydroxylase levels. Steroidogenic enzymes which are located mainly in the leydig cells, decay much faster than microsomal protein, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 12}$ days, which represents mainly decay of tubular protein. The similarity between the major half-life of cytochrome P-450, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 3.3}$ days, 17α-hydroxylase, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 2.3}$ days and the C₁₇–C₂₀ lyase, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 3.4}$ days and the uniformity of their response to human chorionic gonadotrophin (HCG) provides additional evidence that these two steroidogenic enzymes require cytochrome P-450. Both the 17α-hydroxylase and the C₁₇–C₂₀ lyase were shown to have a constant activity per nmole of cytochrome P-450 during a sixfold change in the level of cytochrome P-450 brought about by HCG treatment of rats with intact pituitaries. The decay of 17β-hydroxysteroid dehydrogenase, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4.5}$ days, was slower than P-450 dependent enzymes. Rats with intact pituitaries are not under maximal stimulation by endogenous LH because addition of HCG increases the levels of microsomal and mitochondrial cytochrome P-450 220 and 1620%, respectively. The rates of synthesis during the increase from one cytochrome P-450 level to another was calculated at $\text{0.118}\text{2}$ testes/day for microsomal cytochrome P-450 and 0.10 nmoles/2 testes/day for mitochondrial cytochrome P-450. Treatment of hypophysectomized rats with HCG results in large increases of cytochrome P-450, 17α-hydroxylase, C₁₇–C₂₀ lyase and 5α-reductase, but not cytochrome b₅, microsomal protein, 7α-hydroxylase, or the 17β-hydroxysteroid dehydrogenase. While it is clear that the two cytochrome P-450 dependent hydroxylases involved in steroidogenesis and the 5α-reductase are under the control of gonadotrophin, it is not clear how 17β-hydroxysteroid dehydrogenase levels are maintained or in what manner the 5α-reductase level is controlled in mature animals.     

Distribution of NAD(P)H-dependent cytochrome P-450 mixed function oxidase system in the brush border membrane of rabbit kidney cortex

Optical and magnetic studies were made on subfractions of rabbit kidney cortex. Cytochrome $\text{P-450}$ and cytochrome $\text{b}{}_5\text{-}\text{dependent}$ mixed function oxidase systems were localized mainly in the brush border membranes and microsomes. Cytochrome $\text{P-450-}\text{dependent}$ mixed function oxidases in the membranes comprised both an NADPH-dependent system and an NADH-dependent system.     

The binding of cytochrome b5 to plasma membranes of rat liver. Its implication for membrane specificity and biogenesis

The in vitro incorporation of cytochrome $\text{b}{}_5$ into purified plasma membranes was investigated by biochemical and immunological methods. Plasma membrane preparations incorporated three times less cytochrome $\text{b}{}_5$ than did microsomal preparations; 60% of this cytochrome $\text{b}{}_5$ could not be reduced by the NADH-cytochrome $\text{b}{}_5$ reductase and was considered as being bound to the plasma membrane. The morphological observations made after the immunochemical labeling of cytochrome $\text{b}{}_5$ clearly showed a good but asymmetrical distribution of the ferritin labeling: only the inner face of the plasma membrane incorporated cytochrome $\text{b}{}_5$. These results are discussed with respect to theories which concern the subcellular membrane relationships in the cell.     

Aliphatic hydroxylation by highly purified liver microsomal cytochrome P-450. Evidence for a carbon radical intermediate

The oxidation of norbornane by a reconstituted liver cytochrome P-450 system affords $\text{exo}$- and $\text{endo}$-2-norborneol in a ratio of 3.4:1. The ratio of these products was found to be 0.76:1 when $\text{exo,exo,exo,exo}$-2,3,5,6-tetradueteronorbornane was oxidized. Analysis of the mass spectra of the products from the deuterated hydrocarbon showed that 25% of the $\text{exo}$-norborneol contained four deuterium atoms whereas 9% of the $\text{endo}$-norborneol contained three deuterium atoms. These results, which indicate a very large isotope effect ($\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}\text{= 11.5±1}$) and a significant amount of epimerization for the hydroxylation of norbornane by cytochrome P-450, suggest an initial hydrogen abstraction to give a carbon radical intermediate.     

