Günter Lassmann

Displacement of the tyrosyl radical cofactor in ribonucleotide reductase obtained by single-crystal high-field EPR and 1.4-Å 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•). We further report on subsequent high-field EPR experiments on the radical-containing crystals. A full rotational pattern of the spectra was collected and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical in the crystal frame. The EPR data are discussed in comparison with a 1.42-Å x-ray structure of the met (oxidized) form of the protein, also presented in this paper. Comparison of the orientation of the radical Y122• obtained from high-field EPR with that of the reduced tyrosine Y122-OH reveals a significant rotation of the tyrosyl side chain, away from the diiron center, in the active radical state. Implications for the radical transfer connecting the diiron site in R2 with the substrate-binding site in R1 are discussed. In addition, the present study demonstrates that structural and functional information about active radical states can be obtained by combined x-ray and high-field EPR crystallography.

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. coli protein R2 is k2 = 0.43 M-1 s-1 at 25 degrees C. The reaction follows pseudo-first-order kinetics up to 450 mM hydroxyurea indicating that no saturation by hydroxyurea takes place even at this high concentration. Transient nitroxide-like radicals from hydroxyurea have been detected for the first time in the reaction of hydroxyurea with protein R2 from E. coli and mouse, indicating that 1-electron transfer from hydroxyurea to the tyrosyl radical is the dominating mechanism in the inhibitor reaction. The hydroxyurea radicals appear in low steady-state concentrations during 2-3 half-decay times of the tyrosyl radical and disappear thereafter.

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. Half-times of the back-exchange for spin-labeled and fluorescent analogs were <3.5 and <9.5 s, respectively. The unprecedented time resolution of the assay revealed that the transbilayer movement of spin-labeled analogs is much faster than previously reported. The half-time of the movement was about 16 s or even less at room temperature. Transmembrane movement of NBD-labeled analogs was six- to eightfold slower than that of spin-labeled analogs.

The unique 3-neighborly 4-manifold with few vertices

Triangulated 4-dimensional manifolds with n vertices are considered such that any triple of vertices determines a triangle belonging to the triangulation. It is shown that for n ⩽ 13 there is exactly one such triangulation (besides the boundary of a 5-simplex). It has nine vertices and it is a triangulation of the complex projective plane.

Effect of poly(ethylene glycol) on the polarity of aqueous solutions and on the structure of vesicle membranes.

The partitioning of TEMPO into phosphatidylcholine vesicle membranes is reduced upon addition of poly(ethylene glycol). This is caused by reduced polarity of the aqueous phase as well as decreased membrane fluidity in the presence of poly(ethylene glycol). The isotropic hyperfine splitting of TEMPO in aqueous poly(ethylene glycol) solutions was used as a measure of solvent polarity. The alterations of the membrane fluidity were detected by means of two different fatty acid spin labels. The influences of physicochemical properties of an aqueous poly(ethylene glycol) phase on the membrane structure of cells and vesicles are discussed in the light of membrane fusion.

Adsorption of small molecules to bovine serum albumin studied by the spin-probe method

A spin-probe technique is used for quantitative EPR studies of adsorption of small molecules on globular proteins, of the rigidity of their binding to the protein and of the polarity of the environment. In the case of bovine serum albumin it is shown that nitroxyl radical (2, 2, 6, 6-tetramethyl-4-oxy-1-oxyl-piperidine)-stearate (I) has an adsorption behaviour similar to that of the fatty acids, nitroxyl radical (2, 2,4, 4-tetramethyl-1, 2, 3, 4-tetrahydro-5, 6-benzo-gamma-carboline-3-oxyl) (II) to that of the tryptophan molecule. Radical I rotates relative to the protein molecule, while Radical II is rigidly bound to the protein.

Characterization of hydrophobic regions in proteins by spin labeling technique

1. 1.Hydrophobic forces in proteins have been studied by the spin labeling technique. A change in the polarity of the environment of the bound label causes a change of the hyperfine tensor of the label. This is measured as a change of the distance (2Azz) between the high-field and low-field line of the ESR spectrum in the limiting case of strong immobilization. 2. 2.Using 2Azz, a dimensionless parameter h has been proposed which represents a quantitative measure of the nonpolarity of the protein part surrounding the label. 3. 3.In the case of leucine aminopeptidase (EC 3.4.1.1) covalently labeled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl (I), it is shown that the hydrophobic environment near the spin label changes with temperature. 4. 4.With leucine aminopeptidase at room temperature, a rotational correlation time τ = (1.6 ± 0.4) · 10−8s was determined for the bound label in the hydrophobic pocket.

