A study of the electron transfer properties of the heme undecapeptide from cytochrome c by 1H nmr spectroscopy

Nuclear magnetic resonance (nmr) spectroscopy has been used to investigate the heme undecapeptide from cytochrome c. Assignments of resonances to specific residues have been made based on spin decoupling, redox titration, and the pH and temperature dependence of resonance lines. An outline structure is presented based on the assignments, secondary shift data, and the x-ray crystal structure of cytochrome c. An equation is derived to relate the width of an nmr line during a redox titration to the percentage of each oxidation state. Using this equation the self-exchange rate constant for electron transfer for the heme peptide is 1.3 x 10(7) M-1 sec-1 at 330 degrees K. Discussion of the self-exchange rate constants of cytochrome c, cytochrome c3, and cytochrome c551 is related to this constant for the heme undecapeptide.     

Studies on cytochrome C. XIII. Synthesis of the protected undecapeptide (sequence 77-87) of horse heart cytochrome c.

A solution synthesis of Z-Gly-Thr-Lys (Tfa)-Met-Ile-Phe-Ala-Gly-Ile-Lys (Tfa)-Lys (Tfa)-NHNH-Boc corresponding to the sequence 77-87 of horse heart cytochrome c is described. The protected undecapeptide was obtained from intermediate hepta- and tetrapeptide fragments by an azide coupling.     

The purification and Mössbauer parameters of the haem undecapeptide of cytochrome c

A new method of preparing and purifying the haem undecapeptide of cytochrome c is reported. The Mössbauer spectra of solid samples, lyophilized at pH 7 from water, show mainly the presence of low-spin ferric iron, in contrast with earlier reports. No evidence of temperature dependent spin-spin equilibria was observed. A small proportion of the haem (～ 15%) inhabits an environment distinctly different from that of the majority. These observations are discussed.     

Evidence for an arginine residue at the coenzyme-binding site of Escherichia coli isocitrate dehydrogenase.

Immobilization of biological systems in solid matrices is presently of great interest, in view of the many potential advantages associated with both the higher stability of the immobilized macromolecules and the potential utilization for biotechnology. In the present paper the electrochemical behaviour of the undecapeptide from cytochrome c (called microperoxidase) tightly entrapped in cellulose triacetate membrane is reported; its utilization as 'solid-state' promoter in the electrochemistry of soluble metalloproteins is presented. The results obtained indicate that: (i) membrane-entrapped microperoxidase undergoes rapid reversible electron transfer at a glassy carbon electrode; (ii) the electrochemical process is diffusion-controlled; (iii) entrapped microperoxidase acts as 'solid-state' promoter in the electrochemistry of soluble cytochrome c and of azurin.     

A kinetic study of the pH-dependent properties of the ferric undecapeptide of cytochrome c (microperoxidase).

The ferric form of the haem undecapeptide, derived from horse cytochrome c by peptic digestion, undergoes at least three pH-induced transitions with pK values of 3.4, 5.8 and 7.6. Temperature-jump experiments suggest that the first of these is due to the binding of a deprotonated imidazole group to the feric iron while the second and third arise from the binding of the two available amino groups present (the alpha-NH2 of valine and the epsilon-NH2 of lysine). Molecular models indicate that steric retraints on the peptide dictate that these amino groups may only coordinate to iron atoms via intermolecular bonds, thus leading to the polymerization of the peptide. Cyanide binding studies are in agreement with these conclusions and also yield a value of 3.6 X 10(6) M-1 s-1 for the intrinsic combination constant of CN- anion with the haem. A model is proposed which describes the pH-dependent properties of the ferric undecapeptide.     

Magnetic studies on the ferri-haem undecapeptide of cytochrome c.

The pH-dependence of the magnetic moment of a ferri-haem undecapeptide, produced by peptic digestion of cytochrome c, has been measured in aqueous solution using a nuclear magnetic resonance method. Below pH 3 the magnetic moment is consistent with the presence of hydroxo-bridged dimers of high-spin iron(III). Above pH 7 the iron(III) is low-spin, the spin crossover conforming to a simple titration curve with pK 6.3 (n=1). This transition is discussed with reference to spectrophotometric studies of ligand binding at the haem.     

