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At least 1,135 records · Page 63Linked to original sources
PubMed1998

Steps toward constructing a cytochrome b6 f complex in the purple bacterium Rhodobacter sphaeroides: an example of the structural plasticity of a membrane cytochrome.

We have modified the cytochrome b subunit of the cytochrome bc1 complex from the purple bacterium Rhodobacter sphaeroides to introduce two distinctive features of cytochrome b6 f complexes. In the first one, we have split cyt b into two polypeptides thus mimicking the organization of cyt b6 and subunit IV in the b6 f complexes. In the second, an extra residue was added between His198 and Phe199, thus extending the span between the histidine ligands for the two b-hemes in helix D. The properties of the mutant strains were determined using thermodynamic and kinetic analysis. The two mutant enzymes were assembled and functioned so as to allow the photosynthetic growth of the mutant strains. For the split enzyme, we show that two independently translated fragments of cyt b are inserted in the membrane. Our results indicate a decrease in the stability of the semiquinone formed at the quinone reduction (Qi) site in this mutant. This property, characteristic for b6 f complexes, indicates the functional importance of the connecting span between helices D and E. The presence of the inserted threonine in helix D modified the spectrum and redox potential of the bL-heme, shifting the potential difference between the two b-hemes from 140 mV in the wild-type to 55 mV in the mutant strain. This change in the driving force of electron transfer through the membrane was reflected in an inability of the mutant strain to accumulate a large transmembrane electrical potential on successive flashes.

Amino Acid Sequence↗
PubMed1999

Role of configurational gating in intracomplex electron transfer from cytochrome c to the radical cation in cytochrome c peroxidase.

Electron transfer within complexes of cytochrome c (Cc) and cytochrome c peroxidase (CcP) was studied to determine whether the reactions are gated by fluctuations in configuration. Electron transfer in the physiological complex of yeast Cc (yCc) and CcP was studied using the Ru-39-Cc derivative, in which the H39C/C102T variant of yeast iso-1-cytochrome c is labeled at the single cysteine residue on the back surface with trisbipyridylruthenium(II). Laser excitation of the 1:1 Ru-39-Cc-CcP compound I complex at low ionic strength results in rapid electron transfer from RuII to heme c FeIII, followed by electron transfer from heme c FeII to the Trp-191 indolyl radical cation with a rate constant keta of 2 x 10(6) s-1 at 20 degrees C. keta is not changed by increasing the viscosity up to 40 cP with glycerol and is independent of temperature. These results suggest that this reaction is not gated by fluctuations in the configuration of the complex, but may represent the elementary electron transfer step. The value of keta is consistent with the efficient pathway for electron transfer in the crystalline yCc-CcP complex, which has a distance of 16 A between the edge of heme c and the Trp-191 indole [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. Electron transfer in the complex of horse Cc (hCc) and CcP was examined using Ru-27-Cc, in which hCc is labeled with trisbipyridylruthenium(II) at Lys-27. Laser excitation of the Ru-27-Cc-CcP complex results in electron transfer from RuII to heme c FeII with a rate constant k1 of 2.3 x 10(7) s-1, followed by oxidation of the Trp-191 indole to a radical cation by RuIII with a rate constant k3 of 7 x 10(6) s-1. The cycle is completed by electron transfer from heme c FeII to the Trp-191 radical cation with a rate constant k4 of 6.1 x 10(4) s-1. The rate constant k4 decreases to 3.4 x 10(3) s-1 as the viscosity is increased to 84 cP, but the rate constants k1 and k3 remain the same. The results are consistent with a gating mechanism in which the Ru-27-Cc-CcP complex undergoes fluctuations between a major state A with the configuration of the hCc-CcP crystalline complex and a minor state B with the configuration of the yCc-CcP complex. The hCc-CcP complex, state A, has an inefficient pathway for electron transfer from heme c to the Trp-191 indolyl radical cation with a distance of 20.5 A and a predicted value of 5 x 10(2) s-1 for k4A. The observed rate constant k4 is thus gated by the rate constant ka for conversion of state A to state B, where the rate of electron transfer k4B is expected to be 2 x 10(6) s-1. The temperature dependence of k4 provides activation parameters that are consistent with the proposed gating mechanism. These studies provide evidence that configurational gating does not control electron transfer in the physiological yCc-CcP complex, but is required in the nonphysiological hCc-CcP complex.

2,2'-Dipyridyl↗
PubMed1988

The soluble cytochromes c of methanol-grown Hyphomicrobium X. Evidence against the involvement of autoreduction in electron-acceptor functioning of cytochrome cL.

