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PubMed1982

Laser flash photolysis studies of electron transfer between semiquinone and fully reduced free flavins and the cytochrome c-cytochrome oxidase complex.

Laser flash photolysis has been used to determine the rate constants for the reduction of bovine cytochrome oxidase and the cytochrome c-cytochrome oxidase complex by the semiquinone and fully reduced forms of various flavin analogues (FH. and FH-, respectively). Under the condition used, the reaction of FH. with free cytochrome oxidase is too slow to compete with FH. disproportionation whereas FH- reacts measurably. Both FH. and FH- are effective in reducing the complex. The reduction of heme a in the complex is shown to proceed via cytochrome c, and a limiting first-order rate is observed in the case of FH- at high complex concentrations. The data indicate that the interaction site for electron transfer to cytochrome c is the same in the complex as with the free protein, and although a tight complex exists, at least small reactants like the flavins are not sterically hindered in their access to the bound cytochrome c. Moreover, the results also establish that intramolecular electron transfer between cytochrome c and cytochrome oxidase within the complex occurs with a first-order rate constant of greater than 700 s-1. Thus, the presence of cytochrome c greatly enhances electron transfer from reduced flavins to cytochrome oxidase.

Animals↗
PubMed1982

Cytochrome c is cross-linked to subunit II of cytochrome c oxidase by a water-soluble carbodiimide.

Modification of beef heart cytochrome c oxidase with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) or 1-ethyl-3-[3-(trimethylamino)propyl]carbodiimide (CH3EDC) has been found to significantly inhibit the high-affinity phase of the reaction of this enzyme with cytochrome c. Reaction conditions leading to a 70% inhibition of Vmax resulted in a 16-fold increase in the Km for cytochrome c. The loss in activity was accompanied by modification of subunit II to form a new species, II', which migrated somewhat more rapidly than the unmodified subunit during sodium dodecyl sulfate (NaDodSO4) gel electrophoresis. This new species was the major site of radiolabeling when cytochrome c oxidase was treated with [14C]CH3EDC, indicating covalent incorporation of the carbodiimide. Equimolar concentrations of cytochrome c dramatically protected cytochrome c oxidase from inhibition by the carbodiimide and in approximately the same proportion shielded subunit II from modification to the labeled II' species. In addition, cytochrome c was cross-linked to subunit II to form a new species migrating somewhat faster than subunit I during NaDodSO4 gel electrophoresis. This cross-linked species was shown to contain subunit II by using subunit-specific antibodies. We propose that EDC or CH3EDC reacts with one or more partially buried carboxyl groups on subunit II to form a positively charged N-acylurea which inhibits cytochrome c binding. In the presence of cytochrome c, EDC promotes formation of amide cross-links between lysine amino groups on cytochrome c and their complementary carboxyl groups on cytochrome c oxidase.

Animals↗
PubMed1998

Cytochrome c-induced increase of motionally restricted lipid in reconstituted cytochrome c oxidase membranes, revealed by spin-label ESR spectroscopy.

Cytochrome c oxidase isolated from beef heart mitochondria was reconstituted in bilayer membranes of the anionic lipid dimyristoylphosphatidylglycerol (DMPG) with varying enzyme/DMPG ratio. Lipid-protein interactions in the reconstituted membrane complexes were studied in the presence and absence of saturating amounts of bound cytochrome c, by both chemical binding assays and spin-label ESR spectroscopy. The ESR spectra from a phosphatidylglycerol probe spin-labeled on C-14 of the sn-2 chain revealed two distinct lipid populations differing in their rotational mobility. The stoichiometry of lipids that were restricted in their rotational motion by direct interaction with the integral protein was 50-60 lipids/cytochrome c oxidase monomer, in the absence of cytochrome c, independent of the total lipid/protein ratio. Cytochrome c alone did not induce a motionally restricted population in the lipid ESR spectra, when bound to bilayers of negatively charged DMPG alone, in the fluid phase (at 36 degreesC). However, the motionally restricted lipid population associated with reconstituted cytochrome c oxidase/DMPG membranes increased on binding cytochrome c, indicating structural/dynamic changes taking place in the membrane. Depending on the DMPG/cytochrome c oxidase ratio, apparent stoichiometries of up to 115 motionally restricted lipid molecules/cytochrome c oxidase monomer were found, when saturating amounts of cytochrome c were bound. Under these conditions, cytochrome c binds to approximately 9 negatively charged DMPG molecules, independent of the cytochrome c oxidase content in the reconstituted system. A likely explanation for these results is that the surface binding of cytochrome c propagates the motional restriction of the lipid chains beyond the first boundary shell of cytochrome c oxidase, possibly creating microscopic in-plane domains.

