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Quantum information technology puts stringent demands on the quality of materials and interfaces in the pursuit of increased device coherence. Yet, little is known about the chemical structure and origins of paramagnetic impurities that produce flux/charge noise that causes decoherence of fragile quantum states and impedes the progress toward large-scale quantum computing. Here, we perform high magnetic field electron paramagnetic resonance (HFEPR) and hyperfine multispin spectroscopy on α-Al 2 O 3 , a common substrate for quantum devices. In its amorphous form, α-Al 2 O 3 is also unavoidably present in aluminum-based superconducting circuits and qubits. The detected paramagnetic centers are immanent to the surface and have a well-defined but highly complex structure that extends over multiple hydrogen, aluminum, and oxygen atoms. Modeling reveals that the radicals likely originate from well-known reactive oxygen chemistry common to many metal oxides. We discuss how EPR spectroscopy might benefit the search for surface passivation and decoherence mitigation strategies.

International audience; The 285 GHz EPR spectra of perchlorotriphenylmethyl and tetrathiatriarylmethyl radicals in frozen solution have been accurately measured. The relationship between their molecular structures and their g-tensors has been investigated with the aid of DFT calculations, revealing that the degree of spin density delocalization away from the central methylene carbon is an important determining factor of the g-anisotropy. In particular, the small amount of spin densities on the Cl or S heteroatoms at the 2 and 6 positions with respect to the central carbon have the strongest influence. Furthermore, the amount of spin densities on these heteroatoms and thus the anisotropy can be modulated by the protonation (esterification) state of the carboxylate groups at the 4 position. These results provide unique insights into the g-anisotropy of persistent trityl radicals and how it can be tuned.

We have generated a mutant of C. elegans manganese superoxide dismutase at histidine 30 by site-directed mutagenesis. The structure was solved at a resolution of 1.52 Å by X-ray crystallography (pdb: 6S0D). His30 was targeted, as it forms as a gateway residue at the top of the solvent access funnel to the active site, together with Tyr34. In the wild-type protein, these gateway residues are involved in the hydrogen-bonding network providing the protons necessary for the catalytic reaction at the metal center. However, biophysical characterization and cell viability experiments reveal that a mutation from histidine to asparagine in the H30N mutant modifies metal selectivity in the protein, favoring the uptake of iron over manganese in minimal media conditions, alters active-site coordination from the characteristic trigonal bipyramidal to octahedral geometry, and encourages cellular proliferation in K562 cells, when added exogenously to the cells. Supplementary Information The online version contains supplementary material available at 10.1007/s00249-021-01544-2.

The 285 GHz EPR spectra of perchlorotriphenylmethyl and tetrathiatriarylmethyl radicals in frozen solution have been accurately measured. The relationship between their molecular structures and their g-tensors has been investigated with the aid of DFT calculations, revealing that the degree of spin density delocalization away from the central methylene carbon is an important determining factor of the g-anisotropy. In particular, the small amount of spin densities on the Cl or S heteroatoms at the 2 and 6 positions with respect to the central carbon have the strongest influence. Furthermore, the amount of spin densities on these heteroatoms and thus the anisotropy can be modulated by the protonation (esterification) state of the carboxylate groups at the 4 position. These results provide unique insights into the g-anisotropy of persistent trityl radicals and how it can be tuned.

VUV photons from a synchrotron source were used to record the gas-phase absorption spectrum of isoquinoline over the range 3.5 to 10.7 eV. The rich spectrum exhibits both broad and sharp features, of varying intensities, that are analyzed into eight valence and eight Rydberg transitions. Previous data on the valence transitions of isoquinoline were essentially limited to solution spectra up to 5.4 eV. Our study increases their number considerably. The features in the 3.96 eV region are discussed in terms of vibronic coupling between the nπ∗ 1 1 A′′ and ππ∗ 2 1 A′ valence electronic states. The intensities of some spectral features are augmented by collective π-electron modes considered to be of plasmon-type. Assignments of the valence transitions were facilitated by our DFT calculations and by earlier Pariser-Parr-Pople MO calculations. The calculation results are compared and their relative value is discussed. The DFT calculations reproduce very well a number of experimentally determined properties of the ground state of isoquinoline, in particular its bond distances and angles, rotational constants, vibrational frequencies and dipole moment. No Rydberg series of isoquinoline have previously been observed. Three of the newly observed Rydberg series converge to the D 0 electronic ground state of the ion, while two converge to the D 1 and three to the D 3 excited electronic states of the cation. Astrophysical applications of the VUV absorption spectrum of isoquinoline, in particular the measured absorption cross-sections, are briefly discussed. A comparison between the absorption spectra of isoquinoline and quinoline highlights their similarities and differences, related to their respective molecular orbitals.

