Formation of Complex Organic and Prebiotic Molecules in H2O:NH3:CO2 Ices at Temperatures Relevant to Hot Cores, Protostellar Envelopes, and Planet-Forming Disks.

Photochemistry in H2O:NH3:CO2 cosmic ice analogues was studied at temperatures of 75, 120, and 150 K, relevant to hot cores and warmer regions in protostellar envelopes and planet-forming disks. A combination of two triggers of surface chemistry in cosmic ice analogues, heat and UV irradiation, compared to using either just heat or UV irradiation, leads to a larger variety and an increased production of complex organic molecules, including potential precursors of prebiotic molecules. In addition to complex organic molecules detected in previous studies of H2O:NH3:CO2 ices, ammonium carbamate, carbamic acid, ammonium formate and formamide, we detected acetaldehyde, urea, and, tentatively, glycine, the simplest amino acid. Water ice hampers reactions at low temperature (75 K) but allows the parent molecules, CO2 and NH3, to stay in the solid state and react at higher temperatures (120 and 150 K, above their desorption temperatures). The experiments were performed on the surface of KBr substrates and amorphous silicate grains, analogs of cosmic silicate dust. The production of complex molecules on the silicate surface is decreased compared to KBr. This result suggests that the larger surface area and/or surface properties of the silicate grains play a role in controlling the chemistry, preventing it taking place to the same extent as on the flat KBr substrate. This is further evidence of the fact that cosmic dust grains play an important role in the chemistry taking place on their surface.

Structure of the human Ccr4-Not nuclease module using X-ray crystallography and electron paramagnetic resonance spectroscopy distance measurements.

Regulated degradation of mature, cytoplasmic mRNA is a key step in eukaryotic gene regulation. This process is typically initiated by the recruitment of deadenylase enzymes by cis-acting elements in the 3' untranslated region resulting in the shortening and removal of the 3' poly(A) tail of the target mRNA. The Ccr4-Not complex, a major eukaryotic deadenylase, contains two exoribonuclease subunits with selectivity toward poly(A): Caf1 and Ccr4. The Caf1 deadenylase subunit binds the MIF4G domain of the large subunit CNOT1 (Not1) that is the scaffold of the complex. The Ccr4 nuclease is connected to the complex via its leucine-rich repeat (LRR) domain, which binds Caf1, whereas the catalytic activity of Ccr4 is provided by its EEP domain. While the relative positions of the MIF4G domain of CNOT1, the Caf1 subunit, and the LRR domain of Ccr4 are clearly defined in current models, the position of the EEP nuclease domain of Ccr4 is ambiguous. Here, we use X-ray crystallography, the AlphaFold resource of predicted protein structures, and pulse electron paramagnetic resonance spectroscopy to determine and validate the position of the EEP nuclease domain of Ccr4 resulting in an improved model of the human Ccr4-Not nuclease module.

Astrochemical Pathways to Complex Organic and Prebiotic Molecules: Experimental Perspectives for In Situ Solid-State Studies.

A deep understanding of the origin of life requires the physical, chemical, and biological study of prebiotic systems and the comprehension of the mechanisms underlying their evolutionary steps. In this context, great attention is paid to the class of interstellar molecules known as "Complex Organic Molecules" (COMs), considered as possible precursors of prebiotic species. Although COMs have already been detected in different astrophysical environments (such as interstellar clouds, protostars, and protoplanetary disks) and in comets, the physical-chemical mechanisms underlying their formation are not yet fully understood. In this framework, a unique contribution comes from laboratory experiments specifically designed to mimic the conditions found in space. We present a review of experimental studies on the formation and evolution of COMs in the solid state, i.e., within ices of astrophysical interest, devoting special attention to the in situ detection and analysis techniques commonly used in laboratory astrochemistry. We discuss their main strengths and weaknesses and provide a perspective view on novel techniques, which may help in overcoming the current experimental challenges.

Application of DNP-enhanced solid-state NMR to studies of amyloid-ß peptide interaction with lipid membranes.

