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Calcium arsenates such as pharmacolite (CaHAsO

The insoluble organic material (IOM) in primitive chondritic meteorites is very enriched in D (up to δD ≈ 3500‰ in bulk). Based largely on a series of electron paramagnetic resonance (EPR) studies of IOM from three meteorites (Orgueil, Murchison and Tagish Lake), it has been suggested that these enrichments are the result of exchange with H2D+ in the solar nebula and that exchange with radicals in the IOM was particularly facile so that they are enormously enriched in D (δD ≥ 95000‰). To try to test whether radicals are largely responsible for the D enrichments in IOM, we have used EPR to measure the radical concentrations (spins/g) and g-factors of 18 IOM separates from C1-2 chondrites of varying petrologic type and chemical group that have a much wider range of H isotopic compositions (δD ≈ 600–3500‰) than in previous studies. We confirm the previous studies findings that IOM exhibits non-Curie law behavior and that it does not completely saturate even at microwave excitation powers of 200 mW. We also have obtained similar g-factor values. However, our IOM samples typically exhibit a lower and more limited range of spin concentrations, and smaller deviations from Curie law behavior than in previous studies. Nor do we observe correlations between bulk δD and either spins/g or non-Curie law behavior that would be expected if exchange between H2D+ and radicals, as previously proposed, was the cause of the D-enrichments in IOM. Indeed, in general the radical concentrations and the degree of non-Curie law behavior do not seem to correlate with any of the measured IOM properties, with chondrite group or parent body history (e.g., degree of aqueous alteration). The only exceptions are the IOM in four Tagish Lake lithologies whose spin concentrations increase with increasing degree of thermal processing as indicated by decreasing H/C and δD, and increasing aromaticity.

Mononitrosyl and dinitrosyl iron species, such as {FeNO}7, {FeNO}8 and {Fe(NO)2}9, have been proposed to play pivotal roles in the nitrosylation processes of nonheme iron centers in biological systems. Despite their importance, it has been difficult to capture and characterize them in the same scaffold of either native enzymes or their synthetic analogs due to the distinct structural requirements of the three species, using redox reagents compatible with biomolecules under physiological conditions. Here, we report the realization of stepwise nitrosylation of a mononuclear nonheme iron site in an engineered azurin under such conditions. Through tuning the number of nitric oxide equivalents and reaction time, controlled formation of {FeNO}7 and {Fe(NO)2}9 species was achieved, and the elusive {FeNO}8 species was inferred by EPR spectroscopy and observed by Mössbauer spectroscopy, with complemental evidence for the conversion of {FeNO}7 to {Fe(NO)2}9 species by UV-Vis, resonance Raman and FT-IR spectroscopies. The entire pathway of the nitrosylation process, Fe(ii) → {FeNO}7 → {FeNO}8 → {Fe(NO)2}9, has been elucidated within the same protein scaffold based on spectroscopic characterization and DFT calculations. These results not only enhance the understanding of the dinitrosyl iron complex formation process, but also shed light on the physiological roles of nitric oxide signaling mediated by nonheme iron proteins., Stepwise nitrosylation from Fe(ii) to {FeNO}7, {FeNO}8 and then to {Fe(NO)2}9 is reported for the first time in the same protein scaffold, providing deeper understanding of the detailed mechanism of dinitrosyl iron complex formation.

Type 1 copper (T1Cu) proteins play important roles in electron transfer in biology, largely due to the unique structure of the T1Cu center, which is reflected by its spectroscopic properties. Previous reports have suggested a correlation between a high ratio of electronic absorbance at ∼450 nm to that at ∼600 nm (

Gypsum is the most common sulfate mineral on Earth's surface and is the dominant solid byproduct in a wide variety of mining and industrial processes, thus representing a major source for heavy metal(loid) contamination, including selenium. Gypsum crystals grown from the gel diffusion technique in 0.02 M Na2SeO4 solution at pH 7.5 and 0.02 M Na2SeO3 solutions at pH 7.5 and 9.0 contain 828, 5198, and 5955 ppm Se, respectively. Synchrotron Se K-edge X-ray absorption spectroscopic analyses show that selenite and selenate are the dominant species in Se4+- and Se6+-doped gypsum, respectively. The single-crystal EPR spectra of Se4+- and Se6+-doped gypsum after gamma-ray irradiation reveal five selenium-centered oxyradicals: SeO2-(I), SeO2-(II), SeO2-(III), SeO3-, and HSeO42-. The former three radicals provide unequivocal evidence for the substitution of their paramagnetic precursor SeO32- for SO42- in the gypsum structure, while the latter two confirm the replacement of SeO42- for SO42-. These results demonstrate that gypsum has a significant capacity for sequestrating both selenite and selenate in the structure but has a marked preference for the former, thus confirming important controls on the mobility and bioavailability of selenium oxyanions and pointing to optimal applications of gypsum for remediating selenium contamination under neutral to alkaline conditions.

