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PubMed2001

Unreliability of the cytochrome c-enhanced green fluorescent fusion protein as a marker of cytochrome c release in cells that overexpress Bcl-2.

A cytochrome c-enhanced green fluorescent protein chimera (cyt-c.EGFP) was used to monitor the release of cytochrome c from mitochondria in Bcl-2-negative and Bcl-2-positive MDA-MB-468 breast cancer cells. A comparison was made with the intracellular distribution of endogenous cytochrome c based on Western blotting of cell fractions and immunocytochemistry. The release of endogenous cytochrome c from mitochondria into the cytoplasm was detected in Bcl-2-negative cells treated with the kinase inhibitor staurosporine or the calcium-ATPase inhibitor thapsigargin. No release of endogenous cytochrome c was evident in Bcl-2-positive cells, consistent with earlier evidence that Bcl-2 overexpression inhibits cytochrome c release from mitochondria. Cyt-c.EGFP appeared to be localized to the mitochondria in Bcl-2-negative cells and to be released into the cytoplasm following treatment with either staurosporine or thapsigargin. However, in Bcl-2-positive cells the pattern of distribution of cytochrome c-EGFP was inconsistent with that of endogenous cytochrome c, due to accumulation of both cyt-c.EGFP and free EGFP in the cytoplasm of both treated and untreated cells. In summary, cyt-c.EGFP may be useful for monitoring cytochrome c release in living cells that do not express high levels of Bcl-2 but is an unreliable marker of cytochrome c release in cells that overexpress Bcl-2.

Apoptosis↗
PubMed2004

The role of extra fragment at the C-terminal of cytochrome b (Residues 421-445) in the cytochrome bc1 complex from Rhodobacter sphaeroides.

Sequence alignment of cytochrome b of the cytochrome bc1 complex from various sources reveals that bacterial cytochrome b contain an extra fragment at the C terminus. To study the role of this fragment in bacterial cytochrome bc1 complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc1 complexes with progressive deletion from this fragment (residues 421-445) were generated and characterized. The cytbDelta-(433-445) bc1 complex, in which 13 residues from the C-terminal end of this fragment are deleted, has electron transfer activity, subunit composition, and physical properties similar to those of the complement complex, indicating that this region of the extra fragment is not essential. In contrast, the electron transfer activity, binding of cytochrome b, ISP, and subunit IV to cytochrome c1, redox potentials of cytochromes b and c1 in the cytbDelta-(427-445), cytbDelta-(425-445), and cytbDelta-(421-445) mutant complexes, in which 19, 21, or all residues of this fragment are deleted, decrease progressively. EPR spectra of the [2Fe-2S] cluster and the cytochromes b in these three deletion mutant bc1 complexes are also altered; the extent of spectral alteration increases as this extra fragment is shortened. These results indicate that the first 12 residues (residues 421-432) from the N-terminal end of the C-terminal extra fragment of cytochrome b are essential for maintaining structural integrity of the bc1 complex.

Amino Acid Sequence↗
PubMed1957

Intracellular localization of enzymes in spleen. I. Reduced diphosphopyridine nucleotide cytochrome c reductase, cytochrome c oxidase, and succinic dehydrogenase in the rat and guinea pig.