The cellular membrane disruption induced by the aggregation of Aß peptide has been proposed as a plausible cause of neuronal cell death during Alzheimer's disease. The molecular-level details of the Aß interaction with cellular membranes were previously probed using solid state NMR (ssNMR), however, due to the limited sensitivity of the latter, studies were limited to samples with high Aß-to-lipid ratio. The dynamic nuclear polarization (DNP) is a technique for increasing the sensitivity of NMR. In this work we demonstrate the feasibility of DNP-enhanced ssNMR studies of Aß40 peptide interacting with various model liposomes: (1) a mixture of zwitterionic 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG); (2) a mixture of POPC, POPG, cholesterol, sphingomyelin and ganglioside GM1; (3) the synaptic plasma membrane vesicles (SPMVs) extracted from rat brain tissues. In addition, DNP-ssNMR was applied to capturing changes in Aß40 conformation taking place upon the peptide insertion into POPG liposomes. The signal enhancements under conditions of DNP allow carrying out informative 2D ssNMR experiments with about 0.25 mg of Aß40 peptides (i.e. reaching Aß40-to-lipid ratio of 1:200). In the studied liposome models, the 13C NMR chemical shifts at many 13C-labelled sites of Aß40 are characteristic of ß-sheets. In addition, in POPG liposomes the peptide forms hydrophobic contacts F19-L34 and F19-I32. Both the chemical shifts and hydrophobic contacts of Aß40 in POPG remain the same before and after 8 h of incubation. This suggests that conformation at the 13C-labelled sites of the peptide is similar before and after the insertion process. Overall, our results demonstrate that DNP helps to overcome the sensitivity limitation of ssNMR, and thereby expand the applicability of ssNMR for charactering the Aß peptide interacting with lipids.

Application of spherical harmonics for DEER data analysis in systems with a conformational distribution.

Double electron-electron resonance (DEER) and other pulse electron paramagnetic resonance (EPR) techniques are valuable tools for determining distances between paramagnetic centres. DEER theory is well developed for a scenario where relative orientations of paramagnetic centres do not affect the DEER data. In particular, such theory enables a number of approaches for extracting distance distributions. However, in a more general case, when orientation selection effects become substantial, the analytical theory of DEER is less well developed, therefore quite commonly researchers rely on a comparison of some model-based simulations with experimental data. This work elaborates the theory of DEER with orientation selection effects, focusing on a scenario of a moderate conformational disorder, leading to an orientation distribution in a pair of paramagnetic centres. The analytical treatment based on expansions into spherical harmonics, provides important insights into the structure of DEER data. As follows from this treatment, DEER spectra with orientation selection can be represented as a linear combination of modified Pake pattern (MPP) components. The conformational disorder has a filtering effect on the weights of MPP components, specifically by significantly suppressing MPP components of higher degrees. The developed theory provides a pathway for model-based simulations of DEER data where orientation distribution is defined by analytical functions with parameters. The theory based on spherical harmonics expansions was also applied to develop an iterative processing algorithm based on Tikhonov regularization, which disentangles the distance and orientation information in a model-free manner. As an input, this procedure takes several DEER datasets measured at various positions of an EPR line, and outputs a distance distribution and orientation distribution information encoded in a set of coefficients related to the weights of MPP components. The model-based and model-free approaches based on the developed theory were validated for a nitroxide biradical and a spin-labelled protein.

Ice Coverage of Dust Grains in Cold Astrophysical Environments.

Surface processes on cosmic solids in cold astrophysical environments lead to gas-phase depletion and molecular complexity. Most astrophysical models assume that the molecular ice forms a thick multilayer substrate, not interacting with the dust surface. In contrast, we present experimental results demonstrating the importance of the surface for porous grains. We show that cosmic dust grains may be covered by a few monolayers of ice only. This implies that the role of dust surface structure, composition, and reactivity in models describing surface processes in cold interstellar, protostellar, and protoplanetary environments has to be reevaluated.

Heteronuclear DNP of 1H and 19F nuclei using BDPA as a polarizing agent.