Type 1 copper proteins have a conserved ligand set of one cysteine and two histidines, with many proteins, such as azurin, also containing an axial methionine. While the cysteine and methionine in azurin have been replaced with their respective isostructural analogues of unnatural amino acids to reveal their roles in tuning electronic structures and functional properties, such as reduction potentials (E°'), the histidine ligands have not been probed in this way. We herein report the substitution of His117 in azurin with three unnatural isostructural analogues, 5-nitrohistidine(Ntr), thiazolylalanine(SHis) and 1-methylhistidine(MeH) by expressed protein ligation. While UV-vis absorption and electron paramagnetic resonance spectroscopies confirm that isostructural replacement results in minimal structural change in the Cu(II) state, the E°' of these variants increases with increasing pKa of the δ nitrogens of the imidazole. This counter-intuitive relationship between E°' of the protein and pKa of the sidechain group suggests additional factors may play a role in tuning E°'.

Rare earth elements (REE) in gypsum have long attracted interest for geochemical applications from the reconstruction of paleoclimate, paleoenvironment and paleohydrology to the monitoring of volcanic and seismic activities. Resurgent interests in REE in gypsum stem from the potential recovery of these high-tech metals from gypsum-rich tailings. Development of new geochemical applications and invention of effective recovery technologies all require knowledge about the mechanism of REE uptake in gypsum. In this contribution, we have investigated Gd-doped gypsum from the gel diffusion technique at ambient conditions in the pH range from 6.5 to 9.5. Inductively coupled plasma mass spectrometry analyses of Gd-doped gypsum yielded 752 ppm Gd at pH = 6.5 down to 2.8 ppm Gd at pH = 9.5. Synchrotron Gd L3-edge X-ray absorption spectra show that Gd3+ has a local environment similar to that of Ca2+ in gypsum. Single-crystal electron paramagnetic resonance (EPR, also known as electron spin resonance or ESR) spectra reveal a dominant Gd3+ center with weak satellites. The dominant Gd3+ center arises from a substitutional Gd3+ ion at the Ca site retaining its C2 local symmetry, which is also confirmed by pulsed electron nuclear double resonance (ENDOR) data for proton hyperfine structures up to 5.2 A away from Gd3+. The satellites are attributable to a Gd3+-Gd3+ pair (dimer) along the crystallographic a-axis with a dipolar coupling constant Dd/geβe = −13 G, which corresponds to a distance of 12.6 A between the two Gd3+ ions and provides the first experimental confirmation for the substitution 2Gd3+ + □ = 3Ca2+ in gypsum. Hole trapping at cation vacancies associated with the incorporation of Gd3+ (and other trivalent REE) in gypsum offers a new class of radiation-induced paramagnetic defects in this mineral for EPR/ESR dating. These results also have implications for developing new technologies for effective recovery of REE from gypsum-rich tailings and for understanding the behavior of REE in evaporites and aqueous solutions.

Gypsum is the most common sulfate mineral on Earth's surface and is the dominant solid byproduct in a wide variety of mining and industrial processes, thus representing a major source for heavy metal(loid) contamination, including selenium. Gypsum crystals grown from the gel diffusion technique in 0.02 M Na

Metals brought together do more Enzymatic reduction of oxyanions such as sulfite (SO 3 2− ) requires the delivery of multiple electrons and protons, a feat accomplished by cofactors tailored for catalysis and electron transport. Replicating this strategy in protein scaffolds may expand the range of enzymes that can be designed de novo. Mirts et al. selected a scaffold protein containing a natural heme cofactor and then engineered a cavity suitable for binding a second cofactor—an iron-sulfur cluster (see the Perspective by Lancaster). The resulting designed enzyme was optimized through rational mutation into a catalyst with spectral characteristics and activity similar to that of natural sulfite reductases. Science , this issue p. 1098 ; see also p. 1071

Photoacoustic (PA) tomography is a noninvasive technology that utilizes near-infrared (NIR) excitation and ultrasonic detection to image biological tissue at centimeter depths. While several activatable small-molecule PA sensors have been developed for various analytes, the use of PA molecules for deep-tissue analyte delivery and monitoring remains an underexplored area of research. Herein, we describe the synthesis, characterization, and in vivo validation of photoNOD-1 and photoNOD-2, the first organic, NIR-photocontrolled nitric oxide (NO) donors that incorporate a PA readout of analyte release. These molecules consist of an aza-BODIPY dye appended with an aryl N-nitrosamine NO-donating moiety. The photoNODs exhibit chemostability to various biological stimuli, including redox-active metals and CYP450 enzymes, and demonstrate negligible cytotoxicity in the absence of irradiation. Upon single-photon NIR irradiation, photoNOD-1 and photoNOD-2 release NO as well as rNOD-1 or rNOD-2, PA-active products that enable ratiometric monitoring of NO release. Our in vitro studies show that, upon irradiation, photoNOD-1 and photoNOD-2 exhibit 46.6-fold and 21.5-fold ratiometric turn-ons, respectively. Moreover, unlike existing NIR NO donors, the photoNODs do not require encapsulation or multiphoton activation for use in live animals. In this study, we use PA tomography to monitor the local, irradiation-dependent release of NO from photoNOD-1 and photoNOD-2 in mice after subcutaneous treatment. In addition, we use a murine model for breast cancer to show that photoNOD-1 can selectively affect tumor growth rates in the presence of NIR light stimulation following systemic administration.

