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At least 811 records · Page 45Linked to original sources
PubMed1994

The cydD gene product, component of a heterodimeric ABC transporter, is required for assembly of periplasmic cytochrome c and of cytochrome bd in Escherichia coli.

The cydD gene of Escherichia coli encodes a protein which, together with the CydC protein, probably constitutes a heterodimeric, ABC-family membrane transporter, necessary for biosynthesis of the cytochrome bd quinol oxidase. Here, we demonstrate that a cydD mutant also fails to synthesise periplasmic c-type cytochrome(s), suggesting that the transporter exports haem or some other component involved in assembly of cytochromes that are found in, or exposed to, the periplasm. The CydDC system appears to be the first example of a transporter required for periplasmic cytochrome assembly processes requiring more than one type of haem. A mutant defective in trxB (adjacent to the cydDC operon, and encoding thioredoxin reductase) was unaffected in cytochrome c or bd assembly.

Adenosine Triphosphatases↗
PubMed1996

Cytochrome bd-type quinol oxidase in a mutant of Bacillus stearothermophilus deficient in caa3-type cytochrome c oxidase.

Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as a terminal oxidase in the respiratory chain. To identify alternative oxidases, we isolated B. stearothermophilus mutants defective in the caa3-type oxidase activity. One mutant contained little cytochrome a and had low cytochrome c oxidase activity. However, growth and the respiratory activity of membranes in the presence of NADH were close to normal, suggesting that the mutant contains an alternative electron transfer pathway. A novel oxidase was isolated from the membrane fraction of the mutant. The enzyme is a cytochrome bd-type quinol oxidase composed of two subunits of 52 and 40 kDa, whose N-terminal regions show sequence similarity to polypeptides of the bd-type oxidase from Escherichia coli and Azotobacter vinelandii. This is the first report of a bd-type terminal oxidase purified from a Gram-positive bacterium.

Amino Acid Sequence↗
PubMed1996

Cytochrome b5 Is Essential for Cytochrome P450 6D1-Mediated Cypermethrin Resistance in LPR House Flies

Cytochrome P450 monooxygenase-mediated detoxification is a very important mechanism of resistance to insecticides. In the LPR strain of house fly, the major mechanism of resistance to pyrethroids is monooxygenase-mediated metabolism due to the overexpression of cytochrome P450 6D1 (CYP6D1). Although the role of CYP6D1 in pyrethroid resistance has been demonstrated, there is no information as to whether cytochrome b5 is involved in this reaction and what the major CYP6D1-specific metabolite is. Using a cytochrome b5 antiserum we demonstrate that cytochrome b5 is required for the elevated level of CYP6D1-mediated cypermethrin metabolism observed in microsomes from the resistant LPR strain compared to a susceptible strain. Therefore, cytochrome b5 is directly involved in this monooxygenase-mediated resistance. NADH has no significant effect on this metabolism, suggesting that in vitro the second electron source for CYP6D1 is NADPH when cypermethrin is the substrate. Using GC-MS, the primary CYP6D1 specific metabolite formed in vitro was identified as 4'-OH cypermethrin.

Journal Article↗
PubMed1993

Proton translocation in cytochrome c oxidase: redox linkage through proximal ligand exchange on cytochrome a3.

An analysis of resonance Raman scattering data from CO-bound cytochrome c oxidase and from the photodissociated enzyme indicates that histidine may not be coordinated to the iron atom of cytochrome a3 in the CO-bound form of the enzyme. Instead, the data suggest that either a water molecule or a different amino acid residue occupies the proximal ligand position. From these data, it is postulated that ligand exchange on cytochrome a3 can occur under physiological conditions. Studies of mutant hemoglobins have demonstrated that tyrosinate binds preferentially to histidine in the ferric forms of the proteins. In cytochrome c oxidase tyrosine residues are located near the histidine residues recently implicated in coordination to cytochrome a3 (Shapleigh et al., 1992; Hosler et al., this volume). Expanding on these concepts, we propose a model for proton translocation at the O2-binding site based on proximal ligand exchange between tyrosine and histidine on cytochrome a3. The pumping steps take place at the level of the peroxy intermediate and at the level of the ferryl intermediate in the catalytic cycle and are thereby consistent with the recent results of Wilkstrom (1989) who found that proton pumping occurs only at these two steps. It is shown that the model may be readily extended to account for the pumping of two protons at each of the steps.

Binding Sites↗
PubMed1984

Decreased cytochrome oxidase activity in hepatic mitochondria after chronic ethanol consumption and the possible role of decreased cytochrome aa3 content and changes in phospholipids.