International audience; To elucidate biochemical mechanisms leading to seed deterioration, we studied 23 wheat genotypes after exposure to seed bank storage for 6-16 years compared to controlled deterioration (CD) at 45 °C and 14 (CD14) and 18% (CD18) moisture content (MC) for up to 32 days. Under two seed bank storage conditions, seed viability was maintained in cold storage (CS) at 0 °C and 9% seed MC, but significantly decreased in ambient storage (AS) at 20 °C and 9% MC. Under AS and CS, organic free radicals, most likely semiquinones, accumulated, detected by electron paramagnetic resonance, while the antioxidant glutathione (GSH) was partly lost and partly converted to glutathione disulphide (GSSG), detected by HPLC. Under AS the glutathione half-cell reduction potential (EGSSG/2GSH) shifted towards more oxidizing conditions, from -186 to -141 mV. In seeds exposed to CD14 or CD18, no accumulation of organic free radicals was observed, GSH and seed viability declined within 32 and 7 days, respectively, GSSG hardly changed (CD14) or decreased (CD18) and EGSSG/2GSH shifted to -116 mV. The pH of extracts prepared from seeds subjected to CS, AS and CD14 decreased with viability, and remained high under CD18. Across all treatments EGSSG/2GSH correlated significantly with seed viability (r = 0.8, p \textless 0.001). Data are discussed with a view that the cytoplasm is in a glassy state in CS and AS, but during the CD treatments, underwent transition to a liquid state. We suggest that enzymes can be active during CD but not under the seed bank conditions tested. However, upon CD, enzyme-based repair processes were apparently outweighed by deteriorative reactions. We conclude that seed ageing by CD and under seed bank conditions are accompanied by different biochemical reactions.

Mn(2+) often serves as a paramagnetic substitute to Mg(2+), providing means for exploring the close environment of Mg(2+) in many biological systems where it serves as an essential co-factor. This applies to proteins with ATPase activity, where the ATP hydrolysis requires the binding of Mg(2+)-ATP to the ATPase active site. In this context, it is important to distinguish between the Mn(2+) coordination mode with free ATP in solution as compared to the protein bound case. In this work, we explore the Mn(2+) complexes with ATP, the non-hydrolysable ATP analog, AMPPNP, and ADP free in solution. Using W-band (31)P electron-nuclear double resonance (ENDOR) we obtained information about the coordination to the phosphates, whereas from electron-electron double resonance (ELDOR) – detected NMR (EDNMR) we determined the coordination to an adenosine nitrogen. The coordination to these ligands has been reported earlier, but whether the nitrogen and phosphate coordination is within the same nucleotide molecules or different ones is still under debate. By applying the correlation technique, THYCOS (triple hyperfine correlation spectroscopy), and measuring (15)N-(31)P correlations we establish that in Mn-ATP in solution both phosphates and a nitrogen are coordinated to the Mn(2+) ion. We also carried out DFT calculations to substantiate this finding. In addition, we expanded the understanding of the THYCOS experiment by comparing it to 2D-EDNMR for (55)Mn-(31)P correlation experiments and through simulations of THYCOS and 2D-EDNMR spectra with (15)N-(31)P correlations.

WOS:000442065200016; Mn2+ often serves as a paramagnetic substitute to Mg2+, providing means for exploring the close environment of Mg2+ in many biological systems where it serves as an essential co-factor. This applies to proteins with ATPase activity, where the ATP hydrolysis requires the binding of Mg2+-ATP to the ATPase active site. In this context, it is important to distinguish between the Mn2+ coordination mode with free ATP in solution as compared to the protein bound case. In this work, we explore the Mn2+ complexes with ATP, the non-hydrolysable ATP analog, AMPPNP, and ADP free in solution. Using W-band P-31 electron-nuclear double resonance (ENDOR) we obtained information about the coordination to the phosphates, whereas from electron-electron double resonance (ELDOR) - detected NMR (EDNMR) we determined the coordination to an adenosine nitrogen. The coordination to these ligands has been reported earlier, but whether the nitrogen and phosphate coordination is within the same nucleotide molecules or different ones is still under debate. By applying the correlation technique, THYCOS (triple hyperfine correlation spectroscopy), and measuring N-15-P-31 correlations we establish that in Mn-ATP in solution both phosphates and a nitrogen are coordinated to the Mn2+ ion. We also carried out DFT calculations to substantiate this finding. In addition, we expanded the understanding of the THYCOS experiment by comparing it to 2D-EDNMR for Mn-55-P-31 correlation experiments and through simulations of THYCOS and 2D-EDNMR spectra with N-15-P-31 correlations. (C) 2018 Elsevier Inc. All rights reserved.

