Cytochrome b5 and a recombinant protein containing the cytochrome b5 hydrophobic domain spontaneously associate with the plasma membranes of cells

Both cytochrome b5, isolated from rabbit liver microsomes, and LacZ:HP, a recombinant protein consisting of enzymatically active Escherichia coli beta-galactosidase coupled to the C-terminal membrane-anchoring hydrophobic domain of cytochrome b5, were shown to spontaneously associate with the plasma membranes of erythrocytes and 3T3 cells. Association was promoted by low pH values, but proceeded satisfactorily over several hours at physiological pH and temperature. About 150,000 cytochrome b5 molecules or 100,000 LacZ:HP molecules could be associated per erythrocyte. These proteins were not removed from the membrane by extensive washing, even at high ionic strength. After incubation with fluorescently labeled cytochrome b5 or LacZ:HP, cells displayed fluorescent membranes. The lateral mobility of fluorescently labeled cytochrome b5 and LacZ:HP was measured by photo-bleaching techniques. In the plasma membrane of erythrocytes and 3T3 cells, the apparent lateral diffusion coefficient D ranged from 1.0.10(-9) to 8.10(-9) cm2 s-1 with a mobile fraction M between 0.4 and 0.6. The lateral mobility of these proteins closely resembled that reported for lipid-anchored proteins and was much higher than that reported for Band 3, an erythrocyte membrane-spanning protein with a large cytoplasmic domain. These results suggest that the hydrophobic domain of cytochrome b5 could be employed as a universal, laterally mobile membrane anchor to associate a variety of diagnostically and therapeutically useful recombinant proteins with cells.     

Distinct roles of endoplasmic reticulum cytochrome b5 and fused cytochrome b5-like domain for rat Delta6-desaturase activity

The Delta6-desaturase catalyzes key steps in long-chain polyunsaturated fatty acid biosynthesis. Although the gene coding for this enzyme has been isolated in diverse animal species, the protein structure remains poorly characterized. In this work, rat Delta6-desaturase expressed in COS-7 cells was shown to localize in the endoplasmic reticulum. As the enzyme contains an N-terminal cytochrome b5-like domain, we investigated by site-directed mutagenesis the role of this domain in the enzyme activity. The typical HPGG motif of the cytochrome b5-like domain, and particularly histidine in this motif, is required for the activity of the enzyme, whatever the substrate. Neither endogenous COS-7 cytochrome b5 nor coexpressed rat endoplasmic reticulum cytochrome b5 could rescue the activity of mutated forms of Delta6-desaturase. Moreover, when rat endoplasmic reticulum cytochrome b5 was coexpressed with wild-type desaturase, both proteins interacted and Delta6-desaturase activity was significantly increased. The identified interaction between these proteins is not dependent on the desaturase HPGG motif. These data suggest distinct and essential roles for both the desaturase cytochrome b5-like domain and free endoplasmic reticulum cytochrome b5 for Delta6-desaturase activity.     

The role of the hydrophobic fragment of cytochrome b5 in the interaction with cytochrome P-450

The interaction of highly purified liver microsomal cytochrome P-450 from phenobarbital-induced rabbits and cytochrome b5 has been investigated by the difference and second derivative difference spectroscopy. The addition of cytochrome b5 to cytochrome P-450 results in transition of cytochrome P-450 heme iron from low to high spin state. The interaction is accompanied by the changes in the second derivative spectrum of cytochrome P-450, which point to the participation of tryptophanyl residues in this process. The hydrophilic fragment of cytochrome b5 is unable to form a complex with cytochrome P-450 as judged by the absence of the difference spectrum and any changes in the second derivative UV-spectrum of cytochrome P-450. The evidence obtained indicates that the hydrophobic tail of the cytochrome b5 molecule responsible for its binding to membrane is also indispensable for forming a functional cytochrome P-450-cytochrome b5 complex.     

The interaction of NADH-cytochrome b5 reductase and cytochrome b5 bound to egg lecithin liposomes.