In ethanol-fed baboons, hepatic mitochondrial cytochrome oxidase activity and cytochrome aa3 content were significantly decreased by 58.3 and 50.5%, respectively, compared to their pair-fed controls. However, there was no significant correlation between the two, suggesting that other factors in addition to cytochrome aa3 may be responsible for the depression in cytochrome oxidase activity. The total phospholipid content of the mitochondrial membranes was significantly decreased (0.24 +/- 0.03 mumol of phospholipid phosphorus/mg of protein vs. 0.32 +/- 0.04 in controls). This change was accounted for, in part, by the significant decrease in the levels of phosphatidylcholine and cardiolipin. In addition, the fatty acid pattern of the phospholipids was changed. There was a marked increase in the relative amounts of oleic and linoleic acids and a decrease in arachidonic acid. These changes were associated with an increase in the activity of phospholipase A2. The reactivation rate of phospholipid-depleted cytochrome oxidase by endogenous phospholipids from ethanol-fed baboons was significantly lower than that by phospholipid from pair-fed controls, when measured at an optimal phospholipid to protein ratio. Thus, it appears that alterations in the phospholipid composition of the mitochondrial membranes are responsible, at least in part, for the depression of cytochrome oxidase activity produced by chronic ethanol consumption.

Animals↗
PubMed1990

Active site of bovine adrenocortical cytochrome P-450(11) beta studied by resonance Raman and electron paramagnetic resonance spectroscopies: distinction from cytochrome P-450scc.

Cytochrome P-45011 beta was purified as the 11-deoxycorticosterone-bound form from bovine adrenocortical mitochondria and its active site was investigated by resonance Raman and EPR spectroscopies. Resonance Raman spectra of the purified sample revealed that the heme iron adopts the pure pentacoordinated ferric high-spin state on the basis of the nu 10 (1629cm-1) and nu 3 (1490 cm-1) mode frequencies, which are higher than those of the hexacoordinated ferric high-spin cytochrome P-450scc-substrate complexes. In the ferrous-CO state, a Fe2(+)-CO stretching mode was identified at 481.5 cm-1 on the basis of an isotopic substitution technique; this frequency is very close to that of cytochrome P-450scc in the cholesterol-complexed state (483 cm-1). The EPR spectra of the purified sample at 4.2 K showed ferric high-spin signals (at g = 7.98, 3.65, and 1.71) that were clearly distinct from the cytochrome P-450scc ferric high-spin signals (g = 8.06, 3.55, and 1.68) and confirmed previous assignments of ferric high-spin signals in adrenocortical mitochondria. The EPR spectra of the nitric oxide (NO) complex of ferrous cytochrome P-45011 beta showed EPR signals with rhombic symmetry (gx = 2.068, gz = 2.001, and gy = 1.961) very similar to those of the ferrous cytochrome P-450scc-NO complex in the presence of 22(S)-hydroxycholesterol and 20(R),22-(R)-dihydroxycholesterol at 77 K.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1980

A two-subunit cytochrome c oxidase (cytochrome aa3) from Paracoccus dentrificans.

Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, EC 1.9.3.1) was purified from the cytoplasmic membrane of the bacterium Paracoccus denitrificans. The enzyme contains two heme groups (a and a3) and two copper atoms per minimal unit, oxidizes mammalian cytochrome c at a high rate, and, when incorporated into liposomes, generates an electrochemical proton gradient during cytochrome c oxidation. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis reveals only two subunits of apparent molecular weights 45,000 and 28,000; they appear to correspond to the two largest mitochondrially made subunits of the seven-subunit cytochrome c oxidase isolated from yeast mitochondria. Because of its structural simplicity. Paracoccus cytochrome c oxidase offers new possibilities for exploring the mechanism of cytochrome c oxidase function.

Amino Acids↗
PubMed2001

Adrenodoxin-cytochrome P450scc interaction as revealed by EPR spectroscopy: comparison with the putidaredoxin-cytochrome P450cam system.

The cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc comprises three consecutive monooxygenase reactions (22R-hydroxylation, 20S-hydroxylation, and C(20)-C(22) bond scission) that produces pregnenolone. The electron equivalents necessary for the oxygen activation are supplied from a 2Fe-2S type ferredoxin, adrenodoxin. We found that 1:1 stoichiometric binding of oxidized adrenodoxin to oxidized cytochrome P450scc complexed with cholesterol or 25-hydroxycholesterol caused shifts of the high-spin EPR signals of the heme moiety at 5 K. Such shifts were not observed for the low-spin EPR signals. Ligation of CO or NO to the reduced heme of cytochrome P450scc complexed with reduced adrenodoxin and various steroid substrates did not cause any change in the axial EPR spectrum of the reduced iron-sulfur center at 77 K. These results are in remarkable contrast to those obtained for the cytochrome P450cam-d-camphor-putidaredoxin ternary complex, suggesting that the mode of cross talk between adrenodoxin and cytochrome P450scc is very different from that in the Pseudomonas system. The difference may be primarily due to the location of the charged amino acid residues of the ferredoxins important for the interaction with the partner cytochrome P450.