This work explores the dynamic nuclear polarization (DNP) of 1H and 19F nuclei in a sample of 25/75 (% v/v) fluorobenzene/toluene containing the radical 1,3-bisphenylene-2-phenylallyl radical (BDPA) as a polarizing agent. Previously, heteronuclear effects in DNP were studied by analysing the shapes of DNP spectra, or by observing cross-relaxation between nuclei of different types. In this work, we report a rather specific DNP spectrum, where 1H and 19F nuclei obtain polarizations of opposite signs upon microwave (MW) irradiation. In order to explain this observation, we introduce a novel mechanism called heteronuclear thermal mixing (hn-TM). Within this mechanism the spectra of opposite signs can then be explained due to the presence of four-spin systems, involving a pair of dipolar coupled electron spins and hyperfine coupled nuclear spins of 1H and 19F, such that a condition relating their Larmor frequencies |ω1e - ω2e| ≈ ωH - ωF is satisfied. Under this condition, a strong mixing of electron and nuclear states takes place, enabling simultaneous four-spin flip-flops. Irradiation of electron spin transitions with MW followed by such four-spin flip-flops produces non-equilibrium populations of |αHßF and |ßHαF states, thus leading to the enhancements of opposite signs for 1H and 19F. Signal enhancements, build-up times and DNP-spectra as a function of MW power and polarizing agent concentration, all provide additional support for assigning the observed DNP mechanism as hn-TM and distinguishing it from other possible mechanisms. We also develop a quantum mechanical model of hn-TM based on averaging of spin Hamiltonians. Simulations based on this model show very good qualitative agreement with experimental data. In addition, the system exhibits cross-relaxation between 1H and 19F induced by the presence of BDPA, which was detected by measuring the 19F signal build-up upon saturation of 1H nuclei with a train of radio-frequency pulses. We demonstrate that such cross-relaxation most likely originates due to the same electron and nuclear states mixing in the four-spin systems.

Gas-phase reactivity of CH3OH toward OH at interstellar temperatures (11.7-177.5 K): experimental and theoretical study.

The reactivity of methanol (CH3OH) toward the hydroxyl (OH) radical was investigated in the temperature range 11.7-177.5 K using the CRESU (French acronym for Reaction Kinetics in a Uniform Supersonic Flow) technique. In the present study, the temperature dependence of the rate coefficient for the OH + CH3OH reaction, k(T), has been revisited and additional experimental and computational data are reported. New kinetic measurements were performed to fill the existing gaps (<22 K, 22-42 K and 88-123 K), reporting k(T < 20 K) for the first time. The lowest temperature ever achieved by a pulsed CRESU has been obtained in this work (11.7 K). k(T) abruptly increases by almost 2 orders of magnitude from 177.5 K to around 100 K. At T < 100 K, this increase is less pronounced, reaching the capture limit at temperatures below 22 K. The pressure dependence of k(T) has been investigated for selected temperatures and gas densities (1.5 × 1016 to 4.3 × 1017 cm-3), combining our results with those previously reported. No dependence was observed within the experimental uncertainties below 110 K. The high- and low-pressure rate coefficients, kHPL(T) and kLPL(T), were also studied in detail using high-level quantum chemical and theoretical kinetic methodologies, closely reproducing the experimental data between 20 and 400 K. The results suggest that the experimental data are near the high pressure limit at the lowest temperatures, but that the reaction remains a fast and effective source of CH2OH and CH3O at the low pressures and temperatures prevalent in the interstellar medium.

Uniform Supersonic Chemical Reactors: 30†Years of Astrochemical History and Future Challenges.

The interstellar medium is of great interest to us as the place where stars and planets are born and from where, probably, the molecular precursors of life came to Earth. Astronomical observations, astrochemical modeling, and laboratory astrochemistry should go hand in hand to understand the chemical pathways to the formation of stars, planets, and biological molecules. We review here laboratory experiments devoted to investigations on the reaction dynamics of species of astrochemical interest at the temperatures of the interstellar medium and which were performed by using one of the most popular techniques in the field, CRESU. We discuss new technical developments and scientific ideas for CRESU, which, if realized, will bring us one step closer to understanding of the astrochemical history and the future of our universe.

Conformational landscape of the SF6 dimer as revealed by high resolution infrared spectroscopy and complexation with rare gas atoms.

Taking advantage of a versatile set-up, combining pulsed pin hole or slit nozzle supersonic expansion with an external cavity quantum cascade laser, the rovibrational absorption spectrum of the SF6 dimer in the ν3 mode region has been revisited at high resolution under various experimental conditions in SF6:He mixtures. Two new rotationally resolved spectral bands have been identified in the range of the parallel band of the dimer spectrum in addition to that previously reported. Among these three spectral features, two of them are assigned to conformations of the dimer (noted #1 and #2), clearly distinguished from their different S-S interatomic distances, i.e. 474 and 480 pm respectively. The third one is assigned to a (SF6)2-He complex, from comparison with additional experiments in which (SF6)2-Rg heterotrimers (Rg = Ne, Ar, Kr, Xe) are observed. A schematic picture of the potential energy landscape of the SF6 dimer in terms of a nearly flat surface is proposed to account for the conformational relaxation observed in the expansions and for the structure of the (SF6)2-Rg heterotrimers, which are exclusively formed from the conformer #2 dimer. Although modelling qualitatively supports this picture, much effort has still to be achieved from a theoretical point of view to reach a quantitative agreement with the present benchmark experimental data both in terms of structure and energetics.