The intermediacy of a reduced nickel-iron hydride in hydrogen evolution catalyzed by Ni-Fe complexes was verified experimentally and computationally. In addition to catalyzing hydrogen evolution, the highly basic and bulky (dppv)Ni(μ-pdt)Fe(CO)(dppv) ([1](0); dppv = cis-C2H2(PPh2)2) and its hydride derivatives have yielded to detailed characterization in terms of spectroscopy, bonding, and reactivity. The protonation of [1](0) initially produces unsym-[H1](+), which converts by a first-order pathway to sym-[H1](+). These species have C1 (unsym) and Cs (sym) symmetries, respectively, depending on the stereochemistry of the octahedral Fe site. Both experimental and computational studies show that [H1](+) protonates at sulfur. The S = 1/2 hydride [H1](0) was generated by reduction of [H1](+) with Cp*2Co. Density functional theory (DFT) calculations indicate that [H1](0) is best described as a Ni(I)-Fe(II) derivative with significant spin density on Ni and some delocalization on S and Fe. EPR spectroscopy reveals both kinetic and thermodynamic isomers of [H1](0). Whereas [H1](+) does not evolve H2 upon protonation, treatment of [H1](0) with acids gives H2. The redox state of the "remote" metal (Ni) modulates the hydridic character of the Fe(II)-H center. As supported by DFT calculations, H2 evolution proceeds either directly from [H1](0) and external acid or from protonation of the Fe-H bond in [H1](0) to give a labile dihydrogen complex. Stoichiometric tests indicate that protonation-induced hydrogen evolution from [H1](0) initially produces [1](+), which is reduced by [H1](0). Our results reconcile the required reductive activation of a metal hydride and the resistance of metal hydrides toward reduction. This dichotomy is resolved by reduction of the remote (non-hydride) metal of the bimetallic unit.

S-Nitrosylation has emerged as an important pathway for dynamic post-translational regulation of many classes of proteins. Now, the reversible insertion of NO into a copper–thiolate bond has been observed under physiologically relevant conditions using an engineered azurin. DFT calculation indicates that the reaction proceeds via a radical combination mechanism.

Two new paramagnetic defects ( and ) have been revealed in neutron-irradiated natural -quartz by using electron paramagnetic resonance (EPR) spectroscopy. The EPR spectra of the center as well as a previously reported but incompletely characterized center demonstrate that their super-hyperfine structures arise from interaction with 27 Al, the first-ever examples of Al-associated centers in crystalline quartz. The matrices g and , of , g and of the center, have been determined.

The reduction potential (E°′) is a critical parameter in determining the efficiency of most biological and chemical reactions. Biology employs three classes of metalloproteins to cover the majority of the 2-V range of physiological E°′s. An ultimate test of our understanding of E°′ is to find out the minimal number of proteins and their variants that can cover this entire range and the structural features responsible for the extreme E°′. We report herein the design of the protein azurin to cover a range from +970 mV to −954 mV vs. standard hydrogen electrode (SHE) by mutating only five residues and using two metal ions. Spectroscopic methods have revealed geometric parameters important for the high E°′. The knowledge gained and the resulting water-soluble redox agents with predictable E°′s, in the same scaffold with the same surface properties, will find wide applications in chemical, biochemical, biophysical, and biotechnological fields.

As an early visitor to the injured loci, neutrophil-derived human Myeloperoxidase (hMPO) offers an attractive protein target to modulate the inflammation of the host tissue through suitable inhibitors. We describe a novel methodology of using low temperature ESR spectroscopy (6 K) and FAST™ technology to screen a diverse series of small molecules that inhibit the peroxidase function through reversible binding to the native state of MPO. Our initial efforts to profile molecules on the inhibition of MPO-initiated nitration of the Apo-A1 peptide (AEYHAKATEHL) assay showed several potent (with sub-micro molar IC50s) but spurious inhibitors that either do not bind to the heme pocket in the enzyme or retain high (>50 %) anti oxidant potential. Such molecules when taken forward for X-ray did not yield inhibitor-bound co-crystals. We then used ESR to confirm direct binding to the native state enzyme, by measuring the binding-induced shift in the electronic parameter g to rank order the molecules. Molecules with a higher rank order—those with g-shift R relative ≥15—yielded well-formed protein-bound crystals (n = 33 structures). The co-crystal structure with the LSN217331 inhibitor reveals that the chlorophenyl group projects away from the heme along the edges of the Phe366 and Phe407 side chain phenyl rings thereby sterically restricting the access to the heme by the substrates like H2O2. Both ESR and antioxidant screens were used to derive the mechanism of action (reversibility, competitive substrate inhibition, and percent antioxidant potential). In conclusion, our results point to a viable path forward to target the native state of MPO to tame local inflammation.