Animals↗
PubMed1985

Cytochrome c-551 and azurin oxidation catalysed by Pseudomonas aeruginosa cytochrome oxidase. A steady-state kinetic study.

The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a 'half-hearted' behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady-state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c-551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism.

Apoproteins↗
PubMed1989

Ionic-strength-dependence of the oxidation of native and pyridoxal 5'-phosphate-modified cytochromes c by cytochrome c oxidase.

The ionic-strength-dependences of the rate constants (log k plotted versus square root of 1) for oxidation of native and pyridoxal 5'-phosphate-modified cytochromes c by three different preparations of cytochrome c oxidase have complex non-linear character, which may be explained on the basis of present knowledge of the structure of the oxidase and the monomer-dimer equilibrium of the enzyme. The wave-type curve (with a minimum and a maximum) for oxidation of native cytochrome c by purified cytochrome c oxidase depleted of phospholipids may reflect consecutively inhibition of oxidase monomers (initial descending part), competition between this inhibition and dimer formation, resulting in increased activity (second part with positive slope), and finally inhibition of oxidase dimers (last descending part of the curve). The dependence of oxidation of native cytochrome c by cytochrome c oxidase reconstituted into phospholipid vesicles is a curve with a maximum, without the initial descending part described above. This may reflect the lack of pure monomers in the vesicles, where equilibrium is shifted to dimers even at low ionic strength. Subunit-III-depleted cytochrome c oxidase does not exhibit the maximum seen with the other two enzyme preparations. This may mean that removal of subunit III hinders dimer formation. The charge interactions of each of the cytochromes c (native or modified) with the three cytochrome c oxidase preparations are similar, as judged by the similar slopes of the linear dependences at I values above the optimal one. This shows that subunit III and the phospholipid membrane do not seem to be involved in the specific charge interaction of cytochrome c oxidase with cytochrome c.

Cytochrome c Group↗
PubMed2002

Crystal structure of the yeast cytochrome bc1 complex with its bound substrate cytochrome c.

Small diffusible redox proteins facilitate electron transfer in respiration and photosynthesis by alternately binding to integral membrane proteins. Specific and transient complexes need to be formed between the redox partners to ensure fast turnover. In respiration, the mobile electron carrier cytochrome c shuttles electrons from the cytochrome bc1 complex to cytochrome c oxidase. Despite extensive studies of this fundamental step of energy metabolism, the structures of the respective electron transfer complexes were not known. Here we present the crystal structure of the complex between cytochrome c and the cytochrome bc1 complex from Saccharomyces cerevisiae. The complex was crystallized with the help of an antibody fragment, and its structure was determined at 2.97-A resolution. Cytochrome c is bound to subunit cytochrome c1 of the enzyme. The tight and specific interactions critical for electron transfer are mediated mainly by nonpolar forces. The close spatial arrangement of the c-type hemes unexpectedly suggests a direct and rapid heme-to-heme electron transfer at a calculated rate of up to 8.3 x 10(6) s(-1). Remarkably, cytochrome c binds to only one recognition site of the homodimeric multisubunit complex. Interestingly, the occupancy of quinone in the Qi site is higher in the monomer with bound cytochrome c, suggesting a coordinated binding and reduction of both electron-accepting substrates. Obviously, cytochrome c reduction by the cytochrome bc1 complex can be regulated in response to respiratory conditions.

Benzoquinones↗
PubMed1976

Interaction of integral and peripheral membrane proteins: affinity labeling of yeast cytochrome oxidase by modified yeast cytochrome c.