Antibodies against the COOH-terminal undecapeptide of subunit II, but not those against the NH2-terminal decapeptide, immunoprecipitate the whole human cytochrome c oxidase complex

Antibodies against synthetic peptides derived from the DNA sequence of human cytochrome c oxidase subunit II (COII) have been tested for their capacity to immunoprecipitate the whole enzyme complex. Antibodies against the COOH-terminal undecapeptide of COII (anti-COII-C), when incubated with a Triton X-100 mitochondrial lysate from HeLa cells pulse-labeled with [35S]methionine under conditions selective for mitochondrial protein synthesis and chased for 18 h in unlabeled medium, precipitated the pulse-labeled three largest subunits (mitochondrially synthesized) of cytochrome c oxidase in proportions close to equimolarity. Antibodies against the NH2-terminal decapeptide of COII (anti-COII-N), although equally reactive as the anti-COII-C antibodies with the sodium dodecyl sulfate-solubilized COII, did not precipitate any of the three labeled subunits from the Triton X-100 mitochondrial lysate. In other experiments, all the 13 subunits which have been identified in the mammalian cytochrome c oxidase were immunoprecipitated from a Triton X-100 mitochondrial lysate of cells long-term labeled with [35S]methionine by anti-COII-C antibodies, but not by anti-COII-N antibodies. By contrast, in immunoblots of total mitochondrial proteins dissociated with sodium dodecyl sulfate, the anti-COII-C antibodies reacted specifically only with COII. These results strongly suggest that, in the native cytochrome c oxidase complex, the epitope recognized by the anti-COII-C antibodies is in the COII subunit and that, therefore, in such complex, the COOH-terminal peptide of COII is exposed to antibodies, whereas the NH2-terminal peptide is not accessible.     

Polymethionyl,-valyl and-glycyl derivatives of equine heart cytochromec heme octapeptide

Polymethionyl ((Met)_5·3-H8PT), polyvalyl ((Val)_4·4-H8PT) and polyglycyl ((Gly)_3·2-H8PT) derivatives of the heme octapeptide of equine heart cytochrome c were prepared with the aid of the N-carboxyl anhydrides of their respective amino acids. Only for the (Met)_5·3-H8PT did the γ-peak in the absorption spectrum of the reduced form shift from 412.5 nm, the value for the unsubstituted octapeptide, to 414 nm, the value close to the γ-peak of intact ferrocytochrome c. The γ-peak of the oxidized form of the octapeptide did not shift significantly from 397 nm. Amino acids attached to the N-terminal cysteine of the octapeptide, with methionine excepted, have little or no effect on the spectrum, and apparently cannot serve as either an intramolecular or intermolecular ligand for the iron. This result is in marked contrast to those previously obtained with the substituted cytochrome c heme undecapeptide (ref. 8) where all three derivatives had altered spectra. For the octapeptide, the ratio, absorbance of the γ-peak of the oxidized form to absorbance of the γ-peak of the reduced form, did not change appreciably for any of the derivatives, although when the reaction products from excess methionine anhydride were present (before centrifugation and dialysis) the ratio was lower and approached the ratio for cytochrome c.     

State of heme in heme c systems: Cytochrome c and heme c models

Polarized resonance Raman spectra of horse heart ferricytochrome c as a function of pH in the range 1.0–12, in the presence of the extrinsic ligands imidazole, cyanide, and azide, and in 4 M urea, are reported, as are resonance Raman spectra of heme undecapeptide in the presence of imidazole, pH 6.8 and pH 2.0, and with cyanide at pH 6.8. The range of investigation is 140–1700 cm^?1, using the 5145-, 4880-, and 4579-Å excitations. The spectra have been analyzed in terms of complexity, sensitivity, and the conformation-heme energetics of the systems. The state of heme in various forms is analyzed with regard to heme energetics, core size, nature of planarity, and coordination configuration. All low-spin forms of heme c systems, cytochrome c, and heme models are concluded to be hexacoordinated, in-plane heme iron systems. The effect of the location of the heme in the protein environment is found to be a slight expansion of the porphyrin core, ～0.01 Å, while the covalent linkage of heme to protein and a mixed nature of axial coordination configuration seem to have little effect on the energetics of the heme group. Complex formation with extrinsic ligand, imidazole, cyanide, or azide, results in a slight contraction of the heme core. The formation of cytochrome c form IV, the alkaline form, is shown to follow a process with apK _a of about 8.4, and similarly, acidic form II is created following the prior formation of an intermediate form with apK _a of about 3.6. The precursor to form IV is interpreted as containing perturbation of the pyrrol rings, whereas the precursor to the acidic form seems to reflect alteration of the energetics of the C_αC_{m α} structures of the heme group. The acidic form of heme undecapeptide is a hexacoordinated high-spin heme with an estimated displacement of 0.25 Å from the heme plane. The pH 2 form of cytochrome c is also a hexacoordinated high-spin form with two weak axial ligands, but iron is in the plane of the porphyrin ring.     

