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Simon de Vries

Publications and source records attributed to Simon de Vries.

16 recordsLinked to original sources
PubMed2006

Specific vectorial immobilization of oligonucleotide-modified yeast cytochrome C on carbon nanotubes.

Iso-1-cytochrome c from the yeast Saccharomyces cerevisiae (YCC) contains a surface cysteine residue, Cys102, that is located opposite to the lysine-rich side containing the exposed heme edge, which is the docking site for enzymes. Site-specific vectorial immobilization of YCC via Cys102 on single-walled carbon nanotubes (SWNT) thus provides a selective interface between nanoscopic electronic devices and complex enzymes. We have achieved this by modification of Cys102 with an oligonucleotide (dT(18)). Atomic force microscopy, fluorescence imaging, and cyclic voltammetry show the specific adsorption of YCC, modified with dT(18), on the SWNT sidewall with retention of its native properties. Pretreatment of the SWNT with Triton-X405 blocks the nonspecific binding of untreated YCC but does not interfere with binding of the oligonucleotide-modified YCC.

Adsorption↗
PubMed2005

Purification and characterization of the tungsten enzyme aldehyde:ferredoxin oxidoreductase from the hyperthermophilic denitrifier Pyrobaculum aerophilum.

A tungsten-containing aldehyde:ferredoxin oxidoreductase (AOR) has been purified to homogeneity from Pyrobaculum aerophilum. The N-terminal sequence of the isolated enzyme matches a single open reading frame in the genome. Metal analysis and electron paramagnetic resonance (EPR) spectroscopy indicate that the P. aerophilum AOR contains one tungsten center and one [4Fe-4S](2+/1+) cluster per 68-kDa monomer. Native AOR is a homodimer. EPR spectroscopy of the purified enzyme that has been reduced with the substrate crotonaldehyde revealed a W(V) species with g(zyx) values of 1.952, 1.918, 1.872. The substrate-reduced AOR also contains a [4Fe-4S](1+) cluster with S=3/2 and zero field splitting parameters D=7.5 cm(-1) and E/D=0.22. Molybdenum was absent from the enzyme preparation. The P. aerophilum AOR lacks the amino acid sequence motif indicative for binding of mononuclear iron that is typically found in other AORs. Furthermore, the P. aerophilum AOR utilizes a 7Fe ferredoxin as the putative physiological redox partner, instead of a 4Fe ferredoxin as in Pyrococcus furiosus. This 7Fe ferredoxin has been purified from P. aerophilum, and the amino acid sequence has been identified using mass spectrometry. Direct electrochemistry of the ferredoxin showed two one-electron transitions, at -306 and -445 mV. In the presence of 55 microM ferredoxin the AOR activity is 17% of the activity obtained with 1 mM benzyl viologen as an electron acceptor.

Aldehyde Oxidoreductases↗
PubMed2004

Two CO molecules can bind concomitantly at the diiron site of NO reductase from Bacillus azotoformans.

CO complexes formed in reduced nitric oxide reductase from Bacillus azotoformans were investigated with resonance Raman and FTIR techniques. These experiments shows the presence of two nu(C-O) bands, one at approximately 1970 cm-1 assigned to the heme-CO complex, and one at approximately 2070 cm-1 from the non-heme iron, FeBCO. At cryogenic temperatures, the heme-CO complex adopts a semi-bridging configuration with FeB which decreases its stretching frequency to approximately 1910 cm-1 and decreases the nu(C-O) of FeBCO by approximately 20 cm-1. The concomitant binding of two CO molecules, one per iron(II) at the active site, is consistent with the formation of a [{FeNO}7]2 iron-nitrosyl dimer during substrate turnover. This study strongly supports the notion that this family of enzymes utilizes a reaction mechanism based on catalysis by proximity, where the formation of two iron-nitrosyl groups promotes N-N bond formation.

Bacillus↗
PubMed2004

NO reductase from Bacillus azotoformans is a bifunctional enzyme accepting electrons from menaquinol and a specific endogenous membrane-bound cytochrome c551.