While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pKa and reduction potential in O2 reduction have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O2 reduction mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pKa of the phenol ring and increasing reduction potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O2 reduction activity and the fraction of H2O formation with the pKa of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H2O2, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivatives incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymatic activity beyond that of natural Tyr.

Complexes containing manganese-oxygen bonds have been implicated in a variety of biological and synthetic processes. Herein, we describe the synthesis of a family of stable, high-spin trigonal bipyramidal manganese complexes of the electronically flexible ligand tris(5-cyclohexylimino-pyrrol-2-ylmethyl)amine [H3N(pi(Cy))3] featuring apical water, hydroxyl, and oxo ligands. Terminal Mn(III)-O complexes are rare and the formation of this species was achieved from a variety of reagents including O2, PhIO and NO2(-). Described herein is the preparation, structural and electronic properties of these manganese complexes.

Gem-quality danburite containing 269 ppm As, from Charcas, San Luis Potosi, Mexico, has been investigated by synchrotron X-ray absorption spectroscopy (XAS) and single-crystal electron paramagnetic resonance (EPR) spectroscopy. Arsenic K -edge X-ray absorption near-edge-structure (XANES) spectra show that the dominant oxidation state is +3, and modeling of the extended X-ray absorption fine structure (EXAFS) spectra suggests that As 3+ mainly occupies the Si site. Single-crystal continuous-wave EPR spectra, measured before and after gamma-ray irradiation, reveal three arsenic-associated electron centers (I, II and III). Centers I and II have similar principal electron Zeeman g values and principal 75 As hyperfine constants [I: A 1 /h = 732.8(2) MHz, A 2 /h = −274.3(3) MHz and A 3 /h = −299.8(3) MHz; II: A 1 /h = 743.8(2) MHz, A 2 /h = −306.3(2) MHz and A 3 /h = −325.8(2) MHz]. These parameters suggest that Centers I and II are varieties of the [AsO 2 ] 2− radical, formed from electron trapping on substitutional As 3+ ions at the Si site. This model for the [AsO 2 ] 2− radical is further supported by 11 B (and 10 B) superhyperfine parameters determined from pulsed electron spin echo envelope modulation (ESEEM) spectroscopy. Center III is the [AsO 3 ] 2− radical on the basis of its characteristic 75 As hyperfine constants [A 1 /h = 2406.0(1) MHz, A 2 /h = 1903.4(1) MHz and A 3 /h = 1892.1(1) MHz]. The [AsO 3 ] 2− radical with its A 1 axis along the Si – O4 bond direction originated from electron trapping on a [AsO 4 ] 3− group after removal of the O4 atom during gamma-ray irradiation. Therefore, arsenic in the borosilicate danburite is present in both the +3 and +5 oxidation states and preferentially occupies the Si site. This study also highlights the advantages (and need) of multiple spectroscopic techniques for determining speciation of trace elements such as arsenic in minerals.

A major barrier to understanding the mechanism of nitric oxide reductases (NORs) is the lack of selective probe of NO binding to the non-heme FeB center. By replacing the heme in a biosynthetic model of NORs (L29H/F43H/V68E Mb), that structurally and functionally mimics NORs, with isostructural ZnPP, we report herein a study where the electronic structure and functional properties of the FeB-nitrosyl complex has been probed selectively. This approach allowed us to observe the first S=3/2 non-heme {FeNO}7 complex in a protein system. Such feats are not achievable in native NORs as these are complex membrane proteins containing multiple hemes. Detailed spectroscopic and computational studies show that the electronic state of the {FeNO}7 complex is best described as a HS ferrous iron (S=2) antiferromagnetically coupled to NO radical (S=1/2) [Fe2+-NO•]. The radical nature of the FeB-bound NO would facilitate N-N bond formation by radical coupling with the heme-bound NO. This finding, therefore, supports the proposed trans mechanism of NO reduction by NORs.

Tyrosine is a conserved redox-active amino acid that plays important roles in heme-copper oxidases (HCO). Despite the widely proposed mechanism that involves a tyrosyl radical, its direct observation under O2 reduction conditions remains elusive. Using a functional oxidase model in myoglobin called F33Y-Cu(B)Mb that contains an engineered tyrosine, we report herein direct observation of a tyrosyl radical during both reactions of H2O2 with oxidized protein and O2 with reduced protein by electron paramagnetic resonance spectroscopy, providing a firm support for the tyrosyl radical in the HCO enzymatic mechanism.