Hyphomicrobium X, grown on methanol with O2 or nitrate as electron acceptor, contains two major soluble cytochromes c. These were isolated in electrophoretically homogeneous form. They are related to cytochromes c already described for other methylotrophic bacteria and designated cytochromes cH and cL (properties indicated in that order) in view of the following characteristics: absorption maxima of the reduced forms (414, 520 and 551 nm and 414, 520 and 550 nm); molar absorption coefficients of the alpha-bands (23,700 M-1.cm-1 and 21,600 M-1.cm-1); maxima of the alpha-bands (no splitting) at 77 K (547.6 nm and 548.5 nm); Mr values of the native proteins (15,000 and 19,500); pI values (7.4 and 7.5, and 4.3); midpoint potentials at pH 7.0 (+292 mV and +270 mV). Both were monomers containing 1 haem c group per protein molecule, the oxidized forms binding cyanide at high pH. Autoreduction also occurred at high pH but at a rate significantly lower than that reported for other ferricytochromes c. On the other hand, the reverse situation applies to the reduction of ferricytochrome cL by reduced methanol dehydrogenase, the reduction occurring instantaneously at pH 7 but much more slowly at pH 9 (ferricytochrome cH was reduced at a 7-fold lower rate, but the rates at pH 7 and 9 were similar). Insignificant reduction was observed with cyclopropanol-inactivated enzyme or with enzyme in the presence of EDTA. In view of the dissimilarities, it is concluded that different mechanisms operate in the autoreduction of ferricytochrome cL and in its reduction by reduced methanol dehydrogenase.

Alcohol Oxidoreductases↗
PubMed1989

The cytochromes of anaerobically grown Escherichia coli. An electron-paramagnetic-resonance study of the cytochrome bd complex in situ.

The e.p.r. signals attributable to a cytochrome bd-type ubiquinol:O2 oxidoreductase (cytochrome b-558-b-595-d) were studied in a cytoplasmic membrane preparation of Escherichia coli that had been grown on glycerol with fumarate as respiratory-chain oxidant. Two major high-spin ferric haem signals were resolved on the basis of their potentiometric behaviour: a rhombic high-spin species (gx = 6.25, gy = 5.54) was assigned to haem b-595, and an axial high-spin (gx = 5.97, gy = 5.96) species was assigned to the haem d. These signals titrated with Em.7 values of 154 and 261 mV respectively, corresponding closely to optically determined values for haem b-595 and haem d. At high potentials (greater than 300 mV) the rhombic species attributable to haem b-595 underwent a partial transition to a second rhombic species with g-values of 6.24 (gx) and 5.67 (gy). The high-spin ferric haem spectra were affected by O2, CO, cyanide and pH. A low-spin ferric haem signal was observed at g = 3.3 (gz), which titrated with an Em.7 of 226 mV, and this was assigned to haem b-558. The data support a model for cytochrome bd with two ligand-binding sites, a single haem d and a single haem b-595.

Carbon Monoxide↗
PubMed2004

Crystal structure and characterization of a cytochrome c peroxidase-cytochrome c site-specific cross-link.

A specific covalently cross-linked complex between redox partners yeast cytochrome c peroxidase (CCP) and cytochrome c (cyt. c) has been made by engineering cysteines into CCP and cyt. c that form an intermolecular disulfide bond in high yield. The crystal structure of the cross-linked complex has been solved to 1.88-A resolution and closely resembles the structure of the noncovalent complex [Pellitier, H. & Kraut, J. (1992) Science 258, 1748-1755]. The higher resolution of the covalent complex has enabled the location of ordered water molecules at the peroxidase-cytochrome c interface that serve to bridge between the two proteins by hydrogen bonding. As in the noncovalent complex, direct electrostatic interactions between protein groups appear not to be critical in complex formation. UV-visible spectroscopic and stopped-flow studies indicate that CCP in the covalent complex reacts normally with H(2)O(2) to give compound I. Stopped-flow kinetic studies also show that intramolecular electron transfer between the cross-linked ferrocytochrome c and the Trp-191 cation radical site in CCP compound I occurs fast and is nearly complete within the dead time ( approximately 2 ms) of the instrument. These results indicate that the structure of the covalent complex closely mimics the physiological electron transfer complex. In addition, single-turnover and steady-state experiments reveal that CCP compound I in the covalent complex oxidizes exogenously added ferrocytochrome c at a slow rate (t(1/2) approximately 2 min), indicating that CCP does not have a second independent site for physiologically relevant electron transfer.

