RESUMEN
The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes adenosylcobalamin (AdoCbl)-dependent nucleotide reduction, as well as exchange of the 5' hydrogens of AdoCbl with solvent. A protein-based thiyl radical is proposed as an intermediate in both of these processes. In the presence of RTPR containing specifically deuterated cysteine residues, the electron paramagnetic resonance (EPR) spectrum of an intermediate in the exchange reaction and the reduction reaction, trapped by rapid freeze quench techniques, exhibits narrowed hyperfine features relative to the corresponding unlabeled RTPR. The spectrum was interpreted to represent a thiyl radical coupled to cob(II)alamin. Another proposed intermediate, 5'-deoxyadenosine, was detected by rapid acid quench techniques. Similarities in mechanism between RTPR and the Escherichia coli ribonucleotide reductase suggest that both enzymes require a thiyl radical for catalysis.
Asunto(s)
Radicales Libres , Ribonucleótido Reductasas/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Catálisis , Cobamidas/metabolismo , Desoxiadenosinas/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Lactobacillus/enzimología , Ligandos , Modelos Químicos , Datos de Secuencia Molecular , Oxidación-Reducción , Ribonucleótido Reductasas/química , Solventes , Vitamina B 12/análogos & derivados , Vitamina B 12/metabolismoRESUMEN
Large dynamic nuclear polarization signal enhancements (up to a factor of 100) were obtained in the solid-state magic-angle spinning nuclear magnetic resonance (NMR) spectra of arginine and the protein T4 lysozyme in frozen glycerol-water solutions with the use of dynamic nuclear polarization. Polarization was transferred from the unpaired electrons of nitroxide free radicals to nuclear spins through microwave irradiation near the electron paramagnetic resonance frequency. This approach may be a generally applicable signal enhancement scheme for the high-resolution solid-state NMR spectroscopy of biomolecules.
Asunto(s)
Arginina/química , Espectroscopía de Resonancia Magnética/métodos , Muramidasa/química , Bacteriófago T4/enzimología , Óxidos N-Cíclicos , Electrones , Radicales Libres , Congelación , Microondas , Soluciones , Marcadores de SpinRESUMEN
The design, construction, and performance of a multifrequency pulsed EPR and ENDOR probe for use at cryogenic temperatures are described. Interchangeable resonators based on a folded strip line design allow variation of the resonance frequency over a range of 5-11 GHz. Variable coupling to the resonator is achieved capacitively via a simple mechanical adjustment which is thermally and mechanically stable. The entire assembly is robust and easily fabricated. Common methods of analyzing the resonator parameters such as the Q-factor and coupling coefficient are discussed quantitatively. Probe performance data and multifrequency pulsed ENDOR spectra are presented.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/instrumentación , Diseño de Equipo , TemperaturaRESUMEN
A proton dynamic nuclear polarization (DNP) NMR signal enhancement (epsilon) close to thermal equilibrium, epsilon = 0.89, has been obtained at high field (B(0) = 5 T, nu(epr) = 139.5 GHz) using 15 mM trityl radical in a 40:60 water/glycerol frozen solution at 11 K. The electron-nuclear polarization transfer is performed in the nuclear rotating frame with microwave irradiation during a nuclear spin-lock pulse. The growth of the signal enhancement is governed by the rotating frame nuclear spin-lattice relaxation time (T(1rho)), which is four orders of magnitude shorter than the nuclear spin-lattice relaxation time (T(1n)). Due to the rapid polarization transfer in the nuclear rotating frame the experiment can be recycled at a rate of 1/T(1rho) and is not limited by the much slower lab frame nuclear spin-lattice relaxation rate (1/T(1n)). The increased repetition rate allowed in the nuclear rotating frame provides an effective enhancement per unit time(1/2) of epsilon(t) = 197. The nuclear rotating frame-DNP experiment does not require high microwave power; significant signal enhancements were obtained with a low-power (20 mW) Gunn diode microwave source and no microwave resonant structure. The symmetric trityl radical used as the polarization source is water-soluble and has a narrow EPR linewidth of 10 G at 139.5 GHz making it an ideal polarization source for high-field DNP/NMR studies of biological systems.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Compuestos de Tritilo/química , Fenómenos Químicos , Química Física , Estructura MolecularRESUMEN
We describe a spectrometer for pulsed ENDOR at 140 GHz, which is based on microwave IMPATT diode amplifiers and a probe consisting of a TE011 cavity with a high-quality resonance circuit for variable radiofrequency irradiation. For pulsed EPR we obtain an absolute sensitivity of 3x10(9) spins/Gauss at 20 K. The performance of the spectrometer is demonstrated with pulsed ENDOR spectra of a standard bis-diphenylene-phenyl-allyl (BDPA) doped into polystyrene and of the tyrosyl radical from E. coli ribonucleotide reductase (RNR). The EPR spectrum of the RNR tyrosyl radical displays substantial g-anisotropy at 5 T and is used to demonstrate orientation-selective Davies-ENDOR.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , Ribonucleótido Reductasas/química , Compuestos Alílicos/química , Anisotropía , Derivados del Benceno/química , Simulación por Computador , Espectroscopía de Resonancia por Spin del Electrón/instrumentación , Escherichia coli/enzimología , Radicales Libres , Magnetismo , Poliestirenos , Protones , Ondas de Radio , Sensibilidad y Especificidad , Análisis Espectral , Marcadores de Spin , Transductores , TirosinaRESUMEN
