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M Tegoni

Publications and source records attributed to M Tegoni.

50 records · Page 3Linked to original sources
PubMed1993

Preliminary crystal structure studies of a ternary electron transfer complex between a quinoprotein, a blue copper protein, and a c-type cytochrome.

A ternary electron transfer protein complex has been crystallized and a preliminary structure investigation has been carried out. The complex is composed of a quinoprotein, methylamine dehydrogenase (MADH), a blue copper protein, amicyanin, and a c-type cytochrome (c551i). All three proteins were isolated from Paracoccus denitrificans. The crystals of the complex are orthorhombic, space group C222(1) with cell dimensions a = 148.81 A, b = 68.85 A, and c = 187.18 A. Two types of isomorphous crystals were prepared: one using native amicyanin and the other copper-free apo-amicyanin. The diffraction data were collected at 2.75 A resolution from the former and at 2.4 A resolution from the latter. The location of the MADH portion was determined by molecular replacement. The copper site of the amicyanin molecule was located in an isomorphous difference Fourier while the iron site of the cytochrome was found in an anomalous difference Fourier. The MADH from P. denitrificans (PD-MADH) is an H2L2 hetero-tetramer with the H subunit containing 373 residues and the L subunit 131 residues, the latter containing a novel redox cofactor, tryptophan tryptophylquinone (TTQ). The amicyanin of P. denitrificans contains 105 residues and the cytochrome c551i contains 155 residues. The ternary complex consists of one MADH tetramer with two molecules of amicyanin and two of c551i, forming a hetero-octamer; the octamer is located on a crystallographic diad. The relative positions of the three redox centers--i.e., the TTQ of MADH, the copper of amicyanin, and the heme group of c55li--are presented.

Bacterial Proteins↗
PubMed1993

A hypothetical complex between crystalline flavocytochrome b2 and cytochrome c.

Flavocytochrome b2 and cytochrome c are physiological electron transfer partners in yeast mitochondria. The formation of a stable complex between them has been demonstrated both in solution and in the crystalline state. On the basis of the three-dimensional structures, using molecular modeling and energy minimization, we have generated a hypothetical model for the interaction of these redox partners in the crystal lattice. General criteria such as good charge and surface complementarity, plausible orientation, and separation distance of the prosthetic groups, as well as more specific criteria such as the stoichiometry determined in the crystal, and the involvement of both domains and of more than one subunit of flavocytochrome b2 led us to discriminate between several possible interaction sites. In the hypothetical model we present, four cytochrome c molecules interact with a tetramer of flavocytochrome b2. The b2 and c hemes are coplanar, with an edge-to-edge distance of 14 A. The contact surface area is ca. 800 A2. Several electrostatic interactions involving the flavin and the heme domains of flavocytochrome b2 stabilize the binding of cytochrome c.

Computer Graphics↗
PubMed1990

Inhibition of L-lactate: cytochrome-c reductase (flavocytochrome b2) by product binding to the semiquinone transient. Loss of reactivity towards monoelectronic acceptors.

Pyruvate has previously been shown to slow down the rate of intramolecular electron transfer from the flavosemiquinone (Fs) to the cytochrome b2 moiety of flavocytochrome b2 [Tegoni, M., Silvestrini, M. C., Labeyrie, F. & Brunori, M. (1984) Eur. J. Biochem. 140, 39-45] and to stabilize markedly the Fs state of the prosthetic flavin, relative to the oxidized (Fo) and the reduced (Fh) states [Tegoni, M., Janot, J. M. & Labeyrie, F. (1986) Eur. J. Biochem. 155, 491-503]. In the present study, we have determined the dissociation constants of pyruvate for the three redox forms of the prosthetic flavin and demonstrated that the Fs-pyruvate complex is actually much more stable than the Fo-pyruvate and Fh-pyruvate complexes. The inhibition produced by pyruvate has been characterized under steady-state conditions using both ferricytochrome c and ferricyanide as external acceptor. A detailed analysis and simulations of the suitable reaction scheme, taking into consideration all data from rapid kinetic studies of partial reactions previously published, show that the experimental noncompetitive inhibition results from the sum of a competitive effect due to binding of pyruvate to Fo and an uncompetitive effect due to binding to the Fs intermediate in a dead-end complex. Pyruvate binding to the semiquinone transient results in a marked loss of the reactivity of this donor in electron transfers to its specific partner, the cytochrome b2 present in the same active site, as to ferricyanide, an external acceptor. A critical evaluation of the parameters involved in the control of such reactivities is presented.

Cytochrome c Group↗
PubMed1989

Flavin and heme structures in lactate:cytochrome c oxidoreductase: a resonance Raman study.

