An advanced EPR stopped-flow apparatus based on a dielectric ring resonator.

A novel EPR stopped-flow accessory is described which allows time-dependent cw-EPR measurements of rate constants of reactions involving paramagnetic species after rapid mixing of two liquid reagents. The EPR stopped-flow design represents a state-of-the-art, computer controlled fluid driving system, a miniresonant EPR structure with an integrated small ball mixer, and a stopping valve. The X-band EPR detection system is an improved version of that reported by Sienkiewicz et al.

A new tyrosyl radical on Phe208 as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H. Combined x-ray diffraction and EPR/ENDOR studies.

The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E.

Electronic structure of the cysteine thiyl radical: a DFT and correlated ab initio study.

The electronic structure and the unusual EPR parameters of sulfur-centered alkyl thiyl radical from cysteine are investigated by density functional theory (DFT) and correlated ab initio calculations. Three geometry-optimized, staggered conformations of the radical are found that lie within 630 cm(-1) in energy. The EPR g-values are sensitive to the energy difference between the nearly-degenerate singly occupied orbital and one of the lone-pair orbitals (excitation energies of 1732, 1083, and 3429 cm(-1) from Multireference Configuration Interaction calculations for the structures corresponding to the three minima), both of which are almost pure sulfur 3p orbitals.

Displacement of the tyrosyl radical cofactor in ribonucleotide reductase obtained by single-crystal high-field EPR and 1.4-A x-ray data.

The R2 protein of class I ribonucleotide reductase generates and stores a tyrosyl radical essential for ribonucleotide reduction and, thus, DNA synthesis. X-ray structures of the protein have enabled detailed mechanistic suggestions, but no structural information has been available for the active radical-containing state of the protein. Here we report on methods to generate the functional tyrosyl radical in single crystals of R2 from Escherichia coli (Y122(*)).

Tryptophan and tyrosine radicals in ribonucleotide reductase: a comparative high-field EPR study at 94 GHz.

Tryptophan radicals, which are generated in the reconstitution reaction of mutants Y122F and Y177W of subunit R2 apoprotein of E. coli and mouse ribonucleotide reductase (RNR), respectively, with Fe(2+) and oxygen, are investigated by high-field EPR at 94 GHz and compared with the tyrosine radicals occurring in the respective wild-type proteins. For the first time, accurate g-values are obtained for protein-associated neutral tryptophan free radicals, which show only a small anisotropy.

Rapid flip-flop of phospholipids in endoplasmic reticulum membranes studied by a stopped-flow approach.

The transbilayer movement of short-chain spin-labeled and fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) phospholipid analogs in rat liver microsomes is measured by stopped-flow mixing of labeled microsomes with bovine serum albumin (BSA) solution. Extraction of analogs from the outer leaflet of microsomes to BSA can be directly monitored in conjunction with electron paramagnetic resonance or fluorescence spectroscopy by taking advantage of the fact that the signal of spin-labeled or fluorescent analogs bound to BSA is different from that of the analogs inserted into membranes. From the signal kinetics, the transbilayer movement and the distribution of analogs in microsomal membranes can be derived provided the extraction of analogs by BSA is much faster in comparison to the transbilayer movement of analogs.

The iron-oxygen reconstitution reaction in protein R2-Tyr-177 mutants of mouse ribonucleotide reductase. Epr and electron nuclear double resonance studies on a new transient tryptophan radical.

The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a mu-oxo-bridged diferric iron center, both necessary for enzyme activity. We have studied the reconstitution reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse RNR to investigate if other residues at the site of the radical forming Tyr-177 can harbor free radicals. In Y177W we observed for the first time the formation of a tryptophan radical in protein R2 of mouse RNR with a lifetime of several minutes at room temperature.

Rapid kinetics of insertion and accessibility of spin-labeled phospholipid analogs in lipid membranes: a stopped-flow electron paramagnetic resonance approach.

Spin-labeled phospholipid analogs have been employed to probe the transbilayer distribution of endogenous phospholipids in various membrane systems. To determine the transmembrane distribution of the spin-labeled analogs, the analogs are usually inserted into the membrane of interest and subsequently the amount of analog in the outer membrane leaflet is determined either by chemical reduction with ascorbate or by back-exchange to bovine serum albumin (BSA). For accurate determination of the transbilayer distribution of analogs, both the kinetics of incorporation and those of accessibility of analogs to ascorbate or BSA have to be fast in comparison to their transbilayer movement.