Successive Stages of Amyloid-ß Self-Assembly Characterized by Solid-State Nuclear Magnetic Resonance with Dynamic Nuclear Polarization.

Self-assembly of amyloid-ß (Aß) peptides in human brain tissue leads to neurodegeneration in Alzheimer's disease (AD). Amyloid fibrils, whose structures have been extensively characterized by solid state nuclear magnetic resonance (ssNMR) and other methods, are the thermodynamic end point of Aß self-assembly. Oligomeric and protofibrillar assemblies, whose structures are less well-understood, are also observed as intermediates in the assembly process in vitro and have been implicated as important neurotoxic species in AD. We report experiments in which the structural evolution of 40-residue Aß (Aß40) is monitored by ssNMR measurements on frozen solutions prepared at four successive stages of the self-assembly process. Measurements on transient intermediates are enabled by ssNMR signal enhancements from dynamic nuclear polarization (DNP) at temperatures below 30 K. DNP-enhanced ssNMR data reveal a monotonic increase in conformational order from an initial state comprised primarily of monomers and small oligomers in solution at high pH, to larger oligomers near neutral pH, to metastable protofibrils, and finally to fibrils. Surprisingly, the predominant molecular conformation, indicated by (13)C NMR chemical shifts and by side chain contacts between F19 and L34 residues, is qualitatively similar at all stages. However, the in-register parallel ß-sheet supramolecular structure, indicated by intermolecular (13)C spin polarization transfers, does not develop before the fibril stage. This work represents the first application of DNP-enhanced ssNMR to the characterization of peptide or protein self-assembly intermediates.

Vibrational dynamics. Experimental ground-state combination differences of CH5⁺.

Protonation of methane (CH4), a rather rigid molecule well described by quantum mechanics, produces CH5(+), a prototypical floppy molecule that has eluded definitive spectroscopic description. Experimental measurement of high-resolution spectra of pure CH5(+) samples poses a formidable challenge. By applying two types of action spectroscopy predicated on photoinduced reaction with CO2 and photoinhibition of helium cluster growth, we obtained low-temperature, high-resolution spectra of mass-selected CH5(+). On the basis of the very high accuracy of the line positions, we determined a spectrum of combination differences. Analysis of this spectrum enabled derivation of equally accurate ground state-level schemes of the corresponding nuclear spin isomers of CH5(+), as well as tentative quantum number assignment of this enfant terrible of molecular spectroscopy.

Total power millimeter-wave spectrometer for measurements of dust opacity at cryogenic temperatures.

A highly sensitive total power millimeter-wave spectrometer has been built to investigate the opacity of important interstellar-dust analogues in the 10-300 K temperature range. The key elements of the spectrometer are a frequency agile synthesizer followed by a microwave amplifier and a subsequent frequency multiplier. In a first step, the frequency range of 72-120 GHz is covered by the spectrometer, and a room temperature Schottky detector is employed as a detector. A newly developed two channel (sample/reference) copper sample holder is cryogenically cooled for the 10-300 K range. Here we present the technical details of the spectrometer including examples of the obtained results. The analysis of these results will be published elsewhere.

Dynamic nuclear polarization-enhanced 13C NMR spectroscopy of static biological solids.

We explore the possibility of using dynamic nuclear polarization (DNP) to enhance signals in structural studies of biological solids by solid state NMR without sample spinning. Specifically, we use 2D (13)C-(13)C exchange spectroscopy to probe the peptide backbone torsion angles (φ, ψ) in a series of selectively (13)C-labeled 40-residue ß-amyloid (Aß(1-40)) samples, in both fibrillar and non-fibrillar states. Experiments are carried out at 9.39 T and 8 K, using a static double-resonance NMR probe and low-power microwave irradiation at 264 GHz. In frozen solutions of Aß(1-40) fibrils doped with DOTOPA-TEMPO, we observe DNP signal enhancement factors of 16-21. We show that the orientation- and frequency-dependent spin polarization exchange between sequential backbone carbonyl (13)C labels can be simulated accurately using a simple expression for the exchange rate, after experimentally determined homogeneous (13)C lineshapes are incorporated in the simulations. The experimental 2D (13)C-(13)C exchange spectra place constraints on the φ and ψ angles between the two carbonyl labels. Although the data are not sufficient to determine φ and ψ uniquely, the data do provide non-trivial constraints that could be included in structure calculations. With DNP at low temperatures, 2D (13)C-(13)C exchange spectra can be obtained from a 3.5 mg sample of Aß(1-40) fibrils in 4 h or less, despite the broad (13)C chemical shift anisotropy line shapes that are observed in static samples.