International audience; The absorption spectrum of quinoline was measured in the gas phase between 3.5 and 10.7 eV using a synchrotron photon source. A large number of sharp and broad spectral features were observed, some of which have plasmon-type collective π-electron modes contributing to their intensities. Eight valence electronic transitions were assigned, considerably extending the number of π–π* transitions previously observed mainly in solution. The principal factor in solution red-shifts is found to be the Lorentz–Lorenz polarizability parameter. Rydberg bands, observed for the first time, are analyzed into eight different series, converging to the D0 ground and two excited electronic states, namely, D3 and D4, of the quinoline cation. The R1 series limit is 8.628 eV for the first ionization energy of quinoline, a value more precise than previously published. This value, combined with cation electronic transition data, provides precise energies, respectively, 10.623 and 11.355 eV, for the D3 and D4 states. The valence transition assignments are based on density functional theory (DFT) calculations as well as on earlier Pariser–Parr–Pople (P–P–P) self-consistent field linear combination of atomic orbitals molecular orbital results. The relative quality of the P–P–P and DFT data is discussed. Both are far from spectroscopic accuracy concerning electronic excited states but were nevertheless useful for our assignments. Our time-dependent DFT calculations of quinoline are excellent for its ground-state properties such as geometry, rotational constants, dipole moment, and vibrational frequencies, which agree well with experimental observations. Vibrational components of the valence and Rydberg transitions mainly involve C–H bend and C═C and C═N stretch modes. Astrophysical applications of the vacuum UV absorption of quinoline are briefly discussed.

International audience; Manganous phosphates have been postulated to play an important role in cells as antioxidants. In situ Mn(II) electron-nuclear double resonance (ENDOR) spectroscopy has been used to measure their speciation in cells. The analyses of such ENDOR spectra and the quantification of cellular Mn(II) phosphates has been based on comparisons to in vitro model complexes and heuristic modeling. In order to put such analyses on a more physical and theoretical footing, the Mn(II)-(31)P hyperfine interactions of various Mn(II) phosphate complexes have been measured by 95 GHz ENDOR spectroscopy. The dipolar components of these interactions remained relatively constant as a function of pH, esterification, and phosphate chain length, while the isotropic contributions were significantly affected. Counterintuitively, although the manganese-phosphate bonds are weakened by protonation and esterification, they lead to larger isotropic values, indicating higher unpaired-electron spin densities at the phosphorus nuclei. By comparison, extending the phosphate chain with additional phosphate groups lowers the spin density. Density functional theory calculations of model complexes quantitatively reproduced the measured hyperfine couplings and provided detailed insights into how bonding in Mn(II) phosphate complexes modulates the electron-spin polarization and consequently their isotropic hyperfine couplings. These results show that various classes of phosphates can be identified by their ENDOR spectra and provide a theoretical framework for understanding the in situ (31)P ENDOR spectra of cellular Mn(II) complexes.

International audience; The ability to characterize the structure of metal centers beyond their primary ligands is important to understanding their chemistry. High-magnetic-field pulsed electron double resonance detected NMR (ELDOR-NMR) is shown to be a very sensitive approach to measuring the multinuclear NMR spectra of the nuclei surrounding Mn(II) ions. Resolved spectra of intact organisms with resonances arising from (55)Mn, (31)P, (1)H, (39)K, (35)Cl, (23)Na, and (14)N nuclei surrounding Mn(2+) centers were obtained. Naturally abundant cellular (13)C could be routinely measured as well. The amplitudes of the (14)N and (2)H ELDOR-NMR spectra were found to be linearly dependent on the number of nuclei in the ligand sphere. The evolution of the Mn(II) ELDOR-NMR spectra as a function of excitation time was found to be best described by a saturation phenomenon rather than a coherently driven process. Mn(II) ELDOR-NMR revealed details about not only the immediate ligands to the Mn(II) ions but also more distant nuclei, providing a view of their extended structures. This will be important for understanding the speciation and chemistry of the manganese complexes as well as other metals found in organisms.

International audience; TrmFO is a tRNA methyltransferase that uses methylenetetrahydrofolate (CH2THF) and flavin adenine dinucleotide hydroquinone as cofactors. We have recently shown that TrmFO from Bacillus subtilis stabilizes a TrmFO-CH2-FADH adduct and an ill-defined neutral flavin radical. The adduct contains a unique N-CH2-S moiety, with a methylene group bridging N5 of the isoalloxazine ring and the sulfur of an active-site cysteine (Cys53). In the absence of tRNA substrate, this species is remarkably stable but becomes catalytically competent for tRNA methylation following tRNA addition using the methylene group as the source of methyl. Here, we demonstrate that this dormant methylating agent can be activated at low pH, and we propose that this process is triggered upon tRNA addition. The reaction proceeds via protonation of Cys53, cleavage of the C-S bond, and generation of a highly reactive [FADH(N5)═CH2]+ iminium intermediate, which is proposed to be the actual tRNA-methylating agent. This mechanism is fully supported by DFT calculations. The radical present in TrmFO is characterized here by optical and EPR/ENDOR spectroscopy approaches together with DFT calculations and is shown to be the one-electron oxidized product of the TrmFO-CH2-FADH adduct. It is also relatively stable, and its decomposition is facilitated by high pH. These results provide new insights into the structure and reactivity of the unique flavin-dependent methylating agent used by this class of enzymes.