Bacillus azotoformans is a Gram-positive denitrifying soil bacterium, which is capable of respiring nitrate, nitrite, nitric oxide, and nitrous oxide under anaerobic conditions. It contains a unique menaquinol-dependent nitric oxide reductase (qCu(A)NOR) with a Cu(A) center in its small subunit. The qCu(A)NOR exhibits menaquinol-dependent NO reductase activity, whereas reduced horse heart cytochrome c was inactive. Here we describe the purification of three membrane-bound c cytochromes from B. azotoformans. Their apparent molecular masses on SDS-PAGE are approximately 11 kDa. At neutral pH, these c cytochromes are negatively charged and the E(m) for all is close to 150 mV. Only one of these c cytochromes, which exhibits an alpha-band maximum at 551 nm, acts as a direct electron donor to qCu(A)NOR. Further investigation demonstrated that this cytochrome c(551) possesses two lipoyl moieties, which presumably function to anchor it to the membrane. Steady-state kinetic studies reveal that cytochrome c(551) is a noncompetitive inhibitor of NO reduction when menaquinol is used as an electron donor. This finding points to the presence of two different electron donation pathways in qCu(A)NOR. The ability of qCu(A)NOR to accept electrons from both menaquinol and cytochrome c(551) might be related to the regulation of the rate of NO reduction especially as a defense mechanism of B. azotoformans against the toxicity of NO. Growth experiments in batch culture indeed show that B. azotoformans is highly NO tolerant, in contrast to, for example, Paracoccus denitrificans that has a monofunctional cytochrome c-dependent NOR. We propose that the menaquinol pathway, which has a 4-fold greater maximal activity than the pathway via cytochrome c(551), is used for NO detoxification, whereas electron donation via the endogenous cytochrome c involves the cytochrome b(6)f complex serving the bioenergetic needs of the organism.

Amino Acid Sequence↗
PubMed2004

An oxo-ferryl tryptophan radical catalytic intermediate in cytochrome c and quinol oxidases trapped by microsecond freeze-hyperquenching (MHQ).

The pre-steady state reaction kinetics of the reduction of molecular oxygen catalyzed by fully reduced cytochrome oxidase from Escherichia coli and Paracoccus denitrificans were studied using the newly developed microsecond freeze-hyperquenching mixing-and-sampling technique. Reaction samples are prepared 60 and 200 micros after direct mixing of dioxygen with enzyme. Analysis of the reaction samples by low temperature UV-Vis spectroscopy indicates that both enzymes are trapped in the PM state. EPR spectroscopy revealed the formation of a mixture of two radicals in both enzymes. Based on its apparent g-value and lineshape, one of these radicals is assigned to a weakly magnetically coupled oxo-ferryl tryptophan cation radical. Implications for the catalytic mechanism of cytochrome oxidases are discussed.

Bacterial Proteins↗
PubMed2004

Direct immobilization of native yeast iso-1 cytochrome C on bare gold: fast electron relay to redox enzymes and zeptomole protein-film voltammetry.

Cyclic voltammetry shows that yeast iso-1-cytochrome c (YCC), chemisorbed on a bare gold electrode via Cys102, exhibits fast, reversible interfacial electron transfer (k(0) = 1.8 x 10(3) s(-1)) and retains its native functionality. Vectorially immobilized YCC relays electrons to yeast cytochrome c peroxidase, and to both cytochrome cd(1) nitrite reductase (NIR) and nitric oxide reductase from Paracoccus denitrificans, thereby revealing the mechanistic properties of these enzymes. On a microelectrode, we measured nitrite turnover by approximately 80 zmol (49 000 molecules) of NIR, coadsorbed on 0.65 amol (390 000 molecules) of YCC.

Catalysis↗
PubMed2004

Bidirectional catalysis by copper-containing nitrite reductase.

