scholarly journals The role of tryptophan 97 of cytochrome P450 BM3 from Bacillus megaterium in catalytic function. Evidence against the ‘covalent switching’ hypothesis of P-450 electron transfer

1994 ◽  
Vol 303 (2) ◽  
pp. 423-428 ◽  
Author(s):  
A W Munro ◽  
K Malarkey ◽  
J McKnight ◽  
A J Thomson ◽  
S M Kelly ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Bacillus Megaterium ◽  
Electron Flow ◽  
Tryptophan Residue ◽  
Prosthetic Group ◽  
Catalytic Function ◽  
Redox Partners ◽  
Cast Doubt ◽  
Cytochrome P450 Bm3 ◽  
Cytochrome P 450

The ‘Covalent Switching’ hypothesis suggests that a strongly conserved tryptophan residue acts as a mediator of electron-transfer flow between redox partners in cytochrome P-450 systems [Baldwin, Morris and Richards (1991) Proc. R. Soc. London B 245, 43-51]. We have investigated the effect of alteration of the conserved tryptophan (Trp-97) in cytochrome P-450 BM3 (P-450 102) from Bacillus megaterium. Replacement of Trp-97 with Ala, Phe or Tyr results in a decrease in the natural haem content and alters the resting spin state of the remaining haem in the purified mutant enzymes. However, kinetic analyses indicate that the mutant enzymes retain high levels of catalytic activity. C.d. and e.p.r. spectroscopy also reveal little alteration in secondary structure or change in the pattern of haem ligation. These findings cast doubt on the covalent switching mechanism of intermolecular electron flow in the P-450s, but indicate that this residue plays a role in the association of the haem prosthetic group.

scholarly journals Minireview: Regulation of Steroidogenesis by Electron Transfer

Endocrinology ◽  
2005 ◽  
Vol 146 (6) ◽  
pp. 2544-2550 ◽  
Author(s):  
Walter L. Miller
Keyword(s):  
Electron Transfer ◽  
Electron Flow ◽  
Cytochrome B5 ◽  
Serine Phosphorylation ◽  
Molar Ratio ◽  
Type I ◽  
Type Ii ◽  
Cytochrome P450 Enzymes ◽  
Redox Partners ◽  
P450 Enzyme

Abstract Cytochrome P450 enzymes catalyze the degradation of drugs and xenobiotics, but also catalyze a wide variety of biosynthetic processes, including most steps in steroidogenesis. The catalytic rate of a P450 enzyme is determined in large part by the rate of electron transfer from its redox partners. Type I P450 enzymes, found in mitochondria, receive electrons from reduced nicotinamide adenine dinucleotide (NADPH) via the intermediacy of two proteins—ferredoxin reductase (a flavoprotein) and ferredoxin (an iron/sulfur protein). Type I P450 enzymes include the cholesterol side-chain cleavage enzyme (P450scc), the two isozymes of 11-hydroxylase (P450c11β and P450c11AS), and several vitamin D-metabolizing enzymes. Disorders of these enzymes, but not of the two redox partners, have been described. Type II P450 enzymes, found in the endoplasmic reticulum, receive electrons from NADPH via P450 oxidoreductase (POR), which contains two flavin moieties. Steroidogenic Type II P450 enzymes include 17α-hydroxylase/17,20 lyase (P450c17), 21-hydroxylase (P450c21), and aromatase (P450aro). All P450 enzymes catalyze multiple reactions, but P450c17 appears to be unique in that the ratio of its activities is regulated at a posttranslational level. Three factors can increase the degree of 17,20 lyase activity relative to the 17α-hydroxylase activity by increasing electron flow from POR: a high molar ratio of POR to P450c17, serine phosphorylation of P450c17, and the presence of cytochrome b5, acting as an allosteric factor to promote the interaction of POR with P450c17. POR is required for the activity of all 50 human Type II P450 enzymes, and ablation of the Por gene in mice causes embryonic lethality. Nevertheless, mutation of the human POR gene is compatible with life, causing multiple steroidogenic defects and a skeletal dysplasia called Antley-Bixler syndrome.

scholarly journals Histidine residues in rabbit liver microsomal cytochrome P-450 2B4 control electron transfer from NADPH-cytochrome P-450 reductase and cytochrome b5