High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxide spin-labeled peptide in fluid aqueous solution. The peptide, which follows the single letter sequence, was reacted with the methanethiosulfonate spin label at the cysteine sulfur. The spin sensitivity of high-frequency EPR is excellent with less than 20 pmol of sample required to obtain spectra with good signal-to-noise ratios. Simulation of the temperature-dependent spectral lineshapes reveals the existence of local anisotropic motion about the nitroxide N-O bond with a motional anisotropy tau( perpendicular)/tau( parallel) ( identical with N) approaching 2.6 at 306 K. Comparison with previous work on rigidly labeled peptides suggests that the spin label is reorienting about its side-chain tether. This study demonstrates the feasibility of performing 140-GHz EPR on biological samples in fluid aqueous solution.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón , Péptidos/química , Secuencia de Aminoácidos , Anisotropía , Conformación Proteica , Marcadores de Spin , TemperaturaRESUMEN
Dilute frozen solutions of the single molecule magnet Ni(4) (S=4) have been studied using 130 GHz electron paramagnetic resonance (EPR). Despite the random orientation of the molecules, well defined EPR absorption peaks are observed due to the strong variation of the splittings between the different spin states on magnetic field. Temperature dependent studies above 4 K and comparison with simulations enable identification of the spin transitions and determination of the Hamiltonian parameters. The latter are found to be close to those of Ni(4) single crystals. No echo was detected from Ni(4) in pulsed experiments, which sets an upper bound of about 50 ns on the spin coherence time.
RESUMEN
As a molecular switch, the ras protein p21 undergoes structural changes that couple recognition sites on the protein surface to the guanine nucleotide-divalent metal ion binding site. X-ray crystallographic studies of p21 suggest that coordination between threonine-35 and the divalent metal ion plays an important role in these conformational changes. Recent ESEEM studies of p21 in solution, however, place threonine-35 more distant from the metal and were interpreted as weak or indirect coordination of this residue. We report high frequency (139.5 GHz) EPR spectroscopy of p21.Mn(II) complexes of two guanine nucleotides that probes the link between threonine-35 and the divalent metal ion. By analysis of high-frequency EPR spectra, we determine the number of water molecules in the first coordination sphere of the manganous ion to be four in p21.Mn(II).GDP, consistent with prior low-frequency EPR and X-ray crystallographic studies. In the complex of p21 with a GTP analog, p21.Mn(II).GMPPNP, we determine the hydration number to be 2, also consistent with crystal structures. This result rules out indirect coordination of threonine-35 in the solution structure of p21.Mn(II).GMPPNP, and implicates direct, weak coordination of this residue as suggested by Halkides et al. [(1994) Biochemistry 33,4019]. The 17O hyperfine coupling constant of H2(17)O is determined as 0.25 mT in the GDP from and 0.28 mT in the GTP form. These values are similar to reported values for 17O-enriched aquo ligands and some phosphato ligands in Mn(II) complexes. The high magnetic field strength (4.9 T) employed in these 139.5 GHz EPR measurements leads to a narrowing of the Mn(II) EPR lines that facilitates the determination of 17O hyperfine interactions.
Asunto(s)
Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón/métodos , Guanilil Imidodifosfato/metabolismo , Manganeso , Modelos Químicos , Isótopos de Oxígeno , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Treonina , AguaRESUMEN
A series of 2H- and 13C-labeled glutamates were used as substrates for coenzyme B12-dependent glutamate mutase, which equilibrates (S)-glutamate with (2S,3S)-3-methylaspartate. These compounds contained the isotopes at C-2, C-3, or C-4 of the carbon chain: [2-2H], [3,3-2H2], [4,4-2H2], [2,3,3,4,4-2H5], [2-13C], [3-13C], and [4-13C]glutamate. Each reaction was monitored by electron paramagnetic resonance (EPR) spectroscopy and revealed a similar signal characterized by g'xy = 2.1, g'z = 1.985, and A' = 5.0 mT. The interpretation of the spectral data was aided by simulations which gave close agreement with experiment. This approach underpinned the idea of the formation of a radical pair, consisting of cob(II)alamin interacting with an organic radical at a distance of 6.6 +/- 0.9 A. Comparison of the hyperfine couplings observed with unlabeled glutamate with those from the labeled glutamates enabled a principal contributor to the radical pair to be identified as the 4-glutamyl radical. These findings support the currently accepted mechanism for the glutamate mutase reaction, i.e., the process is initiated through hydrogen atom abstraction from C-4 of glutamate by the 5'-deoxyadenosyl radical, which is derived by homolysis of the Co-C sigma-bond of coenzyme B12.