The copper-containing nitrite reductase from Alcaligenes faecalis S-6 was found to catalyze the oxidation of nitric oxide to nitrite, the reverse of its physiological reaction. Thermodynamic and kinetic constants with the physiological electron donor pseudoazurin were determined for both directions of the catalyzed reaction in the pH range of 6-8. For this, nitric oxide was monitored by a Clark-type electrode, and the redox state of pseudoazurin was measured by optical spectroscopy. The equilibrium constant (K(eq)) depends on the reduction potentials of pseudoazurin and nitrite/nitric oxide, both of which vary with pH. Above pH 6.2 the formation of NiR substrates (nitrite and reduced pseudoazurin) is favored over the products (NO and oxidized pseudoazurin). At pH 8 the K(eq) amounts to 10(3). The results show that dissimilatory nitrite reductases catalyze an unfavorable reaction at physiological pH (pH = 7-8). Consequently, nitrous oxide production by copper-containing nitrite reductases is unlikely to occur in vivo with a native electron donor. With increasing pH, the rate and specificity constant of the forward reaction decrease and become lower than the rate of the reverse reaction. The opposite occurs for the rate of the reverse reaction; thus the catalytic bias for nitrite reduction decreases. At pH 6.0 the k(cat) for nitrite reduction was determined to be 1.5 x 10(3) s(-1), and at pH 8 the rate of the reverse reaction is 125 s(-1).

Alcaligenes faecalis↗
PubMed2003

Purification and characterization of the MQH2:NO oxidoreductase from the hyperthermophilic archaeon Pyrobaculum aerophilum.

The membrane-bound NO reductase from the hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum was purified to homogeneity. The enzyme displays MQH2:NO oxidoreductase (qNOR) activity, consists of a single subunit, and contains heme and nonheme iron in a 2:1 ratio. The combined results of EPR, resonance Raman, and UV-visible spectroscopy show that one of the hemes is bis-His-coordinated low spin (gz = 3.015; gy = 2.226; gx = 1.45), whereas the other heme adopts a high spin configuration. The enzyme also contains one nonheme iron center, which in the oxidized enzyme is antiferromagnetically coupled to the high spin heme. This binuclear high spin heme/nonheme iron center is EPR-silent and the site of NO reduction. The reduced high spin heme is bound to a neutral histidine and can bind CO to form of a low spin complex. The oxidized high spin heme binds NO, yielding a ferric nitrosyl complex, the intermediate causing the commonly found substrate inhibition in NO reductases (Ki(NO) = 7 microm). The qNOR as present in the membrane is, in contrast to the purified enzyme, quite thermostable, incubation at 100 degrees C for 86 min leading to 50% inhibition. The pure enzyme lacks heme b and instead contains stoichiometric amounts of hemes Op1 and Op2, ethenylgeranylgeranyl and hydroxyethylgeranylgeranyl derivatives of heme b, respectively. The archaeal qNOR is the first example of a NO reductase, which contains modified hemes reminiscent of cytochrome bo3 and aa3 oxidases. This report is the first describing the purification and structural and spectroscopic properties of a thermostable NO reductase.

Cell Division↗
PubMed2003

Biophysical and structural analysis of a novel heme B iron ligation in the flavocytochrome cellobiose dehydrogenase.

The fungal extracellular flavocytochrome cellobiose dehydrogenase (CDH) participates in lignocellulose degradation. The enzyme has a cytochrome domain connected to a flavin-binding domain by a peptide linker. The cytochrome domain contains a 6-coordinate low spin b-type heme with unusual iron ligands and coordination geometry. Wild type CDH is only the second example of a b-type heme with Met-His ligation, and it is the first example of a Met-His ligation of heme b where the ligands are arranged in a nearly perpendicular orientation. To investigate the ligation further, Met65 was replaced with a histidine to create a bis-histidyl ligated iron typical of b-type cytochromes. The variant is expressed as a stable 90-kDa protein that retains the flavin domain catalytic reactivity. However, the ability of the mutant to reduce external one-electron acceptors such as cytochrome c is impaired. Electrochemical measurements demonstrate a decrease in the redox midpoint potential of the heme by 210 mV. In contrast to the wild type enzyme, the ferric state of the protoheme displays a mixed low spin/high spin state at room temperature and low spin character at 90 K, as determined by resonance Raman spectroscopy. The wild type cytochrome does not bind CO, but the ferrous state of the variant forms a CO complex, although the association rate is very low. The crystal structure of the M65H cytochrome domain has been determined at 1.9 A resolution. The variant structure confirms a bis-histidyl ligation but reveals unusual features. As for the wild type enzyme, the ligands have a nearly perpendicular arrangement. Furthermore, the iron is bound by imidazole N delta 1 and N epsilon 2 nitrogen atoms, rather than the typical N epsilon 2/N epsilon 2 coordination encountered in bis-histidyl ligated heme proteins. To our knowledge, this is the first example of a bis-histidyl N delta 1/N epsilon 2-coordinated protoporphyrin IX iron.