Incubation of liposomes prepared by sonication of egg lecithin with the amphipathic form of cytochrome b5 results in the binding of a maximum of 244 molecules of cytochrome b5 per liposomal vesicle. Interactions of the phospholipid with the hydrophobic segment of cytochrome b5 are involved in this binding which does not disrupt the liposome. When a small amount of NADH-cytochrome b5 reductase is bound liposomes simultaneously with cytochrome b5, the two proteins catalyze the reduction of cytochrome c by NADH. A qualitative kinetic analysis reveals that all of the cytochrome b5 interacts with reductase, a result consistent with these protein undergoing translational diffusion in the plane of the membrane. This system and the purified stearyl coenzyme A desaturase provide a model to study the dynamics of protein andlipid interactions in this membrane-bound oxidative sequence.     

The hydrophobic domain of cytochrome b5 is capable of anchoring beta-galactosidase in Escherichia coli membranes.

Cytochrome b5 is inserted posttranslationally into membranes in vivo and spontaneously into liposomes in vitro by a short carboxyl-terminal hydrophobic membrane-anchoring sequence. DNA corresponding to this hydrophobic sequence has been synthesized, and two gene fusions with the Escherichia coli enzyme beta-galactosidase have been constructed by locating the hydrophobic domain in one case at the EcoRI site near the C terminus and in the other at the normal C terminus of the enzyme. The latter fusion protein was enzymatically active, having approximately 50% of the specific activity of beta-galactosidase, and cells expressing this protein grew normally with lactose as the sole carbon source. Both fusion proteins were localized to the E. coli inner membrane, converting beta-galactosidase from a cytoplasmic enzyme to a membrane-associated enzyme. The hydrophobic domain of cytochrome b5 therefore contains the information required to target polypeptides containing this domain to the membrane. Use of the cytochrome b5 hydrophobic peptide, either alone or in conjunction with other localizing sequences such as signal sequences, provides a general procedure for associating proteins with membranes. Polypeptides bearing this hydrophobic peptide may have considerable use as pharmaceuticals when associated with liposomes or cellular membranes.     

An enzymatically active chimeric protein containing the hydrophilic form of NADPH-cytochrome P450 reductase fused to the membrane-binding domain of cytochrome b5.

The microsomal flavoprotein NADPH-cytochrome P450 reductase (CPR) contains an N-terminal hydrophobic membrane-binding domain required for reconstitution of hydroxylation activities with cytochrome P450s. In contrast, cytochrome b5 (b5) contains a C-terminal hydrophobic membrane-binding domain required for interaction with P450s. We have constructed, expressed and purified a chimeric flavoprotein (hdb5-CPR) where the C-terminal 45 amino acid residues of b5 have replaced the N-terminal 56 amino acid domain of CPR. This hybrid flavoprotein retains the catalytic properties of the native CPR and is able to reconstitute fatty acid and steroid hydroxylation activities with CYP4A1 and CYP17A. However hdb5-CPR is much less effective than CPR for reconstituting activity with CYP3A4. We conclude that differences on the surface of the P450s reflect unique and specific information essential for the recognition needed to establish reactions of intermolecular electron transfer from the flavoprotein CPR.     

Purification by hydrophobic chromatography of soluble cytochrome b5 of human erythrocytes

Soluble cytochrome b5 of human erythrocytes was purified very effectively by hydrophobic chromatography using a butyl-Toyopearl 650 column. Cytochrome b5 was adsorbed tightly on the column in the presence of 60% saturated ammonium sulfate, and was eluted at 40% saturation of ammonium sulfate in the elution buffer. The chromatography gave a good yield of cytochrome b5 of the highest purity so far reported as estimated from the 414 nm to 280 nm absorbance ratio of the oxidized form of the cytochrome b5. The value obtained with the cytochrome b5 purified in this study was 6.57, and is higher than the previously reported highest value of 6.4 (Hultquist, D.E., Dean, R.T. and Douglas, R.H. (1974) Biochem. Biophys. Res. Commun. 60, 28-34). Spectral properties including molecular absorption coefficients were determined using the cytochrome b5 purified by this method.     