1996 ◽  
Vol 318 (3) ◽  
pp. 857-862 ◽  
Author(s):  
Peter HLAVICA ◽  
Michael LEHNERER ◽  
Manfred EULITZ
Keyword(s):  
Electron Transfer ◽  
Cytochrome B5 ◽  
Cumene Hydroperoxide ◽  
Differential Susceptibility ◽  
Histidine Residues ◽  
Nadph Cytochrome ◽  
Fluorescence Measurements ◽  
Redox Partners ◽  
Nadh Cytochrome ◽  
Cytochrome P 450

Treatment of cytochrome P-450 2B4 (P-450 2B4) with diethylpyrocarbonate to introduce 10–11 equivalents of acylating agent per polypeptide chain resulted in the selective derivatization of histidine residues characterized by differential susceptibility toward the modifier. Second-derivative spectral analysis as well as fluorescence measurements disproved gross alterations in P-450 2B4 structure as a consequence of labelling. The modified haemoprotein retained its ability to bind hexobarbital and catalyse cumene hydroperoxide-sustained N-demethylation of the barbiturate. However, there was a steady attenuation of NAD(P)H-driven electron flux with increasing extent of P-450 2B4 carbethoxylation in reconstituted systems fortified with either NADPH-cytochrome P-450 reductase or NADH-cytochrome b5 reductase/cytochrome b5 as the redox partners, with 50% inhibition occurring when 6–7 histidines were blocked. Hampered P-450 2B4 reductase activities recovered to differing degrees upon treatment of the acylated mono-oxygenase with neutral hydroxylamine. Spectral data indicated that docking of the redox components to derivatized P-450 2B4 was not perturbed, so that disruption of the electron flows most likely resulted from some injury of the electron-transfer mechanisms.

scholarly journals Cytochrome c550 from Pseudomonas aeruginosa

1993 ◽  
Vol 289 (1) ◽  
pp. 173-178 ◽  
Author(s):  
P Reichmann ◽  
H Görisch
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Electron Transfer ◽  
Oxygen Consumption ◽  
Electron Transport ◽  
Electron Transport Chain ◽  
Prosthetic Group ◽  
Midpoint Potential ◽  
Transport Chain ◽  
Ethanol Dehydrogenase ◽  
Soluble C

In cells of Pseudomonas aeruginosa A.T.C.C. 17933 grown on ethanol the synthesis of a soluble c-type cytochrome, together with quinoprotein ethanol dehydrogenase, is induced. The cytochrome, with an alpha-absorption band at 550 nm, was purified to homogeneity. The molecular mass of the monomeric protein is 15 kDa, the pI is 4.8, and it contains one haem prosthetic group. The midpoint potential of the autoxidizable, but not autoreducible, cytochrome is 280 mV. Cytochrome c550 mediates electron transfer between quinoprotein ethanol dehydrogenase and ferricyanide. In a system composed of membrane particles with NN‘NN’-tetramethyl-p-phenylenediamine oxidase activity and quinoprotein ethanol dehydrogenase, oxygen consumption is only observed in the presence of cytochrome c550. This indicates the participation of the cytochrome in the electron-transport chain linked to quinoprotein ethanol dehydrogenase in P. aeruginosa. The electron transport from ethanol dehydrogenase to oxygen is inhibited by myxothiazol and antimycin, indicating that a cytochrome bc1-like complex is involved.

scholarly journals NADPH: cytochrome P-450 reductase in olfactory epithelium Relevance to cytochrome P-450-dependent reactions

1986 ◽  
Vol 240 (2) ◽  
pp. 585-592 ◽  
Author(s):  
C J Reed ◽  
E A Lock ◽  
F De Matteis
Keyword(s):  
Olfactory Epithelium ◽  
Direct Evidence ◽  
Reductase Activity ◽  
Electron Flow ◽  
Male Rats ◽  
Male And Female ◽  
Marked Activity ◽  
Nadh Cytochrome ◽  
Olfactory Tissue ◽  
Cytochrome P 450

