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Upon incubation of detergent-solubilized NADPH-cytochrome P-450 reductase and either cytochrome b5 or cytochrome c in the presence of a water-soluble carbodiimide, a 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), covalently cross-linked complex was formed. The cross-linked derivative was a heterodimer consisting of one molecule each of flavoprotein and cytochrome, and it was purified to 90% or more homogeneity. The binary covalent complex between the flavoprotein and cytochrome b5 was exclusively observed following incubation of all three proteins including NADPH-cytochrome P-450 reductase, cytochrome b5, and cytochrome c in L-alpha-dimyristoylphosphatidylcholine vesicles, and no heterotrimer could be identified. The isolated reductase-cytochrome b5 complex was incapable of covalent binding with cytochrome c in the presence of EDC. No clear band for covalent complex formation between PB-1 and reductase was seen with the present EDC cross-linking technique. More than 90% of the cross-linked cytochrome c in the purified derivative was rapidly reduced upon addition of an NADPH-generating system, whereas approximately 80% of the cross-linked cytochrome b5 was rapidly reduced. These results showed that in the greater part of the complexes, the flavin-mediated pathway for reduction of cytochrome c or cytochrome b5 by pyridine nucleotide was intact. When reconstituted into phospholipid vesicles, the purified amphipathic derivative could hardly reduce exogenously added cytochrome c, cytochrome b5, or PB-1, indicating that the cross-linked cytochrome shields the single-electron-transferring interface of the flavoprotein. These results suggest that the covalent cross-linked derivative is a valid model of the noncovalent functional electron-transfer complex.
The preparation, purification, and characterization of four new derivatives of cytochrome c trifluoroacetylated at lysines 72, 79, 87, and 88 are reported. The redox reaction rates of these derivatives with cytochrome b5, cytochrome c1 and cytochrome oxidase indicated that the interaction domain on cytochrome c for all three proteins involves the lysines immediately surrounding the heme crevice. Modification of lysines 72, 79, 87 had a large effect on the rate of all three reactions, while modification of lysine 88 had a very small effect. Even though lysines 87 and 88 are adjacent to one another, lysine 87 is at the top left of the heme crevice oriented towards the front of cytochrome c, while lysine 88 is oriented more towards the back. Since the interaction sites for cytochrome c1 and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.
Cytochrome b5 is required for the cytochrome P-450 LM2 catalyzed oxidation of the anesthetic methoxyflurane. The ability of cytochrome b5 to support methoxyfluorane oxidation is affected by treatment with diethylpyrocarbonate, a reagent that at neutral pH is relatively specific for histidine residues. This inactivation of cytochrome b5 is reversed with hydroxylamine, which also suggests but does not prove histidine involvement. The studies reported in this paper were undertaken to determine whether histidine modification was involved in the decrease in effectiveness of cytochrome b5, or whether the inactivation could be attributed to modification of another amino acid. Our experiments demonstrate that diethylpyrocarbonate inactivates detergent-solubilized cytochrome b5 by modifying the axial histidines and displacing the heme. Because of the unexpected ease with which diethylpyrocarbonate displaced the heme from cytochrome b5, this same process was investigated in two other hemoproteins, cytochrome c and myoglobin. Diethylpyrocarbonate could not dissociate the heme from cytochrome c, whereas the heme was lost from myoglobin even more readily than from cytochrome b5.
Human erythrocytes were divided into age groups according to their density using phthalate esters as separating liquids. The concentration of cytochrome b5 and the activity of NADH-cytochrome b5 reductase decreased exponentially with the age of red cells. The apparent half-life of cytochrome b5 was estimated to be 44 days. The decline of cytochrome b5 seemed to be more rapid than the decline in the activities of glutamate-oxaloacetate transaminase and NADH-cytochrome b5 reductase whose apparent half-lives were 210 and 240 days, respectively. A biphasic decline of cytochrome b5 was observed on storage of erythrocytes at 4 degrees C. It was deduced from the kinetic results that the decrease of cytochrome b5 might be involved in the increase of the concentration of methemoglobin in senescent erythrocytes. Cytochrome b5 may be used as an indicator of mean red cell age.