The alpha-helical membrane spanning domain of cytochrome b5 interacts with cytochrome P450 via nonspecific interactions

Cytochrome b5 (cyt b5) is an amphipathic membrane-bound heme protein found in the endoplasmic reticulum of eukaryotes. It consists of three domains, an N-terminal cytosolic, hydrophilic domain containing the heme, a short flexible linker and an alpha-helical membrane-spanning domain. This study investigated whether there are specific side chain helix-helix packing interactions between the COOH-terminal membrane anchor of cyt b5 and cytochrome P450 (cyt P450) 2B4 in a purified reconstituted system. Alanine was inserted at six positions in the membrane anchor of cyt b5. Insertion of alanine into an alpha-helix causes all amino acids at its carboxyl terminus to be rotated by 100 degrees . The ability of the alanine insertion mutants of cyt b5 to bind to cyt P450 2B4 was similar to that of the wild-type protein as was the ability of the mutant cyts b5 to stimulate the metabolism of the anesthetic, methoxyflurane. These results demonstrate that the C-terminal hydrophobic alpha-helix of cyt b5 does not interact with cyt P450 2B4 through a specific stereochemical fit of amino acid side chains, but rather through nonspecific interactions.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b5 has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b5-vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b5 upon the interaction with its redox partners is discussed.     

Membrane-bound progesterone receptors contain a cytochrome b5-like ligand-binding domain

Background

Membrane-associated progesterone receptors (MAPRs) are thought to mediate a number of rapid cellular effects not involving changes in gene expression. They do not show sequence similarity to any of the classical steroid receptors. We were interested in identifying distant homologs of MAPR better to understand their biological roles.

Results

We have identified MAPRs as distant homologs of cytochrome b ₅. We have also found regions homologous to cytochrome b ₅ in the mammalian HERC2 ubiquitin transferase proteins and a number of fungal chitin synthases.

Conclusions

In view of these findings, we propose that the heme-binding cytochrome b ₅ domain served as a template for the evolution of membrane-associated binding pockets for non-heme ligands.     

Fluorescence study of a mutant cytochrome b5 with a single tryptophan in the membrane-binding domain

Fluorescence studies of cytochrome b5 are complicated by the presence of three tryptophans, at positions 108, 109, and 112, in the membrane-binding domain. The cDNA for rabbit liver cytochrome b5, isolated from a lambda gt11 library, was used to generate a mutated mRNA where the codons for tryptophans-108 and -112 were replaced by codons for leucine. The sequence was expressed in Escherichia coli and the mutant protein was isolated. This mutant protein had the expected absorption spectrum, and its amino acid composition was confirmed by amino acid analysis and by DNA sequencing of the construct. The fluorescence emission spectrum of the mutant is blue-shifted and is narrower than that of the native protein. The quantum yield of the mutant protein, per molecule, is only 60% of that of the native protein, and the enhancement when bound to lipid vesicles or detergent micelles is higher for the mutant. Fluorescence anisotropy measurements and quenching studies using brominated lipids suggest that the fluorescence of the native protein is due to tryptophans-109 and -108 while tryptophan-112 does not emit but undergoes nonradiative energy transfer to tryptophan-108. With this mutant, it was shown that incomplete energy transfer from tyrosines-126 and -129 to tryptophan-109 occurs when the membrane binding domain is inserted into lipid vesicles, which suggests that the membrane-binding domain does not exist in a tight hairpin loop.     

NMR studies of the association of cytochrome b5 with cytochrome c

In an effort to gain greater insight into the molecular mechanism of the electron-transfer reactions of cytochrome b(5), the bovine cytochrome b(5)-horse cytochrome c complex has been investigated by high-resolution multidimensional NMR spectroscopy using (13)C, (15)N-labeled cytochrome b(5) expressed from a synthetic gene. Chemical shifts of the backbone (15)N, (1)H, and (13)C resonances for 81 of the 82 residues of [U-90% (13)C,U-90% (15)N]-ferrous cytochrome b(5) in a 1:1 complex with ferrous cytochrome c were compared with those of ferrous cytochrome b(5) in the absence of cytochrome c. A total of 51% of these residues showed small, but significant, changes in chemical shifts (the largest shifts were 0.1 ppm for the amide (1)H, 1.15 for (13)C(alpha), 1.03 ppm for the amide (15)N, and 0.15 ppm for the (1)H(alpha) resonances). Some of the residues exhibiting chemical shift changes are located in a region that has been implicated as the binding surface to cyt c [Salemme, F. R. (1976) J. Mol. Biol. 10, 563-568]. Surprisingly, many of the residues with changes are not located on this surface. Instead, they are located within and around a cleft observed to form in a molecular dynamics study of cytochrome b(5) [Storch, E. M., and Daggett, V. (1995) Biochemistry 34, 9682-9693](.) The rim of this cleft can readily accommodate cytochrome c. Molecular dynamics simulations of the Salemme and cleft complexes were performed for 2 ns and both complexes were stable.     