A common feature of a large majority of the manganese metalloenzymes, as well as many synthetic biomimetic complexes, is the bonding between the manganese ion and imidazoles. This interaction was studied by examining the nature and structure of manganese(II) imidazole complexes in frozen aqueous solutions using 285 GHz high magnet-field continuous-wave electron paramagnetic resonance (cw-HFEPR) and 95 GHz pulsed electron-nuclear double resonance (ENDOR) and pulsed electron-double resonance detected nuclear magnetic resonance (PELDOR-NMR). The (55)Mn hyperfine coupling and isotropic g values of Mn(II) in frozen imidazole solutions continuously decreased with increasing imidazole concentration. ENDOR and PELDOR-NMR measurements demonstrated that the structural basis for this behavior arose from the imidazole concentration-dependent distribution of three six-coordinate and two four-coordinate species: [Mn(H2O)6](2+), [Mn(imidazole)(H2O)5](2+), [Mn(imidazole)2(H2O)4](2+), [Mn(imidazole)3(H2O)](2+), and [Mn(imidazole)4](2+). The hyperfine and g values of manganese proteins were also fully consistent with this imidazole effect. Density functional theory methods were used to calculate the structures, spin and charge densities, and hyperfine couplings of a number of different manganese imidazole complexes. The use of density functional theory with large exact-exchange admixture calculations gave isotropic (55)Mn hyperfine couplings that were semiquantitative and of predictive value. The results show that the covalency of the Mn-N bonds play an important role in determining not only magnetic spin parameters but also the structure of the metal binding site. The relationship between the isotropic (55)Mn hyperfine value and the number of imidazole ligands provides a quick and easy test for determining whether a protein binds an Mn(II) ion using histidine residues and, if so, how many are involved. Application of this method shows that as much as 40% of the Mn(II) ions in Deinococcus radiodurans are ligated to two histidines (Tabares, L. C.; Un, S. J. Biol. Chem 2013, in press).

High magnetic field high frequency electron paramagnetic resonance techniques were used to measure in situ Mn(II) speciation in Deinococcus radiodurans, a radiation-resistant bacteria capable of accumulating high concentrations of Mn(II). It was possible to identify and quantify the evolution of Mn(II) species in intact cells at various stages of growth. Aside from water, 95-GHz high field electron nuclear double resonance showed that the Mn(II) ions are bound to histidines and phosphate groups, mostly from fructose-1,6-bisphosphate but also inorganic phosphates and nucleotides. During stationary growth phase, 285-GHz continuous wave EPR measurements showed that histidine is the most common ligand to Mn(II) and that significant amounts of cellular Mn(II) in D. radiodurans are bound to peptides and proteins. As much as 40% of the total Mn(II) was in manganese superoxide dismutase, and it is this protein and not smaller manganese complexes, as has been suggested recently, that is probably the primary defense against superoxide. Background: Mn(II) plays a role in cellular defense against oxidative stress. Results: The Mn(II) speciation in D. radiodurans cells has been determined in situ. Conclusion: Manganese superoxide dismutase is the predominant Mn(II) species and is likely to be more important than small Mn(II) complexes for regulating superoxide. Significance: This work establishes a method for studying Mn(II) speciation and homeostasis in intact cells.

Double electron–electron resonance (DEER) can be used to study the structure of a protein in its native cellular environment. Until now, this has required isolation, in vitro labeling, and reintroduction of the protein back into the cells. We describe a completely biosynthetic approach that avoids these steps. It exploits genetically encodable lanthanide-binding tags (LBT) to form self-assembling GdIIImetal-based spin labels and enables direct in-cell measurements. This approach is demonstrated using a pair of LBTs encoded one at each end of a 3-helix bundle expressed in E. coli grown on GdIII-supplemented medium. DEER measurements directly on these cells produced readily detectable time traces from which the distance between the GdIIIlabels could be determined. This work is the first to use biosynthetically produced self-assembling metal-containing spin labels for non-disruptive in-cell structural measurements. Fil: Mascali, Florencia Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Ching, H. Y. Vincent. Université Paris-Saclay; Francia. Université Paris Sud; Francia. Centre National de la Recherche Scientifique; Francia Fil: Rasia, Rodolfo Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Un, Sun. Université Paris-Saclay; Francia. Université Paris Sud; Francia. Centre National de la Recherche Scientifique; Francia Fil: Tabares, Leandro Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Université Paris-Saclay; Francia. Université Paris Sud; Francia. Centre National de la Recherche Scientifique; Francia