Inactivation of ribonucleotide reductase in tumour cells and inhibition of tumour cell growth by p-alkoxyphenols.

A significant correlation between the inactivation of the growth-regulating enzyme ribonucleotide reductase (RR) with the growth inhibition of four different tumour cell lines has been found for seven different p-alkoxyphenol derivatives with varying lengths of alkyl side chain. In Novikoff hepatoma and human leukaemia cells, inactivation of RR by p-alkoxyphenols was monitored by electron paramagnetic resonance (EPR) spectroscopy of the catalytically essential tyrosyl radical in the subunit R2 of RR. A significant inhibition of cellular growth of Novikoff hepatoma cells, human leukaemia cells and two human melanoma cell lines (MeWo and M5) by p-alkoxyphenols was also observed by growth inhibition assays.

Detection of a stable free radical in the B2 subunit of the manganese ribonucleotide reductase (Mn-RRase) of Corynebacterium ammoniagenes.

Ribonucleotide reductases catalyze the irreversible reductive formation of 2'-deoxyribonucleotides required for DNA replication and cell proliferation, and a radical mechanism was assumed to be involved in this reaction. In order to search for a radical in the aerobic manganese ribonucleotide reductase (Mn-RRase) by electron paramagnetic resonance (EPR) the native metal-containing 100 kDa B2 subunit was deliberately prepared from the wild type strain Corynebacterium ammoniagenes ATCC 6872. Enrichment by 2'5'-ADP Sepharose 4B affinity chromatography, fast protein liquid chromatography (FPLC) with SuperoseTM12 and concentration by vacuum evaporation allowed for the first time the detection of a stable free radical by EPR spectroscopy at 77 K.

Structure of transient radicals from cytostatic-active p-alkoxyphenols by continuous-flow EPR.

Para-alkoxyphenols are of medical significance as futural cytostatic drugs in antimelanoma chemotherapy. They take part in a radical redox-reaction in which the catalytically essential protein-linked tyrosyl radical in the functional subunit R2 of the growth-regulating enzyme ribonucleotide reductase (RR) is quenched. EPR spectroscopy has been employed in conjunction with a continuous-flow system to study the structure of transient radicals from p-alkoxyphenols with different alkyl chain lengths.

Reduction of the tyrosyl radical and the iron center in protein R2 of ribonucleotide reductase from mouse, herpes simplex virus and E. coli by p-alkoxyphenols.

The rate of reduction of the tyrosyl radical in the small subunit of ribonucleotide reductase (protein R2) from E. coli, mouse, and herpes simplex virus (HSV-2) by a series of p-alkoxyphenols with different alkyl chains, have been studied by stopped-flow UV-vis and stopped-flow EPR spectroscopy. The reduction and release of iron in R2 by the inhibitors was followed using bathophenanthroline as chelator of Fe2+.

Transient free radicals in iron/oxygen reconstitution of mutant protein R2 Y122F. Possible participants in electron transfer chains in ribonucleotide reductase.

Ferrous iron/oxygen reconstitution of the mutant R2 apoprotein Y122F leads to formation of a diferric center similar to that of the wild-type R2 protein of Escherichia coli ribonucleotide reductase. This reconstitution reaction requires two extra electrons, supplied or transferred by the protein matrix of R2. We observed several transient free radical species using stopped flow and freeze quench EPR and stopped flow UV-visible spectroscopy.

Tryptophan radicals formed by iron/oxygen reaction with Escherichia coli ribonucleotide reductase protein R2 mutant Y122F.

The active state of the small subunit, protein R2, of ribonucleotide reductase is formed by the reaction of apoprotein with Fe2+ and O2, whereby the diferric site and a stable phenoxy free radical on a tyrosyl residue (Tyr122) is formed. The corresponding reaction was studied in the mutant Y122F R2. It leads to a normal iron site, but the reduction equivalent from Tyr122 now has to be supplied from elsewhere.

p-Alkoxyphenols, a new class of inhibitors of mammalian R2 ribonucleotide reductase: possible candidates for antimelanotic drugs.