To identify possible substrate-binding subunit(s) of yeast cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1-9-3-1), the purified enzyme was reacted with yeast iso-1-cytochrome c whose single free sulfhydryl group at position 107 had been activated with 5,5'-dithiobis(2-nitrobenzoate). The resulting cytochrome c derivative appeared to function as an "affinity-label" of cytochrome oxidase, since it rapidly inactivated the enzyme. Inactivation was competitively prevented by underivatized cytochrome c. When the "affinity-labeled" oxidase was analyzed by two-dimensional polyacrylamide electrophoresis in dodecyl sulfate (separation in the second dimension being carried out in the presence of excess sulfhydryl compound), it was found that the derivatized cytochrome c had specifically formed a mixed disulfide with the mitochondrially made subunit III (apparent molecular weight 24,000) of the oxidase. Similar results were obtained when underivatized iso-I-cytochrome c was crosslinked to the oxidase by oxidative disulfide bridge formation in the presence of ortho-phenanthroline and Cu++. These data indicate that the hydrophobic mitochondrially made subunit III of yeast cytochrome c oxidase is in close proximity to the cytochrome c binding site on the enzyme. Since cytochrome c and the mitochondrially made cytochrome oxidase subunit III are typical peripheral and integral membrane proteins, respectively, the present study suggests a useful approach for analyzing specific interactions between these different classes of membrane proteins.

Affinity Labels↗
PubMed1999

Equilibrium unfolding of a small low-potential cytochrome, cytochrome c553 from Desulfovibrio vulgaris.

To understand general aspects of stability and folding of c-type cytochromes, we have studied the folding characteristics of cytochrome c553 from Desulfovibrio vulgaris (Hildenborough). This cytochrome is structurally similar but lacks sequence homology to other heme proteins; moreover, it has an abnormally low reduction potential. Unfolding of oxidized and reduced cytochrome c553 by guanidine hydrochloride (GuHCl) was monitored by circular dichroism (CD) and Soret absorption; the same unfolding curves were obtained with both methods supporting that cytochrome c553 unfolds by an apparent two-state process. Reduced cytochrome c553 is 7(3) kJ/mol more stable than the oxidized form; accordingly, the reduction potential of unfolded cytochrome c553 is 100(20) mV more negative than that of the folded protein. In contrast to many other unfolded cytochrome c proteins, upon unfolding at pH 7.0 both oxidized and reduced heme in cytochrome c553 become high-spin. The lack of heme misligation in unfolded cytochrome c553 implies that its unfolded structure is less constrained than those of cytochromes c with low-spin, misligated hemes.

Circular Dichroism↗
PubMed1989

Purification and characterization of cytochrome P-450sca from Streptomyces carbophilus. ML-236B (compactin) induces a cytochrome P-450sca in Streptomyces carbophilus that hydroxylates ML-236B to pravastatin sodium (CS-514), a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase.

Pravastatin sodium (CS-514) is a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme in cholesterol biosynthesis. This compound is obtained by microbial hydroxylation of sodium ML-236B (compactin) carboxylate. The soluble cytochrome P-450 was induced by sodium ML-236B carboxylate in Streptomyces carbophilus of Actinomycetes as detected in its cell-free extract. This cytochrome P-450 was designated as cytochrome P-450sca after its origin. Cytochrome P-450sca was purified by successive chromatography on anion-exchange, gel filtration and hydroxyapatite columns. On hydroxyapatite cytochrome P-450sca was further separated into minor and major peaks, designated cytochrome P-450sca-1 and cytochrome P-450sca-2, respectively. Each peak yielded a single band on sodium dodecyl sulfate/polyacrylamide gels with molecular masses of 46 +/- 1 kDa. The activity hydroxylating sodium ML-236B carboxylate to pravastatin sodium was reconstituted in the presence of an electron transport system, an NADPH-generating system and oxygen. The Ks values of the cytochromes P-450sca-1 and P-450sca-2 for sodium ML-236B carboxylate were 179 microM and 229 microM, respectively. The CO versus reduced difference spectra of both cytochromes P-450 showed an absorption maximum at 448.5 nm. Their substrate difference spectra with sodium ML-236B carboxylate showed an absorption maximum at 386 nm. Amino acid analysis indicated that cytochrome P-450sca-1 and P-450sca-2 contained 46% and 47% hydrophobic residues, respectively. On Western blotting, cytochromes P-450sca-1 and P-450sca-2 were immunologically identical.