1. The intracellular distribution of nitrogen, DPNH cytochrome c reductase, succinic dehydrogenase, and cytochrome c oxidase has been studied in fractions derived by differential centrifugation from rat and guinea pig spleen homogenates. 2. In the spleens of each species, the nuclear fraction accounted for 40 to 50 per cent of the total nitrogen content of the homogenate, and the mitochondrial, microsome, and supernatant fractions contained about 8, 12, and 30 per cent of the total nitrogen, respectively. 3. Per mg. of nitrogen, DPNH cytochrome c reductase was concentrated in the mitochondria and microsomes of both rat and guinea pig spleens. Seventy per cent of the total DPNH cytochrome c reductase activity was recovered in these two fractions. The reductase activity associated with the nuclear fraction was lowered markedly by isolating nuclei from rat spleens with the sucrose-CaCl(2) layering technique. The lowered activity was accompanied by the recovery of about 90 per cent of the homogenate DNA in the isolated nuclei, indicating that little, if any, of the reductase is present in spleen cell nuclei. 4. Per mg. of nitrogen, succinic dehydrogenase was concentrated about 10-fold in the mitochondria of rat spleen, and 65 per cent of the total activity was recovered in this fraction. 5. Cytochrome c oxidase was concentrated, per mg. of nitrogen, in the mitochondria of both rat and guinea pig spleens. The activity associated with the nuclear fraction was greatly diminished when this fraction was isolated from rat spleens by the sucrose-CaCl(2) layering technique. Only 50 to 70 per cent of the total cytochrome c oxidase activity of the original homogenates was recovered among the four fractions from both rat and guinea pig spleens, while the specific activities of reconstructed homogenates were only 55 to 75 per cent of those of the original whole homogenates. This was in contrast to the results with DPNH cytochrome c reductase and succinic dehydrogenase where the recovery of total enzyme activity approached 100 per cent, and the specific activities of reconstructed homogenates equalled those of the original homogenates. The recovery of cytochrome c oxidase was greatly improved when only the nuclei were separated from rat spleen homogenates. 6. Data were presented comparing the concentrations (ratio of activity per mg. of nitrogen of the fraction to activity per mg. of nitrogen of the homogenate) of DPNH cytochrome c reductase in mitochondria and microsomes derived from different organs of different animals. 7. Data were presented comparing the activities per mg. of nitrogen of DPNH cytochrome c reductase in homogenates from several organs of various animals.

Animals↗
PubMed2002

Importance of hydrophobic interaction between a SoxB-type cytochrome c oxidase with its natural substrate cytochrome c-551 and its mutants.

Cytochrome c-551, the electron donor of SoxB-type cytochrome c oxidase in thermophilic bacilli, can be over-expressed in Bacillus thermodenitrificans cells by tranformation with pSTEc551. Several mutant cytochromes c-551 were prepared by site-directed mutagenesis to this expression plasmid. Among them, several Lys residues were changed to Ala/Ser, and we found that these mutant cytochromes retained their activity as substrates, although their K(m) values were 0.04-0.12 microM, depending on the site replaced. In contrast, the C19A mutant cytochrome, which was produced in Brevibacillus choshinensis as a secretion protein, lost its activity as a substrate, suggesting that the fatty acyl-glyceryl residue covalently bound to the cysteine residue of the wild-type c-551 plays a very important role in the activity. The importance of the hydrophobic fatty acid residue for the binding of cytochrome c-551 to the oxidase was also shown by the loss of substrate activity in deacylated cytochrome c-551. These results show the importance of the hydrophobic interaction between this cytochrome and SoxB-type oxidase, despite the fact that the importance of an electrostatic interaction between cytochrome c and mitochondrial cytochrome aa(3) oxidase has already been established.

Amino Acid Sequence↗
PubMed1979

Cytochrome c oxidase subunits in nuclear and extranuclear cytochrome-aa3-deficient mutants of Neurospora crassa.

The mitochondria of cytochrome-aa3-deficient Neurospora crassa mutants were screened for the seven polypeptide constiuents of cytochrome c oxidase. The polypeptides of the holoenzyme and the unassembled or partially assembled subunits were detected by sodium dodecyl sulfate/acrylamide gel electrophoresis of immunoprecipitates obtained with antiserum to the holoenzyme as well as to several individual subunits. With respect to the mitochondrially synthesized polypeptides of the oxidase, subunits 1 to 3, the results obtained from the analysis of immunoprecipitates were confirmed through the direct electrophoretic analysis of mitochondrial translation products. The results were as follows. 1. The mitochondria of the cya-2-8 and cya-3-16 nuclear mutants and the [exn-5] cytoplasmic mutant contained a protein complex immunoprecipitated by anti-holoenzyme antibody and composed of the complete set of the seven cytochrome oxidase polypeptides. Only the oxidase subunits 5 and 6 were immunoprecipitated by anti-holoenzyme antibody from the mitochondria of the cyt-2-1 and 299-1 nuclear mutants, even though at least some of the mitochondrially synthesized polypeptides were detected in both mutants by subunit specific immunoprecipitation. 2. A 'subunit 1' polypeptide larger than the authentic subunit-1 polypeptide of wild-type cytochrome oxidase was found in the mitochondria from two nuclear mutants, cyt-2-1, and 299-1 and the [mi-3] cytoplasmic mutant. This larger polypeptide may be an unprocessed precursor of the 'mature' subunit 1 protein of the holoenzyme. No changes in the apparent molecular weights were found for the polypeptide subunits of cytochrome oxidase in mitochondria of the [exn-5] cytoplasmic mutant and the cya-2-8 and cya-4-23 nuclear mutants. 3. A nuclear mutant, 299-1, lacks the mitochondrially synthesized subunit-2 polypeptide of cytochrome oxidase. When cells were labelled in the presence of cycloheximide, the subunit 2 content of mitochondria from mutants [exn-5], cya-2-8, cya-3-16 and cya-4-23 was lower than in mitochondria from wild-type. This deficiency, however, does not appear to be sufficiently severe to fully account for the lack of cytochrome aa3 in these mutants. The cya-4-23 nuclear mutant either is severely deficient in or lacks cytochrome oxidase subunits 5 and 6. On the basis of these and previously reported observations, it is proposed that the cytochrome oxidase deficiencies of as many as seven of the eight N. crassa cytochrome-aa3-deficient mutants could be caused by genetically imposed alterations in regulatory systems controlling the production of different components of the enzyme.

