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C Veeger

Publications and source records attributed to C Veeger.

At least 109 records · Page 6Linked to original sources
PubMed1980

Fluorescence polarization and energy-transfer studies on the pyruvate dehydrogenase complex of Escherichia coli.

We have attached eosin maleimide specifically to the lipoyl group of the pyruvate dehydrogenase complex isolated from Escherichia coli. Using this as the fluorescence acceptor and the intrinsic FAD of the lipoamide dehydrogenase subunit as the fluorescence donor, we confirmed previous measurements with other probes, in which it was suggested that the flavin moiety is at a substantial distance (over 4.5 nm) from the labeled lipoyl group. Since the lipoyl group must apply electrons to the FAD during the catalytic decarboxylation of pyruvate, we have investigated several potential mechanisms whereby this could happen. Movement within the complex, possibly triggered by the presence of substrate, seemed to be a strong possibility. Complex labeled with fluorophores on the accessible sulfhydryls, or on the lipoyl functions, did not give evidence of such triggering upon addition of substrate as judged by both static and dynamic fluorescence depolarization. The mobility of the subunits of labeled lipoamide dehydrogenase exceeded that expected for the total complex. Pyrene maleimide bound to the lipoyl functions also exhibited considerably faster rotations than the predicted one of the whole complex (tau c > 3 micros). This suggests that a constant movement within the complex, coupled with the rotation of the lipoyl group, may bring the active sites of the complex transiently close enough together to interact on a time scale much faster than enzyme turnover. At the same time, the lipoyl group and the active sites of the complex can spend most of their time at points which are rather distant from each other.

Chemical Phenomena↗
PubMed1979

Purification and properties of hydrogenase from Megasphaera elsdenii.

A hydrogenase has been purified to homogeneity from the soluble fraction of the rumen bacterium Megasphaera elsdenii, the overall purification is 200 times with a yield of 14%. The pure enzyme consists of a single polypeptide chain with Mr approximately 50 000 which contains 12 atoms of non-haem iron and 12 atoms of acid-labile sulphide. The enzyme is rapidly inactivated by O2 and it is therefore purified under nitrogen and in the presence of sodium dithionite. The optical spectrum of the enzyme, after removal of the dithionite with air, shows a peak at 275 nm (epsilon 275 nm = 143 mM-1 cm-1) and a shoulder between 350 nm and 400 nm (epsilon 400 nm = 46 mM-1 cm-1). The enzyme catalyses hydrogen production from sodium dithionite at a low rate. The rate is greatly enhanced by addition of the electron donors flavodoxin, ferredoxin and methyl viologen. The kinetic data with these three electron donors suggest co-operativity, but no indication of self-association of the enzyme was obtained. Sodium chloride enhances the rate of hydrogen production with methyl viologen semiquinone and changes the kinetic behaviour of the enzyme with this electron donor, but causes inhibition of the reactions mediated by ferredoxin and flavodoxin. Two kinetic models were developed which are consistent with the kinetic data of the three electron donors tested. The apparent co-operativity for the hydrogen production can be fitted with the mathematical form of those models. The identical kinetic behaviour of the hydrogenase with the one-electron donors flavodoxin and methyl viologen semiquinone monomer and the two-electron donor ferredoxin indicates that the hydrogenase accepts two electrons in two separate, independent steps and further indicates that the two (4Fe-4S) clusters of the donor ferredoxin are independent. The interpretation of the kinetic data with methyl viologen semiquinone is complicated by the fact that the semiquinone dimerises, and that the formation of the dimer is enhanced by salt. Taking into account the association of this donor, the activity of the enzyme with methyl viologen semiquinone can be described by the sum of the activities of the enzyme with methyl viologen monomer and methyl viologen dimer. The enzyme catalyses the oxidation of hydrogen gas with methyl and benzyl viologen as electron acceptors to their semiquinone forms; both electron acceptors show Michaelis-Menten kinetics. The hydrogen oxidation activity with both electron acceptors is stimulated by addition of sodium chloride. The kinetic data of the oxidation of hydrogen with the two-electron acceptors used are consistent with the porposed models, if it is assumed that the pathway followed is compulsory. At this moment no choice can be made between the models proposed.

Computers↗
PubMed1979

Fluorescence properties of reduced flavins and flavoproteins.