Solid state nuclear magnetic resonance with magic-angle spinning and dynamic nuclear polarization below 25 K.

We describe an apparatus for solid state nuclear magnetic resonance (NMR) with dynamic nuclear polarization (DNP) and magic-angle spinning (MAS) at 20-25 K and 9.4 Tesla. The MAS NMR probe uses helium to cool the sample space and nitrogen gas for MAS drive and bearings, as described earlier, but also includes a corrugated waveguide for transmission of microwaves from below the probe to the sample. With a 30 mW circularly polarized microwave source at 264 GHz, MAS at 6.8 kHz, and 21 K sample temperature, greater than 25-fold enhancements of cross-polarized (13)C NMR signals are observed in spectra of frozen glycerol/water solutions containing the triradical dopant DOTOPA-TEMPO when microwaves are applied. As demonstrations, we present DNP-enhanced one-dimensional and two-dimensional (13)C MAS NMR spectra of frozen solutions of uniformly (13)C-labeled l-alanine and melittin, a 26-residue helical peptide that we have synthesized with four uniformly (13)C-labeled amino acids.

Dynamic nuclear polarization-enhanced ¹H-¹³C double resonance NMR in static samples below 20 K.

We demonstrate the feasibility of one-dimensional and two-dimensional ¹H-¹³C double resonance NMR experiments with dynamic nuclear polarization (DNP) at 9.4 T and temperatures below 20 K, including both ¹H-¹³C cross-polarization and ¹H decoupling, and discuss the effects of polarizing agent type, polarizing agent concentration, temperature, and solvent deuteration. We describe a two-channel low-temperature DNP/NMR probe, capable of carrying the radio-frequency power load required for ¹H-¹³C cross-polarization and high-power proton decoupling. Experiments at 8 K and 16 K reveal a significant T2 relaxation of ¹³C, induced by electron spin flips. Carr-Purcell experiments and numerical simulations of Carr-Purcell dephasing curves allow us to determine the effective correlation time of electron flips under our experimental conditions. The dependence of the DNP signal enhancement on electron spin concentration shows a maximum near 80 mM. Although no significant difference in the absolute DNP enhancements for triradical (DOTOPA-TEMPO) and biradical (TOTAPOL) dopants was found, the triradical produced greater DNP build-up rates, which are advantageous for DNP experiments. Additionally the feasibility of structural measurements on ¹³C-labeled biomolecules was demonstrated with a two-dimensional ¹³C-¹³C exchange spectrum of selectively ¹³C-labeled ß-amyloid fibrils.

Spin delocalization over type zero copper.

Hard-ligand, high-potential copper sites have been characterized in double mutants of Pseudomonas aeruginosa azurin (C112D/M121X (X = L, F, I)). These sites feature a small A(zz)(Cu) splitting in the EPR spectrum together with enhanced electron transfer activity. Due to these unique properties, these constructs have been called "type zero" copper sites. In contrast, the single mutant, C112D, features a large A(zz)(Cu) value characteristic of the typical type 2 Cu(II). In general, A(zz)(Cu) comprises contributions from Fermi contact, spin dipolar, and orbital dipolar terms. In order to understand the origin of the low A(zz)(Cu) value of type zero Cu(II), we explored in detail its degree of covalency, as manifested by spin delocalization over its ligands, which affects A(zz)(Cu) through the Fermi contact and spin dipolar contributions. This was achieved by the application of several complementary EPR hyperfine spectroscopic techniques at X- and W-band (∼9.5 and 95 GHz, respectively) frequencies to map the ligand hyperfine couplings. Our results show that spin delocalization over the ligands in type zero Cu(II) is different from that of type 2 Cu(II) in the single C112D mutant. The (14)N hyperfine couplings of the coordinated histidine nitrogens are smaller by about 25-40%, whereas that of the (13)C carboxylate of D112 is about 50% larger. From this comparison, we concluded that the spin delocalization of type zero copper over its ligands is not dramatically larger than in type 2 C112D. Therefore, the reduced A(zz)(Cu) value of type zero Cu(II) is largely attributable to an increased orbital dipolar contribution that is related to its larger g(zz) value, as a consequence of the distorted tetrahedral geometry. The increased spin delocalization over the D112 carboxylate in type zero mutants compared to type 2 C112D suggests that electron transfer paths involving this residue are enhanced.