Resonance Raman spectra of Hansenula anomala L-lactate:cytochrome c oxidoreductase (or flavocytochrome b2), of its cytochrome b2 core, and of a bis(imidazole) iron-protoporphyrin complex were obtained at the Soret preresonance from the oxidized and reduced forms. Raman contributions from both the isoalloxazine ring of flavin mononucleotide (FMN) and the heme b2 were observed in the spectra of oxidized flavocytochrome b2. Raman diagrams showing frequency differences of selected FMN modes between aqueous and proteic environments were drawn for various flavoproteins. These diagrams were closely similar for flavocytochrome b2 and for flavodoxins. This showed that the FMN structure must be very similar in both types of proteins, despite their very different proteic pockets. However, the electron density at this macrocycle was found to be higher in flavocytochrome b2 than in these electron transferases. No significant difference was observed between the heme structures in flavocytochrome b2 and in cytochrome b2 core. The porphyrin center-N(pyrrole) distances in the oxidized and reduced heme b2 were estimated to be 1.990 and 2.022 A from frequencies of porphyrin skeletal modes, respectively. The frequency of the vinyl stretching mode of protoporphyrin was found to be very affected in resonance Raman spectra of flavocytochrome b2 and of cytochrome b2 core (1634-1636 cm-1) relative to those observed in the spectra of iron-protoporphyrin [bis(imidazole)] complexes (1620 cm-1). These specificities were interpreted as reflecting a near coplanarity of the vinyl groups of heme b2 with the pyrrole rings to which they are attached. The low-frequency regions of resonance Raman indicated that the iron atoms of the four hemes b2 are in the porphyrin plane whatever their oxidation state. The histidine-Fe-histidine symmetric stretching mode was located at 205 cm-1 in the spectra of flavocytochrome b2 and of cytochrome b2 core. It was insensitive to the iron oxidation state and indicated strong Fe-His bonds in both states.

Flavin Mononucleotide↗
PubMed1988

Crystallographic study of the complex between sulfite and bakers' yeast flavocytochrome b2.

The complex between Saccharomyces cerevisiae flavocytochrome b2 and the sulfite anion has been analyzed by x-ray diffraction. A map of the difference in electron density between the complex and the native protein has been computed. One positive peak of electron density is visible at the active site of each of the two subunits in the asymmetric unit, very close to the N-5 of the flavin. The molecular fragment SO3(2-) can account for the shape of this difference in electron density. A third peak is visible in the subunit containing pyruvate, the reaction product. It is a peak of negative electron density localized at the position where the pyruvate usually is in the native form. These results are interpreted on the basis of the mechanism defined in solution for the reaction between flavins and sulfite.

Binding Sites↗
PubMed1986

Kinetics of electron transfer between two Hansenula anomala flavocytochrome b2 derivatives and two simple copper proteins (azurin and stellacyanin).

Two derivatives of Hansenula anomala flavocytochrome b2 have been prepared, one deprived of the flavin prosthetic group (deflavocytochrome b2), and the other consisting of the heme-b-carrying globule (b2 core). The redox potential of the heme in the two derivatives is -5 (+/- 5) mV and -10 (+/- 5) mV respectively, fairly similar to the value of -20 (+/- 5) mV reported for the holoenzyme, indicating a minor effect of the flavin and of the flavodehydrogenase domain on heme potential. The kinetics of azurin and stellacyanin reduction by both derivatives have been investigated. At pH 7.0, I = 0.2 M and 20 degrees C the second-order rate constants are: k = 8 X 10(5) M-1 S-1 for azurin reduction by deflavocytochrome b2; k = 1.6 X 10(6) M-1 S-1 for azurin reduction by b2 core; k = 1 X 10(7) M-1 S-1 for stellacyanin reduction by deflavocytochrome b2; k = 3 X 10(7) M-1 S-1 for stellacyanin reduction by b2 core. The change in pH markedly affects the kinetics in the case of azurin, but has no effect on stellacyanin reduction. The change in ionic strength has a significant effect when deflavocytochrome b2 is the reductant, indicating that the flavodehydrogenase domain plays a role in the stabilization of the transient kinetic complex by means of electrostatic interactions. The kinetic results are discussed in the framework of the Marcus theory.

Azurin↗
PubMed1986

Regulation of dehydrogenases/one-electron transferases by modification of flavin redox potentials. Effect of product binding on semiquinone stabilization in yeast flavocytochrome b2.

Spectroscopic and potentiometric measurements have been carried out, at room temperature, during anaerobic titrations of Hansenula anomala L-lactate cytochrome c oxidoreductase (or flavocytochrome b2) both in the presence and in the absence of pyruvate (the physiological reaction product). Under the same conditions, the flavin spectral contribution was estimated and the flavosemiquinone proportion was directly determined by electron paramagnetic resonance measurements. In the present study, we show the visible light absorption and paramagnetic characteristics of the flavin radical at 18 degrees C and also the dramatic effect of pyruvate on the redox potential of each monoelectronic couple of the flavin. Thermodynamic stabilization of the semiquinone form, in the presence of pyruvate, is interpreted as a mode of regulation of flavocytochrome b2 activity. Taking into account that analogous controls have been observed with two other flavoenzymes belonging to this class of dehydrogenases/one-electron transferases, we suggest that redox potential modulation could be a type of regulation effective for the whole class of enzymes in which a semiquinone is an obligate intermediate.