Cytochromes↗
PubMed1976

Alterations in enzyme and cytochrome profiles of Rana catesbeiana liver organelles during thyroxine-induced metamorphosis. Changes in membrane-localized phosphohydrolases, oxidoreductases, and cytochrome levels in response to in vivo thyroxine administration.

A primary objective of the present study has been to determine the changes which occur in Rana catesbeiana liver organelle membranes during thyroxine-induced metamorphosis. To this end, enzyme and cytochrome profiles were determined for mitochondria, microsomes, and nuclear membrane fractions isolated from livers of R. catesbeiana tadpoles which had been fasted for 6 days at 15 +/- 0.5 degrees and then immersed in thyroxine, 2.6 X 10(-8) M, for periods of up to 12 days at 23.5 +/- 0.4 degrees. The ratio of total succinate-cytochrome c reductase activity in the initial homogenate fraction to the total activity of this mitochondrial "marker" enzyme recovered in the final mitochondrial fraction remained constant, approximately 0.5, throughout the course of thyroxine treatment; however, after a 3- to 4-day latency the mitochondrial protein mass recovered per unit mass of initial homogenate protein was found to increase significantly (approximately 2-fold by Day 10 of thyroxine treatment). A similar increase was also observed in the yield of microsomal, but not nuclear membrane, protein mass as a function of thyroxine treatment. Prolonged thyroxine treatment (12 days) resulted in approximately 50% decreases in tadpole liver homogenate and microsomal NADH-cytochrome c reductase specific activities; in contrast, mitochondrial and nuclear membrane NADH-cytochrome c reductase specific activities were not altered under the same conditions. In addition, homogenate and microsomal NADPH-cytochrome c reductase specific activities were found to have increased significantly after 12 days of thyroxine treatment; however, the specific activity of NADPH-cytochrome c reductase in the mitochondrial fraction was unchanged. It was also observed that thyroxine treatment resulted in increases in homogenate and microsomal glucose-6-phosphatase specific activities, whereas the mitochondrial as well as nuclear membrane glucose-6-phosphatase specific activities remained unchanged. Furthermore, in contrast to homogenate and mitochondrial monoamine oxidase specific activities, which decreased 30 and 40%, respectively, as a consequence of thyroxine treatment (12 days), the succinate-cytochrome c reductase and oligomycin-sensitive Mg2+ ATPase specific activities determined for these fractions increased significantly. In all instances, changes as a result of thyroxine treatment in membrane-localized homogenate or organelle enzyme specific activities were apparent only after a 3- to 4-day initial latent period. The in vitro effects of thyroxine (10(-10) - 10(-5) M) on the membrane-localized enzyme activities examined in this study were either negligible or, as in the case of mitochondrial succinate-cytochrome c reductase and microsomal NADH-cytochrome c reductase, opposite to the changes observed in response to in vivo thyroxine treatment, with the exception of microsomal NADPH-cytochrome c reductase activity which was enhanced approximately 2-fold by 10(-5) M thyroxine...

Animals↗
PubMed1988

Hepatic mitochondrial cytochrome P-450 system. Purification and characterization of two distinct forms of mitochondrial cytochrome P-450 from beta-naphthoflavone-induced rat liver.

