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D Pompon

Publications and source records attributed to D Pompon.

65 records · Page 4Linked to original sources
PubMed1984

On the mechanism of action of cytochrome P-450. Oxidation and reduction of the ferrous dioxygen complex of liver microsomal cytochrome P-450 by cytochrome b5.

The effects of cytochrome b5 on the decay of the ferrous dioxygen complexes of P-450LM2 and P-450LM4 from rabbit liver microsomes were studied by stopped-flow spectrophotometry. The P-450 (FeIIO2) complexes accept an electron from reduced cytochrome b5 and, in a reaction not previously described, donate an electron to oxidized cytochrome b5 to give ferric P-450. A comparison with the electron-transferring properties of ferrous P-450 under anaerobic conditions allowed determination of the limiting steps of the two reactions involving the oxygenated complex. The rate of decay of the dioxygen complex was increased in all cases with b5 present; however, with oxidized b5 a large increase in the rate was observed with P-450 isozyme 4 but not with isozyme 2, whereas the opposite situation was found when reduced b5 was used. The reactions between b5 and ferrous dioxygen P-450 were not at thermodynamic equilibrium under the conditions employed. From the results obtained, a model is proposed in which the ferrous dioxygen complex decomposes rapidly into another species differing from ferric P-450 in its spectral properties and from the starting complex in its electron-transferring properties. A scheme is presented to indicate how competition among spontaneous decay, cytochrome b5 oxidation, and cytochrome b5 reduction by the ferrous O2 complex may influence substrate hydroxylation.

Aerobiosis↗
PubMed1983

A cysteine cluster critical for flavin binding in flavocytochrome b2 from Baker's yeast.

We have recently described the addition of 2-keto-3-butynoic acid to flavin-free flavocytochrome b2, a reaction which leads to the loss of flavin-binding capacity ('inactivation') [D. Pompon and F. Lederer (1982) Eur. J. Biochem. 129, 143-137]. For total inactivation, the extrapolated incorporation value was 0.9 mol reagent/mol subunit. In this work we report the results of sequence studies which elucidate the nature of the modification. The modified protein was cleaved with cyanogen bromide and the peptides separated on Sephadex G-100 and SP-Sephadex C-25. 14C-labeled peptides were digested with trypsin and chymotrypsin and smaller labeled fragments purified by chromatography on Sephadex G-50 and thin-layer fingerprinting. It is shown that three cysteine residues are fractionally labeled with nearly complete mutual exclusion. Furthermore, a fraction of the modified peptides is found under the form of cross-linked fragments, where two cysteines have added to the same ketobutynoate molecule. Only two of the possible cross-links were found. These results show that the three cysteines are close to one another in space in the flavin-free enzyme and hence probably also in the holoenzyme. These results, combined with those obtained in the affinity labeling reaction of holoenzyme with bromopyruvate [Alliel et al. (1982) Eur. J. Biochem. 122, 553-558], show that the three residues are located in or close to the active site. Their possible role is discussed.

Binding Sites↗
PubMed1982

Reconstitution of liver NADH: cytochrome b5 oxidoreductase and of Desulfovibvio vulgaris flavodoxin with 1-carba-1-deazaflavin.

Flavin-free cytochrome b5 reductase was reconstituted with 1-deazaflavin and 5-deazaflavin mononucleotides and dinucleotides. The 5-deazaenzyme functioned in transhydrogenation reactions but lacked electron transferase activity. The 1-deazaenzyme was fully competent for both input and output reactions. The flavin reduction rate was lowered about sevenfold upon N-1 substitution of FAD, but hydrogen abstraction from NADH remained the limiting step. Autoxidation of the reduced enzyme was more rapid than with the normal cofactor. Oxidation was accompanied by appearance of a transient blue-type semiquinone and superoxide ion production. Flavin-free apoflavodoxin was reconstituted with 1-deaza-1-carbaflavin mononucleotide (1-deaza-FMN). Its behaviour toward dithionite and oxygen was qualitatively highly similar to that of native flavodoxin. These observations contrast with the fact that apoflavocytochrome b2 could not be reconstituted with 1-deaza-FMN [Pompon. D. and Lederer, F. (1979) Eur. J. Biochem. 96. 571-579]. These results, as well as other data from the literature, are discussed in the light of existing hypotheses, which try to correlate flavin protein interactions and flavoprotein function.

Anaerobiosis↗
PubMed1982

A residue critical for flavin binding in flavocytochrome b2 from Baker's yeast. Inactivation and labeling of flavin-free enzyme by 2-keto-3-butynoate.