A genetically encodable paramagnetic spin-label capable of self-assembly from naturally available components would offer a means for studying the in-cell structure and interactions of a protein by electron paramagnetic resonance (EPR). Here, we demonstrate pulse electron-electron double resonance (DEER) measurements on spin-labels consisting of Mn(II) ions coordinated to a sequence of histidines, so-called His-tags, that are ubiquitously added by genetic engineering to facilitate protein purification. Although the affinity of His-tags for Mn(II) was low (800 μM), Mn(II)-bound His-tags yielded readily detectable DEER time traces even at concentrations expected in cells. We were able to determine accurately the distance between two His-tag Mn(II) spin-labels at the ends of a rigid helical polyproline peptide of known structure, as well as at the ends of a completely cell-synthesized 3-helix bundle. This approach not only greatly simplifies the labeling procedure but also represents a first step towards using self-assembling metal spin-labels for in-cell distance measurements. Fil: Ching, H. Y. Vincent. Université Paris-Saclay; Francia Fil: Mascali, Florencia Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Bertrand, Hélène C.. Ecole Normale Supérieure; Francia. Centre National de la Recherche Scientifique; Francia. Universite de Paris VI; Francia Fil: Bruch, Eduardo Marcos. Université Paris-Saclay; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Demay Drouhard, Paul. Ecole Normale Supérieure; Francia. Centre National de la Recherche Scientifique; Francia. Universite de Paris VI; Francia Fil: Rasia, Rodolfo Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Policar, Clotilde. Ecole Normale Supérieure; Francia. Centre National de la Recherche Scientifique; Francia. Universite de Paris VI; Francia Fil: Tabares, Leandro Cesar. Université Paris-Saclay; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Un, Sun. Université Paris-Saclay; Francia

International audience; Pulsed EPR dipolar spectroscopy is a powerful tool for determining the structure and conformational dynamics of biological macromolecules, as it allows precise measurements of distances in the range of 1.5–10 nm. Utilization of high-spin Mn 2+ species as spin probes for distance measurements is of significant interest, because they are biologically compatible and endogenous in numerous biological systems. However, to date dipolar spectroscopy experiments with this kind of species have been underexplored. Here we present pulsed electron electron double resonance (PELDOR also called DEER) and relaxation-induced dipolar modulation enhancement (RIDME) experiments, which have been performed at W-band (94 GHz) and J-band frequencies (263 GHz) on a bis-MnDOTA (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) model system. The distances obtained from these experiments are in good agreement with predictions. RIDME experiments reveal a significantly higher modulation depth compared to PELDOR, which is an important consideration for biological samples. These experiments also feature higher harmonics of the dipolar coupling frequency due to effective multiple-quantum relaxation of high-spin Mn 2+ as well as the multiple-component background function. Harmonics of the dipolar coupling frequency were taken into account by including additional terms in the kernel function of Tikhonov regularization analysis.

International audience; The flavoprotein TrmFO catalyzes the C5 methylation of uridine 54 in the TΨC loop of tRNAs using 5,10-methylenetetrahydrofolate (CH(2)THF) as a methylene donor and FAD as a reducing agent. Here, we report biochemical and spectroscopic studies that unravel the remarkable capability of Bacillus subtilis TrmFO to stabilize, in the presence of oxygen, several flavin-reduced forms, including an FADH(*) radical, and a catalytic intermediate endowed with methylating activity. The FADH(*) radical was characterized by high-field electron paramagnetic resonance and electron nuclear double-resonance spectroscopies. Interestingly, the enzyme exhibited tRNA methylation activity in the absence of both an added carbon donor and an external reducing agent, indicating that a reaction intermediate, containing presumably CH(2)THF and FAD hydroquinone, is present in the freshly purified enzyme. Isolation by acid treatment, under anaerobic conditions, of noncovalently bound molecules, followed by mass spectrometry analysis, confirmed the presence in TrmFO of nonmodified FAD. Addition of formaldehyde to the purified enzyme protects the reduced flavins from decay by probably preventing degradation of CH(2)THF. The absence of air-stable reduced FAD species during anaerobic titration of oxidized TrmFO, performed in the absence or presence of added CH(2)THF, argues against their thermodynamic stabilization but rather implicates their kinetic trapping by the enzyme. Altogether, the unexpected isolation of a stable catalytic intermediate suggests that the flavin-binding pocket of TrmFO is a highly insulated environment, diverting the reduced FAD present in this intermediate from uncoupled reactions.