We have purified two distinct isoforms of mitochondrial cytochrome P-450 from beta-naphthoflavone (beta-NF)-induced rat liver to greater than 85% homogeneity and characterized their molecular and catalytic properties. One of these isoforms showing an apparent molecular mass of 52 kDa is termed P-450mt1 and the second isoform with 54-kDa molecular mass is termed P-450mt2. Cytochrome P-450mt2 comigrates with similarly induced microsomal P-450c (the major beta-NF-inducible form) on sodium dodecyl sulfate-polyacrylamide gels and cross-reacts with polyclonal antibody monospecific for cytochrome P-450c. Cytochrome P-450mt2, however, represents a distinct molecular species since it failed to react with a monoclonal antibody to P-450c and produced V8 protease fingerprints different from P-450c. Cytochrome P-450mt1, on the other hand, did not show any immunochemical homology with P-450c or P-450mt2 as well as partially purified P-450 from control mitochondria. Electrophoretic comparisons and Western blot analysis show that both P-450mt1 and P-450mt2 are induced forms not present in detectable levels in control liver mitochondria. A distinctive property of mitochondrial P-450mt1 and P-450mt2 was that their catalytic activities could be reconstituted with both NADPH-cytochrome P-450 reductase as well as mitochondrial specific ferredoxin and ferredoxin reductase electron transfer systems, while P-450c showed exclusive requirement for NADPH-cytochrome P-450 reductase. Cytochromes P-450mt1 and P-450mt2 were able to metabolize xenobiotics like benzo(a)pyrene and dimethyl benzanthracene at rates only one-tenth with cytochrome P-450c. Furthermore, P-450mt1, P-450mt2, as well as partially purified P-450 from control liver, but not P-450c, showed varying activities for 25- and 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3. These results provide evidence for the presence of at least two distinct forms of beta-NF-inducible cytochrome P-450 in rat hepatic mitochondria.

Animals↗
PubMed1977

Topographic determinants on cytochrome c. I. The complete antigenic structures of rabbit, mouse, and guanaco cytochromes c in rabbits and mice1.

Rabbit, mouse, and guanaco cytochromes c differ from each other by only two amino acid residues. The identification is described of all of the antigenic determinants of mouse and guanaco cytochrome c that elicit an antibody response in rabbits, and those of the rabbit and guanaco proteins that elicity antibodies in the mouse. All except one of these sites center around single amino acid residue differences between the antigen and the host cytochrome c. The corresponding antibody popylations bind only to the areas of the protein in which the substitutions occur. Such antigenic determinants manifested in rabbits by quanaco and mouse cytochromes c are centered around residues 62 and 89, and residues 44 and 89, respectively. Similarly, the mouse recognizes sites containing residues 44 and 62 in guanaco cytochrome c, and residues 44 and 89 in rabbit cytochrome c. In none of these instances has a change in sequence failed to produce an antibody response. Each of these determinants appears to elicit and bind to its antibody, independently of other determinants present on the protein. In addition, two different autoantigenic responses have been detected. The antibodies produced against the determinant formed by glutamyl residue 62 of the guanaco protein in both rabbits and mice, the cytochromes c of which carry an aspartyl residue in that position, also bind to the aspartyl-containing region but with lower affinity. However, mouse and rabbit cytochrome c also elicit antibodies to the area of residue 62 in rabbits and mice, respectively, and these antibodies still bind more strongly to the glutamyl-than to the aspartyl-containing determinant. This last response occurs only when there are residue substitutions elsewhere in the molecule, because mice and rabbits fail to respond to their own cytochrome c. Antibodies produced in mice against the change from alanyl to valyl residue 44 by rabbit and guanaco cytochromes c also bind to the alanyl-containing determinant, except less tightly than to the valyl region. Conversely, antibodies raised in rabbits against the change from valyl to alanyl residue 44 only bind to this region when it carries an alanine. It is suggested that antigenic determinants that arise as a result of amino acid residue substitutions between the immunizing and the corresponding host protein, without a change in the spatial arrangement of the polypeptide backbone, be termed topographic determinants.

Amino Acid Sequence↗
PubMed1980

Studies on the synthesis of cytochrome P-450 and cytochrome P-448 in rat liver.