Metal-sulfenate centers are known to play important roles in biology and yet only limited examples are known due to their instability and high reactivity. Herein we report a copper-sulfenate complex characterized in a protein environment, formed at the active site of a cavity mutant of an electron transfer protein, type 1 blue copper azurin. Reaction of hydrogen peroxide with Cu(I)-M121G azurin resulted in a species with strong visible absorptions at 350 and 452 nm and a relatively low electron paramagnetic resonance gz value of 2.169 in comparison with other normal type 2 copper centers. The presence of a side-on copper-sulfenate species is supported by resonance Raman spectroscopy, electrospray mass spectrometry using isotopically enriched hydrogen peroxide, and density functional theory calculations correlated to the experimental data. In contrast, the reaction with Cu(II)-M121G or Zn(II)-M121G azurin under the same conditions did not result in Cys oxidation or copper-sulfenate formation. Structural and computational studies strongly suggest that the secondary coordination sphere noncovalent interactions are critical in stabilizing this highly reactive species, which can further react with oxygen to form a sulfinate and then a sulfonate species, as demonstrated by mass spectrometry. Engineering the electron transfer protein azurin into an active copper enzyme that forms a copper-sulfenate center and demonstrating the importance of noncovalent secondary sphere interactions in stabilizing it constitute important contributions toward the understanding of metal-sulfenate species in biological systems.

Electron paramagnetic resonance (EPR) spectroscopy, also known as electron spin resonance (ESR) spectroscopy, is a group of techniques used to study paramagnetic species that contain one or more unpaired electrons. The basic principles of EPR are analogous to those of nuclear magnetic resonance (NMR) spectroscopy, because they both deal with interactions between electromagnetic radiation and magnetic moments. However, the former is based on the excitation of electron spins, whereas nuclear spins are excited in the latter. EPR as a structural probe provides a wealth of information about the local structures and dynamic processes of the paramagnetic species studied, and is known for its unique sensitivity (~1012 spins/cm3 or parts per billion; Pan et al. 2002a; Weil and Bolton 2007), unmatched by any other structural techniques. In addition, quantitative EPR, provided that sufficient calibration and standardization are carried out, is possible and is useful for chemical analysis, dosimetry and geochronology, with applications to not only rocks and minerals but also other Earth and planetary materials such as coals, crude oils and meteorites (Ikeya 1993; Dyrek et al. 1996, 2003; Eaton et al. 2009). The basic principles of EPR spectroscopy can be found in various textbooks and monographs (Abragam and Bleaney 1970; Poole and Farah 1999; Schweiger and Jeschke 2001; Weil and Bolton 2007; Brustolon and Giamello 2009; Eaton et al. 2009; Misra 2011). Excellent reviews with emphasis on applications of EPR spectroscopy to minerals can be found in Marfunin (1979), Calas (1988), Vassilikou-Dova (1993), and Goodman and Hall (1994). However, most previous reviews on applications to minerals focused almost exclusively on conventional continuous-wave (CW) techniques, whereas more advanced techniques such as pulse electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) …

The synthesis of a novel dipodal ligand framework, H2[(Me)N(pi(Cy))2], is summarized. Upon metalation with MCl2 salts (M = Fe, Cu), the ligand undergoes a conformational change, resulting in the formation of a trigonal bipyramidal metal center with a pseudoplanar, meridionally-bound ligand framework. This tautomerization positions pendant amines in the metal's secondary coordination sphere. Metalation with M(OTf)2 in coordinating solvent yields octahedral metal complexes, where two solvent molecules bind in the apical positions with one outer sphere counter ion. Reactivity of these complexes, ((Me)N(afa(Cy))2)M(X)2 (X = Cl, OTf), with 2,2'-bypyridine results in ligand reorganization, yielding a facial coordination geometry of the dipodal framework. The described complexes have been characterized by (1)H NMR, EPR, IR, Mössbauer and electronic absorption spectroscopies as well as X-ray crystallography.

Gypsum (CaSO 4 ·2H 2 O) is a major by-product of mining and milling processes of borate, phosphate and uranium deposits worldwide and, therefore, potentially plays an important role in the stability and bioavailability of heavy metalloids, including As, in tailings and surrounding areas. Gypsum containing 1900 and 185 ppm As, synthesized with Na 2 HAsO 4 ·7H 2 O and NaAsO 2 in the starting materials, respectively, have been investigated by synchrotron X-ray absorption spectroscopy (XAS), single-crystal electron paramagnetic resonance spectroscopy (EPR), and pulsed electron nuclear double resonance spectroscopy (ENDOR). Quantitative analyses of As K edge XANES and EXAFS spectra show that arsenic occurs in both +3 and +5 oxidation states and the As 3+ /As 5+ value varies from 0.35 to 0.79. Single-crystal EPR spectra of gamma-ray-irradiated gypsum reveal two types of arsenic-associated oxyradicals: [AsO 3 ] 2− and an [AsO 2 ] 2− . The [AsO 3 ] 2− center is characterized by principal 75 As hyperfine coupling constants of A 1 = 1952.0(2) MHz, A 2 = 1492.6(2) MHz and A 3 = 1488.7(2) MHz, with the unique A axis along the S–O1 bond direction, and contains complex 1 H superhyperfine structures that have been determined by pulsed ENDOR. These results suggest that the [AsO 3 ] 2− center formed from electron trapping on the central As 5+ ion of a substitutional (AsO 4 ) 3− group after removal of an O1 atom. The [AsO 2 ] 2− center is characterized by its unique A ( 75 As) axis approximately perpendicular to the O1–S–O2 plane and the A 2 axis along the S–O2 bond direction, consistent with electron trapping on the central As 3+ ion of a substitutional (AsO 3 ) 3− group after removal of an O2 atom. These results confirm lattice-bound As 5+ and As 3+ in gypsum and point to potential application of this mineral for immobilization and removal of arsenic pollution.