The effect of chloride concentration on Mn2+ (S = 5/2, I = 5/2) ions in frozen aqueous solutions is studied by high-field high-frequency electron paramagnetic resonance (HFEPR). The usually six sharp lines characteristic of Mn2+ ions, arising from the m s = −1/2 → 1/2 transition, is modified by the addition of Cl− anions and the six resonances become much broader and more complex. This new feature likely arises from the ligation of one Cl− anion to a hydrated Mn2+ ion forming a [Mn(H2O)5Cl]− complex. This complex increases linearly with Cl− concentration with an association constant of K a, apparent = 61 M−1. The structure of the putative chloride complex was studied using density functional theory calculations and the expected zero-field interaction of such a manganese center was calculated using the superposition model. The predicted values were similar to those determined from the simulation of the spectrum of the m s = −5/3 → −3/2 transition of the chloride complex. This effect of Cl− anions occurs at biologically relevant concentration and can be used to probe the Mn2+ ions in cellular and protein environments.

A high-field electron paramagnetic resonance (HFEPR) study of oxalate decarboxylase (OxdC) is reported. OxdC breaks down oxalate to carbon dioxide and formate and possesses two distinct manganese(II) binding sites, referred to as site-1 and -2. The Mn(II) zero-field interaction was used to probe the electronic state of the metal ion and to examine chemical/mechanistic roles of each of the Mn(II) centers. High magnetic-fields were exploited not only to resolve the two sites, but also to measure accurately the Mn(II) zero-field parameters of each of the sites. The spectra exhibited surprisingly complex behavior as a function of pH. Six different species were identified based on their zero-field interactions, two corresponding to site-1 and four states to site-2. The assignments were verified using a mutant that only affected site-1. The speciation data determined from the HFEPR spectra for site -2 was consistent with a simple triprotic equilibrium model, while the pH dependence of site-1 could be described by a single pK(a). This pH dependence was independent of the presence of the His-tag and of whether the preparations contained 1.2 or 1.6 Mn per subunit. Possible structures of the six species are proposed based on spectroscopic data from model complexes and existing protein crystallographic structures obtained at pH 8 are discussed. Although site-1 has been identified as the active site and no role has been assigned to site-2, the pronounced changes in the electronic structure of the latter and its pH behavior, which also matches the pH-dependent activity of this enzyme, suggests that even if the conversion of oxalate to formate is carried out at site-1, site-2 likely plays a catalytically relevant role.

The catalytic cycle of numerous enzymes involves the coupling between proton transfer and electron transfer. Yet, the understanding of this coordinated transfer in biological systems remains limited, likely because its characterization relies on the controlled but experimentally challenging modifications of the free energy changes associated with either the electron or proton transfer. We have performed such a study here in Photosystem II. The driving force for electron transfer from Tyr(Z) to P(680)(*+) has been decreased by approximately 80 meV by mutating the axial ligand of P(680), and that for proton transfer upon oxidation of Tyr(Z) by substituting a 3-fluorotyrosine (3F-Tyr(Z)) for Tyr(Z). In Mn-depleted Photosystem II, the dependence upon pH of the oxidation rates of Tyr(Z) and 3F-Tyr(Z) were found to be similar. However, in the pH range where the phenolic hydroxyl of Tyr(Z) is involved in a H-bond with a proton acceptor, the activation energy of the oxidation of 3F-Tyr(Z) is decreased by 110 meV, a value which correlates with the in vitro finding of a 90 meV stabilization energy to the phenolate form of 3F-Tyr when compared to Tyr (Seyedsayamdost et al. J. Am. Chem. Soc. 2006, 128,1569-1579). Thus, when the phenol of Y(Z) acts as a H-bond donor, its oxidation by P(680)(*+) is controlled by its prior deprotonation. This contrasts with the situation prevailing at lower pH, where the proton acceptor is protonated and therefore unavailable, in which the oxidation-induced proton transfer from the phenolic hydroxyl of Tyr(Z) has been proposed to occur concertedly with the electron transfer to P(680)(*+). This suggests a switch between a concerted proton/electron transfer at pHs7.5 to a sequential one at pHs7.5 and illustrates the roles of the H-bond and of the likely salt-bridge existing between the phenolate and the nearby proton acceptor in determining the coupling between proton and electron transfer.

Superoxide dismutases (SODs) catalyze the disproportionation of superoxide to dioxygen and hydrogen peroxide. The active metal sites of iron and manganese superoxide dismutases are structurally indistinguishable from each other. Despite the structural homology, these enzymes exhibit a high degree of metal selective activity suggesting subtle redox tuning of the active site. The redox tuning model, however, up to now has been challenged by the existence of so-called cambialistic SODs that function with either metal ion. We have prepared and investigated two sets of manganese complexes in which groups of varying electron-withdrawing character, as measured by their Hammett constants sigma Para, have been introduced into the ligands. We observed that the Mn(III)/Mn(II) reduction potential for the series based on 4'-X-terpyridine ligands together with the corresponding values for the iron-substituted 4'-X-terpyridine complexes changed linearly with sigma Para. The redox potential of the iron and manganese complexes could be varied by as much as 600 mV by the 4'-substitution with the manganese complexes being slightly more sensitive to the substitution than iron. The difference was such that in the case where the 4'-substituent was a pyrrolidine group both the manganese and the iron complex were thermodynamically competent to catalytically disproportionate superoxide, making this particular ligand "cambialistic". Taking our data and those available from the literature together, it was found that in addition to the electron-withdrawing capacity of the 4'-substituents the overall charge of the Mn(II) complexes plays a major role in tuning the redox potential, about 600 mV per charge unit. The ion selectivity in Mn and FeSODs and the occurrence of cambialistic SODs are discussed in view of these results. We conclude that the more distant electrostatic contributions may be the source of metal specific enzymatic activity.