The reagent 2-keto-3-butynoic acid is the product formed in the reaction between the suicide reagent 2-hydroxy-3-butynoate and a number of flavoproteins. We describe in this paper the inactivation of flavin-free flavocytochrome b2 by 2-keto-3-butynoate, in a rapid reaction which introduces 0.9 mol reagent for total inactivation. The modification results in loss of affinity for flavin and affects a cysteine residue. We also describe in this paper a simple enzymatic method for preparing 2-keto-3-butynoate, as well as some properties of the reagent, in particular its stability and susceptibility to nucleophilic attack. We show that at neutral pH it is highly specific for thiol compounds. Some properties of the adduct formed with glutathione are described. These experiments should pave the way for the use of 2-keto-3-butynoate with other proteins.

Binding Sites↗
PubMed1980

Flavocytochrome b2 (Baker's yeast). Deuterium isotope effect studied by rapid-kinetic methods as a probe for the mechanism of electron transfer.

The use of DL-[2-2H]lactate in steady-state measurements of ferricyanide reduction by flavocytochrome b2 at 30 degrees C has previously yielded an isotope effect of 5 [F. Lederer (1974) Eur. J. Biochem. 46, 393--399]. We report here studies carried out at 5 degrees C with L-[2-2H]lactate, where flavin and heme reduction were observed in the stopped-flow apparatus, in the absence of acceptor. The generally biphasic reduction curves were analysed according to a new mathematical treatment which allowed us to derive microscopic constants from initial reduction rates. It has thus been possible to determine an isotope effect of 8 on flavin reduction, 6 on heme reduction, compared to 4 in the steady state. Consequently, two slightly rate-limiting steps occur after the first one where the alpha-hydrogen is abstracted. It has also been possible to calculate the substrate association and dissociation rate constants for intact enzyme. The studies were carried out in parallel on intact and cleaved cytochrome b2. The results suggest that proteolysis affects essentially the steps involved in flavin reduction, and not intramolecular electron transfer steps. Moreover, the experimental data obtained at low rates of electron entry have led us to reexamine a previously proposed scheme for electron transfer [Capeillère-Blandin, Bray, Iwatsubo and Labeyrie (1975) Eur. J. Biochem. 54, 549--566]. An alternative model based on computer-simulation studies will be presented in a paper in this journal.

Deuterium↗
PubMed1980

Binding of Cibacron blue F3GA to the flavin and NADH sites in cytochrome b5 reductase.

The behaviour of cytochrome b5 reductase holoenzyme and apoenzyme toward blue-dextran--Sepharose has been studied. Holoenzyme was adsorbed at low ionic strength and could be eluted with 100 microM NADH or NAD+. Flavin-free enzyme was even more strongly bound and could be eluted with 1 M NaCl, or 100 microM NADH + 10 microM FAD. Separately the cofactors were without effect. FMN was less effective than FAD. ADP and AMP eluted nothing. Cibacron blue F3GA was found to exert a mixed inhibition on NADH oxidation. Dye binding to holoenzyme elicited a characteristic red shift in its spectrum. Comparison of the difference spectrum amplitude at 680 and 585 nm showed the presence of a second binding mode at higher dye concentrations. These results point to the existence for cytochrome b5 reductase of two binding sites with high affinity for blue-dextran--Sepharose: the NADH binding site and flavin binding site. For the latter it is clear that isoalloxazine pocket must play a role in dye binding. Cytochrome b5 reductase is the second flavoenzyme which has been shown to have affinity for immobilized dye at the flavin site, the first one being flavocytochrome b2, and FMN-dependent enzyme [D. Pompon and F. Lederer (1978) Eur. J. Biochem. 90, 563--569].

Animals↗
PubMed1980

Flavocytochrome b2 from baker's yeast. Computer-simulation studies of a new scheme for intramolecular electron transfer.