The mixed-valence diiron hydrido complex (μ-H)Fe2(pdt)(CO)2(dppv)2 ([H1](0), where pdt =1,3-propanedithiolate and dppv = cis-1,2-C2H2(PPh2)2), was generated by reduction of the differous hydride [H1](+) using decamethylcobaltocene. Crystallographic analysis shows that [H1](0) retains the stereochemistry of its precursor, where one dppv ligand spans two basal sites and the other spans apical and basal positions. The Fe---Fe bond elongates to 2.80 from 2.66 Å, but the Fe-P bonds only change subtly. Although the Fe-H distances are indistinguishable in the precursor, they differ by 0.2 Å in [H1](0). The X-band electron paramagnetic resonance (EPR) spectrum reveals the presence of two stereoisomers, the one characterized crystallographically and a contribution of about 10% from a second symmetrical (sym) isomer wherein both dppv ligands occupy apical-basal sites. The unsymmetrical (unsym) arrangement of the dppv ligands is reflected in the values of A((31)P), which range from 31 MHz for the basal phosphines to 284 MHz for the apical phosphine. Density functional theory calculations were employed to rationalize the electronic structure of [H1](0) and to facilitate spectral simulation and assignment of EPR parameters including (1)H and (31)P hyperfine couplings. The EPR spectra of [H1](0) and [D1](0) demonstrate that the singly occupied molecular orbital is primarily localized on the Fe center with the longer bond to H, that is, Fe(II)-H···Fe(I). The coupling to the hydride is A((1)H) = 55 and 74 MHz for unsym- amd sym-[H1](0), respectively. Treatment of [H1](0) with H(+) gives 0.5 equiv of H2 and [H1](+). Reduction of D(+) affords D2, leaving the hydride ligand intact. These experiments demonstrate that the bridging hydride ligand in this complex is a spectator in the hydrogen evolution reaction.

Mononuclear cupredoxin proteins usually contain a coordinately saturated type 1 copper (T1Cu) center and function exclusively as electron carriers. Here we report a cupredoxin isolated from the nitrifying archaeon Nitrosopumilus maritimus SCM1, called Nmar1307, that contains a T1Cu center with an open binding site containing water. It displays a deep purple color due to strong absorptions around 413 nm (1880 M(-1) cm(-1)) and 558 nm (2290 M(-1) cm(-1)) in the UV-vis electronic spectrum. EPR studies suggest the protein contains two Cu(II) species of nearly equal population, one nearly axial, with hyperfine constant A∥ = 98 × 10(-4) cm(-1), and another more rhombic, with a smaller A∥ value of 69 × 10(-4) cm(-1). The X-ray crystal structure at 1.6 Å resolution confirms that it contains a Cu atom coordinated by two His and one Cys in a trigonal plane, with an axial H2O at 2.25 Å. Both UV-vis absorption and EPR spectroscopic studies suggest that the Nmar1307 can oxidize NO to nitrite, an activity that is attributable to the high reduction potential (354 mV vs SHE) of the copper site. These results suggest that mononuclear cupredoxins can have a wide range of structural features, including an open binding site containing water, making this class of proteins even more versatile.

The synthesis and characterization of a series of cobalt complexes featuring a pincer bis(carbene) ligand of the meta-phenylene-bridged bis-N-heterocyclic carbene ((Ar)CCC, Ar = 2,6-diispropylphenyl or mesityl) are reported. Cleavage of the aryl C-H bond of the ligand was achieved in a one-pot metalation procedure using Co(N(SiMe3)2)2(py)2, an equivalent of exogenous base, and trityl chloride to form the ((DIPP)CCC)CoCl2py complex. This species could be reduced to the Co(ii) and Co(i)-N2 molecules with the appropriate equivalents of reductant. Subsequent generation of ((Mes)CCC)Co(I-III) derivatives with the mesityl ligand proceeded in good yields. A suite of characterization techniques and the interconversion between all three oxidation states of the cobalt complexes is described.