Amino Acids↗
PubMed1990

Flavin-photosensitized oxidation of reduced c-type cytochromes. Reaction mechanism and comparison with photoreduction of oxidized cytochromes by flavin semiquinones.

In order to compare the oxidation and reduction reactions of c-type cytochromes (cytochrome c552 from the green alga Monoraphidium braunii and horse heart cytochrome c) by different flavins (lumiflavin, riboflavin and FMN), laser flash photolysis studies have been carried out using either reduced or oxidized protein in the presence of triplet or semiquinone flavin, respectively. The reaction kinetics clearly demonstrate that cytochrome oxidation is mediated by the flavin triplet state. The rate constants for reduction are 20-100 times smaller than those for oxidation, indicating that the triplet state is a more effective reactant than is the semiquinone. This is attributed to its excited state nature and correspondingly high free energy content. The rate constants for both the reduction and oxidation of cytochrome c552 by riboflavin are significantly smaller than those obtained with lumiflavin, suggesting a steric interference of the ribityl side chain in the flavin-cytochrome interaction. The comparison between oxidation and reduction indicates that the former process is less affected by steric hindrance than the latter. Both reduction and oxidation of cytochrome c552 by FMN show an ionic strength dependence with the same sign, consistent with a negatively charged reaction site on the cytochrome. The magnitude of the electrostatic effect is slightly smaller for reduction than it is for oxidation. A pattern quite similar to that observed with cytochrome c552 was obtained when parallel experiments were carried out with horse cytochrome c, although differences were observed in the steric and electrostatic properties of the electron transfer site(s) in these two cytochromes. These results suggest that the same or closely adjacent sites on the proteins are involved in the oxidation and reduction reactions. The biochemical implications of this are discussed.

Cytochrome c Group↗
PubMed1996

A high-potential soluble cytochrome c-551 from the purple phototrophic bacterium Chromatium vinosum is homologous to cytochrome c8 from denitrifying pseudomonads.

A minor cytochrome c-551 component of Chromatium vinosum was previously found to efficiently couple electron transfer between the cytochrome bc1 complex and the photosynthetic reaction center. We have now determined the amino acid sequence of this cytochrome c-551 and find that it is homologous to cytochrome c8 (formerly called Pseudomonas cytochrome c-551). It is most similar to Methylophilus methylotrophus, Rhodocyclus tenuis, and Azotobacter vinelandii cytochromes c8 (respectively, 57%, 52% and 51%). The C. vinosum cytochrome c8 has a single residue insertion relative to Pseudomonas and Azotobacter cytochromes c8. It has fewer charged residues than its homologs and is essentially neutral, which may explain why it is less soluble than the others. The cytochromes c8 are only very distantly related to the cytochromes c2 found in other species of purple bacteria which are much larger in size and which usually mediate electron transfer between the cytochrome bc1 complex and the reaction center. The photosynthetic pathway in Chromatium thus appears to be radically different from that in purple non-sulfur bacteria.

Amino Acid Sequence↗
PubMed1989

Studies on covalent binding of (-)trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites to cytochromes P-450 LM2 and LM4 and NADPH-cytochrome P-450 reductase.