Isolation of an acid protease from rabbit reticulocytes and evidence for its role in processing redox proteins during erythroid maturation

A protease which generates a soluble hemepeptide from bovine liver microsomal cytochrome $\text{b}{}_5$ has been isolated from the membrane fraction of rabbit reticulocytes. Inhibition by pepstatin and an acidic pH optimum indicate that the protease belongs to the acid protease class. Little cytochrome $\text{b}{}_5$-processing activity is observed in rabbit erythrocytes. We suggest that the protease may be involved in the processing which generates the proteins of the methemoglobin reduction system from their membrane-bound precursors during the maturation of the erythroid cell.     

DNA complementary to rabbit globin mRNA made by E. coli polymerase I

Incubation of liver microsomes with cytochrome $\text{b}{}_5$, purified after solubilization with detergents, caused an effective incorporation of the cytochrome into the microsomal membranes. The incorporated cytochrome was reducible by NADH and could not be removed by repeated washing with 0.3 M KCl or 10 mM EDTA. The incorporation was much more efficient at 37°C than at 0°C. Trypsin-solubilized cytochrome $\text{b}{}_5$, which lacks the hydrophobic tail of the native protein, could not be inserted into the membranes. These findings confirm the view that the hydrophobic tail of the cytochrome molecule is responsible for its tight binding to the microsomal membranes.     

Constraint on the substrate cytochrome P-450 binding reaction in bovine adrenocortical microsomes at physiological temperature

The addition of cholate to the microsomes at 37.5°C resulted in a striking decrease in the apparent substrate dissociation constant ($\text{K′}{}_{\mathrm{<mtext>s</mtext>}}$) and its temperature dependency. The microsomal membranes depleted of 80% of the lipids preserved the temperature dependency of the $\text{K}{}_{\mathrm{<mtext>s</mtext>}}$ and exhibited breaks in the Van't Hoff plot at the characteristic temperature of the lipids phase transition. The results indicate that the cytochrome $\text{P-450}$ is considerably restrained from expressing its maximum substrate binding potential at physiological temperature. In addition, the results indicate that the majority of the lipids apparently do not play a significant role in imposing constraint on the substratecytochrome $\text{P-450}$ binding reaction and in the temperature dependency of the $\text{K}{}_{\mathrm{<mtext>s</mtext>}}$.     

Lipid-protein interactions in membrane models fluorescence polarization study of cytochrome b5-phospholipids complexes

According to previous authors, cytochrome $\text{b}{}_5$, when extracted from bovine liver by a detergent method, is called cytochrome $\text{d-b}{}_5$. On the other hand, the protein obtained after trypsin action, which eliminates an hydrophobic peptide of about 54 residues, is called cytochrome $\text{t-b}{}_5$.Fluorescence polarization of the dansyl phosphatidylethanolamine probe inserted into phospholipid vesicles is very senstive to the binding of proteins, and so is a useful method to study lipid-protein interactions.The chromophore mobility, $\text{R}$, decreases markedly when dipalmitoyl phosphatidylcholine vesicles are incubated with cytochrome $\text{d-b}{}_5$, whereas $\text{R}$ does not change for cytochrome $\text{c}$ and cytochrome $\text{t-b}{}_5$. This can be interpreted as a strengthening of the bilayer, only due to the interaction of the hydrophobic peptide tail.Interaction of dipalmitoyl phosphatidylcholine vesicles with cytochrome $\text{d-b}{}_5$ occurs either below or above the melting temperature of the aliphatic chains (41 °C). Even for a high protein to lipid molar ratio (1 molecule of protein for 40 phospholipid molecules), the melting temperature is apparently unaffected.Phosphatidylserine and phosphatidylinositol do not interact at pH 7.7 with cytochrome $\text{d-b}{}_5$, because electrostatic forces prevent formation of complexes. At low pH, the interaction with the protein occurs, but the binding is mainly of electrostatic nature.     