Blotting, Western↗
PubMed2003

Microbial ferric iron reductases.

Almost all organisms require iron for enzymes involved in essential cellular reactions. Aerobic microbes living at neutral or alkaline pH encounter poor iron availability due to the insolubility of ferric iron. Assimilatory ferric reductases are essential components of the iron assimilatory pathway that generate the more soluble ferrous iron, which is then incorporated into cellular proteins. Dissimilatory ferric reductases are essential terminal reductases of the iron respiratory pathway in iron-reducing bacteria. While our understanding of dissimilatory ferric reductases is still limited, it is clear that these enzymes are distinct from the assimilatory-type ferric reductases. Research over the last 10 years has revealed that most bacterial assimilatory ferric reductases are flavin reductases, which can serve several physiological roles. This article reviews the physiological function and structure of assimilatory and dissimilatory ferric reductases present in the Bacteria, Archaea and Yeast. Ferric reductases do not form a single family, but appear to be distinct enzymes suggesting that several independent strategies for iron reduction may have evolved.

Archaea↗
PubMed2003

Inhibition of nicotinoprotein (NAD+-containing) alcohol dehydrogenase by trans-4-(N,N-dimethylamino)-cinnamaldehyde binding to the active site.

Ethanol oxidation by nicotinoprotein alcohol dehydrogenase (np-ADH) from the bacterium Amycolatopsis methanolica is inhibited by trans-4-(N,N-dimethylamino)-cinnamaldehyde through direct binding to the catalytic zinc ion in a substrate-like geometry. This binding is accompanied by a characteristic red shift of the aldehyde absorbance from 398 nm to 467 nm. Np-ADH is structurally related to mammalian ADH class I, and a model of np-ADH shows how the cinnamaldehyde derivative can be accommodated in the active site of the nicotinoprotein, correlating the structural and enzymological data.

Actinobacteria↗
PubMed2003

Kinetics and thermodynamics of nick sealing by T4 DNA ligase.

T4 DNA ligase is an Mg2+-dependent and ATP-dependent enzyme that seals DNA nicks in three steps: it covalently binds AMP, transadenylates the nick phosphate, and catalyses formation of the phosphodiester bond releasing AMP. In this kinetic study, we further detail the reaction mechanism, showing that the overall ligation reaction is a superimposition of two parallel processes: a 'processive' ligation, in which the enzyme transadenylates and seals the nick without dissociating from dsDNA, and a 'nonprocessive' ligation, in which the enzyme takes part in the abortive adenylation cycle (covalent binding of AMP, transadenylation of the nick, and dissociation). At low concentrations of ATP (<10 microM) and when the DNA nick is sealed with mismatching base pairs (e.g. five adjacent), this superimposition resolves into two kinetic phases, a burst ligation (approximately 0.2 min(-1)) and a subsequent slow ligation (approximately 2x10(-3) min(-1)). The relative rate and extent of each phase depend on the concentrations of ATP and Mg2+. The activation energies of self-adenylation (16.2 kcal.mol(-1)), transadenylation of the nick (0.9 kcal.mol(-1)), and nick-sealing (16.3-18.8 kcal.mol(-1)) were determined for several DNA substrates. The low activation energy of transadenylation implies that the transfer of AMP to the terminal DNA phosphate is a spontaneous reaction, and that the T4 DNA ligase-AMP complex is a high-energy intermediate. To summarize current findings in the DNA ligation field, we delineate a kinetic mechanism of T4 DNA ligase catalysis.

Adenosine Monophosphate↗
PubMed2002

Dynamic mechanism of nick recognition by DNA ligase.