Base Sequence↗
PubMed1975

Apparent dependence of interactions between cytochrome b5 and cytochrome b5 reductase upon translational diffusion in dimyristoyl lecithin liposomes.

Dimyristoyl lecithin liposomes, containing cytochrome b5 reductase (NADH:ferricytochrome b5 oxidoreductase, EC 1.6.2.2) and varying amounts of cytochrome b5, were used to measure flavoprotein catalysis alone and catalysis requiring electron transfer between the reductase and cytochrome as a function of temperature. Whereas flavoprotein catalysis showed a simple linear temperature dependence in an Arrhenius plot, the reaction involving electron transfer between the two bound enzymes showed a marked, 4-fold, change in rate at the crystalline-liquid crystalline phase transition of the hydrocarbon chains of the lecithin vesicles and a second, minor change involving the minor transition. These data represent strong evidence that protein-protein interactions in this membrane model system are dependent upon translational diffusion of nonpolar segments of the proteins in the hydrocarbon region of the phospholipid bilayer.

Animals↗
PubMed1999

A cytochrome b5 is required for full activity of flavonoid 3', 5'-hydroxylase, a cytochrome P450 involved in the formation of blue flower colors.

The substitution pattern of anthocyanin pigments is a main determinant of flower color. Flavonoid 3',5'-hydroxylase (F3'5'H) is a cytochrome P450 enzyme (Cyt P450) that catalyzes the 3', 5'-hydroxylation of dihydroflavonols, the precursors of purple anthocyanins. Species such as rose and carnation lack F3'5'H activity and are, therefore, unable to generate purple or blue flowers. Petunia, on the other hand, contains two loci, termed hf1 and hf2, that encode a Cyt P450 with F3'5'H activity. Here we report the identification of an additional petunia gene that is required for 3',5' substitution of anthocyanins and purple flower colors. It encodes a cytochrome b5 and is expressed exclusively in the flower. Inactivation of the gene by targeted transposon mutagenesis reduced F3'5'H enzyme activity and the accumulation of 5'-substituted anthocyanins, resulting in an altered flower color. However, no phenotypic effect on the activity of other Cyt P450s, involved in the synthesis of hormones or general phenylpropanoids, was observed. These data provide in vivo evidence for the regulation of the activity of specific Cyt P450s by a cytochrome b5.

Amino Acid Sequence↗
PubMed2001

Structure of the bound dioxygen species in the cytochrome oxidase reaction of cytochrome cd1 nitrite reductase.

Reduction of dioxygen to water is a key process in aerobic life, but atomic details of this reaction have been elusive because of difficulties in observing active oxygen intermediates by crystallography. Cytochrome cd(1) is a bifunctional enzyme, capable of catalyzing the one-electron reduction of nitrite to nitric oxide, and the four-electron reduction of dioxygen to water. The latter is a cytochrome oxidase reaction. Here we describe the structure of an active dioxygen species in the enzyme captured by cryo-trapping. The productive binding mode of dioxygen in the active site is very similar to that of nitrite and suggests that the catalytic mechanisms of oxygen reduction and nitrite reduction are closely related. This finding has implications to the understanding of the evolution of oxygen-reducing enzymes. Comparison of the dioxygen complex to complexes of cytochrome cd(1) with stable diatomic ligands shows that nitric oxide and cyanide bind in a similar bent conformation to the iron as dioxygen whereas carbon monoxide forms a linear complex. The significance of these differences is discussed.

Crystallization↗
PubMed2006

Identification of the interactions between cytochrome P450 2E1 and cytochrome b5 by mass spectrometry and site-directed mutagenesis.