1. Metabolism of 14C-labelled benzo[a]pyrene (-)trans-7,8-dihydrodiol to protein- and DNA-binding products in a reconstituted enzyme system proceeds 5 to 10 times faster with rabbit cytochrome P-450 LM4 than with LM2. 2. Either cytochrome converts the substrate to ethyl acetate- and water-soluble metabolites, identified by h.p.l.c. Water-soluble metabolites comprise 78% of the total products with cytochrome P-450 LM2, but only 50% of those formed by LM4. The relative proportion of the two types of metabolites is differentially affected by certain modifiers such as 7,8-benzoflavone. 3. Half of the radioactivity in the aqueous phase of reaction mixtures containing cytochrome P-450 LM4 represents (-)trans-7,8-diol metabolites in complex primarily with NADPH and phosphate. The remaining water-soluble products are bound covalently to proteins in the reconstituted system. 4. Polyacrylamide gel electrophoresis, autoradiography, and measurement of the radioactivity in individual bands indicate that a larger fraction of metabolites is bound to cytochrome P-450 LM4 than to NADPH-cytochrome P-450 reductase, and only marginal binding to cytochrome P-450 LM2 is seen. Metabolite binding to added DNA is likewise substantially greater in magnitude when cytochrome P-450 LM4, as opposed to LM2, catalyses (-)trans-7,8-diol oxygenation. Thus, the degree of metabolite binding to monoxygenase proteins and to DNA correlates well with the catalytic activity of cytochrome P-450 LM4 and LM2 towards (-)trans-7,8-diol. 5. DNA causes a dramatic enhancement in the activity of cytochrome P-450 LM4 with (-)trans-7,8-diol, indicating that the cytochrome and/or the reductase may be functionally impaired by metabolites of this substrate. Such an effect may alter the balance between detoxication and activation of the carcinogenic benzo[a]pyrene.

Animals↗
PubMed1989

Induction of microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450IVA1 (P-450LA omega) by dehydroepiandrosterone in rats: a possible peroxisomal proliferator.

Dehydroepiandrosterone (DHEA) is a naturally occurring C19-steroid that is found in the peripheral circulation of mammals, including humans. The feeding of DHEA to rodents has been shown to inhibit chemical carcinogenesis in colon, liver, and lung. Therefore, the effect of DHEA on hepatic enzyme activities that are associated with carcinogen metabolism was assessed. Microsomal NADPH-cytochrome P-450 reductase activity and the content of cytochrome b5 were induced 1.8- and 1.4-fold, respectively, upon feeding male Sprague-Dawley rats a synthetic diet containing 0.45% DHEA (w/w). No significant changes in total content of microsomal cytochrome P-450 or the activities of microsomal NADH-cytochrome b5 reductase and cytosolic or microsomal NAD(P)H-quinone oxidoreductase were noted at day 7 of feeding. Cytosolic glutathione S-transferase activity was decreased to 68% of control activity. Administration of DHEA p.o. or by i.p. injection for 5 days led to the same extent of induction of NADPH-cytochrome P-450 reductase activity. Maximal induction of this flavoprotein reductase was noted between days 3 and 4 of feeding or at a dose of 80-120 mg/kg i.p. A small but statistically significant increase in total microsomal cytochrome P-450 was observed after DHEA administration i.p. Rats fed DHEA had a slower growth rate compared with rats fed control diet, whereas rats treated with DHEA i.p. had growth rates identical to those of controls. The liver weights of rats given DHEA by p.o. or i.p. routes were increased significantly compared to those of control rats. Pair feeding of rats with DHA-containing or control diets served to demonstrate that the levels of induction of hepatic microsomal NADPH-cytochrome P-450 reductase and at least one form of cytochrome P450 (P-450IVA1) were the same as those seen in livers of rats fed DHEA ad libitum. This finding suggested that the induction of the flavoprotein and at least one form of the cytochrome was not due to caloric restriction. The increase in NADPH-cytochrome P-450 reductase content of liver microsomes prepared from rats either fed or treated i.p. with DHEA was also observed by Western blotting techniques. DHEA did not appear to induce any of the major forms of rat liver microsomal cytochrome P-450 that are normally increased by either phenobarbital, beta-naphthoflavone, or dexamethasone pretreatment of rats in vivo. However, the measurement of androstenedione and testosterone metabolism in vitro showed pronounced decreases in the 16 alpha-hydroxylase activities of liver microsomes following DHEA feeding.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Metabolism of 32-hydroxy-24,25-dihydrolanosterol by purified cytochrome P-45014DM from yeast. Evidence for contribution of the cytochrome to whole process of lanosterol 14 alpha-demethylation.