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 μ-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. We assign it to an oxidized neutral tryptophan radical on Trp-177, based on selective deuteration and EPR and electron nuclear double resonance spectroscopy in H2O and D2O solution. The reconstitution reaction at 22 °C in both Y177F and Y177C leads to the formation of a so-called intermediate X which has previously been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster. Surprisingly, in both mutants that do not have successor radicals as Trp⋅ in Y177W, this cluster exists on a much longer time scale (several seconds) at room temperature than has been reported forX in E. coli Y122F or native mouse protein R2. All three mouse R2 mutants were enzymatically inactive, indicating that only a tyrosyl radical at position 177 has the capability to take part in the reduction of substrates.

Protein thiyl radicals directly observed by EPR spectroscopy

[1].Theevidencefortheirexis-tence,however,isonlyindirect.WiththeexceptionofathiylradicalthatisstronglycoupledtoacobaltcomplexinclassIIRNR[1],thereissofarnospectroscopicevidenceneitherforthiylradicalappearancenorfortheircatalyticcompetence.InclassIaRNR,thethiylradicalisproposedtobegeneratedattheactivesiteintheR1proteincomponentviaatyrosylrad-icalintheR2protein[1].Becauseofthesmallchancetoob-servethiylradicalsspectroscopicallyundernormalturnoverconditionsinRNR(R1/R2/substrateassay),modelstudieswithartificiallygeneratedthiylradicalsinR1(withoutR2)arenecessarytoanalyzetheminR1and,finally,toassesstheircatalyticcompetence.ThisrequiresknowledgeoftheEPRspectroscopicpropertiesofthiylradicalsinproteinsingeneralwhichhassofarbeencompletelylacking.Recently,were-portedonartificialgenerationofshort-livedproteinthiylradicalsinbovineserumalbumin(BSA)andR1ofEscherichiacoliRNRatroomtemperaturebychemicaloxidationofthiolsusingCe(IV)andbyphotochemicalreleaseofNOfromnit-rosylatedthiolsandtheirindirectEPRdetectionusingthespintrapphenyl-N-t-butylnitrone(PBN)[2].Spintrapping,how-ever,cannotprovideinformationonspectroscopicpropertieslikelineshape,g-andhfs-tensors,relaxation,andlifetimeoftheactualthiylradical.Sofar,thereareonlyfewexamplesofEPRstudiesonthiylradicalsinlowmolecularweightthiolcompounds,suchascysteine[3–5].HerewereportforthefirsttimeonthedirectEPRdetectionofprotein-basedthiylradi-calsandexploretheirunusualspectroscopicproperties.Inthiswork, thiyl radicals from proteins have been generatedphotolyticallybyUVirradiationat77Koffrozenaqueoussolutionsoftwodifferentproteins,BSAasaproteinmodel(onesinglecysteineatC34andseveraldisulfides)andreducedR1proteinofclassIaRNRfromE. coli (11cysteinespermonomericunitoftheR1homodimer).Thiylradicalsfromlow-molecular-weightthiolsinafrozencysteinesolutionhavealsobeenstudiedforcomparison.

Permuted difference cycles and triangulated sphere bundles

Abstract For any dimension d and any k = 1, …, d we construct a 2-neighborly triangulation of a d -manifold M k d which is invariant under the action of the dihedral group D n on n = 2 d − k ( k + 3) − 1 vertices. M k d is the boundary of a ( d + 1)-manifold M k d + 1 with the same properties. Special cases in this family have been observed before: M d d is the boundary of a ( d + 1)-simplex, M d − 1 d + 1 is an orientable or nonorientable 1-handle depending on the parity of d , M 1 d is a d -dimensional torus. Topologically, M k d (or M k d + 1 ) is the total space of a sphere bundle (or disc bundle) over a ( d − k )-dimensional torus. The construction of the triangulation itself is purely combinatorial. It is based on permutations of certain difference cycles encoding all the information about the triangulation in d (or d + 1) integers.