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R Boelens

Publications and source records attributed to R Boelens.

137 records · Page 8Linked to original sources
PubMed1984

Sequential resonance assignments in 1H NMR spectra of oligonucleotides by two-dimensional NMR spectroscopy.

A sequential assignment procedure is outlined, based on two-dimensional NOE ( NOESY ) and two-dimensional J-correlated spectroscopy ( COSY ), for assigning the nonexchangeable proton resonances in NMR spectra of oligonucleotides. As presented here the method is generally applicable to right-handed helical oligonucleotides of intermediate size. We applied it to a lac operator DNA fragment consisting of d( TGAGCGG ) and d( CCGCTCA ) and obtained complete assignments for the adenine H8, guanine H8, cytosine H6 and H5, thymine H6 and 5-methyl, and the deoxyribose H1', H2', H2", H3', and H4' resonances, as well as some H5', H5" (pairwise) assignments. These assignments are required for the analysis of two-dimensional NOE and J-coupling data in terms of the solution structure of oligonucleotides.

Hydrogen Bonding↗
PubMed1983

An EPR study of the photodissociation reactions of oxidised cytochrome c oxidase-nitric oxide complexes.

Complexes of oxidised cytochrome c oxidase with NO in the absence and presence of ligands such as formate, fluoride and cyanide are photodissociable. After photodissociation at 10 K the EPR spectrum of the high-spin cytochrome a3+3 in the absence of ligands or in the presence of fluoride or formate disappears - as does the EPR spectrum of the low-spin cytochrome a3+3 in the presence of cyanide. The action spectra of the photodissociation reaction of these complexes show slight differences but all have maxima at 640-660 nm and below 400 nm, and are assigned to a diamagnetic Cu+B-NO+ complex. The differences in the action spectra in the presence of various ligands are due to binding of these anions to the cytochrome (a3-CuB) couple. The disappearance of the cytochrome a3 signal upon photodissociation of the Cu+B-NO+ complex is explained by a magnetic interaction between cytochrome a3+3 and Cu2+B in the photodissociated complex. The temperature at which NO recombines with Cu2+B is about 30 K and slightly affected by the presence of added ligands. It is suggested that in the oxidised ligand-cytochrome c oxidase complexes the coupling ligand between cytochrome a3+3 and Cu2+B is cyanide, fluoride and formate. The observation that two ligands may bind simultaneously to the cytochrome a3-CuB couple leads to further support for the notion that during turnover of cytochrome c oxidase both metal ions are involved in binding and reduction of oxygen.

Animals↗
PubMed1982

The peroxidation of thiocyanate catalysed by myeloperoxidase and lactoperoxidase.

Peroxidation of SCN- to OSCN-, catalysed by myeloperoxidase and lactoperoxidase, was studied. The rate of this reaction showed sharp optima between pH 5 and 7.5, the position of which is determined by the concentrations of both SCN- and H2O2. At low pH values, both SCN- and H+ inhibited myeloperoxidase and lactoperoxidase competitively with respect to H2O2. The inhibition constants of SCN- for myeloperoxidase and lactoperoxidase (2 and 6 mM, respectively) are independent of pH. For these enzymes a Ki for H+ of 1 microM was found that corresponded to an ionisable group on the enzymes (pKa = 6) which controls the enzymic activity. A kinetic expression is proposed that explains most of the data. The physiological consequences of the corresponding mechanism are discussed.

Animals↗
PubMed1982

Electron transfer after flash photolysis of mixed-valence carboxycytochrome c oxidase.