Adrenal Cortex↗
PubMed1983

Mechanism of the increase in cytochrome c oxidase activity in pea cotyledons during seed hydration. The presence of free cytochrome-c-oxidase subunits in dry cotyledons and their probable assembly into the holoenzyme during seed hydration.

The increase in cytochrome c oxidase activity in pea cotyledons during seed hydration was investigated with two kinds of antibodies against the enzymes purified from mitochondria of pea shoot and sweet potato root. The antibody against the pea enzyme recognized only the largest of the enzyme subunits (subunit I), while that against the sweet potato one did other three of the pea enzyme subunits: the second largest (subunit II) and the two smallest (subunits IV and V). There was no increase in the amount of protein immunoprecipitated with the anti-(pea enzyme) antibody in a crude fraction of cotyledon membranes during seed hydration, despite the marked increase in cytochrome c oxidase activity. Neither cycloheximide nor chloramphenicol had any effect on the activity or the amount. The crude membrane fraction from dry cotyledons contained an enzymatically inert protein precipitated with the anti-(pea enzyme) antibody. The inert protein could be separated from the active enzyme protein through solubilization of the protein from the membranes with various concentrations of Triton X-100. It consisted of only one kind of polypeptide with the same molecular weight as subunit I and decreased with the concomitant increase in the active enzyme protein during seed hydration. Dry pea cotyledons also contained a soluble protein that was immunoprecipitated by antibody to the sweet potato enzyme. The soluble form of the immunoreactive protein decreased with the concomitant increase in active cytochrome c oxidase protein in the crude membrane fraction from the cotyledons during seed hydration. None of these changes during seed hydration were inhibited by cycloheximide or chloramphenicol. The soluble, immunoreactive protein contained only one kind of polypeptide, of which the molecular weight was identical to that of subunit IV. The mitochondrial inner membranes of dry cotyledons were separated into three fractions by sucrose density gradient centrifugation. The lightest was the richest of the three in the enzymatically inert protein immunoprecipitated with the anti-(pea enzyme)antibody; cytochrome c oxidase activity in this fraction increased immediately after the onset of seed hydration. These results indicate that a membrane-bound, free form of subunit I and a soluble, free form of subunit IV of cytochrome c oxidase exist in dry pea cotyledons. We propose that these free forms of subunits are assembled with the other subunits to form the active cytochrome c oxidase during seed hydration.

Electron Transport Complex IV↗
PubMed1994

Mutagenesis of a gene encoding a cytochrome o-like terminal oxidase of Azotobacter vinelandii: a cytochrome o mutant is aero-tolerant during nitrogen fixation.

The amino acid sequence obtained by translating the nucleotide sequence of a 0.55 kb fragment, amplified from Azotobacter vinelandii chromosomal DNA by PCR, was 57% identical to part of the Escherichia coli cyoB gene, encoding subunit I of the cytochrome bo-type quinol oxidase. This fragment was mutated in vitro by insertion of a kanamycin-resistance cassette and introduced into the chromosome of A. vinelandii by homologous recombination. The mutant contained no spectrally detectable cytochrome o. However, in the stationary phase of growth, the level of the alternative oxidase (cytochrome bd) was 11-fold higher than in the wild-type strain. Respiration of the mutant was insensitive to chlorpromazine, an inhibitor thought to act specifically on cytochrome o. Cytochrome o-deficient mutants fixed nitrogen in air, clearly distinguishing the role of this oxidase from that of cytochrome bd, which is required for respiratory protection of oxygen-labile nitrogenase.

Amino Acid Sequence↗
PubMedDate not supplied

Comparative study on cytochrome P-450 of yeasts using specific antibodies to cytochromes P-450alk and P-450(14DM).

The occurrence of cytochrome P-450(14DM) (lanosterol 14 alpha-demethylase) and cytochrome P-450alk (long-chain alkane terminal hydroxylase) in various yeast strains was determined with immunological procedures. Cytochrome P-450(14DM), which is constitutive or housekeeping enzyme playing an essential role in ergosterol biogenesis, was found in all yeast strains so far tested. Cytochromes P-450(14DM) from different species of yeast were immunologically different, although they may have had a few common antigenic sites. In contrast, cytochrome P-450alk was detected only in the alkane-assimilating yeasts.