The effects of cytochrome b5 with manganese-protoporphyrin IX substituted for heme were compared with those of native cytochrome b5 and the apoenzyme on the oxygenation of substrates in the reconstituted system containing liver microsomal cytochrome P-450, NADPH-cytochrome P-450 reductase, and phosphatidylcholine. Mn-b5, unlike b5, remains essentially fully oxidized in the presence of NADPH and NADPH-cytochrome P-450 reductase under aerobic conditions. The effects of various concentrations of b5 and its derivatives were determined at constant P-450 and reductase concentrations. Cytochrome b5 inhibits benzphetamine demethylation by isozyme 2, the effect increasing up to the highest concentrations tested, and stimulates 7-ethoxycoumarin deethylation by isozyme 2 and acetanilide p-hydroxylation by isozyme 4, the optimal b5:P-450 molar ratio being about 2. In contrast, Mn-b5 inhibits all three reactions and apo-b5 is either inactive or slightly inhibitory. The activities of the three substrates as well as testosterone were determined with P-450 isozymes 2, 3b, 3c, and 4 in the reconstituted system with no additions or with b5 or Mn-b5 present. Cytochrome b5 is stimulatory, inhibitory, or without any effect, the result depending on both the substrate and P-450 isozyme present, whereas Mn-b5 is inhibitory in most instances. Both b5 and its manganese derivative alter the rates of testosterone 6 beta- or 16 alpha-hydroxylation by most of the P-450 cytochromes. The activities are influenced by the molar ratio of reductase to P-450. The Km values of benzphetamine, ethoxycoumarin, and acetanilide are, with one exception, significantly decreased in the presence of b5 or Mn-b5. We conclude that some of the effects of b5 on the oxygenase system are not accounted for by its role as an electron donor to cytochrome P-450.
NADH-cytochrome b5 reductase readily binds to preformed phospholipid vesicles either below or above the phase transition temperature of the lipid and in the absence of detergents. The isolated vesicles are free of unbound reductase, and the lipid is present as small, closed bilayers (250 to 400 A in diameter) as indicated by gel filtration, density gradient centrifugation, and internal volume measurements with [3H]glucose. The order of substrate specificity of the bound reductase is: ferricyanide = cytochrome b5 bound to reductase vesicles (100%) greater than cytochrome b5 heme peptide (13%) greater than unbound cytochrome b5 (4.5%) greater than cytochrome b5 vesicles (0.1%). This indicates that a specific orientation of cytochrome b5 and reductase in the bilayer is required for optimal interaction. Protein transfer occurs between reductase vesicles and cytochrome b5 vesicles. The transfer is time-dependent (40 to 70% complete in 2 h), does not involve vesicle fusion, is most rapid at the phase transition temperature of the phospholipid, and appears to require a fluid bilayer.
Age-dependent decrease in cytochrome b5 was observed in erythrocytes from both a normal person and a patient with hereditary methaemoglobinaemia without neurological symptoms. With aging, concentrations of cytochrome b5 in erythrocytes from the patient were almost the same as those in the control. Age-dependent decrease in cytochrome b5 reductase activity in the control erythrocytes was also shown; however, the reductase activity was very low in erythrocytes from the patient over the whole age range. Our studies show that methaemoglobin content of erythrocytes seems to be dependent on the content of cytochrome b5 in the cells, both in the control subject and in the patient.
Dimyristoyl lecithin liposomes, containing cytochrome b5 reductase (NADH:ferricytochrome b5 oxidoreductase, EC 1.6.2.2) and varying amounts of cytochrome b5, were used to measure flavoprotein catalysis alone and catalysis requiring electron transfer between the reductase and cytochrome as a function of temperature. Whereas flavoprotein catalysis showed a simple linear temperature dependence in an Arrhenius plot, the reaction involving electron transfer between the two bound enzymes showed a marked, 4-fold, change in rate at the crystalline-liquid crystalline phase transition of the hydrocarbon chains of the lecithin vesicles and a second, minor change involving the minor transition. These data represent strong evidence that protein-protein interactions in this membrane model system are dependent upon translational diffusion of nonpolar segments of the proteins in the hydrocarbon region of the phospholipid bilayer.