The light-induced difference spectra of the fully reduced (a2+ a23+-CO) complex and the mixed-valence carboxycytochrome c oxidase (a3+ a23+-CO) during steady-state illumination and after flash photolysis showed marked differences. The differences appear to be due to electron transfer between the redox centres in the enzyme. The product of the absorbance coefficient and the quantum yield was found to be equal in both enzyme species, both when determined from the rates of photolysis and from the values of the dissociation constants of the cytochrome a23+-CO complex. This would confirm that the spectral properties of cytochrome a3 are not affected by the redox state of cytochrome a and CuA. When the absorbance changes after photolysis of cytochrome a23+-CO with a laser flash were followed on a time scale from 1 mus to 1 s in the fully reduced carboxycytochrome c oxidase, only the CO recombination reaction was observed. However, in the mixed-valence enzyme an additional fast absorbance change (k = 7 X 10(3) s-1) was detected. The kinetic difference spectrum of this fast change showed a peak at 415 nm and a trough at 445 nm, corresponding to oxidation of cytochrome a3. Concomitantly, a decrease of the 830 nm band was observed due to reduction of CuA. This demonstrates that in the partially reduced enzyme a pathway is present between CuA and the cytochrome a3-CuB pair, via which electrons are transferred rapidly.

Animals↗
PubMed1982

EPR studies of the photodissociation reactions of cytochrome c oxidase-nitric oxide complexes.

Three complexes of NO with cytochrome c oxidase are described which are all photodissociable at low temperatures as measured by EPR. The EPR parameters of the cytochrome a2+(3)-NO complex are the same both in the fully reduced enzyme and in the mixed-valence enzyme. The kinetics of photodissociation of cytochrome a2+(3)-NO and recombination of NO with cytochrome a2+(3) (in the 30-70 K region) revealed no differences in structure between cytochrome a2+(3) in the fully reduced and the mixed-valence states. The action spectrum of the photodissociation of cytochrome a2+(3)-NO as measured by EPR has maxima at 595, 560 and 430 nm, and corresponds to the absorbance spectrum of cytochrome a2+(3)-NO. Photodissociation of cytochrome a2+(3)-NO in the mixed-valence enzyme changes the EPR intensity at g 3.03, due to electron transfer from cytochrome a2+(3) to cytochrome a3+. The extent of electron transfer was found to be temperature dependent. This suggests that a conformational change is coupled to this electron transfer. The complex of NO with oxidized cytochrome c oxidase shows a photodissociation reaction and recombination of NO (in the 20-40 K region) which differ completely from those observed in cytochrome a2+(3)-NO. The observed recombination occurs at a temperature 15 K lower than that found for the cytochrome a2+(3)-NO complex. The action spectrum of the oxidized complex shows a novel spectrum with maxima at 640 and below 400 nm; it is assigned to a Cu2+B-NO compound. The triplet species with delta ms = 2 EPR signals at g 4 and delta ms = 1 signals at g 2.69 and 1.67, that is observed in partially reduced cytochrome c oxidase treated with azide and NO, can also be photodissociated.

Electron Spin Resonance Spectroscopy↗
PubMed1981

The effect of pH and ionic strength on the pre-steady-state reaction of cytochrome c and cytochrome aa3.

(1) In the pH range between 5.0 and 8.0, the rate constants for the reaction of ferrocytochrome c with both the high- and low-affinity sites on the cytochrome aa3 increased by a factor of approx. 2 per pH unit. (2) The pre-steady-state reaction between ferrocytochrome c and cytochrome aa3 did nt cause a change in the pH of an unbuffered medium. Furthermore, it was found that this reaction and the steady-state reaction are equally fast in H2O and 2H2O. From these results it was concluded that no protons are directly involved in a rate-determining reaction step. (3) Arrhenius plots show that the reaction between ferrocytochrome c and cytochrome aa3 requires a higher enthalpy of activation at temperatures below 20 degrees C (15--16 kcal/mol) as compared to that at higher temperature (9 kcal/mol). We found no effect of ionic strength on the activation enthalpy of the pre-steady-state reaction, nor on that of the steady-state reaction. This suggests that ionic strength does not change the character of these reactions, but merely affects the electrostatic interaction between both cytochromes.

Animals↗
PubMed1979

Electron-transfer processes in carboxy-cytochrome c oxidase after photodissociation of cytochrome a3 2+ . CO.