Electron Spin Resonance Spectroscopy↗
PubMed1984

A temperature-jump study of the electron transfer reactions in Hansenula anomala flavocytochrome b2.

Temperature-jump experiments on flavocytochrome b2 were carried out at different levels of heme reduction at pH 7.0 and 6.0, and as a function of pyruvate concentration. The relaxation, corresponding to an increase in the concentration of reduced heme, is in no case a simple process. AtpH 7.0 the mean reciprocal relaxation time is 1/tau* = 190 s-1, independent of enzyme concentration, wavelength of observation and percentage of heme reduction. Flavin semiquinone has been identified as the major electron donor to the heme in this process. At the same pH the presence of pyruvate in the millimolar concentration range increases the relaxation rate and affects its amplitude. The latter effect could be accounted for by a change in redox equilibria between heme and flavin upon pyruvate binding. At pH 6.0 the relaxation pattern depends more clearly on the level of heme reduction. A rapid process (tau-1 = 2500 s-1), predominant at high percentages of reduced heme, has been assigned to the reduction of heme by flavin hydroquinone, while the slower process (tau-1 = 350 s-1), essentially the only one present at or below 50% of heme reduction, has been ascribed to the reduction of heme by flavin semiquinone. These results are discussed in relation to the catalytic mechanism of the enzyme.

Ascomycota↗
PubMed1984

Modifications of redox equilibria with semiquinone stabilization upon pyruvate binding to L-lactate cytochrome c oxidoreductase (flavocytochrome b2).

Spectral redox titrations of flavin and cytochrome b2 moieties of flavocytochrome b2 were achieved in the absence and in the presence of pyruvate under equilibrium conditions at 18 degrees C; direct measurements of spin flavosemiquinone proportions have been carried out by EPR determinations at the same temperature. Our results show that the equilibria involving flavin are largely affected by the presence of pyruvate; the semiquinone proportion markedly increases almost till unit near half-reduction of cytochrome b2; at 10 mM pyruvate, the dismutation constant, Kdism = (Fs)2/(Fo)*(Fr) increases by a factor greater than or equal to 10.

Electron Spin Resonance Spectroscopy↗
PubMed1984

Modification of redox equilibria between heme and flavin within yeast flavocytochrome b2 (L-lactate cytochrome c reductase) upon binding of pyruvate, the reaction product.

Direct determinations of the concentration of semiquinone spin in redox equilibrium with the cytochrome b2 moiety were carried out at room temperature, in the presence of added pyruvate or in its absence. Results show that redox potentials of the one-electron couples of the prosthetic flavin are markedly affected by binding of pyruvate, the reaction product in the oxidation of L-lactate. The proportion of flavin semiquinone nearly reaches then 100 per cent.

Electron Spin Resonance Spectroscopy↗
PubMed1983

Complex formation and intermolecular electron transfer between flavocytochrome b2 in the crystal and cytochrome c.

The present study addresses the question whether tetrameric flavocytochrome b2 in the crystal is catalytically competent and, if so, whether it is possible to prepare a functional complex of the crystalline enzyme with its physiological electron acceptor cytochrome c. By single crystal microspectrophotometry we show that the native reduced enzyme can be oxidized by oxygen or ferricyanide and that the oxidized enzyme can be reduced by the electron donor L-lactate. Reduced cytochrome c appears to diffuse through the liquid channels of flavocytochrome b2 crystals and, at low ionic strength, to accumulate in amounts stoichiometrically equivalent to the enzyme protomers. Both cytochromes can be oxidized by ferricyanide. In the presence of L-lactate, both cytochromes become reduced. Since reduction of cytochrome c by L-lactate requires the catalytic action of flavocytochrome b2, it is concluded that the structure of the crystalline enzyme not only allows for electron transfer from L-lactate to flavin and intramolecular electron transfer from flavin to heme, but also for the formation of a productive complex with cytochrome c.

Crystallization↗
PubMed1980

Spontaneous dissociation of a cytochrome core and a biglobular flavoprotein after mild trypsinolysis of the bifunctional Saccharomyces cerevisiae flavocytochrome b2.

Saccharomyces cerevisiae flavocytochrome b2 is known as a bifunctional enzyme which behaves as the association of an FMN flavodehydrogenase with its specific acceptor, a b5-like cytochrome. Mild trypsinolysis gives rise to three complementary fragments (n, X, beta'), both prosthetic groups being still bound. After such proteolysis the separation of a biglobular flavoprotein domain (carrying FMN) from a cytochrome domain (with the heme) is obtained by molecular sieving under non-denaturing conditions. The marked lack of affinity between the tetrameric flavoprotein (X, beta')4 and the monomeric cytochrome core (n) leads to the hypothesis that the two domains are not tightly associated in the native molecule and might more relative to each other. Their respective mobility is possibly required for the catalytic mechanism. The comparison with previous trypsinolysis studies on the flavocytochrome b2 from Hansenula anomala suggests the presence of two common zones of hypersensitivity to proteases, along the protomeric polypeptide chain, and strongly supports the validity of the triglobular model for both flavocytochromes.

Flavin Mononucleotide↗