Fluorescence lifetimes and polarized emission properties of reduced flavin were measured using several model compounds and flavoproteins. Depending on the conditions of solvent and temperature or reduction method the lifetimes vary between 1 and 15 ns. The longer lifetime values are found in several forms of reduced lactate oxidase, in which a good correlation exists between fluorescence intensity and lifetime. In practically all flavoproteins the fluorescence is heterogeneous. Several mechanisms are proposed to explain the observed heterogeneity in lifetimes. The reduced models in glycerol at subzero temperature exhibit high degrees of polarization of the fluorescence, whereas distinct depolarization is encountered in several reduced flavoproteins suggesting a certain mobility of the flavin chromophor.

Flavins↗
PubMed1979

Structure of pyridine nucleotide transhydrogenase from Azotobacter vinelandii.

1. Pyridine nucleotide transhydrogenase of Azotobacter vinelandii purified by affinity chromatography consists of a mixture of polydisperse rods at neutral pH. No other structures are seen by electron microscopy. 2. At high pH (8.5--9.0) the rods depolymerize. Complete depolymerization can be achieved in 0.1 M Tris-Cl pH 9.0. The depolymerized enzyme has a molecular weight of 421000 (sedimentation equilibrium), its sedimentation coefficient s20, w = 15 S and its Stokes' radius Rs = 7 nm. Since gel electrophoresis in the presence of sodium dodecyl sulphate shows that transhydrogenase consists of a single polypeptide chain of molecular weight (54 +/- 2) X 10(3) it follows that the depolymerized enzyme has an octameric quaternary structure. We propose that this octamer serves as the functional monomeric unit ('unimer') from which the polymeric form of transhydrogenase is constructed. 3. Gel filtration and sucrose gradient centrifugation studies of cell-free extracts from A. vinelandii show the unimer to be the predominant active species.

Azotobacter↗
PubMed1978

Involvement of the cytoplasmic membrane in nitrogen fixation by Rhizobium leguminosarum bacteroids.

1. The nitrogen-fixing efficiency of freshly prepared suspensions of Rhizobium leguminosarum bacteroids from pea root nodules was considerably enhanced by addition of bovine serum albumin. Evidence was found that during preparation of bacteroids the cell membrane is exposed to the uncoupling effect of free fatty acids and to plant phospholipase D activity. Both effects could be counteracted by bovine serum albumin. 2. A technique was developed by which concentrations of free O2 and nitrogenase activity could be measured simultaneously under conditions of steady-state respiration. By means of this system it could be shown that in contrast to previous claims, high ATP/ADP ratios can be achieved in bacteroids even with a high concentration of O2 in the medium. 3. Nitrogen fixation was found to be controlled by the ATP/ADP ratio, the generation of reducing equivalents and the switch-off phenomenon. It was demonstrated that the generation of reducing equivalents for nitrogenase is regulated by the energized state and the integrity of the bacteroid cell membrane. The data indicate that the process of aerobic nitrogen fixation in R. leguminosarum bacteroids resembles that of Azotobacter vinelandii.

Adenosine Diphosphate↗
PubMed1978

Membrane energization in relation with nitrogen fixation in Azotobacter vinelandii and Rhizobium leguminosarum bacteroids.

Nitrogen fixation in A. vinelandii and R. leguminosarum bacteroides shows identical characteristics with respect to the dependence on membrane energization, the sensitivity to uncouplers, the ATP/ADP-ratio, and the dependences on flavodoxinhydroquinone as electrondonor. Although we have been successful in preparing inside-out vesicles which can be energized, attempts to couple these membranes to N2-ase were still unsuccessful. One of the major problems could be the failure to energize these vesicles directly by ATP. Although subject to polymerisation after addition of MgCl2, it could be shown that the actual mol.wt. of the O2-stable N2-ase complex is about 300,000 in agreement with a 1:1:1 stoichiometry of the three constituent proteins, namely, component I, component II and the 2Fe-2S protein.

Adenosine Diphosphate↗
PubMed1978

Fluorescence energy-transfer studies on the pyruvate dehydrogenase complex isolated from Azotobacter vinelandii.