Copper transfer to cuproproteins located in vesicular compartments of the secretory pathway depends on activity of the copper translocating ATPase (ATP7A or ATP7B) but the mechanism of transfer is largely unexplored. Copper-ATPase ATP7A is unique in having a sequence rich in histidine and methionine residues located on the lumenal side of the membrane. The corresponding fragment binds Cu(I) when expressed as a chimera with a scaffold protein, and mutations or deletions of His and/or Met residues in its sequence inhibit dephosphorylation of the ATPase, a catalytic step associated with copper release. Here we present evidence for a potential role of this lumenal region of ATP7A in copper transfer to cuproenzymes. Both Cu(II) and Cu(I) forms were investigated since the form in which copper is transferred to acceptor proteins is currently unknown. Analysis of Cu(II) using EPR demonstrated that at Cu:P ratios below 1:1, 15N-substituted protein had Cu(II) bound by 4 His residues, but this coordination changed as the Cu(II) to protein ratio increased towards 2:1. XAS confirmed this coordination via analysis of the intensity of outer-shell scattering from imidazole residues. The Cu(II) complexes could be reduced to their Cu(I) counterparts by ascorbate, but here again, as shown by EXAFS and XANES spectroscopy, the coordination was dependent on copper loading. At low copper Cu(I) was bound by a mixed ligand set of His + Met while at higher ratios His coordination predominated. The copper-loaded loop was able to transfer either Cu(II) or Cu(I) to peptidylglycine monooxygenase in the presence of chelating resin, generating catalytically active enzyme in a process that appeared to involve direct interaction between the two partners. The variation of coordination with copper loading suggests copper-dependent conformational change which in turn could act as a signal for regulating copper release by the ATPase pump.

Single crystals of the p-type semiconductor CuAlO2, synthesized from the CuO flux method, have been investigated by electron paramagnetic resonance (EPR) spectroscopy at temperatures from 3.5 to 294 K. EPR spectra reveal an axial Cr3+ center and an axial Fe3+ center, presumably arising from impurities in the starting materials. The Cr3+ center at 294 K is characterized by g = 1.979(1) and D = − 7803(3) × 10−4 cm−1. The Fe3+ center at 294 K has g// = 1.9942(1), g⟂ = 1.9964(1), D = 261.9(3) × 10−4 cm−1, , and . These spin-Hamiltonian parameters, evaluated by superposition model analyses and periodic density functional theory (DFT) calculations, suggest that both Cr3+ and Fe3+ occupy the Al3+ site but the latter involves distortion related to perturbation from a substitutional Cu+ ion at the nearest Al site. This difference in the local structure between Fe3+ and Cr3+ explains their contrasting effects on the p-type conductivity in CuAlO2.

A series of mixed-valence nickel-iron dithiolates is described. Oxidation of (diphosphine)Ni(dithiolate)Fe(CO)(3) complexes 1, 2, and 3 with ferrocenium salts affords the corresponding tricarbonyl cations [(dppe)Ni(pdt)Fe(CO)(3)](+) ([1](+)), [(dppe)Ni(edt)Fe(CO)(3)](+) ([2](+)) and [(dcpe)Ni(pdt)Fe(CO)(3)](+) ([3](+)), respectively, where dppe = Ph(2)PCH(2)CH(2)PPh(2), dcpe = Cy(2)PCH(2)CH(2)PCy(2), (Cy = cyclohexyl), pdtH(2) = HSCH(2)CH(2)CH(2)SH, and edtH(2) = HSCH(2)CH(2)SH. The cation [2](+) proved unstable, but the propanedithiolates are robust. IR and EPR spectroscopic measurements indicate that these species exist as C(s)-symmetric species. Crystallographic characterization of [3]BF(4) shows that Ni is square planar. Interaction of [1]BF(4) with P-donor ligands (L) afforded a series of substituted derivatives of type [(dppe)Ni(pdt)Fe(CO)(2)L]BF(4) for L = P(OPh)(3) ([4a]BF(4)), P(p-C(6)H(4)Cl)(3) ([4b]BF(4)), PPh(2)(2-py) ([4c]BF(4)), PPh(2)(OEt) ([4d]BF(4)), PPh(3) ([4e]BF(4)), PPh(2)(o-C(6)H(4)OMe) ([4f]BF(4)), PPh(2)(o-C(6)H(4)OCH(2)OMe) ([4g]BF(4)), P(p-tol)(3) ([4h]BF(4)), P(p-C(6)H(4)OMe)(3) ([4i]BF(4)), and PMePh(2) ([4j]BF(4)). EPR analysis indicates that ethanedithiolate [2](+) exists as a single species at 110 K, whereas the propanedithiolate cations exist as a mixture of two conformers, which are proposed to be related through a flip of the chelate ring. Mössbauer spectra of 1 and oxidized S = 1/2 [4e]BF(4) are both consistent with a low-spin Fe(I) state. The hyperfine coupling tensor of [4e]BF(4) has a small isotropic component and significant anisotropy. DFT calculations using the BP86, B3LYP, and PBE0 exchange-correlation functionals agree with the structural and spectroscopic data, suggesting that the SOMOs in complexes of the present type are localized in an Fe(I)-centered d(z(2)) orbital. The DFT calculations allow an assignment of oxidation states of the metals and rationalization of the conformers detected by EPR spectroscopy. Treatment of [1](+) with CN(-) and compact basic phosphines results in complex reactions. With dppe, [1](+) undergoes quasi-disproportionation to give 1 and the diamagnetic complex [(dppe)Ni(pdt)Fe(CO)(2)(dppe)](2+) ([5](2+)), which features square-planar Ni linked to an octahedral Fe center.