Conformation of horse heart ferricytochrome c. III. Comparative optical rotatory dispersion study of the protein with its derivative heme undecapeptide

The optical rotatory dispersion of horse heart ferricytochrome c and of a ferri heme undecapeptide have been determined under various conditions. Analysis of the Soret region makes it possible to characterize three different states of ferricytochrome c. the native state (superposition of a negative and a positive Cotton effect); an intermediate state (single positive Cotton effect whose magnitude Δ[M] is equal to 55,000); a denatured state (single positive Cotton effect whose magnitude Δ[M] is equal to 115,000) in which compared to both the native and intermediate states a more or less important decrease in helix content is observed. The optical rotatory dispersion spectra of the Soret region of the monomeric ferri heme undecapeptide is similar to that of denatured ferricytochrome c. The multiplicity of Cotton effects observed under certain conditions for the hemopeptide is a consequence, resulting from a polymerization, of intermolecular interactions. The comparison of the optical rotatory dispersion spectra of ferricytochrome c and the ferri heme undecapeptide indicates that in the intermediate state interactions remain between the heme group and the portion of the poly pep tide chain absent in the hemopeptide. These interactions disappear in the denatured state.     

A microsomal ecdysone-binding cytochrome P450 from the insect Locusta migratoria purified by sequential use of type-II and type-I ligands.

A dual-affinity method was established to purify, for the first time, a microsomal ecdysone-binding cytochrome P450 protein from locust Malpighian tubules. This method involved, after prepurification on omega-octylamino-agarose and hydroxylapatite, binding of cytochrome P450 to an immobilized triazole-based general P450 inhibitor (type-II ligand) followed by elution with the substrate ecdysone (type-I ligand) of the bound cytochrome. The isolated material showed a typical cytochrome P450 spectrum, a specific heme content of 13 nmol/mg protein, and a prominent protein of about 60 kDa on SDS-PAGE. Based on a tryptic undecapeptide sequence the isolated protein may be identical to CYP6H1, a putative ecdysone 20-monooxygenase recently cloned from the same tissue. Ecdysone 20-monooxygenase activity could be partially reconstituted from microsomal detergent extracts, when supplemented with purified bovine cytochrome P450 reductase and detergent-extracted microsomes; reconstitution was not successful with any chromatographic fraction, however. Therefore, purification of the locust cytochrome P450 was monitored by ecdysone-induced type-I difference spectra, whenever applicable, in addition to carbon monoxide spectra. Affinity columns with matrix-bound diethylstilbestrol and testosterone 3-thiosemicarbazone, but not with the 17beta-hemisuccinate, yielded elution profiles with ecdysone that were comparable to those of the triazole matrix. The concept of dual-affinity chromatography described here may be generally applicable to the isolation of cytochromes P450.     

In vitro effects of corticosteroids, DDT, and 4, 9-dichlorodibenzodioxin on rat liver xanthine oxidase activity. Interactions between xanthine oxidase and cytochrome P450 in rat liver in vivo

It was established that deoxycorticosterone, cortisol, dexamethasone, DDT, and 4,9-dichlorodibenzodioxin inhibit in vitro binding of xanthine to highly purified rat liver xanthine oxidase. They are suggested to be allosteric inhibitors. The corresponding inhibition and binding constants were estimated. Also established was that cortisol and DDT, in dose 20 mg per kg of weight, inhibit xanthine oxidase and increase microsomal cytochrome P450 level in rat liver. There is an inverse relationship between xanthine oxidase activity and cytochrome P450 level in rat liver.     

Hepatic mixed function oxidase system and effect of phenobarbital, 3-methylcholanthrene and 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl)ethane in developing chickens.