Interaction of cytochrome P-450scc with cytochrome b5

Spectrophotometric, affinity chromatography and cross-linking experiments provided evidence that cytochrome P-450scc from bovine adrenocortical mitochondria forms a tight complex with cytochrome b5 from rabbit liver microsomes. In the reconstituted system cholesterol side chain activity of cytochrome P-450scc was enhanced by the addition of cytochrome b5.     

Structure of cytochrome b5 unique to tardigrades

Cytochrome b₅ is an essential electron transfer protein, which is ubiquitously found in living systems and involved in wide variety of biological processes. Tardigrades (also known as water bears), some of which are famous for desiccation resistance, have many proteins unique to them. Here, we report spectroscopic and structural characterization of a cytochrome b₅ like protein from one of the desiccation‐tolerant tardigrades, Ramazzottius varieornatus strain YOKOZUNA‐1 (RvCytb₅). A 1.4 Å resolution crystal structure revealed that RvCytb₅ is a new cytochrome b₅ protein specific to tardigrades.     

Modification of trypsin-solubilized cytochrome b5, apocytochrome b5, and liposome-bound cytochrome b5 by diethylpyrocarbonate

The interactions of diethylpyrocarbonate (DEP) with the various forms of cytochrome b5 were studied to gain a better understanding of the factors that influence the extent of modification of the axial histidines of cytochrome b5. Very low concentrations of DEP were able to decrease the heme binding capacity of apocytochrome b5. Moreover, it was shown that two additional histidines, presumed to be the axial ligands (His 39 and 63), were modified in the apo but not the holo form of a given preparation of cytochrome b5. Trypsin-solubilized bovine cytochrome b5 was resistant to the effects of DEP. A 200-fold molar excess of DEP displaced only 15% of the heme in the trypsin-solubilized protein in contrast to an 84% displacement of the heme in the detergent-solubilized protein. However, detergent-solubilized cytochrome b5 which had been incorporated into phospholipid vesicles exhibited the same reactivity with DEP as did the trypsin-solubilized protein. This is attributed to the fact that the two resistant preparations of cytochrome b5 are monomeric in their respective environments while detergent-solubilized cytochrome b5 is known to exist as an octamer in aqueous solutions. Our studies suggest that dissociation of the octamer to the monomer results in a conformational change that decreases the reactivity of the axial ligands of the hydrophilic heme-containing domain of cytochrome b5. Examination of the cytochrome b5 molecule by computer graphics indicates that a tunnel leads from the surface of the molecule to axial histidine 63 and that axial histidine 39 is buried.     

Interaction of cholesterol hydroxylating cytochrome P-450 with cytochrome b5

Some new relations between cytochrome P-450-dependent monooxygenases were discovered. Cytochrome b5, a representative of "microsomal" monooxygenases, was shown to form a highly specific complex with cytochrome P-450scc, a member of the "ferredoxin" monooxygenase family. This interaction is characterized by a dissociation constant, Kd, of 0.28 microM. The cytochrome P-450scc-cytochrome b5 complex may be cross-linked with water-soluble carbodiimide. Using proteolytic modification of cytochrome b5, it was shown that both hydrophilic and hydrophobic fragments of cytochrome b5 are involved in the interaction with cytochrome P-450scc. Cytochrome b5 immobilized via amino groups is an effective affinity matrix for cytochrome P-450scc purification. The role of some amino acid residues in cytochrome P-450scc interaction with cytochrome b5 was studied. The role and the nature of complexes in cytochrome P-450-dependent monooxygenases as well as interrelationships between "microsomal" and "ferredoxin" monooxygenases are discussed.     