The second-order electron transfer reaction between cytochrome b5 and cytochrome c has been studied by cyclic voltammetry utilizing a gold electrode modified with beta-mercaptopropionate. When cyclic voltammetry is performed on a solution containing a mixture of cytochrome b5, cytochrome c and polylysine, cytochrome b5 undergoes reversible electrochemistry at the electrode surface while cytochrome c discriminates against the electrode surface. The selectivity of the modified electrode for negatively charged proteins makes it possible to selectively reduce a protein possessing a net negative charge and a relatively low reduction potential (outer mitochondrial membrane cytochrome b5, Eo = -102 mV; microsomal cytochrome b5, Eo = 3 mV) in the presence of another protein possessing a net positive charge and a relatively high reduction potential (cytochrome c, Eo = 265 mV). The electrochemical reduction of ferricytochrome b5 at the electrode surface is followed by a second-order electron transfer reaction between ferrocytochrome b5 and ferricytochrome c that yields ferricytochrome b5 and ferrocytochrome c. This fast homogeneous electron transfer reaction which is preceded by a heterogeneous electron transfer reaction results in a characteristic cyclic voltammogram containing a pre-peak to the reduction current. The second-order rate constant for the homogeneous reaction was obtained by invoking the above reaction scheme for digital simulation of a cyclic voltammogram which was subsequently fitted to the experimental data. Second-order rate constants obtained with this method are 2.9 x 10(8) and 8.9 x 10(8) for the homogeneous electron transfer reactions between rat liver outer mitochondrial membrane (OM) ferrocytochrome b5 and beef liver microsomal ferrocytochrome b5, with horse heart ferricytochrome c, respectively. These values are in good agreement with second-order rate constants obtained for the same protein systems by flash photolysis. [Meyer, T. E., Rivera, M., Walker, F. A., Mauk, M. R., Mauk, A. G., Cusanovich, M. A., & Tollin, G. (1993) Biochemistry 32, 622-627].
Cytochrome P-450coh from pyrazole-treated mice was shown to form a tight and specific complex with cytochrome b5 from mouse liver microsomes. The complex formation was found to result in type I spectral changes indicating a spin shift from the low to the high spin form. When added to a reconstituted system containing cytochrome P-450coh, NADPH-cytochrome P-450 reductase and phospholipid, cytochrome b5 stimulates hydroxylation of coumarin and O-deethylation of 7-ethoxycoumarin. The maximal stimulating effect is reached at a 1:1 stoichiometry. Mouse liver cytochrome b5 stimulates hydroxylation and deethylation by 100% and 60%, respectively. The stimulating effect of cytochrome b5 was found to result from the increase of the maximal rate of oxidation, being practically without effect on Km. Cytochrome b5 purified from rat and rabbit liver microsomes interacts with cytochrome P-450coh but fails to stimulate the oxidation reaction. At large excess, cytochrome b5 inhibits the oxidations catalyzed by cytochrome P-450coh. Immobilized cytochrome b5 either from mouse or rat and rabbit microsomes proved to be an efficient affinity matrix for cytochrome P-450coh purification.