Contents of hepatic microsomal protein, aminopyrine N-demethylase, acetanilide hydroxylase, aniline hydroxylase, hydrogen peroxide formation, cytochrome-c-reductase, cytochrome b5 and cytochrome P-450 were examined in control, phenobarbital (PB), 3-methylcholanthrene (3-MC) and 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl)ethane (DDT) treated group of 1-28 days old chickens. Increase in aminopyrine N-demethylase, acetanilide hydroxylase, aniline hydroxylase, cytochrome-c-reductase, cytochrome b5 and cytochrome P-450 was noticed at all stages of development during administration of PB and 3-MC. But these enzyme activities were not always paralleled by increase in age. Aminopyrine N-demethylase was increased in early stages only during DDT administration, which indicates that the form of cytochrome P-450, responsible for aminopyrine N-demethylation is present in early stages only. However, acetanilide hydroxylase was decreased in all stages of development, in postnatal development the basal activities of the enzymes for various substrates do not exhibit identical pattern, the degree of inducibility by inducers varied in relation to age of animal. Hydrogen peroxide formation increased in all stages of developing chickens due to the administration of PB and DDT. It however decreased due to 3-MC administration which may be due to induction of high spin cytochrome P-450.     

Effect of DDT on hepatic mixed-function oxidase activity in normal and vitamin A-deficient rats

Feeding of vitamin A-deficient diet to male weanling rats for 10 weeks resulted in significant decrease in the body weight and marked reduction in the hepatic vitamin A content. The levels of hepatic phase I microsomal enzymes cytochrome P-450, cytochrome b5, aminopyrine N-demethylase and arylhydrocarbon hydroxylase were found to be substantially reduced by vitamin A-deficiency. Also, the activity of phase II microsomal UDP - glucuronyl transferase enzyme was significantly decreased in deficient animals. Following repeated oral administration of DDT (15 mg/kg/body wt/day) for 21 days, the phase I microsomal enzymes were induced to a greater extent in controls as compared to deficient animals. UDP - glucuronyltransferase remained insensitive to DDT induction. The results imply that the capacity for induction of the hepatic mixed-function oxidase enzyme system is impaired in deficient animals concurrently exposed to DDT.     

Phosphorylation in the carboxyl-terminal domain of the capsid protein of hepatitis B virus: evaluation with a monoclonal antibody.

The capsid protein of hepatitis B virus (p21c) is made of 183 amino acids coded for by the C gene. By using p21c isolated from Dane particles (hepatitis B virus) as an immunogen, a monoclonal antibody (no. 2212) which recognized an epitope dependent on the phosphorylation of p21c was raised. The binding of no. 2212 antibody to authentic p21c was completely inhibited by a synthetic undecapeptide with a sequence of RRRSQSPRRRR, representing amino acids 165 to 175 of p21c, only when the peptide was phosphorylated. Either or both of Ser-168 and Ser-170 were phosphorylated in p21c in vivo, therefore, and contributed to the manifestation of the epitope. No. 2212 antibody bound to p21c from core particles derived from Dane particles or hepatocellular carcinoma tissues (PLC/342) propagated in nude mice but did not bind to p21c from core particles expressed in Escherichia coli or yeast cells, indicating different states of phosphorylation in them. Nonphosphorylated p21c showed a higher affinity for the viral DNA than did phosphorylated p21c. Since the serum from an asymptomatic carrier, with a high titer for antibody to hepatitis B core antigen, specifically bound to phosphorylated undecapeptide (amino acids 165 to 175), the epitope would stimulate humoral antibody responses in the human host.     

Phytoremediation of trichloroethylene and dichlorodiphenyltrichloroethane-polluted water using transgenic Sesbania grandiflora and Arabidopsis thaliana plants harboring rabbit cytochrome p450 2E1

Sesbania grandiflora (L.) pers (Fabaceae) and Arabidopsis thaliana (L.) (Brassicaceae) were genetically engineered to constitutively express the rabbit cytochrome p450 2E1 enzyme aiming at increasing their activity toward trichloroethylene (TCE) and dichlorodiphenyltrichloroethane (DDT) removal Successful generation of Sesbania and Arabidopsis transgenic plants was verified using p450 2E1 specific PCR and confirmed by western blot analysis. Gas chromatography (GC) analysis revealed that small cuttings of Sesbania and third generation (F3) Arabidopsis transgenic plants exposed to TCE and DDT in small hydroponics' vessels accumulated more TCE and DDT compared to plants transformed with the empty vector. Furthermore, both transgenic plants were more effective in breaking down TCE and DDT with a 2-fold increase in TCE metabolism. Two independent Arabidopsis lines showed that DDT was metabolized about 4-fold higher than that detected in non transformed plants. Similarly, S. grandiflora cuttings removed 51 to 90% of the added DDT compared with only 3% removal in controls transformed with the null vector. Notably, stability of rabbit cytochrome p450 2E1 was confirmed using third generation Arabidopsis plants that displayed higher potential for the removal of two important pollutants, TCE and DDT compared with the controls.     