The Mn4Ca cluster of photosystem II (PSII) goes through five sequential oxidation states (S0-S4) in the water oxidation process that also involves a tyrosine radical intermediate (TyrZ*). An S2TyrZ* state in which the Mn4Ca cluster and TyrZ* are magnetically coupled to each other and which is characterized by a distinct "split-signal" EPR spectrum can be generated in acetate-treated PSII. This state was examined by high-field EPR (HFEPR) in PSII from Thermosynechococcus elongatus isolated from a D2-Tyr160Phe mutant to avoid spectral contributions from TyrD*. In contrast to the same state in plants, both antiferromagnetic and ferromagnetic spin-spin couplings were observed. The intrinsic g values of TyrZ* in the coupled state were directly measured from the microwave frequency dependence of the HFEPR spectrum. The TyrZ* gx value in the antiferromagnetic centers was 2.0083, indicating that the coupled radical was in a less electropositive environment than in Mn-depleted PSII. Two gx values were found in the ferromagnetically coupled centers, 2.0069 and 2.0079. To put these values in perspective, the second redox-active tyrosine, TyrD*, was examined in various electrostatic environments. The TyrD* gx value changed from 2.0076 in the wild type to 2.0095 when the hydrogen bond from histidine 189 to TyrD* was removed using the D2-His189Leu mutant, indicating a change to a significantly less electropositive environment. BLY3P/6-31+G** density functional calculations on the hydrogen-bonded p-ethylphenoxy radical-imidazole supermolecular model complex showed that the entire range of Tyr* gx values, from 2.0065 to 2.0095, could be explained by the combined effects of hydrogen bonding and the dielectric constant of the local protein environment.

International audience; Pulse electron-electron double resonance (PELDOR) is a versatile technique for probing the structures and functions of complex biological systems. Despite the recent interest in high-spin metal-ions for high field/frequency applications, PELDOR measurements of Mn(II) remain relatively underexplored. Here we present Mn(II)-Mn(II) PELDOR distance measurements at 94 GHz on polyproline II (PPII) helices doubly spin-labeled with Mn(II) DOTA, which are distinguished by their small zero-field interaction. The measured Mn-Mn distances and distribution profiles were in good agreement with the expected values from molecular models. Additional features in the frequency-domain spectra became apparent at certain combinations of detect and pump frequencies. Spin-Hamiltonian calculations showed that they likely arose from contributions from the pseudo-secular component of the dipolar interaction that were found to be non-negligible for Mn(II) DOTA. However, the influence of the pseudo-secular component on the distance distribution profiles apparently was limited. The results show the potential of Mn(II) DOTA spin labels for high-field PELDOR distance measurements in proteins and other biological systems.

The manganese(II) speciation in intact cells of D. radiodurans, E. coli, S. cerevisiae and Arabidopsis thaliana seeds was measured using high-field electron paramagnetic resonance techniques. The majority of the Mn(II) ions in these organisms were six-coordinate, bound predominately by water, phosphates and nitrogen-based molecules. The relative distribution of the different phosphates in bacteria and S. cerevisiae was the same and dominated by monophosphate monoesters. Mn(II) was also found bound to the phosphate backbone of nucleic acids in these organisms. Phosphate ligation in Arabidopsis seeds was dominated by phytate. The extent of nitrogen ligation in the four organisms was also determined. On average, the Mn(II) in D. radiodurans had the most nitrogen ligands followed by E. coli. This was attributed to higher concentrations of Mn(II) bound to proteins in these species. Although constitutively expressed in all four organisms, MnSOD was only detected in D. radiodurans. As previously reported, D. radiodurans also accumulates a second abundant Mn containing protein species. The high concentration of proteinaceous Mn(II) is a unique feature of D. radiodurans.