Fluorescence energy transfer has been employed to estimate the minimum distance between each of the active sites of the 4 component enzymes of the pyruvate dehydrogenase multienzyme complex from Azotobacter vinelandii. No energy transfer was seen between thiochrome diphosphate, bound to the pyruvate decarboxylase active site, and the FAD of the lipoamide dehydrogenase active site. Likewise, several fluorescent sulfhydryl labels, which were specifically bound to the lipoyl moiety of lipoyl transacetylase, showed no energy transfer to either the flavin or thiochrome diphosphate. These observations suggest that all the active centers of the complex are quite far apart (greater than or equal to 40 nm), at least during some stages of catalysis. These results do not preclude the possibility that the distances change during catalysis. Several of the fluorescent probes used possessed multiple fluorescent lifetimes, as shown by determination of lifetime averages by both phase and modulation measurements on a phase fluorimeter. These lifetimes are shown to result from multiple factors, not necessarily related to multiple protein conformations.

Acetylcysteine↗
PubMed1976

Symmetry and asymmetry of the pyruvate dehydrogenase complexes from Azotobacter vinelandii and Escherichia coli as reflected by fluorescence and spin-label studies.

Fluorescence-lifetime measurements of FAD bound to lipoamide dehydrogenase from Azotobacter vinelandii and Escherichia coli were performed. It is shown from these results that the two FAD groups in the isolated dimeric enzyme, as well as in the enzyme in the intact complex of E. coli, are in non-equivalent surroundings. This contrasts with the near equivalence of the FAD groups of both the enzyme and complex isolated from A. vinelandii. Reduction of the complex with Mg2+, thiamine pyrophosphate and pyruvate or with NADH enables the attachment of a maleimide analogue specifically to the lipoyl moieties of the transacetylase(s). Spin label [N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide] introduced in such a way proves the existence of at least two different micro-environments around the lipoyl moieties in complex isolated from A. vinelandii. Electron paramagnetic resonance spectra of the specifically spin-labelled complexes from E. coli and A. vinelandii, when dissolved in tricine [N-tris(hydroxymethyl)-methylglycine] buffer, show interactions of at least two electron spins with each other, which indicate that the lipoyl moieties are rather close together. Fluorescent label [N-(1-anilinonaphthyl-4)maleimide] is specifically attached to the lipoyl moiety of the high-Mr transacetylase of the freshly isolated complex from A. vinelandii. From the large differences in the apparent lifetimes tau p and tau m, as detected by phase fluorimetry, it is shown that this fluorscent label is distributed in different micro-environments. The differences observed in energy transfer between fluorescent label, attached to the lipoyl moiety of the high-Mr transacetylase, indicate different conformations of the complex from A. vinelandii. Upon introduction of the label after reduction with NADH a much larger energy transfer, thus a shorter distance, is observed between the label and FAD than when reduction is performed with Mg2+, thiamine pyrophosphate and pyruvate. A similar conformation dependence upon reduction is found for the pyruvate dehydrogenase complex from E. coli. It is thus proposed that the transacetylase of E. coli and the high-Mr transacetylase of A. vinelandii are both non-symmetrically distributed within the complex.

Acetyltransferases↗
PubMed1976

Studies on the binding of FMN by apoflavodoxin from Peptostreptococcus elsdenii, pH and NaCl concentration dependence.

1. The pH and ionic strength dependence of the interaction of FMN with apoflavodoxin has been studied by fluorometry in the pH region 2-5, at 22 degrees C. 2. The rate constant of dissociation and the dissociation constant were experimentally determined; the rate constants of association were claculated at a given pH value. These constants depend on the ionic strength. The plots of these constants against the square root of the ionic strength are straight. 3. Our data have been interpreted in terms of the Brönsted theory, which relates chemical reaction rates to ionic strength. The data indicate that the apoenzyme reaches its maximum net positive charge at pH 2.0-2.6. The calculated net charge in this pH region is between 11 and 12 and is in agreement with the theoretical value of 12 as deduced from the primary structure of the protein. The isoelectric point of the holoenzyme is about 4. 4. The rate constant of association extrapolated to zero ionic strength is 3.2-10(5)M-1-s-1 and is pH-independent. 5. The rate constant of dissociation and the dissociation constant extrapolated to zero ionic strength depend on the pH. The results are explained by assuming that there are two protein ionizations with a pK value of 3.4; these ionizing groups are possibly close to the FMN binding site.

Apoproteins↗