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 Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding

2011 ◽  
Vol 286 (18) ◽  
pp. 16246-16260 ◽  
Author(s):  
Chuanwu Xia ◽  
Djemel Hamdane ◽  
Anna L. Shen ◽  
Vivian Choi ◽  
Charles B. Kasper ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Cytochrome P450 ◽  
Disulfide Bond ◽  
Conformational Changes ◽  
Wild Type ◽  
Disulfide Linkage ◽  
Nadph Cytochrome ◽  
Redox Partners ◽  
Domain Movement ◽  
Flavin Domain

The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp147 and Arg514 in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP+ revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP+ shows movement of the Gly631–Asn635 loop. In the NADP+-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP+ shows movement of the Gly631–Asn635 loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly631–Asn635 loop movement controls NADPH binding and NADP+ release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners.

scholarly journals Characterization of Saccharomyces cerevisiae CYP51 and a CYP51 fusion protein with NADPH cytochrome P-450 oxidoreductase expressed in Escherichia coli.

1997 ◽  
Vol 41 (4) ◽  
pp. 776-780 ◽  
Author(s):  
K Venkateswarlu ◽  
D E Kelly ◽  
S L Kelly
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Reductase Activity ◽  
Spectral Response ◽  
Cumene Hydroperoxide ◽  
Type Ii ◽  
Nadph Cytochrome ◽  
Binding Spectra ◽  
Cytochrome P 450 ◽  
Fus Protein

Saccharomyces cerevisiae CYP51, target of azole antifungal agents, and CYP51 fused with S. cerevisiae cytochrome P-450 oxidoreductase (FUS protein) were expressed in active forms in Escherichia coli by cloning into pET15b. The expression was monitored immunologically, catalytically, and by using reduced carbon monoxide difference and type II binding spectra. CYP51 and FUS enzymes were located in membranes and produced a Soret peak at 448 nm in the reduced CO difference spectrum. The cytochrome P-450 contents in the membrane fractions containing CYP51 and FUS proteins were 12.8 +/- 2.6 and 17.4 +/- 3.7 pmol/mg of protein, respectively. The NADPH cytochrome P-450 oxidoreductase (CPR) content was estimated to be 15.7 +/- 1.1 pmol/mg of protein in FUS membrane fractions. FUS protein catalyzed the demethylation of substrate at the 14alpha position, with a turnover number of 1.96 +/- 0.37 min(-1) in the presence of NADPH. No reductase activity was observed in membrane fractions containing CYP51, and therefore, CYP51 did not function catalytically in the presence of NADPH, but in the presence of an artificial electron donor, cumene hydroperoxide, activity was comparable to that of the FUS enzyme. Further support for a normal structure for the hemoproteins was obtained from type II binding spectra, in which the spectral response was saturated with an equimolar concentration of ketoconazole.

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.

Contribution of Electrostatics to the Kinetics of Electron Transfer from NADH-Cytochrome b5 Reductase to Fe(III)-Cytochrome b5

2016 ◽  
Vol 120 (33) ◽  
pp. 8193-8207 ◽  
Author(s):  
Sireesha Kollipara ◽  
Shivakishore Tatireddy ◽  
Thusitha Pathirathne ◽  
Lasantha K. Rathnayake ◽  
Scott H. Northrup
Keyword(s):  
Electron Transfer ◽  
Cytochrome B5 ◽  
Cytochrome B5 Reductase ◽  
Nadh Cytochrome ◽  
Kinetics Of

scholarly journals The effects of halothane on hepatic microsomal electron transfer

1975 ◽  
Vol 148 (2) ◽  
pp. 179-186 ◽  
Author(s):  
M C Berman ◽  
K M Ivanetich ◽  
J E Kench
Keyword(s):  
Electron Transfer ◽  
Desaturase Activity ◽  
Anaesthetic Agent ◽  
Reductase Activity ◽  
Electron Flow ◽  
Cytochrome B5 ◽  
Order Rate Constant ◽  
Redox Equilibrium ◽  
Microsomal Cytochrome B5 ◽  
Nadh Cytochrome

1. The effects of halothane (CF3CHBrCl), a volatile anaesthetic agent, on electron transfer in isolated rat liver microsomal preparations were examined. 2. At halothane concentrations achieved in tissues during clinical anaesthesia (1-2mM), halothane shifts the redox equilibrium of microsomal cytochrome b5 in the presence of NADPH towards the oxidized form. Halothane accelerates stoicheiometric consumption of NADPH and O2, increases the rate of reoxidation of NADH-reduced microsomal ferrocytochrom b5, but does not affect NADPH- or NADH-cytochrome c reductase activity. The enhanced microsomal electron flow seen in the presence of halothane is not diminished by CO nor is it increased by pretreatment of the animals with phenobarbital. 3. The effects of halothane are maximum in microsomal preparations isolated from animals fed on a high-carbohydrate diet to induce stearate desaturase activity. Changes in microsomal electron transfer caused by halothane are in all cases abolished by low concentrations (1-2mM) of cyanide. Microsomal stearate desaturase activity is unaffected by halothane. 4. The first-order rate constant for oxidation of membrane-bound ferrocytochrome b5 in the absence of added substrate (k1 equals 1.5 times 10(-3)A-1) is similar to that for autoxidation of purified ferrocytochrome b5(k1 equals 7 times 10(-3)S-1) the rate of autoxidation of soluble ferrocytochrome b5 is unaffected by halothane. 5. It is concluded that the effects of halothane on microsomal electron transfer are not related to cytochrome P-450 linked metabolism but rather arise from the interaction of halothane with the cyanide-sensitive factor of the stearate desaturase pathway.

scholarly journals An enzyme-linked immunoadsorbent assay for measuring cytochrome b5 and NADPH-cytochrome P-450 reductase in rat liver microsomal fractions. Evidence for functionally inactive protein

1984 ◽  
Vol 217 (3) ◽  
pp. 623-632 ◽  
Author(s):  
L K Shawver ◽  
S L Seidel ◽  
P A Krieter ◽  
T K Shires
Keyword(s):  
Rat Liver ◽  
Cytochrome B5 ◽  
Spectral Measurement ◽  
Nadph Cytochrome ◽  
Cytochrome P 450 ◽  
Enzymic Assay ◽  
Inactive Protein

Immunoreactive cytochrome b5 and NADPH-cytochrome P-450 reductase (EC 1.6.2.4) from rat liver microsomal fractions were measured by using an enzyme-linked immunoadsorbent assay (e.l.i.s.a.) as a function of age, sex and type of inducer (phenobarbital or 3-methylcholanthrene), and the values were compared with those obtained by spectral measurement (for cytochrome b5) or enzymic assay (for reductase). In untreated animals, there was more cytochrome b5 and NADPH-cytochrome P-450 reductase when measured by an e.l.i.s.a. than was seen spectrally or enzymically. However, for microsomal preparations from phenobarbital-pretreated animals, spectrally obtained values for cytochrome b5 and immunoreactive-cytochrome b5 values were similar. Values from control animals suggest that there is about 20-30% more immunoreactive cytochrome b5 than that which is spectrally detectable.

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