Identity of the exchangeable sulfur-containing ligand at the Mo(V) center of R160Q human sulfite oxidase.

In our previous study of the fatal R160Q mutant of human sulfite oxidase (hSO) at low pH (Astashkin et al. J. Am. Chem. Soc.2008, 130, 8471-8480), a new Mo(V) species, denoted "species 1", was observed at low pH values. Species 1 was ascribed to a six-coordinate Mo(V) center with an exchangeable terminal oxo ligand and an equatorial sulfate group on the basis of pulsed EPR spectroscopy and (33)S and (17)O labeling. Here we report new results for species 1 of R160Q, based on substitution of the sulfur-containing ligand by a phosphate group, pulsed EPR spectroscopy in K(a)- and W-bands, and extensive density functional theory (DFT) calculations applied to large, more realistic molecular models of the enzyme active site. The combined results unambiguously show that species 1 has an equatorial sulfite as the only exchangeable ligand. The two types of (17)O signals that are observed arise from the coordinated and remote oxygen atoms of the sulfite ligand. A typical five-coordinate Mo(V) site is compatible with the observed and calculated EPR parameters.

Temperature-dependent exchange interaction in molecular magnets Cu(hfac)2L(R) studied by EPR: methodology and interpretations.

Exchange-coupled spin triads nitroxide-copper(II)-nitroxide are the key building blocks of molecular magnets Cu(hfac)(2)L(R). These compounds exhibit thermally induced structural rearrangements and spin transitions, where the exchange interaction between spins of copper(II) ion and nitroxide radicals changes typically by 1 order of magnitude. We have shown previously that electron paramagnetic resonance (EPR) spectroscopy is sensitive to the observed magnetic anomalies and provides information on both inter- and intracluster exchange interactions. The value of intracluster exchange interaction is temperature-dependent (J(T)), that can be accessed by monitoring the effective g-factor of the spin triad as a function of temperature (g(eff)(T)). This paper describes approaches for studying the g(eff)(T) and J(T) dependences and establishes correlations between them. The experimentally obtained g(eff)(T) dependences are interpreted using three different models for the mechanism of structural rearrangements on the molecular level leading to different meanings of the J(T) function. The contributions from these mechanisms and their manifestations in X-ray, magnetic susceptibility and EPR data are discussed.

Probing conformational variations at the ATPase site of the RNA helicase DbpA by high-field electron-nuclear double resonance spectroscopy.

The RNA helicase DbpA promotes RNA remodeling coupled to ATP hydrolysis. It is unique because of its specificity to hairpin 92 of 23S rRNA (HP92). Although DbpA kinetic pathways leading to ATP hydrolysis and RNA unwinding have been recently elucidated, the molecular (atomic) basis for the coupling of ATP hydrolysis to RNA remodeling remains unclear. This is, in part, due to the lack of detailed structural information on the ATPase site in the presence and absence of RNA in solution. We used high-field pulse ENDOR (electron-nuclear double resonance) spectroscopy to detect and analyze fine conformational changes in the protein's ATPase site in solution. Specifically, we substituted the essential Mg(2+) cofactor in the ATPase active site for paramagnetic Mn(2+) and determined its close environment with different nucleotides (ADP, ATP, and the ATP analogues ATPγS and AMPPnP) in complex with single- and double-stranded RNA. We monitored the Mn(2+) interactions with the nucleotide phosphates through the (31)P hyperfine couplings and the coordination by protein residues through (13)C hyperfine coupling from (13)C-enriched DbpA. We observed that the nucleotide binding site of DbpA adopts different conformational states upon binding of different nucleotides. The ENDOR spectra revealed a clear distinction between hydrolyzable and nonhydrolyzable nucleotides prior to RNA binding. Furthermore, both the (13)C and the (31)P ENDOR spectra were found to be highly sensitive to changes in the local environment of the Mn(2+) ion induced by the hydrolysis. More specifically, ATPγS was efficiently hydrolyzed upon binding of RNA, similar to ATP. Importantly, the Mn(2+) cofactor remains bound to a single protein side chain and to one or two nucleotide phosphates in all complexes, whereas the remaining metal coordination positions are occupied by water. The conformational changes in the protein's ATPase active site associated with the different DbpA states occur in remote coordination shells of the Mn(2+) ion. Finally, a competitive Mn(2+) binding site was found for single-stranded RNA construct.