Cytochrome P-450 LM2 purified from rabbit liver microsomes has been shown to be a substrate for cAMP-dependent protein kinase. Cytochrome b5, in contrast, was a very poor substrate for cAMP-dependent protein kinase, although it stimulated the activity of the kinase toward histone. When purified rabbit cytochrome b5 was mixed with purified LM2, phosphorylation of LM2 by cAMP-dependent protein kinase was inhibited approximately 80-90%. Recently, a functional covalent complex of cytochrome b5 and LM2 was prepared and purified to homogeneity (P.P. Tamburini and J.B. Schenkman (1987) Proc. Natl. Acad. Sci. USA 84, 11-15). When present as a covalent complex with cytochrome b5, the phosphorylation of LM2 in the complex by cAMP-dependent protein kinase was also inhibited about 80-90% relative to an equivalent amount of LM2 alone. On the other hand, when the LM2 was phosphorylated prior to interaction with cytochrome b5, the ability of the latter to perturb the spin equilibrium of LM2 and oxidation of p-nitroanisole by the LM2 was diminished to an extent comparable to the degree of phosphorylation. The results suggest either that the phosphorylation site on LM2 may be within the cytochrome b5 binding site or that phosphorylation and cytochrome b5 cause mutually exclusive conformational changes in LM2. In addition, eight different forms of cytochrome P-450 from the rat (RLM2, RLM3, fRLM4, RLM5, RLM5a, RLM5b, RLM6, and PBRLM5) were examined as potential substrates for cAMP-dependent protein kinase under the same conditions. Maximal phosphorylation of about 20 mol% was obtained with LM2, and about half as much with PBRLM5. The low extent of phosphorylation of LM2 was not due to the prior presence of phosphate on the enzyme since LM2, as isolated, contains less than 0.1 mol phosphate/mol of enzyme. The other forms of cytochrome P-450 tested showed little or no phosphorylation in vitro despite the presence of a cAMP-dependent protein kinase phosphorylation sequence on at least two of them.
Cytochrome b5 is composed of two domains that can be isolated after tryptic cleavage as two polypeptide fragments. One fragment is globular and hydrophilic and contains the heme; the other fragment is rich in hydrophobic amino acids and is essential for recombination of cytochrome b5 with microsomal membranes (Ito, A., and Sato, R. (1968), J. Biol. Chem. 243, 4922; Spatz, L., and Strittmatter, P. (1971), Proc. Nat. Acad. Sci. U.S. 68, 1042). Equilibrium dialysis and sedimentation equilibrium measurements of the binding of deoxycholate, Triton X-100 and dodecyl sulfate show that neither intact cytochrome b5 nor its proteolytic fragments possess high affinity binding sites for any of these amphiphiles. However, each detergent binds to the protein in a highly cooperative manner at concentrations near the critical micelle concentration. Binding measurements using the separated tryptic fragments show that deoxycholate and Triton X-100 (both nondenaturing detergents) bind to the hydrophobic fragment to the same extent as to intact cytochrome b5, and not at all to the polar fragment. Sodium dodecyl sulfate (a denaturing detergent) is bound to both tryptic fragments, but 70% of the detergent is bound to the hydrophobic fragment although it comprises only 30% of the protein mass. Less detailed measurements were made with synthetic and natural phosphatidylcholines, and show that the intact protein is quantitatively incorporated into phosphatidylcholine vesicles, but that no interaction with the polar fragment occurs. These results are interpreted in terms of the hydrophobic domain of cytochrome b5 having a diffuse hydrophobic surface that can act as a nonspecific nucleus for the formation of a micelle with a variety of amphiphilic substances. This domain of the molecule will insert into any available hydrophobic environment, whether it be detergent micelles, synthetic phospholipid vesicles, or the microsomal membrane. The incorporation of cytochrome b5 into the microsomal membrane is only a specialized case of the general property.
Cytochrome b5 was purified from detergent solubilized sheep liver microsomes by using three successive DEAE-cellulose, and Sephadex G-100 column chromatographies. It was purified 54-fold and the yield was 23.5% with respect to microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200 +/- 500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b5 gave peaks at 557, 526.5 and 423 nm. The ability of the purified sheep liver cytochrome b5 to transfer electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. The K(m) and Vmax values were calculated to be 0.088 microM cytochrome b5 and 315.8 microM cytochrome c reduced/min/mg enzyme, respectively. Also the reduction of cytochrome b5 by reductase was studied and K(m) and Vmax values were determined to be 5 microM cytochrome b5 and 5200 nmol cytochrome b5 reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating concentration of cytochrome b5 were found to be 0.0017 mM NADH and 6944 nmol cytochrome b5 reduced/min/mg enzyme, respectively. NADH-cytochrome b5 reductase was also partially purified from the same source, detergent solubilized sheep liver microsomes, by using two successive DEAE-cellulose, and 5'-ADP-agarose affinity column chromatographies. It was purified 144-fold and the yield was 7% with respect to microsomes. The apparent monomer Mr of reductase was estimated to be 34,000 by SDS-PAGE. When ferricyanide was used as an electron acceptor, reductase showed maximum activity between 6.8 and 7.5. The K(m) and Vmax values of the enzyme for ferricyanide were calculated as 0.024 mM ferricyanide and 673 mumol ferricyanide reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating amounts of ferricyanide were found to be 0.020 mM NADH and 699 mumol ferricyanide reduced/min/mg enzyme, respectively.