Metabolism of 32-hydroxy-24,25-dihydrolanosterol (lanost-8-ene-3 beta,32-diol), a posturated intermediate of the 14 alpha-demethylation (removal of C-32) of 24,25-dihydrolanosterol (lanost-8-en-3 beta-ol), by a reconstituted system consisting of yeast cytochrome P-450 which catalyzes lanosterol 14 alpha-demethylation (cytochrome P-45014DM) (Yoshida, Y., and Aoyama, Y. (1984) J. Biol. Chem. 259, 1655-1660 and Aoyama, Y., Yoshida, Y., and Sato, R. (1984) J. Biol. Chem. 259, 1661-1666) and NADPH-cytochrome P-450 reductase was studied. The reconstituted system converted both 32-hydroxy-24,25-dihydrolanosterol and 24,25-dihydrolanosterol to 4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol, the 14 alpha-demethylated product of the latter. The metabolism of these compounds was inhibited by a low concentration of ketoconazole which is a potent cytochrome P-45014DM inhibitor. Affinity of cytochrome P-45014DM for 32-hydroxy-24,25-dihydrolanosterol was about 20 times higher than for 24,25-dihydrolanosterol and the cytochrome metabolized the former about 4 times faster than the latter under the experimental conditions. Spectral analysis suggested that the 32-hydroxyl group of 32-hydroxy-24,25-dihydrolanosterol interacted with the heme iron of the oxidized cytochrome and this interaction might support the high affinity of this compound for the cytochrome. These lines of evidence indicate that 32-hydroxy-24,25-dihydrolanosterol is the intermediate of the 14 alpha-demethylation of 24,25-dihydrolanosterol by cytochrome P-45014DM. It is also clear that the cytochrome catalyzes further metabolism of the 32-hydroxylated intermediate to the 14 alpha-demethylated product with higher efficiency than the 32-hydroxylation of the substrate. Cytochrome P-45014DM is thus classified as lanosterol C14-C32 lyase.

Chromatography, Gas↗
PubMed1985

Immunohistochemical localization of cytochrome P-450 and reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase in the rat ventral prostate.

Rabbit antibodies raised against the major isozymes of cytochrome P-450 isolated from hepatic microsomes of beta-naphthoflavone- (BNF) and phenobarbital-treated rats (cytochrome P-450 BNF-B2 and cytochrome P-450 PB-B2, respectively) and against rat liver NADPH-cytochrome P-450 reductase were used to localize these enzymes immunohistochemically in the rat ventral prostate. Using the unlabeled antibody peroxidase-antiperoxidase technique, NADPH-cytochrome P-450 reductase was detected exclusively in the epithelial cells of the gland to the same magnitude in untreated, phenobarbital-, and BNF-treated rats. Cytochrome P-450 BNF-B2-like immunoreactivity was exclusively present in the glandular epithelium in BNF-treated rats, whereas staining could not be visualized in untreated or in phenobarbital-treated rats. The staining for NADPH-cytochrome P-450 reductase was more uniformly distributed within the epithelium than was the cytochrome P-450 BNF-B2-like immunoreactivity. Cytochrome P-450 PB-B2-like immunoreactivity was not found, regardless of animal pretreatment. These findings support our previous results (Haaparanta, T., Halpert, J., Glaumann, H., and Gustafsson, J-A., Cancer Res. 43: 5131-5137, 1983) demonstrating the presence of constitutive NADPH-cytochrome P-450 reductase in the prostate and that an isozyme of cytochrome P-450 is highly inducible by BNF in this gland. The significance of these findings are discussed in view of the essentially unknown etiology of human prostatic cancer.

Animals↗
PubMed1981

Formation of a cytochrome c-like species from horse apoprotein and hemin catalyzed by yeast mitochondrial cytochrome c synthetase.