Catalysis by hemoproteins and their structural organization in reversed micelles of surfactants in octane

The cytochromes $\text{P-450}$ LM-2 and $\text{b}{}_5$ from rabbit liver microsomes have been entrapped into reversed micelles of surfactants in octane. The optimum conditions providing for the maximum stability of the hemoproteins have been found: pH and concentration of the buffer solution, the glycerol addition, the surfactant concentration, the [H₂O]/[surfactant] ratio and, finally, the reversed micelles composition including aerosol OT and its mixture with Triton X-45, Tween 20 and cetyltrimethylammonium bromide (CTAB). The transformation kinetics of the hemoproteins solubilized by detergents has been studied by monitoring the absorbance of hemoproteins in the Soret band region. Significant changes in tryptophan fluorescence of cytochrome $\text{b}{}_5$ and in CD spectra of myoglobin in reversed micelles and their dependence on the [H₂O]/[aerosol OT] ratio have been shown. The three hemoproteins in reversed micelles have been found to exhibit high catalytic activity with respect to their reaction with cumene hydroperoxide. The kinetic and spectral data reveal the structural transformations of the proteins entrapped into the micelles due to the interactions of the lumenal surface of the micelles and the protein molecule surface.     

Purification of bovine liver microsomal NADH-cytochrome b5 reductase using affinity chromatography

Microsomal NADH-cytochrome $\text{b}{}_5$ reductase has been purified from bovine liver by an improved procedure which employs affinity chromatography on ADP-agarose in combination with anion exchange chromatography. The reductase was extracted from a 105,000 × $\text{g}$ microsomal pellet with Triton X-100. The overall purification from isolated microsomes was 98-fold and the yield was 10%. The preparation was nearly homogeneous on SDS-PAGE. This procedure requires less time and effort than previously described procedures. Partially purified cytochrome $\text{b}{}_5$ is also obtained.     

Specificity in the interaction of phospholipids and fatty acids with vesicle reconstituted cytochrome P-450. A spin label study

The association of fatty acids, androstane, phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid with purified and phospholipid-vesicle reconstituted cytochrome $\text{P-450}$ was studied by spin labeling. Spin-labeled fatty acids were found to be motionally restricted by cytochrome $\text{P-450}$ in both phospholipid vesicles and in microsomes to a much greater extent than spin-labeled phospholipids. The equilibrium of spin-labeled fatty acid between the bulk membrane lipid and the protein interface could be shifted towards an increased amount in the bulk phospholipid phase by the addition of oleic acid or lysophosphatidylcholine, but not by sodium cholate. Microsomes from different animals showed a variable extent of motional restriction of fatty acids, independent of pretreatment of the animals with phenobarbital or β-naphthoflavone, of cytochrome $\text{P-450}$ content, of the presence of type I and type II substrates for cytochrome $\text{P-450}$. These differences are attributed to the presence of varying amounts of lipid breakdown products in the microsomal membrane such as lysolipids or fatty acids which compete with the externally added spin-labeled fatty acids, or with spin-labeled androstane for the binding to cytochrome $\text{P-450}$. The negative charge of the fatty acid was found to be involved in its association with the protein. Cytochrome $\text{P-450}$ was shown to interact only with a few spin-labeled phospholipid molecules in such a way that the motional restriction of the spin acyl chains can be detected by electron paramagnetic resonance ($\text{τ}{}_{\mathrm{<mtext>R</mtext>}}\text{> 10}{}^{\mathrm{?8}}\text{s}$). The number of associated lipid molecules per protein probably is too small to form a complete shell around the protein. This lipid-protein interaction could be destroyed by the addition of sodium cholate, in contrast to the fatty acid-protein interaction.     