It has been shown recently that the use of L-(+)-[2-2H]lactate as substrate instead of unlabeled L-(+)-lactate induces a lowering of the flavin reduction rate of cytochrome b2 by a factor of 8 [D. Pompon, M. Iwatsubo, and F. Lederer (1980) Eur. J. Biochem. 104, 479--488]. This high isotope effect enabled us to study electron transfer between prosthetic groups at a very low rate of electron entry. The kinetic scheme for electron transfer in cytochrome b2 proposed by Capeillère-Blandin [Eur. J. Biochem. 56, 91--101 (1975)] is examined here in the light of the kinetic data reported in our preceding paper. This study indicates some disagreements, particularly at a low rate of electron entry. New kinetic schemes capable of explaining data obtained with deuterated lactate are proposed. These new schemes differ from that of Capeillère-Blandin in that: (a) the hypothesis of simultaneous prosthetic group reduction for the two protomers of one given dimer is abandoned; (b) the limiting step of the slow phases of heme and flavin reduction is a slow interprotomer electron exchange between a heme pair, a flavin pair or heme and flavin; (c) a rather fast conformational change controlled by the redox state of heme or flavin of one promoter can modulate the rate of electron transfer in another promoter. These new kinetic schemes allow us to determine the rate of intraprotomer and interprotomer electron transfer and to decide precisely how these steps are modified by the proteolytic cleavage of 'intact' enzyme to 'cleaved' enzyme.

Computers↗
PubMed1978

Binding of Cibacron Blue F3GA to flavocytochrome b2 from baker's yeast.

1. Flavin-free cytochrome b2 has been prepared by rapid Sephadex filtration at acid pH. The method, which yields an apo-enzyme with high reconstitution potential and has several advantages over previously used procedures, is described in detail. 2. Flavin-free cytochrome b2 thus prepared is retained by blue-dextran-bound Sepharose. It can be eluted by an increase in ionic strength, by dilute ethylene glycol and specifically by low concentrations of FMN. The holoenzyme is not retarded at all. 3. Both flavin-free and holocytochrome b2 bind Cibacron blue F3GA with appearance of distinct difference spectra. Cibacron blue is an inhibitor for the holoenzyme, it shows mixed type inhibition with respect to lactate. 4. It is concluded that there are two types of binding sites for Cibacron blue F3GA on flavocytochrome b2. Both possess ionic and hydrophobic character; one of them, which is the flavin binding site, is only available in the absence of the cofactor. Taken together these results may mean that the enzyme possesses a local flavin-binding structure similar to the 'dinucleotide fold'.

Anthracenes↗
PubMed1976

Controlled proteolysis of flavocytochrome b2. Characterization of a 15000-dalton heme-binding core and comparison with detergent solubilized cytochrome b5.

It is known that each subunit of the tetrameric flavocytochrome b2 can be cleaved by yeast proteases to fragments of molecular weight 33-36000 and 21 000, with some modification of catalytic properties, but without destruction of the oligomeric state of the protein. We report here experimental conditions which enabled us to simulate this specific cleavage in a controlled fashion with chymotrypsin and subtilisin. With trypsin and papain, on the other hand, it was not found possible to stop the digestion in such a way as to obtain a homogeneous still active product. A characterization of the enzymatic forms obtained by digestion with chymotrypsin and subtilisin at 0 degrees C shows that modification of enzymatic and solubility properties occurs in a stepwise fashion. It is also ccluded that cleavage by yeast proteases is accompanied by loss of 10 to 25 residues. At 37 degrees C, chymotrypsin digestion yields a heme-binding core of molecular weight 15 000, larger than the already characterized tryptic heme-binding core by about 40 residues. Although the latter is known to be very similar to trypsin-solubilized cytochrome b5, the lack of aggregation of the former in aqueous solution, its amino acid composition and circular dichroism spectra do not point to a similarity of its additional peptide segment with the hydrophobic tail of detergent-solubilized cytochrome b5.

Amino Acids↗
PubMedDate not supplied

Maximizing the expression of mammalian cytochrome P-450 monooxygenase activities in yeast cells.

Cytochrome P-450s constitute a superfamily of mono-oxygenases which require the association with specific redox enzymes bound to the endoplasmic reticulum membrane for their activity. Conditions for the functional expression of these mammalian enzymes in yeast cells and the respective merits and limitations of currently used P-450 expression systems, are considered. The dependence of the mouse P-450 IA1 specific activity on the cytochrome expression level in yeast microsomes is studied and results demonstrate that the low amounts of endogenous NADPH-cytochrome P-450 reductase and cytochrome b5 which are naturally present, are limiting for the heterologous monooxygenase activities. The sequences encoding human liver cytochrome b5, the native and a modified form of the yeast NADPH-cytochrome P-450 reductase were cloned by making use of PCR techniques, over-expressed in yeast as functional forms, and characterized. New vectors allowing a high level of mammalian P-450 expression upon induction were also constructed and tested. A strategy for the construction of a co-expression system allowing maximal activity of mammalian cytochrome P-450s is discussed.

Animals↗