The reaction cycles of cytochrome P450s (P450) require input of two electrons. Electrostatic interactions are considered important driving forces in the association of P450s with their redox partners, which in turn facilitates the transfer of the two electrons. In this study, the cross-linking reagent, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), was used to covalently link cytochrome P450 2E1 (CYP2E1) with cytochrome b(5) (b(5)) through the formation of specific amide bonds between complementary charged residue pairs. Cross-linked peptides in the resulting protein complex were distinguished from non-cross-linked peptides using an (18)O-labeling method on the basis that cross-linked peptides incorporate twice as many (18)O atoms as non-cross-linked peptides during proteolysis conducted in (18)O-water. Subsequent tandem mass spectrometric (MS/MS) analysis of the selected cross-linked peptide candidates led to the identification of two intermolecular cross-links, Lys(428)(CYP2E1)-Asp(53)(b(5)) and Lys(434)(CYP2E1)-Glu(56)(b(5)), which provides the first direct experimental evidence for the interacting orientations of a microsomal P450 and its redox partner. The biological importance of the two ion pairs for the CYP2E1-b(5) interaction, and the stimulatory effect of b(5), was confirmed by site-directed mutagenesis. Based on the characterized cross-links, a CYP2E1-b(5) complex model was constructed, leading to improved insights into the protein interaction. The described method is potentially useful for mapping the interactions of various P450 isoforms and their redox partners, because the method is relatively rapid and sensitive, and is capable of suggesting not only protein interacting regions, but also interacting orientations.

Acetaminophen↗
PubMed1995

Functional expression of fused enzymes between human cytochrome P4501A1 and human NADPH-cytochrome P450 oxidoreductase in Saccharomyces cerevisiae.

The activity of human cytochrome P450 enzymes heterologously expressed in Saccaromyces cerevisiae cells is limited by the yeast endogenous cytochrome P450 oxidoreductase (yOR). To overcome these limitations, we constructed hybrids between human P4501A1 (CYP1A1) and human P450 oxidoreductase (hOR) by combining the cDNA encoding hOR with the CYP1A1 cDNA. In addition, in one construct, the amino terminus of hOR was replaced by the membrane anchor domain of a yeast protein. Anchoring of the fusion constructs in internal membranes either by the amino terminus of hOR or by the yeast peptide resulted in functional hybrid proteins, which were present in similar amounts as the authentic CYP1A1 in microsomal fractions of recombinant cells. Saccharomyces cerevisiae cells transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-CYP1A immunoglobulin (Ig) and anti-oxidoreductase Ig. Saccharomyces cerevisiae yOR-mutant (cpr1-) and wild-type (CPR1+) cells containing the fused enzymes exhibited CYP1A1-specific 7-ethoxyresorufin-O-deethylase activities. Reduced CO-difference spectra of microsomal fractions containing the fused enzymes indicated a proper incorporation of protoheme into the CYP1A1 domains. These results show that the chimeric proteins represent catalytically self-sufficient monooxygenase systems. The hOR domains of the hybrid proteins were also functional as cytochrome c reductases and able to activate the yeast P450 enzyme lanosterol-14 alpha-demethylase, indicating correct insertion of the chimeric proteins in internal membranes.

Cloning, Molecular↗
PubMed1984

Ethanol-inducible cytochrome P-450 is more susceptible to in vitro carbon tetrachloride-mediated destruction than phenobarbital-inducible and beta-naphthoflavone-inducible cytochromes P-450.

Liver microsomes were incubated aerobically in vitro with NADPH and carbon tetrachloride in the presence of 1.5 mM EDTA. The resulting losses of haemoproteins and monooxygenase activities were evaluated after washing. Microsomes from ethanol-pretreated rats lost about 66% of their cytochrome P-450 measured as such, together with 94% of their ethoxycoumarin 0-deethylase activity. Microsomes from control and phenobarbital-pretreated rats lost between 30 and 35% of their cytochrome P-450 and between 65 and 50% of the ethoxycoumarin 0-deethylase activity. Microsomes from beta-naphtoflavone-pretreated rats lost about 10% of their cytochrome P-450 and 13% of their ethoxycoumarin 0-deethylase activity. The inhibition with tetrahydrofuran, metyrapone and alpha-naphtoflavone were also changed after incubation, suggesting selective destruction of isoenzymes with high affinity for tetrahydrofuran and selective sparing of others. Studies of 4-nitroanisole 0-demethylation indicated selective destruction of enzymes with high affinity for the 4-nitroanisole and sparing of enzymes with lower affinity. The properties that were destroyed after incubation with carbon tetrachloride were those that were elevated after chronic ethanol feeding. The destructive action of carbon tetrachloride in microsomes from ethanol-pretreated rats was found to be different from those of allyl alcohol and 2-isopropyl 4-pentenamide. The findings may have relevance to the enhancement of chronic ethanol feeding on the hepatotoxicty of carbon tetrachloride.

7-Alkoxycoumarin O-Dealkylase↗
PubMedDate not supplied

Catalytic and immunologic similarities between monkey and human liver cytochrome P-450db1 (human cytochrome P-450 2D6).