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The interaction and electron transfer between cytochrome b5 and cytochrome P-450B1 were investigated using the reconstituted p-nitroanisole O-demethylase system. Apocytochrome b5 was prepared from detergent-solubilized cytochrome b5 by the acid-butanone method. The apocytochrome b5 thus obtained has been substituted with several metalloporphyrin derivatives. The reconstituted system containing cytochrome b5 substituted with heme derivatives such as proto-, meso-, and deuteroheme exhibited demethylation activity at the maximum turnover rates of 94, 58, 30%, respectively, compared to that containing the native cytochrome b5, while neither apocytochrome b5 nor cobaltic protoporphyrin-cytochrome b5 displayed the activity. Kinetic analysis showed the formation of a 1:1 complex between cytochrome P-450B1 and each of these substituted cytochrome b5's, except for cobaltic protoporphyrin-cytochrome b5; the affinities differed with the cytochrome b5 species used. The synergistic effect with the addition of the NADH-linked electron transport system was more remarkable at the lower reduction levels of cytochrome b5 in the steady state. Interaction between the components involved in NADH- and NADPH-linked electron transport systems was modulated by the existence of Triton X-100. The optimal concentration in the reconstituted system for the demethylation was observed at around 0.03% of Triton X-100, where the reduction rates for cytochrome b5 and cytochrome P-450B1 by the respective reductases were maximal. These results indicate that the two electron transport systems are closely coupled and exhibit the demethylase activity.
A simplified, rapid procedure for the purification of NADH-cytochrome b5 oxidoreductase and cytochrome b5 from either rat or rabbit liver is described. Microsomes were prepared by fractionation with polyethylene glycol and solubilized with Triton X-100. Cytochrome b5 was purified by a two-column procedure, anion exchange chromatography using DEAE-cellulose, and hydrophobic chromatography on phenyl-Sepharose. The final preparation of cytochrome b5 was purified more than a 120-fold from rat or rabbit liver microsomes, with specific content of about 50 nmol per mg protein, and overall yield of 22 to 32%. Only a single band with mol wt of 18,600 was found on sodium dodecyl sulfate (SDS)-gels or on Western blots using a polyclonal antibody raised against the purified b5. NADH-cytochrome b5 oxidoreductase was purified by a three-column procedure, DEAE-cellulose, hydroxylapatite, and ADP-agarose. The final product was purified more than 400-fold from rat or rabbit liver microsomes with a yield of about 25% and final specific activity of about 1600 mumol ferricyanide reduced per minute per milligram of protein. A single band with mol wt of 33, 100 was found on SDS-gels. The reductase catalyzed reduction of ferricyanide, dichlorophenol-indophenol, and cytochrome b5. Cytochrome c was reduced in the presence of reductase plus cytochrome b5, and this was inhibited by the anti-b5 IgG. The reductase catalyzed a rapid rate of reduction of ferric-ATP, which was slightly elevated by cytochrome b5. Ferric-histidine and ferric-ammonium sulfate were slowly reduced by reductase; addition of cytochrome b5 markedly stimulated reduction of these ferric complexes but inhibited reduction of ferric-EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)
Cytochrome P-450scc from bovine adrenal cortex mitochondria was shown to be selectively phosphorylated by protein kinase C. The amino acid residues most accessible to phosphorylation by protein kinase C are located in the N-terminal sequence of cytochrome P-450scc. Adrenodoxin and cytochrome b5 protect cytochrome P-450scc from phosphorylation, this effect being dependent on the protein concentration.