Topography of the membrane-binding domain of cytochrome b5 in lipids by Fourier-transform infrared spectroscopy

Fourier-transform infrared spectroscopy was used to examine the secondary structure of the membrane-binding domain (nonpolar peptide) of rabbit liver cytochrome b5 in D2O and in the presence of phospholipids and deoxycholate. In all situations, the predominant structure was alpha helix, but an examination of the components of the amide I band in the spectrum of the nonpolar peptide showed that the major peak was shifted from 1655 cm-1 in the lipids to 1650 cm-1 in deoxycholate. This shift to lower frequency, together with a decrease in intensity of the amide II band, is indicative of N-H to N-D exchange of the peptide backbone. A semiquantitative analysis indicated that the alpha helix of the peptide is over 95% exchanged in the presence of deoxycholate but is only 10% exchanged in the presence of lipid. These data suggest that the membrane-inserted portion of the peptide is alpha helical and is largely protected from N-H to N-D exchange by the bilayer. We suggest that this technique appears to provide a general method for determining the type of secondary structure involved in membrane interaction and the percentage of this structure which is involved in the interaction.     

A comparative analysis of the structure of the myoglobin, cytochrome b5, and alpha-chymotrypsin hydrophobic nuclei

The myoglobin, cytochrome b5 and alpha-chymotrypsin hydrophobic nucleus sizes were calculated as well as sizes of theoretical spherical nucleus, radii that are equal to the lengths of phenylalanine and tryptophan lateral groups. All calculated values of sizes lie in the (0.99-1.65) nm3 interval. The quantitative estimation of analyzed proteins nucleus heterogeneous composition has been shown.     

Interaction of purified microsomal cytochrome P-450 with cytochrome b5

The interactions between purified microsomal cytochrome P-450 and cytochrome b₅ has been demonstrated by aqueous two-phase partition technique. Major forms of cytochrome P-450 induced by phenobarbital (P-450_LM2) and β-naphthoflavone (P-450_LM4) are almost exclusively distributed in the dextran-rich bottom phase (partition coefficient, K = 0.06), whereas NADPH-cytochrome P-450 reductase and cytochrome b₅ are mainly distributed in the polyethylene glycol-rich top phase (K = 3.5 and 2.5, respectively), when these enzymes were partitioned separately in the dextran-polyethylene glycol two-phase system. The mixing of P-450_LM with cytochrome b₅ changes the partition coefficients of both P-450_LM and cytochrome b₅ indicating that molecular interaction between P-450_LM and cytochrome b₅ occurred. Complex formation was also confirmed by optical absorbance difference spectral titration, and the stimulation of the P-450_LM-dependent 7-ethoxycoumarin and p-nitrophenetole O-deethylase activities by equal molar quantity of detergent-solubilized cytochrome b₅, but not trypsin-solubilized enzyme, in the reconstituted system. Cytochrome b₅ decreases the K_m's of both substrates for P-450_LM2-dependent O-deethylations and increases the V's of both reactions by two- to three-fold. This stimulatory effect requires the presence of phospholipid in the reconstituted enzyme system. These results suggest that cytochrome b₅ plays a role in some reconstituted drug oxidation enzyme systems and that molecular interactions among cytochrome P-450, reductase, and cytochrome b₅ are catalytically competent in the electron transport reactions.     

Chromosomal localization of a cytochrome b5 gene to human chromosome 18 and a cytochrome b5 pseudogene to the X chromosome.

We have isolated cDNA clones that code for human cytochrome b5. Owing to the high degree of evolutionary conservation of cytochrome b5 sequences and the existence of human and rodent cytochrome b5 processed pseudogenes, we were unable to map unambiguously the chromosomal localization of the human gene(s) by Southern blot hybridization of DNA from human-rodent somatic cell hybrids. An alternative approach, based on restriction enzyme digestion of PCR-amplified DNA, enabled us to map the human cytochrome b5 gene(s) to chromosome 18 and one of its processed pseudogenes to the X chromosome. We propose the designations CYB5 and CYB5P1 for the gene and pseudogene loci, respectively.