The synthesis of cytochrome P-450 (phenobarbital inducible) and cytochrome P-448 (3-methylcholanthrene inducible) have been studied in rat liver in vivo and in the wheat germ cell-free system using anti-cytochrome P-450 and anti-cytochrome P-448 antibodies. The major mature forms synthesized in vivo correspond to a molecular weight of 47,000 for cytochrome P-450 and 53,000 for cytochrome P-448. Translation of poly(A)-containing RNA from phenobarbital-treated rats in the wheat germ cell-free system reveals that the cell-free product immunoprecipitated with anti-cytochrome P-450 antibody has a molecular weight close to 47,000. In the case of 3-methylcholanthrene, the cell-free product immunoprecipitated with anti-cytochrome P-448 antibody shows a molecular weight around 59,000. Significant conversion of the 59,000 species to the 53,000 species can be demonstrated when the translation is carried out in the presence of microsomal membranes isolated from rat liver. Phenobarbital and 3-methylcholanthrene enhance the translatable messenger RNA contents for cytochrome P-450 and cytochrome P-448, respectively.

Animals↗
PubMed1988

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.

Animals↗
PubMed1998

Electrostatic interaction between NADH-cytochrome b5 reductase and cytochrome b5 studied by site-directed mutagenesis.

Electrostatic interaction between NADH-cytochrome b5 reductase and cytochrome b5 was studied by site-directed mutagenesis. The target residues for mutagenesis were selected on the basis of the previously reported chemical cross-linking study of these two proteins, which implicated possible charge-pair interactions between Lys-41, Lys-125, Lys-162, and Lys-163 of the enzyme, and Glu-47, Glu-48, Glu-52, Glu-60, Asp-64 (group A), and heme propionate of cytochrome b5. Mutant reductases that lost one of the above-listed Lys residues showed higher K(m) values for cytochrome b5 and lower kcat values than those of the wild type, suggesting that all of the examined Lys residues participate in binding with cytochrome b5 as reported previously. In contrast, a removal of one of (or even all of) the group A residues from cytochrome b5 by mutagenesis caused no significant effect on the catalytic properties of cytochrome b5. Additional elimination of another set of negative residues (Glu-41, Glu-42, Asp-57, and Glu-63 (Group B)), which are also located close to heme, elevated the K(m) value by more than five folds. These results suggest that there should be other acidic residue(s) than group A in cytochrome b5 which participate in binding with NADH-cytochrome b5 reductase.

Binding Sites↗
PubMed1986

Specific overproduction and purification of the cytochrome b558 component of the cytochrome d complex from Escherichia coli.

In Escherichia coli strain GR84N[pNG10], the cloned gene for subunit I of the membrane-bound cytochrome d complex resulted in the overproduction of cytochrome b558 and facilitated purification of this cytochrome. Extracting membranes with 1% Triton X-100 followed by two chromatographic steps yielded a single band on sodium dodecyl sulfate-polyacrylamide gels corresponding to subunit I (Mr 57 000). Purified cytochrome b558 was in its native state as determined by difference absorption spectroscopy and by potentiometric analysis. Both the membranes of strain GR84N[pNG10] and the purified subunit I lacked the other two spectroscopically defined cytochromes, b595 (previously "a1") and d, of the cytochrome d complex. Reconstitution of cytochrome b558 in phospholipid vesicles demonstrated that cytochrome b558 can be reduced by ubiquinol but that it does not reduce molecular oxygen. Heme extraction of cytochrome b558 yielded an extinction coefficient of 22 000 M-1 cm-1 for the wavelength pair of 560 and 580 nm in the reduced-minus-oxidized spectrum. The mutation on pNG10 that eliminates subunit II was mapped to a 250 base pair DNA fragment.

Cell Membrane↗
PubMed1977

Relationship between the reduction of oxygen, artificial acceptors and cytochrome P-450 by NADPH--cytochrome c reductase.

The interaction of NADPH--cytochrome c reductase with oxygen, artificial acceptors and cytochrome P-450 was studied. The generation of superoxide anion radicals (O2-.) from the oxidation of adrenaline to adrenochrome catalysed by NADPH--cytochrome c reductase proceeds independently of the interaction of the enzyme with the artificial anaerobic acceptors cytochrome c or 2,6-dichlorophenol-indophenol. Propyl 3,4,5-trihydroxybenzoate inhibited competitively the adrenaline oxidation by isolated NADPH--cytochrome c reductase (Ki 3.2--4.7 micrometer) and inhibited non-competitively the cytochrome c reduction (Ki 92--109 micrometer). In contrast with the process of electron transfer to cytochrome c, the rate of reduction of cytochrome P-450 and the rate of oxidation of adrenaline in liver microsomal fraction are correlated. Hexobarbital increases the Vmax. of adrenaline oxidation without affecting the Km value, whereas metyrapone, a metabolic inhibitor decreases Vmax. without affecting the Km. From the results obtained, some conclusions about NADPH--cytochrome c reductase function were made.