The inhibition by different p-alkoxyphenol derivatives of the growth-regulating enzyme ribonucleotide reductase (RR) in purified Escherichia coli and mouse R2 protein preparations was studied by EPR spectroscopy. The inhibitor-induced inactivation of the catalytic subunit protein R2 was measured at 77 degrees K by observing the decrease of the typical EPR signal from the functionally essential protein-linked tyrosyl free radical. p-Methoxy-, p-ethoxy-, p-propoxy-, and p-allyloxyphenol were about 2 orders of magnitude more effective in inhibiting mouse R2, compared with E.

ESR studies on reactivity of protein-derived tyrosyl radicals formed by prostaglandin H synthase and ribonucleotide reductase.

Using ESR spectroscopy, the ability of enzyme inhibitors to quench protein-derived tyrosyl radicals was studied in two different enzymes, prostaglandin H synthase and ribonucleotide reductase. The prostaglandin H synthase inhibitors indomethacin, eugenol, and MK-410 effectively prevent the formation of tyrosyl radicals during the oxidation of arachidonic acid by prostaglandin H synthase from ram seminal vesicles. A direct reaction with preformed tyrosyl radicals was observed only with eugenol.

Spectral analysis of the protein-derived tyrosyl radicals from prostaglandin H synthase.

We have analyzed the low temperature EPR spectra of the protein-derived tyrosyl radicals detected upon addition of arachidonic acid or 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP) to prostaglandin H synthase. With either arachidonic acid or PPHP the initial radical detected is a doublet (peak-to-trough = 35 Gauss) that disappears rapidly and is replaced by a broad singlet (peak-to-trough = 30 Gauss) followed by a narrow singlet (peak-to-trough = 26.5 Gauss).

EPR stopped-flow studies of the reaction of the tyrosyl radical of protein R2 from ribonucleotide reductase with hydroxyurea.

The reaction of the functional tyrosyl radical in protein R2 of ribonucleotide reductase from E. coli and mouse with the enzyme inhibitor hydroxyurea has been studied by EPR stopped-flow techniques at room temperature. The rate of disappearance of the tyrosyl radical in E.

Electron spin resonance investigation of tyrosyl radicals of prostaglandin H synthase. Relation to enzyme catalysis.

We have examined, by low temperature ESR, the protein-derived radicals formed by reaction of purified ram seminal vesicle prostaglandin H synthase (PHS). Upon addition of arachidonic acid or 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP) to PHS reconstituted with Fe(III)-protoporphyrin IX (Fe-PHS) at -12 degrees C, an ESR spectrum was observed at -196 degrees C containing a doublet that rapidly converted into a singlet. These protein-derived radicals were identified as tyrosyl radicals.

Quenching of tyrosine radicals of M2 subunit from ribonucleotide reductase in tumor cells by different antitumor agents: an EPR study.

The inhibition of ribonucleotide reductase (RR) of intact Ehrlich ascites tumor cells by different antitumor agents was compared using EPR spectroscopy. The inactivation of M2 subunit was measured via quenching of the functionally essential tyrosine radical. Inhibitors of different classes, for example, hydroxyurea, pyrogallol, and thiosemicarbazones, differ in their efficiency by three orders of magnitude.

Intestinal neuronal degeneration in a patient with chronic idiopathic intestinal pseudoobstruction.

Neuropathological examination of the gut in a patient with chronic idiopathic intestinal pseudoobstruction and temporal lobe epilepsy showed a degeneration of Auerbach's and of Meissner's plexus. The extent of generative changes increased in an aboral to oral direction. Neuronal degeneration was characterized by ballooning of the cytoplasm of the ganglial cells, by a hyperargyrophilia, a shortening and dilation of cell processes, and a progressive fragmentation and loss of axons.

Protection of melanoma cells against superoxide radicals by melanins.

Human melanoma cells transplanted into immunocompetent mice by the 6-day subrenal capsule technique are characterized by high resistance against immunological attack. This resistance is suggested to be the consequence of scavenging of superoxide free radicals by melanin. Scavenging of superoxide radicals by the melanoma cells was clearly demonstrated using electron spin resonance techniques.

ESR studies of structure and kinetics of radicals from hydroxyurea. An antitumor drug directed against ribonucleotide reductase.

Hydroxyurea (HU) is a clinically applied antineoplastic drug, which quenches tyrosine radicals in the active site of ribonucleotide reductase (RR) and inhibits DNA synthesis in proliferating cells. Under oxidizing conditions (Cu2+ or H2O2) long-lived radicals from HU have been found by ESR. The structure of HU radicals was established to be: (formula; see text).