The presence of a very active cytochrome P-450-dependent drug-metabolizing system in the olfactory epithelium has been confirmed by using 7-ethoxycoumarin, 7-ethoxyresorufin, hexobarbitone and aniline as substrates, and the reasons for the marked activity of the cytochrome P-450 in this tissue have been investigated. The spectral interaction of hexobarbitone and aniline with hepatic and olfactory microsomes has been examined. By this criterion there was no evidence for marked differences in the spin state of the cytochromes of the two tissues, or for the olfactory epithelium containing a greater amount of cytochrome capable of binding hexobarbitone, a very actively metabolized substrate. Rates of NADPH and NADH: cytochrome c reductase activity were found to be higher in the olfactory epithelium than in the liver, and direct evidence was obtained for a greater amount of the NADPH-dependent flavoprotein in the olfactory microsomes. Investigation of male rats and male and female mice, as well as male hamsters, demonstrated that, in all cases, the cytochrome P-450 levels of the olfactory epithelium were lower than those of the liver, while the 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities were higher. A correlation was found between 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities for both tissues in all species examined. The ratio of reductase to cytochrome P-450 was found to be considerably higher in the olfactory epithelium (1:2-1:3) than in the liver (1:11-1:15), regardless of the species examined, suggesting that facilitated electron flow may contribute significantly to the cytochrome P-450 catalytic turnover in the olfactory tissue.

scholarly journals Induction of cytochrome P-450BM-3 (CYP 102) by non-steroidal anti-inflammatory drugs in Bacillus megaterium

1996 ◽  
Vol 316 (1) ◽  
pp. 279-283 ◽  
Author(s):  
Neil ENGLISH ◽  
Valerie HUGHES ◽  
C. Roland WOLF
Keyword(s):  
Fatty Acid ◽  
Bacillus Megaterium ◽  
Peroxisome Proliferators ◽  
Induction Process ◽  
Oxygenase Activity ◽  
Operator Sequence ◽  
Inflammatory Drugs ◽  
Anti Inflammatory ◽  
Cytochrome P 450 ◽  
Rational Mechanism

Bacillus megaterium contains a cytochrome P-450 fatty acid mono-oxygenase which is inducible with barbiturate drugs. We have demonstrated that this enzyme system is inducible with peroxisome proliferators. In mammals, peroxisome proliferators also induce mono-oxygenases in the CYP4A gene family. In this paper we demonstrate that the non-steroidal anti-inflammatory drugs ibuprofen, ketoprofen and indomethacin are potent inducers of fatty acid mono-oxygenase activity as well as of P-450BM-3 protein in B. megaterium. The levels of induction of P-450 protein were 11.8-, 3.9- and 3.0-fold respectively. In addition, we demonstrate that these inducing agents interact with a transcriptional repressor, Bm3R1, which leads to its dissociation from its operator sequence. This provides a rational mechanism for the induction process. This is the first report which demonstrates that non-steroidal anti-inflammatory drugs can interact directly with a transcription factor to initiate gene expression, and further substantiates the structure–activity relationships that identify inducers of cytochrome P-450BM-3 and compounds that have the potential to act as peroxisome proliferators and induce CYP4A expression in mammals.

scholarly journals The effect of magnesium ions on the diemthylaniline oxidation rate and electron transfer in liver microsomal fraction

1973 ◽  
Vol 136 (2) ◽  
pp. 371-379 ◽  
Author(s):  
A. I. Archakov ◽  
I. I. Karuzina ◽  
I. S. Kokareva ◽  
G. I. Bachmanova
Keyword(s):  
Electron Transfer ◽  
Fluorescence Quantum Yield ◽  
Sharp Increase ◽  
Microsomal Fraction ◽  
Magnesium Ions ◽  
Total Rate ◽  
Nadph Oxidation ◽  
Liver Microsomal Fraction ◽  
Rate Limiting ◽  
Cytochrome P 450

1. Reactions of N-demethylation, p-hydroxylation and N-oxidation of one substrate, i.e. dimethylaniline, have been used to show that the activating effect of Mg2+ takes place only in the first two reactions. 2. An increase in Vmax. of N-demethylation of dimethylaniline is accompanied by an increase in Km. In the p-hydroxylation of dimethylaniline Vmax. increases whereas Km does not change. A comparison of the changes in the Km values of these reactions with the change in Ks shows that in both cases Km does not characterize the affinity of cytochrome P-450 for dimethylaniline. 3. The rate-limiting site of N-demethylation and p-hydroxylation of dimethylaniline, as well as the total rate of NADPH oxidation in the presence of dimethylaniline, is between cytochromes b5 and P-450. Addition of Mg2+ to the incubation medium changes the hydrophobic environment of phosphatidylcholine in the membrane, the process being accompanied by a sharp increase in the fluorescence quantum yield of 8-anilinonaphthalene-1-sulphonate.

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