DNA ligases are the enzymes responsible for the repair of single-stranded and double-stranded nicks in dsDNA. DNA ligases are structurally similar, possibly sharing a common molecular mechanism of nick recognition and ligation catalysis. This mechanism remains unclear, in part because the structure of ligase in complex with dsDNA has yet to be solved. DNA ligases share common structural elements with DNA polymerases, which have been cocrystallized with dsDNA. Based on the observed DNA polymerase-dsDNA interactions, we propose a mechanism for recognition of a single-stranded nick by DNA ligase. According to this mechanism, ligase induces a B-to-A DNA helix transition of the enzyme-bound dsDNA motif, which results in DNA contraction, bending and unwinding. For non-nicked dsDNA, this transition is reversible, leading to dissociation of the enzyme. For a nicked dsDNA substrate, the contraction of the enzyme-bound DNA motif (a) triggers an opened-closed conformational change of the enzyme, and (b) forces the motif to accommodate the strained A/B-form hybrid conformation, in which the nicked strand tends to retain a B-type helix, while the non-nicked strand tends to form a shortened A-type helix. We propose that this conformation is the catalytically competent transition state, which leads to the formation of the DNA-AMP intermediate and to the subsequent sealing of the nick.

Amino Acid Motifs↗
PubMed2002

Scanning mutagenesis using T4 DNA ligase and short degenerate DNA oligonucleotides containing tri-nucleotide mismatches.

Scanning mutagenesis is an attractive tool for protein structure-function correlation analysis. With one round of this method it is possible to obtain a library containing all possible single-residue mutants of the protein of interest. The practical application of this approach is currently limited by the large number and cost of the required 30-35mer oligonucleotides. As an alternative, we studied the ligation of shorter DNA oligonucleotides (6-11mer) containing a degenerate binding site and a desired mutation mismatch to a nested set of megaprimers annealed to the gene of interest. T4 DNA ligase was able to perform this task, and the obtained ligation products were elongated by DNA polymerase. The effectiveness of ligation depends on the length of the random binding site of the mutagenic oligonucleotide, on its molar excess over the template-primer complex and on the position of the mismatching tri-nucleotide insert with respect to the joining site. The secondary structure of the DNA template close to the joining site also influences the ligation yield. Mismatching oligonucleotides, protected by a 3'-phosphate group, were joined to a nested set of megaprimers, the latter being obtained by a novel procedure called reversible chain termination, i.e., termination of the dsDNA synthesis with ddNTP followed by the subsequent removal of the incorporated ddNMP with exonuclease III. T7 sequenase 2.0 DNA polymerase elongated the ligation products after the 3'-phosphate protection group was removed with T4 polynucleotide kinase, resulting in the incorporation of a specific tri-nucleotide mismatch into dsDNA. This sequence of reactions serves as the basis for a novel scanning mutagenesis procedure.

Bacteriophage T4↗
PubMed2001

Kinetic mechanism of the Mg2+-dependent nucleotidyl transfer catalyzed by T4 DNA and RNA ligases.

The Mg(2+)-dependent adenylylation of the T4 DNA and RNA ligases was studied in the absence of a DNA substrate using transient optical absorbance and fluorescence spectroscopy. The concentrations of Mg(2+), ATP, and pyrophosphate were systematically varied, and the results led to the conclusion that the nucleotidyl transfer proceeds according to a two-metal ion mechanism. According to this mechanism, only the di-magnesium-coordinated form Mg(2)ATP(0) reacts with the enzyme forming the covalent complex E.AMP. The reverse reaction (ATP synthesis) occurs between the mono-magnesium-coordinated pyrophosphate form MgP(2)O(7)(2-) and the enzyme.MgAMP complex. The nucleotide binding rate decreases in the sequence ATP(4-) > MgATP(2-) > Mg(2)ATP(0), indicating that the formation of the non-covalent enzyme.nucleotide complex is driven by electrostatic interactions. T4 DNA ligase shows notably higher rates of ATP binding and of subsequent adenylylation compared with RNA ligase, in part because it decreases the K(d) of Mg(2+) for the enzyme-bound Mg(2)ATP(0) more than 10-fold. To elucidate the role of Mg(2+) in the nucleotidyl transfer catalyzed by T4 DNA and RNA ligases, we propose a transition state configuration, in which the catalytic Mg(2+) ion coordinates to both reacting nucleophiles: the lysyl moiety of the enzyme that forms the phosphoramidate bond and the alpha-beta-bridging oxygen of ATP.

Adenosine Triphosphate↗