Asunto(s)
Carbono/metabolismo , Clostridium/enzimología , Cobamidas/metabolismo , Glutamina/metabolismo , Transferasas Intramoleculares/metabolismo , Isótopos de Carbono , Catálisis , Deuterio/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Glutamatos/metabolismo , Especificidad por SustratoRESUMEN
The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleoside 5'-triphosphates to 2'-deoxynucleoside 5'-triphosphates and uses coenzyme B12, adenosylcobalamin (AdoCbl), as a cofactor. Use of a mechanism-based inhibitor, 2'-deoxy-2'-methylenecytidine 5'-triphosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed identification of the lower axial ligand of cob(II)alamin when bound to RTPR. In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroatom migrations and in contrast to the enzymes catalyzing reversible carbon skeleton rearrangements, the dimethylbenzimidazole moiety of the cofactor is not displaced by a protein histidine upon binding to RTPR.
Asunto(s)
Bencimidazoles/metabolismo , Cobamidas/metabolismo , Lactobacillus/enzimología , Ribonucleótido Reductasas/metabolismo , Vitamina B 12/metabolismo , Catálisis , Nucleótidos de Desoxicitosina/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Inhibidores Enzimáticos/farmacología , Ligandos , Unión Proteica , Ribonucleótido Reductasas/antagonistas & inhibidoresRESUMEN
Recent evidence indicates that the prion protein (PrP) plays a role in copper metabolism in the central nervous system. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds divalent copper ions (Cu(2+)) in vivo. To elucidate the specific mode and site of binding, we have studied a series of Cu(2+)-peptide complexes composed of 1-, 2-, and 4-octarepeats and several sub-octarepeat peptides, by electron paramagnetic resonance (EPR, conventional X-band and low-frequency S-band) and circular dichroism (CD) spectroscopy. At pH 7.45, two EPR active binding modes are observed where the dominant mode appears to involve coordination of three nitrogens and one oxygen to the copper ion, while in the minor mode two nitrogens and two oxygens coordinate. ESEEM spectra demonstrate that the histidine imidazole contributes one of these nitrogens. The truncated sequence HGGGW gives EPR and CD that are indistinguishable from the dominant binding mode observed for the multi-octarepeat sequences and may therefore comprise the fundamental Cu(2+) binding unit. Both EPR and CD titration experiments demonstrate rigorously a 1:1 Cu(2+)/octarepeat binding stoichiometry regardless of the number of octarepeats in a given peptide sequence. Detailed spin integration of the EPR signals demonstrates that all of the bound Cu(2+) is detected thereby ruling out strong exchange coupling that is often found when there is imidazolate bridging between paramagnetic metal centers. A model consistent with these data is proposed in which Cu(2+) is bound to the nitrogen of the histidine imidazole side chain and to two nitrogens from sequential glycine backbone amides.
Asunto(s)
Cobre/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas PrPC/química , Secuencia de Aminoácidos , Sitios de Unión , Cationes Bivalentes , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón/métodos , Humanos , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Fragmentos de Péptidos/síntesis química , Proteínas PrPC/metabolismo , Estructura Terciaria de Proteína , Secuencias Repetitivas de Aminoácido , VolumetríaRESUMEN
Electron paramagnetic resonance spectroscopy at 139.5 GHz has been used to study p21 ras complexed with Mn(II) and guanosine 5'-(beta, gamma-imidotriphosphate), an analog of GTP. The p21 sample studied was selectively labeled with [17O gamma]threonine to a final enrichment of 30%. A Mn(II)-17O hyperfine interaction was observed, but the value of the coupling constant, 0.11 +/- 0.04 mT, is the smallest such value yet reported. Ab initio calculations indicate that this value is consistent with direct coordination of the threonine hydroxyl group and provide an estimate for the Mn(II)-17O bond length of 2.7 A. The measured hyperfine coupling constant and associated bond length starkly contrast with typical values for Mn(II)-17O coordination complexes, namely, approximately 0.25 mT and approximately 2.2 A, respectively. This contrast underscores the peculiar weakness of this Mn(II)-O interaction in p21 and persuasively argues that the nucleotide-induced conformational change, which is known to encompass the region of p21 involving Thr35, is not driven by Mn(II) coordination of the Thr35 hydroxyl group.