Binding of bovine cytochrome b5 to phosphatidycholine liposomes Characterization of the reconstituted lipid-protein vesicles

Cytochrome $\text{b}{}_5$ was extracted and purified from beef liver by a detergent method (cytochrome $\text{d-b}{}_5$). The hydrophilic moiety which carries the heme group (cytochrome $\text{t-b}{}_5$) was prepared by trypsin action upon pure cytochrome $\text{d-b}{}_5$.Single-shelled lecithin liposomes form complexes with cytochromes $\text{d-b}{}_5$ up to a molar ratio of one protein for 35 phospholipids. The lipid-protein complexes were isolated by gel filtration on Sepharose 4B. They are hollow vesicles in which [³H]-glucose can be trapped. Their diameter is greater than that of the initial liposomes.Cytochrome $\text{t-b}{}_5$ does not interact with the vesicles. These results show that the hydrophobic tail is necessary for the binding and that the hydrophilic part of the protein is located on the outer face of the vesicles. This asymmetry is also proved by the action of reducing agents.Experiments with saturated phosphatidylcholines show that the protein interacts with the lipids both below the transition temperature $\text{T}{}_{\mathrm{M}}$. i.e. when the aliphatic chains are in a crystalline state, and above $\text{T}{}_{\mathrm{M}}$, when the alipathic chain are in a fluid state.¹H NMR spectra show that even at the maximum cytochrome $\text{d-b}{}_5$ concentration the presence of the proteins does not markedly change the dynamics to the phospholipid molecules. An asymmetric single-shelled vesicle structure is proposed for the complex.     

Evidence for a protonmotive Q cycle mechanism of electron transfer through the cytochrome b-c1 complex.

A protein named oxidation factor can be reversibly removed from succinate-cytochrome $\text{c}$ reductase complex and shown to be required for electron transfer between succinate and cytochrome $\text{c}$. This protein is required for reduction of cytochrome $\text{c}{}_1$ and, in the presence of antimycin, for reduction of both cytochromes $\text{b}$ and $\text{c}{}_1$. These results are consistent with a protonmotive Q cycle mechanism in which the oxidation factor catalyzes electron transfer from reduced quinone to cytochrome $\text{c}{}_1$ and thus liberates from reduced quinone one of two protons required for energy conservation during electron transfer through the cytochrome $\text{b}\text{-}\text{c}{}_1$ complex.     

Catalytic properties of the resolved flavoprotein and cytochrome B components of the NADPH dependent O2−• generating oxidase from human neutrophils

The resolved flavoprotein and cytochrome b₅₅₉ components of the NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generating oxidase from human neutrophils were the subject of further study. The resolved flavoprotein, depleted of cytochrome b₅₅₉, was reduced by NADPH under anaerobic conditions and reoxidized by oxygen. NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generation by the resolved flavoprotein fraction was not detectable, however it was competent in the transfer of electrons from NADPH to artificial electron acceptors. The resolved cytochrome b₅₅₉, depleted of flavoprotein, demonstrated no measureable NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generating activity and was not reduced by NADPH under anaerobic conditions. The dithionite reduced form of the resolved cytochrome b₅₅₉ was rapidly oxidized by oxygen, as was the cytochrome b₅₅₉ in the intact oxidase.     

Ubisemiquinone radicals from the cytochrome b-c1 complex of the mitochondrial electron transport chain--demonstration of QP-S radical formation

Stable ubisemiquinone radical(s) in the cytochrome $\text{b}\text{?}\text{c}{}_1$-II complex of bovine heart was observed following reduction by succinate in the presence of catalytic amounts of succinate dehydrogenase. The radical was abolished by addition of antimycin A, but a residual radical remained in the presence of excess exogenous Q₂. The radical showed an EPR signal of g = 2.0046 ± .003 at X band (～9.4 GHz) with no resolved hyperfine structure and had a line width of 8.1 ± .5 Gauss at 23°C. The Q band (35 GHz) spectra showed wellresolved $\text{g}$-anisotropy and had a field separation between derivative extrema of 26 ± 1 Gauss. This radical is evidently from QP-C. These observations substantiate that the radical is immobilized and bound to a protein. The QP-S radical was demonstrated in the cytochrome $\text{b}\text{-}\text{c}{}_1$-II complex only in the presence of more than a catalytic amount of succinate dehydrogenase and cytochrome $\text{b}\text{-}\text{c}{}_1$. This signal was not antimycin a inhibitory. The signal amplitude paralleled the reconstitutive enzymic activity of succinate-cytochrome $\text{c}$ reductase from succinate dehydrogenase and the cytochrome $\text{b}\text{-}\text{c}{}_1$-II complex.