Alkanes↗
PubMed1997

Expression of cytochrome P450 3A7 in Escherichia coli: effects of 5' modification and catalytic characterization of recombinant enzyme expressed in bicistronic format with NADPH-cytochrome P450 reductase.

Cytochrome P450 3A7 is the major P450 form present in fetal liver tissue and may be responsible for the detoxification of many drugs that reach the fetal circulation. We report the development of bacterial expression systems for P450 3A7. Maximal yields (up to 50 nmol P450/liter culture) were obtained with a construct in which the 5'-terminus of the 3A7 cDNA was modified to include the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, Proc. Natl. Acad. Sci. USA 88, 5597-5601, 1991) and to incorporate several downstream amino acid substitutions derived from the P450 3A5 sequence. This sequence also appeared optimal for expression of P450 3A4 and 3A5. Recombinant P450 3A7 was partially purified using ion-exchange and hydroxylapatite chromatography and reconstituted with NADPH-cytochrome P450 reductase, cytochrome b5, and lipids. Activity comparable to that of P450 3A4 was demonstrated toward a number of procarcinogens. An alternative approach was used to further characterize recombinant 3A7 due to low yields of recombinant protein in the expression and poor recovery in the purification. P450 3A7 was subcloned into a bicistronic vector containing human NADPH-cytochrome P450 reductase and expressed in bacteria. Recombinant P450 3A7 coexpressed in bacterial membranes with NADPH-cytochrome P450 reductase showed similar levels of activity toward erythromycin (N-demethylation) and ethylmorphine (N-demethylation) to P450 3A4 and 3A5 expressed in the same system, whereas 3A7 was less active toward midazolam (1'- and 4-hydroxylation) and nifedipine (oxidation).

Amino Acid Sequence↗
PubMed1995

Characterization of chimeric heme-copper respiratory oxidases using subunits I of Escherichia coli cytochrome b o and Halobacterium salinarium cytochrome aa3.

We constructed chimeric enzymes with the Escherichia coli cytochrome bo and the Halobacterium salinarium cytochrome aa3 through recombinant DNA techniques and investigated their spectroscopic and biochemical properties. Although most of the chimeras could not retain hemes in the molecule, the chimeric enzyme containing helix VII of subunit I of the H. salinarium cytochrome aa3 showed the spectral properties similar to those of the native E. coli oxidase, suggesting that both the low-spin heme b and the high-spin heme o are associated with the chimeric subunit I. However, CuB was absent in the chimera. Helix VII of subunit I of the H. salinarium cytochrome aa3 is 70% similar to the counterpart of the E. coli cytochrome bo and further contains two invariant histidines which serve as the CuB ligands. These results indicate that helix VII must be arranged properly relative to helix VI which provides the third CuB ligand.

Amino Acid Sequence↗
PubMed1996

Isolation of a new mouse cDNA clone: hybrid form of cytochrome P450 2b10 and NADPH-cytochrome P450 oxidoreductase.

We have isolated a novel cDNA sequence from a male mouse liver library. It consists of an open reading frame containing no stop codon of 1348 bp which share 98% similarity with the cytochrome P450 2b10 followed by 95 nucleotides sharing 88% identity with the mouse NADPH cytochrome P450 oxidoreductase. Proteins are encoded by the opposite DNA strands. Genomic amplification excludes a library artefact or a de novo recombination event. RT-PCR analyses revealed expression of this clone in the liver of male and female mice as well as in kidney, lung and intestine. This cDNA was not, however, induced by phenobarbital. Preliminary results indicate that this cDNA could span more than 2.5 kb. This study is the first description of a cytochrome P450 cDNA sequence encoding part of both a cytochrome P450 and the NADPH cytochrome P450 oxidoreductase in mammalian liver.

Amino Acid Sequence↗
PubMed1994

Structural aspects of the cytochrome b6f complex; structure of the lumen-side domain of cytochrome f.