In vivo pharmacogenetic studies have suggested that the monkey may be an animal model for the human polymorphism of cytochrome P-450 2D6 (also called cytochrome P-450db1). In the present study, the catalytic, immunologic, and electrophoretic properties of cytochrome P-450db1 in liver microsomes from African green monkeys (Cercopithecus aethiops) were examined and compared with P-450db1 in human liver microsomes. Using sparteine as the substrate, the activity of microsomal P-450db1 from the two sources was indistinguishable in terms of the pattern of sparteine metabolites produced, the apparent Ki values of 8 competitive inhibitors (r = 0.94, p less than 0.001), and the extent of immunoinhibition by anti-rat P-450db1 antibody. Kinetic analyses demonstrated that the apparent KM values of the high affinity component of sparteine oxidation in monkey liver microsomes fell within the range observed in human livers; the Vmax of this component was as much as six times greater than the highest value reported for human liver. Western immunoblots showed a protein band in monkey liver microsomes that co-migrated with P-450db1 in human liver. The high degree of similarity observed here between P-450db1 of monkey and human liver microsomes suggests that the monkey will be a good animal model for P-450db1 enzyme studies, and possibly for studies of the role of this enzyme in drug abuse and dependence.

Animals↗
PubMed1991

[Oxidative modification of cytochrome P-450 during its function. I. Comparative study of the inactivation of cytochrome P-450 LM2 in various systems].

The changes in the content of purified isolated cytochrome P-450 LM2 under the action of hydrogen peroxide and during its operation in a soluble reconstituted system were studied. It was found that cytochrome P-450 LM2 inactivation by hydrogen peroxide is accompanied by a decrease in the hemoprotein activity, loss of heme, oxidation of SH-groups and changes in the oligomeric state of the enzyme. There were some differences in the mechanisms of cytochrome P-450 LM2 inactivation under the action of H2O2 and during catalysis.

Animals↗
PubMed1991

[Oxidative modification of cytochrome P-450 during its function. II. Study of the mechanism of cytochrome P-450 LM2 inactivation in a soluble reconstructed monooxygenase system].

Inactivation of cytochrome P-450 LM2 induced by hydrogen peroxide formed in the active site of the enzyme was studied. Catalase did not protect cytochrome P-450 LM2 from inactivation during its operation in a soluble reconstituted system. The hemoprotein inactivation in this system was found to depend on the ratio of hemo- to flavoproteins. It was demonstrated that cytochrome P-450 LM2 inactivation during catalysis is accompanied by cleavage of the hemoprotein molecule. It is probable that this fact plays a key role in regulation of enzyme decay.

Animals↗
PubMed1986

Accessibility of the cytochrome a heme in cytochrome c oxidase to exchangeable protons.

The comparison of the resonance Raman spectrum of cytochrome a2+ from cytochrome oxidase in deuterated buffers to that in protonated buffers reveals many lines that have different frequency or intensity. Some of the frequency differences are very large, e.g. on the order of 10 cm-1. From these differences in the Raman spectra, we infer that the heme pocket is readily accessible to protons and that labile groups are either on the heme or interact strongly with it. These data suggest the possibility of direct participation in proton translocation and/or oxygen protonation by the heme of cytochrome a.

Cytochrome a Group↗
PubMed1982

Role of the hydrophobic tail of cytochrome b5 in the interaction with cytochrome P-450 LM2.

The interaction of cytochrome P-450 LM2 with cytochrome b5 is accompanied by a high spin shift in P-450 LM2 and the improvement of a second derivative spectra in the near ultraviolet region. After incorporation into phospholipid vesicles the interaction between P-450 LM2 and b5 is increased according to a decrease of the apparent binding constant. The involvement of a tryptophanyl residue in the interaction will be discussed. Contrary the tryptic fragment of cytochrome b5 which lacks the membrane binding tail does not show an interaction with P-450 LM2 either in the absence or presence of phospholipids.

Animals↗
PubMed1976

An infrared study of the carbon monoxide complexes of cytochrome P-450 and cytochrome P-420.

The infrared stretch vibrations (upsilonCO) of the CO-complexes of cytochrome P-450 and cytochrome P-420 have been determined from infrared difference spectra. The CO-complexes exhibit IR-bands at 1949 cm-1 and 1966 cm-1 with half widths of approximately 17 cm-1 and approximately 20 cm-1 respectively. These results are compared with the CO-stretch frequencies of other haemoproteins and discussed with respect to specific interactions of the CO-ligand with the protein moiety and to the ligand trans to CO of the cytochromes.

Binding Sites↗