Two different temperature dependences of the manganese(II) high-field electron paramagnetic resonance spectrum of manganese superoxide dismutase from E. coli were observed. In the 25-200 K range, the zero-field interaction steadily decreased with increasing temperature. This was likely due to the thermal expansion of the protein. From these results, it was possible to deduce an approximately r(-)(2.5) dependence of Mn(II) zero-field interaction on ligand-metal distance. At temperatures above 240 K, a distinct six-line component was detected, the amplitude of which decreased with increasing temperature. On the basis of similarities to the six-line spectrum observed for the azide-complexed E. coli manganese superoxide dismutase, the newly detected six-line spectrum was assigned to a hexacoordinate Mn(II) center resulting from the coordination of a nearby water molecule to the normally five-coordinate center. The changes in enthalpy and entropy characterizing the hexacoordinate-pentacoordinate equilibrium in the 240-268 K range were -5 kcal/mol and -24 cal/mol.K, respectively. The structural implications of the zero-field parameters of the newly found hexacoordinate form in comparison to those of the Mn(II) centers in concanavalin-A and manganese-containing R. spheroides photosynthetic reaction centers and the values predicted by the superposition model are discussed.

Electron paramagnetic resonance (EPR) spectroscopy has been extensively used to identify and characterize protein-based redox active amino acid radicals based on their g-values and hyperfine couplings. To better understand how these parameters depend on the electronic structure and environment of the radical, the theoretical g-values and proton hyperfine tensors of three models corresponding to the tyrosyl, tryptophanyl and glycyl radicals were calculated using Gaussian 03. The g-values were determined using the B3LYP/6-31+G(D,P) combination of density functional and basis set, while the hyperfine tensors were determined using the B3LYP/EPR-III and PBE0/EPR-III combinations. Comparisons are made to measured values. It was found that by appropriately accounting for hydrogen bonds and the dielectric constant of the environment, good agreement could be achieved between the calculated and measured g-values. In all three cases, the g-anisotropy arose from significant spin density on a nitrogen or oxygen atom. The calculated hyperfine tensors for the three radicals did not differ significantly from previous calculations. In the case of the tyrosyl radical, it is shown for the first time that the para-position substituent that is opposite of the C-O group can break the symmetry of the phenyl ring, leading to different hyperfine tensors for the two large ortho proton couplings. For the tyrosyl and tryptophanyl models, the calculated hyperfine couplings to hydrogen-bonding protons were in very good agreement with measured values for both the tyrosyl and tryptophanyl models.

Catalase-peroxidases are bifunctional heme enzymes with a high structural homology to peroxidases from prokaryotic origin and a catalatic activity comparable to monofunctional catalases. These unique features of catalase-peroxidases make them good systems to study and understand the role of alternative electron pathways both in catalases and peroxidases. In particular, it is of interest to study the poorly understood role of tyrosyl and tryptophanyl radicals as alternative cofactors in the catalytic cycle of catalases and peroxidases. In this work, we have used a powerful combination of multifrequency EPR spectroscopy, isotopic labeling of tryptophan and tyrosine residues, and site-directed mutagenesis to unequivocally identify the reactive intermediates formed by the wild-type Synechocystis PCC6803 catalase-peroxidase. Selected variants of the heme distal and proximal sides of the Synechocystis enzyme were investigated. Variants on the aromatic residues of the short stretch located relatively close to th...

The g-tensors and hyperfine tensors of the S = (1)/(2) ground state of the mixed valence [LMn(IIImu-O)(2)Mn(IV)L](3+) complex (L = N,N'-dimethyl-N,N'-bis(2-pyridylmethyl)ethane-1,2-diamine) was deter-mined in the solid-state and frozen acetonitrile solution by high-field EPR. Both samples exhibited complex anisotropic temperature behaviors that precluded the use of routine spectrum simulation procedures to extract the spin parameters. To circumvent this problem, the parameters were measured directly by using multifrequency techniques. In the case of the frozen solution, this approach yielded seven of the nine spin parameters with varying uncertainty, the two extreme principal g-values, the four hyperfine couplings associated with each of these two g-values, and the middle g-value. This latter parameter was obtained from a first moment analysis. Unlike simulations, the statistical errors associated with each value could be assigned in a straightforward and rigorous manner. The directly measured g-values were different in frozen solution and polycrystalline powder. The temperature dependence of the high-field EPR spectra of the polycrystalline powder revealed a spin-spin interaction between the neighboring binuclear complexes.

The coupling of proton chemistry with redox reactions is important in many enzymes and is central to energy transduction in biology. However, the mechanistic details are poorly understood. Here, we have studied tyrosine oxidation, a reaction in which the removal of one electron from the amino acid is linked to the release of its phenolic proton. Using the unique photochemical properties of photosystem II, it was possible to oxidize the tyrosine at 1.8 K, a temperature at which proton and protein motions are limited. The state formed was detected by high magnetic field EPR as a high-energy radical intermediate trapped in an unprecedentedly electropositive environment. Warming of the protein allows this state to convert to a relaxed, stable form of the radical. The relaxation event occurs at 77 K and seems to involve proton migration and only a very limited movement of the protein. These reactions represent a stabilization process that prevents the back-reaction and determines the reactivity of the radical.