The following findings concerning the structure of the cytochrome b6f complex and its component polypeptides, cyt b6, subunit IV and cytochrome f subunit are discussed: (1) Comparison of the amino acid sequences of 13 and 16 cytochrome b6 and subunit IV polypeptides, respectively, led to (a) reconsideration of the helix lengths and probable interface regions, (b) identification of two likely surface-seeking helices in cyt b6 and one in SU IV, and (c) documentation of a high degree of sequence invariance compared to the mitochondrial cytochrome. The extent of identity is particularly high (88% for conserved and pseudoconserved residues) in the segments of cyt b6 predicted to be extrinsic on the n-side of the membrane. (2) The intramembrane attractive forces between trans-membrane helices that normally stabilize the packing of integral membrane proteins are relatively weak. (3) The complex isolated in dimeric form has been visualized, along with isolated monomer, by electron microscopy. The isolated dimer is much more active than the monomer, is the major form of the complex isolated and purified from chloroplasts, and is inferred to be a functional form in the membrane. (4) The isolated cyt b6f complex contains one molecule of chlorophyll a. (5) The structure of the 252 residue lumen-side domain of cytochrome f isolated from turnip chloroplasts has been solved by X-ray diffraction analysis to a resolution of 2.3 A.

Amino Acid Sequence↗
PubMed2001

Kinetic studies on the oxidation of cytochrome b(5) Phe35 mutants with cytochrome c, plastocyanin and inorganic complexes.

To illustrate the functions of the aromatic residue Phe35 of cytochrome b(5) and to give further insight into the roles of the Phe35-containing hydrophobic patch and/or aromatic channel of cytochrome b(5), we studied electron transfer reactions of cytochrome b(5) and its Phe35Tyr and Phe35Leu variants with cytochrome c, with the wild-type and Tyr83Phe and Tyr83Leu variants of plastocyanin, and with the inorganic complexes [Fe(EDTA)](-), [Fe(CDTA)](-) and [Ru(NH(3))(6)](3+). The changes at Phe35 of cytochrome b(5) and Tyr83 of plastocyanin do not affect the second-order rate constants for the electron transfer reactions. These results show that the invariant aromatic residues and aromatic patch/channel are not essential for electron transfer in these systems.

Cytochrome c Group↗
PubMed1995

Fatty acid discrimination and omega-hydroxylation by cytochrome P450 4A1 and a cytochrome P4504A1/NADPH-P450 reductase fusion protein.

The omega-hydroxylation of fatty acids by certain cytochrome P450 enzymes shows a degree of chain-length and regionspecificity which is remarkable in view of the conformational flexibility of these substrates, the strong similarity in properties among homologs, and the lack of polar groups (other than the carboxy terminus) with which to guide and strength enzyme-substrate interactions. To investigate the chemical basis for these features of omega-hydroxylation we designed and synthesized a series of lauric acid analogs and evaluated them as substrates and inhibitors of omega-hydroxylation catalyzed by cytochrome P4504A1 and a cytochrome P450 4A1/NADPH-P450 reductase fusion protein. Among n-alkanoic acids, lauric acid was found to have the optimum chain length for the fusion protein, as it does for native cytochrome P450 4A1. With both enzymes, chain shortening caused a precipitous drop in turnover while chain lengthening caused a gradual drop in turnover. The fusion protein omega-hydroxylated methyl laurate and lauryl alcohol about 1/10th as efficiently as lauric acid, but it did not hydroxylate lauramide. 10-Methoxydecanoic acid underwent O-demethylation (via omega-hydroxylation). The branched substrate 11-methyllauric acid was hydroxylated efficiently and selectively at the omega-position. In contrast, the cyclopropyl analog 11,12-methanolauric acid was not detectably hydroxylated, although it induced Type I binding spectrum and inhibited lauric acid omega-hydroxylation by 43% at equimolar concentrations. omega-(Imidazolyl)-decanoic acid induced a Type II heme-binding spectrum and was an especially potent inhibitor of lauric acid hydroxylation. Collectively these data suggest that the active site of cytochrome P450 4A1 has an elongated tubular shape of definite length (ca. 14 A) with a recognition site for polar groups (including but not limited to carboxyl) at its entrance and the (oxo)heme group at its terminus.

Cytochrome P-450 CYP4A↗
PubMed1990

L-Lactate cytochrome c reductase: rapid kinetic studies of electron transfers within the flavocytochrome b2-cytochrome c assembly.

This study is part of a series aimed at the characterization of individual steps of electron transfer taking place between prosthetic flavin, heme b2, heme c within active sites and complexes. After rapid mixing of ferricytochrome c with partially reduced flavocytochrome b2, the reaction is followed at the level of two reactants, cytochrome b2 and cytochrome c. In order to define the proper reactivity of flavosemiquinone, conditions under which this form is highly stabilized (presence of pyruvate) have been chosen. With the help of simulations, it has been possible to characterize a rapid step of electron transfer from cytochrome b2 to cytochrome c within a complex (at approx. 70% saturation) and a slow step k = 5 s-1 assigned to cytochrome b2 reduction by flavosemiquinone within the active site of the pyruvate-liganded enzyme.

Computer Simulation↗