2,6-Dichloroindophenol↗
PubMed1981

Modification of the spectral properties of cytochrome b in mutants of Saccharomyces cerevisiae resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Mapping at two distinct genetic loci of the split mitochondrial gene of cytochrome b.

The effects of five inhibitors of the cytochrome bc1 complex: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), 2-n-heptyl-4-hydroxyquinoline-N-oxide (HpHOQnO), antimycin A, funiculosin and mucidin were measured in submitochondrial particles of strains of the yeast Saccharomyces cerevisiae belonging to two classes of diuron-resistant mutants Diu 1 and Diu 2 which are modified in different exons of the split mitochondrial gene of cytochrome b. 1. The oxidation of NADH and of cytochrome b-561 exhibits a similar resistance to diuron and HpHOQnO in Diu 1 and Diu 2 mutants. 2. No extra reduction of cytochrome b-561 and cytochrome b-565 is observed in the presence of diuron and HpHOQnO. 3. Both Diu 1 and Diu 2 mutants exhibit the red shift of cytochrome b-561 induced by concentrations of HpHOQno 2 -- 3-times higher than those required in the parental strains. 4. The spectral and respiratory effects of antimycin A, funiculosin and mucidin and generally similar in the diuron-resistant mutants and in their parental strains. However a cross-resistance between diuron and antimycin A is indicated in one Diu 2 mutant. 5. From the combined genetic and biochemical data it is concluded that the interaction of diuron and HpHOQnO with cytochrome b is mediated by at least two specific amino acids located apart in the central region of the apocytochrome b peptide coded by mitochondrial DNA. These two amino acids control tightly the extra reduction of cytochromes b-565 and b-561 as well as the flow of electrons through the bc1 complex. However the binding of HpHOQnO required for the expression of the red shift of cytochrome b-561 is only slightly affected by the diu-1 and diu-2 mutations.

Antimycin A↗
PubMed1990

Cloning and mutagenesis of genes encoding the cytochrome bd terminal oxidase complex in Azotobacter vinelandii: mutants deficient in the cytochrome d complex are unable to fix nitrogen in air.

The genome of Azotobacter vinelandii contains DNA sequences homologous to the structural genes for the Escherichia coli cytochrome bd terminal oxidase complex. Two recombinant clones bearing cydA- and cydB-like sequence were isolated from an A. vinelandii gene library and subcloned into the plasmid vector pACYC184. Physical mapping demonstrated that the cydA- and cydB-like regions in A. vinelandii are contiguous. The cydAB and flanking DNA was mutagenized by the insertion of Tn5-B20. Mutations in the cydB-hybridizing region resulted in the loss of spectral features associated with cytochromes b595 and d. A new locus, cydB, encoding cytochromes b595 and d in A. vinelandii is proposed. A second region adjacent to cydB was also involved in expression of the cytochrome bd complex in A. vinelandii, since mutations in this region resulted in an increase in the levels of both cytochrome b595 and cytochrome d. The regions involved in expression of the cytochrome bd complex and cydB are transcribed in the same direction. Mutants deficient in cytochromes b595 and d were unable to grow on N-deficient medium when incubated in air but could fix nitrogen when the environmental O2 concentration was reduced to 1.5% (vol/vol). It is proposed that the branch of the respiratory chain terminated by the cytochrome bd complex supports the high respiration rates required for the respiratory protection of nitrogenase.

Azotobacter↗
PubMed1992

Change of the terminal oxidase from cytochrome a1 in shaking cultures to cytochrome o in static cultures of Acetobacter aceti.