Under continuous illumination the CO binding curve of reduced carboxy-cytochrome c oxidase maintains the shape of the binding curve in the dark. The apparent dissociation constant calculated from the binding curves at various light intensities is a linear function of the light intensity. Marked differences are observed between the light-induced difference spectra of the fully reduced carboxy-cytochrome c oxidase and the mixed-valence carboxy-cytochrome c oxidase. These differences are enhanced in the presence of ferricyanide as an electron acceptor and are explained by partial oxidation of cytochrome a3 in the mixed-valence enzyme after photodissociation. Upon addition of CO to partially reduced formate cytochrome c oxidase (a2+a3 3+ . HCOOH) the cytochrome a3 2+. CO compound is formed completely with a concomitant oxidation of cytochrome a and the Cu associated with cytochrome a. During photodissociation of the CO compound the formate rebinds to cytochrome a3 and cytochrome a and its associated Cu are simultaneously reduced. These electron transfer processes are fully reversible since in the dark the a3 3+ . HCOOH compound is dissociated slowly with a concomitant formation of the a3 2+ . CO compound and oxidation of cytochrome a. When these experiments are carried out in the presence of cytochrome c, both cytochrome c and cytochrome a are reduced upon illumination of the mixed-valence carboxy-cytochrome c oxidase. In the dark both cytochrome c and cytochrome a are reoxidized when formate dissociates from cytochrome a3 and the a2+ 3 . CO compound is formed back. Thus, in this system we are able to reverse and to modulate the redox state of the different components of the final part of the respiratory chain by light.

Animals↗
PubMedDate not supplied

The photoreactivity of the copper-NO complexes in cytochrome c oxidase and in other copper-containing proteins.

The complexes of NO with CuB of cytochrome c oxidase in which cytochrome a3 may or may not be ligated to cyanide or fluoride are photodissociable. NO does not appear to react with CuB in complexes of cytochrome c oxidase in which sulphide or mercaptans are ligated to the haem iron of cytochrome a3. A comparison is made between the photoreactivity of the complexes of NO with cytochrome c oxidase and those with ceruloplasmin, ascorbate oxidase, and haemocyanin. It is shown that the photoreactivity of CuB 2+.NO in cytochrome c oxidase is not unique for this enzyme, but may also be observed in the complexes of NO with type-1 copper-containing enzymes. This would suggest that the ligation of CuB in cytochrome c oxidase shows some similarity to type-1 copper in blue oxidases.

Animals↗
PubMedDate not supplied

NMR studies of interactions between inhibitors and porcine pancreatic phospholipase A2.

Two-dimensional NMR studies were performed on the complexes of porcine pancreatic phospholipase A2, bound to a micellar lipid-water interface of fully deuterated dodecylphosphocholine, with competitive inhibitors derived from the following general structure: [formula: see text] X and Y are alkyl chains with various 'reporter groups'. The interactions between the inhibitor and the enzyme were localized by comparison of 2-D nuclear Overhauser effect spectra using protonated and selectively deuterated inhibitors, and inhibitors with groups having easily identifiable chemical shifts. These experiments led us to the following conclusions for the phospholipase A2/inhibitor/micelle complex: i) the His48 C2 ring proton is in close proximity to both the amide proton and the methylene protons at the sn-1 position of the glycerol skeleton of the inhibitor, ii) the acyl chain of the inhibitor at the sn-2 position makes hydrophobic contacts near Phe5, Ile9, Phe22 and Phe106; iii) no interactions between the acyl chain at the sn-1 position and the protein could be identified. Comparison of our results on the enzyme/inhibitor/micelle ternary complex with the crystal structure of the enzyme-inhibitor complex shows that the mode of inhibitor binding is similar. However, in several cases we found indications that the hydrophobic chains of the inhibitors can have multiple conformations.

Animals↗
PubMedDate not supplied

Determination of protein structures from nuclear magnetic resonance data using a restrained molecular dynamics approach: the lac repressor DNA binding domain.

A procedure is described to determine from NMR data the three-dimensional structure of biomolecules. This procedure combines model building with a restrained Molecular Dynamics algorithm, in which distance information from NOEs is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or "headpiece" (amino acids 1-51) of the lac repressor from E. coli, for which no crystal structure is available. The spatial structure of the headpiece is discussed in terms of known physical and biochemical data and of its DNA binding properties.

Binding Sites↗