Purification of the heme peptide of cytochrome c by affinity chromatography

The ferriporphyrin c-peptide obtained from horse heart cytochrome c by digestion with pepsin can be purified by affinity chromatography on bovine or human serum albumin coupled to Sepharose. Amino acid analysis of the peptide isolated by this method is identical to the undecapeptide isolated by Tsou (4) and sequenced by Tuppy and Paleus (5).     

Effects of dietary carbohydrate and myo-inositol on metabolic changes in rats fed 1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane (DDT)

This study was conducted to examine the effects of dietary carbohydrate [starch or sucrose (500 g/kg diet)] and myo-inositol (2 g/kg diet) on metabolic changes in rats fed 1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane (DDT) (0.7 g/kg diet). Dietary DDT enhanced serum and hepatic lipids and hepatic thiobarbituric acid reactive substances (TBA-RS), elevated hepatic activities of lipogenic enzymes such as malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PD) and fatty acid synthetase (FAS), increased hepatic cytochrome P-450 content and the activities of drug-metabolizing enzymes such as aminopyrine N-demethylase, glutathione S-transferase and 4-nitrophenol-UDP glucuronosyltransferase (4NP-UDPGT) and raised hepatic ascorbic acid and serum copper. Dietary sucrose promoted the increases in hepatic concentrations of total lipids, triglyceride and cholesterol, hepatic activity of ME, hepatic TBA-RS, cytochrome P-450 content and serum copper due to DDT feeding when compared to DDT administered in a starch based diet. Dietary myo-inositol significantly depressed the rises in hepatic concentrations of total lipids, triglyceride and cholesterol and the activities of ME and G6PD due to DDT feeding regardless of dietary carbohydrate quality. Dietary starch supplemented with myo-inositol potentiated the enhancements in hepatic activities of Phase II drug-metabolizing enzymes such as glutathione S-transferase and 4NP-UDPGT due to DDT feeding. These results suggest that dietary starch and myo-inositol can protect DDT fed rats against an accumulation of hepatic lipids, which might be mainly ascribed to the depression of hepatic lipogenesis. In addition, the present study implies that the supplementation of myo-inositol to high starch diet might improve the function of drug-metabolizing enzymes exposed to DDT.     

Antibodies against synthetic peptides reveal that the unidentified reading frame A6L, overlapping the ATPase 6 gene, is expressed in human mitochondria

Antibodies prepared against chemically synthesized peptides predicted from the DNA sequence have been used to detect human mitochondrial gene products. In particular, antibodies directed against either the NH2-terminal decapeptide or the COOH-terminal undecapeptide of cytochrome c oxidase subunit II (COII) were both very effective in immunoprecipitating the previously identified COII polypeptide from an SDS lysate of mitochondria from HeLa cells. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative polypeptide encoded in the unidentified reading frame A6L, which overlaps the ATPase 6 gene, immunoprecipitated specifically a component (#25) of the HeLa cell mitochondrial translation products; antibodies directed against the NH2-terminal octapeptide also precipitated protein 25, although less efficiently. The size of protein 25, as estimated from its electrophoretic mobility, is compatible with its being the unidentified reading frame A6L product. Furthermore, a fingerprinting analysis of this protein after trypsin digestion has given results consistent with this identification.     

On the preparation and mössbauer properties of some heme peptides of cytochrome c

The preparations of a series of peptides derived from horse heart cytochrome c by proteolytic digestions are reported. The Mössbauer spectra of the hexa- and nonapeptides are reported here for the first time and compared with those of the undecapeptide and the parent cytochrome. The nona- and undecapeptides have Mössbauer spectra similar to that of ferricytochrome c and would appear to be useful models for the iron environment. As with free hemes, pyridine may act as a reducing agent, and thus we wish to record a caveat against the use of this compound during peptide preparation.