Acetobacter aceti has an ability to grow under two different culture conditions, on shaking submerged cultures and on static pellicle-forming cultures. The respiratory chains of A. aceti grown on shaking and static cultures were compared, especially with respect to the terminal oxidase. Little difference was detected in several oxidase activities and in cytochrome b and c contents between the respiratory chains of both types of cells. Furthermore, the results obtained here suggested that the respiratory chains consist of primary dehydrogenases, ubiquinone, and terminal ubiquinol oxidase, regardless of the culture conditions. There was a remarkable difference, however, in the terminal oxidase, which is cytochrome a1 in cells in shaking culture but cytochrome o in cells grown statically. Change of the culture condition from shaking to static caused a change in the terminal oxidase from cytochrome a1 to cytochrome o, which is concomitant with an increase of pellicle on the surface of the static culture. In contrast, reappearance of cytochrome a1 in A. aceti was attained only after serial successive shaking cultures of an original static culture; cytochrome a1 predominated after the culture was repeated five times. In the culture of A. aceti, two different types of cells were observed; one forms a rough-surfaced colony, and the other forms a smooth-surfaced colony. Cells of the former type predominated in the static culture, while the cells of the latter type predominated in the shaking culture. Thus, data suggest that a change of the culture conditions, from static to shaking or vice versa, results in a change of the cell type, which may be related to the change in the terminal oxidase from cytochrome a1 to cytochrome o in A. aceti.

Acetobacter↗
PubMed1993

Microsomal cytochrome P450 1A1 dependent monooxygenase activity in guinea pig heart: induction, inhibition, and increased activity by addition of exogenous NADPH-cytochrome P450 reductase.

Characterization of cytochrome P450 1A1 dependent monooxygenases in guinea pig heart revealed low rates of 7-ethoxyresorufin O-deethylation, which are markedly increased (20-fold) by treatment with beta-naphthoflavone, a polycyclic aromatic hydrocarbon. Both 7-ethoxyresorufin O-deethylation and 7-methoxyresorufin O-demethylation were found to be approximately 4-fold higher in microsomes prepared from the ventricle than the atrium of beta-naphthoflavone-induced guinea pigs. The low rates of 7-ethoxyresorufin O-deethylation in cardiac microsomes were due, at least in part, to a deficiency of the flavoprotein NADPH--cytochrome P450 reductase; addition of exogenous NADPH--cytochrome P450 reductase; addition of exogenous NADPH--cytochrome P450 reductase dramatically increased 7-ethoxyresorufin O-deethylation in cardiac microsomes of guinea pigs, before and after treatment with beta-naphthoflavone. N-Benzyl-1-aminobenzotriazole, a suicide substrate of cytochrome P450 1A1 in guinea pig, was able to inhibit almost all of the 7-ethoxyresorufin O-deethylase and 7-methoxyresorufin O-demethylase activities in polycyclic aromatic hydrocarbon induced guinea pig heart (88 and 71%, respectively), suggesting that cytochrome P450 1A1 coupled to NADPH--cytochrome P450 reductase in these microsomes inactivates itself by a suicidal mechanism. Addition of alpha-naphthoflavone, an inhibitor of cytochrome P450 1A isozymes, to cardiac microsomes from beta-naphthoflavone-induced guinea pigs resulted in greater than 95% inhibition of 7-ethoxyresorufin O-deethylase activity. The biological significance of these low levels of cytochrome P450 1A1 monooxygenase activity in guinea pig heart and their induction by polycyclic aromatic hydrocarbons are not currently understood.

Animals↗
PubMed1983

Participation of cytochrome b5 electron transport system coupled with delta 5-3 beta-hydroxysteroid dehydrogenase on cytochrome P-450 monooxygenase reactions of guinea pig adrenal microsomes.

Not only cytochrome P-450 and cytochrome P-450 reductase, but considerable amount of cytochrome b5 and cytochrome b5 reductase were also contained in the adrenal microsomes of guinea pig. Addition of nicotinamide adenine dinucleotide (NADH) stimulates the activities of cytochrome P-450 monooxygenases such as 21-hydroxylase, 17 alpha-hydroxylase and C17,20 lyase supported with nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. In addition, substrate for delta 5-3 beta-hydroxysteroid dehydrogenase plus NAD+ also stimulate the same cytochrome P-450 monooxygenase activities. These observations were suggesting the participation of cytochrome b5 electron transport system on cytochrome P-450 monooxygenase (21-hydroxylase, 17 alpha-hydroxylase and C17,20 lyase) reactions associated with the biosynthesis of corticoids and adrenal androgens. Furthermore, it is also suggesting that cytochrome b5 electron transport system involves the reaction of delta 5-3 beta-hydroxysteroid dehydrogenase.

3-Hydroxysteroid Dehydrogenases↗