Cytochrome c synthetase in yeast mitochondria catalyzes the formation of a yeast cytochrome c-like species from the apoprotein and hemin (Basile, G., DiBello, C., and Taniuchi, H. (1980) J. Biol. Chem. 255, 7181-7191). To test the specificity of this enzyme, 125I-labeled horse apocytochrome c was incubated with the yeast mitochondrial fraction in the presence of hemin, NADPH, and an ethanol extract of the postmitochondrial fraction. A radioactive 125I-labeled cytochrome c-like species was formed in yields of up to 26%. This 125I-labeled species is indistinguishable from horse cytochrome c by ion exchange chromatography (under the conditions which allow separation of horse and yeast cytochrome c), resistance in its reduced form to digestion by trypsin, resistance against autoxidation, reduction by cytochrome b2, and generation of the apoprotein after treatment with silver sulfate and dithiothreitol. With unlabeled horse apoprotein and [59Fe]hemin, the yield of a [59Fe-labeled horse cytochrome c-like species was up to 7% with respect to the apoprotein incubated. The yield of the 59Fe-labeled species was not altered by the addition of unlabeled FeCl3. Conversely, synthesis of the 59Fe-labeled species was not detectable after incubation of yeast mitochondria with unlabeled horse apoprotein, unlabeled hemin, and 59FeCl3. The formation of both 125I- and 59Fe-labeled cytochrome c-like species was sensitive to heat. Thus, we conclude that cytochrome c synthetase catalyzes direct bonding of heme (or hemin) to the apoprotein. Since the amino acid sequences of horse and yeast cytochromes c differ considerably, cytochrome c synthetase may recognize only a limited region(s) of the apoprotein.

Animals↗
PubMed1982

Kinetic characterization of the interaction between cytochrome oxidase and cytochrome c.

The mechanism of electron transfer catalyzed by cytochrome oxidase was investigated by monitoring the reaction of cytochrome oxidase with cytochrome c under carefully controlled anaerobic conditions. The kinetics of the reaction were examined by varying conditions of ionic strength, inhibitor binding, and oxidation-reduction potential. An analogue of cytochrome c in which the iron atom was replaced with cobalt was used to probe the effect of redox potential on the reaction. Under conditions of low ionic strength, there is very rapid oxidation of cytochrome c and reduction of oxidase which occurs at a rate of 3 X 10(7) M-1 s-1. The number of electrons transferred exhibit a hyperbolic dependence on the concentration of cytochrome c reaching a maximum of 2 electrons transferred at the highest concentration of reduced cytochrome c employed. The total number of electrons transferred was always observed to be distributed equally between cytochrome a and a second acceptor which appears to be the associated copper center; electron transfer to cytochrome a3 did not occur in the absence of oxygen. Substitution of cytochrome c by the cobalt analogue (which represents a decrease in oxidation-reduction potential of about 400 mV) yielded identical results indicating that the origin of the lack of reactivity of cytochrome a3 is of a kinetic nature. The effect of increasing the ionic strength on the reaction was 2-fold: a marked decrease in reaction rate and the appearance of biphasic kinetics with the amplitude of the very fast absorbance changes at 605 nm decreasing from 80% to 40% of the total anticipated from static absorbance measurements. Each of the two phases accounted for a maximum of 1 electron at the highest ionic strength employed. These results are simulated in terms of a sample kinetic reaction scheme involving a two-step electron transfer at one binding site.

Anaerobiosis↗
PubMed1983

Complete analysis of the cytochrome components of beef heart mitochondria in terms of spectra and redox properties. The b-type cytochromes.

The b-cytochromes of beef heart mitochondria have been analyzed by procedures which use optical spectra rather than two points for each cytochrome species. The analytical procedures included first derivatives for background behavior, second derivatives for peak features, and singular value decomposition (Shrager, R.I., and Hendler, R.W. (1982) Anal. Chem. 54, 1147-1152) for the entire spectra. Titrations were performed by electrical oxidation and reduction and by chemical reduction. Four b-cytochromes have been identified in terms of their unique difference spectra, midpoint potentials (Em), and number of electrons transferred (n). They are cytochrome bK, Em = 83 mV, n = 1; cytochrome bT2, Em = -17 mV, n = 2; cytochrome bT1; Em = -95 mV, n = 1; and cytochrome b562L, Em = -146 mV, n = 4. The Em values of cytochromes bT1, bT2, and b562L respond to changes in pH indicating a loss of one proton per oxidation. Antimycin and/or ATP do not affect the spectrum or Em value of any of the cytochromes. An apparent red shift for the absorption maximum of cytochrome bK caused by antimycin may be due to an increased level of reduction of cytochrome bT2.

Animals↗