Interactions of the axial ligand with its blue copper center are known to be important in tuning spectroscopic and redox properties of cupredoxins. While conversion of the blue copper center with a weak axial ligand to a green copper center containing a medium strength axial ligand has been demonstrated in cupredoxins, converting the blue copper center to a red copper center with a strong axial ligand has not been reported. Here we show that replacing Met121 in azurin from Pseudomonas aeruginosa with Cys caused an increased ratio (R(L)) of absorption at 447 nm over that at 621 nm. Whereas no axial Cu-S(Cys121) interaction in Met121Cys was detectable by extended X-ray absorption fine structure (EXAFS) spectroscopy at pH 5, similar to what was observed in native azurin with Met121 as the axial ligand, the Cu-S(Cys121) interaction at 2.74 A is clearly visible at higher pH. Despite the higher R(L) and stronger axial Cys121 interaction with Cu(II) ion, the Met121Cys variant remains largely a type 1 copper protein at low pH (with hyperfine coupling constant A( parallel) = 54 x 10(-4) cm(-1) at pH 4 and 5), or distorted type 1 or green copper protein at high pH (A(parallel) = 87 x 10(-4) cm(-1) at pH 8 and 9), attributable to the relatively long distance between the axial ligand and copper and the constraint placed by the protein scaffold. To shorten the distance between axial ligand and copper, we replaced Met121 with a nonproteinogenic amino acid homocysteine that contains an extra methylene group, resulting in a variant whose spectra (R(L)= 1.5, and A(parallel) = 180 x 10(-4) cm(-1)) and Cu-S(Cys) distance (2.22 A) are very similar to those of the red copper protein nitrosocyanin. Replacing Met121 with Cys or homocysteine resulted in lowering of the reduction potential from 222 mV in the native azurin to 95 +/- 3 mV for Met121Cys azurin and 113 +/- 6 mV for Met121Hcy azurin at pH 7. The results strongly support the "coupled distortion" model that helps explain axial ligand tuning of spectroscopic properties in cupredoxins, and demonstrate the power of using unnatural amino acids to address critical chemical biological questions.

Single-crystal electron paramagnetic resonance (EPR) spectra of gamma-ray-irradiated prehnite (Jeffrey mine, Quebec, Canada) measured at 298 and 160 K reveal an aluminum-associated oxygen hole center (Al—O − ). Spin Hamiltonian parameters g and A ( 27 Al) fitted from the 298 K spectra suggest that this Al—O − center represents hole trapping on an apical hydroxyl oxygen atom (after removal of the proton) coordinated to an octahedral Al 3+ ion ( i.e. , an [ • OAlO 4 (OH)] center from the [(OH)AlO 4 (OH)] precursor, where • denotes the unpaired spin). Pulsed electron nuclear double resonance (ENDOR) spectra measured at 25 K allow the identification and quantitative analysis of two sets of 27 Al hyperfine structures and five proton hyperfine structures, which are all consistent with the proposed structural model. Isothermal and isochronal annealing experiments show that this center is bleached out completely at 375 °C, but can be readily restored by gamma-ray irradiation, and exhibits second-order decay kinetics. These results from the Al—O − center in prehnite provide support for and new insights into Clozel et al . (1995)’s VI Al—O − — VI Al model for B-centers in kaolinite.

Unsaturated organoiridium complexes were prepared with amidophenolate ligands derived from 2-(2-trifluoromethyl)amino-4,6-di-tert-butylphenol (H2tBAF) and 2-tert-butylamino-4,6-di-tert-butylphenol (H2tBAtBu). The following 16e complexes were characterized: Cp*M(tBAR) with M = Ir (1F and 1t-Bu), Rh (2F), and (cymene)Ru(tBAF) (3F). These complexes undergo two 1e oxidations at potentials of about 0 and −0.25 V vs Cp2Fe0/+. The magnitude of ΔE1/2 is sensitive to the counteranions, and the reversibility is strongly affected by the presence of Lewis bases, which stabilize the oxidized derivatives. Crystallographic measurements indicate that upon oxidation the amidophenolate ligands adopt quinoid character, as indicated by increased alternation of the C−C bond lengths in the phenylene ring backbone and shortened C−N and C−O bonds. Unlike the charge-neutral precursors, the cationic [Cp*M(tBAR)]+ are Lewis acidic and form well-characterized adducts with PR3 (R = Me, Ph), CN−, MeCN (reversibly), and CO. In the abse...

A hole-like centre present in natural hydroxylapophyllite without any artificial irradiation has been investigated by single-crystal and powder electron paramagnetic resonance (EPR) spectroscopy at 290 K and 90 K, and by three-pulse electron spin echo envelope modulation (ESEEM) spectroscopy at 25 K. Calculated matrices g , A ( 29 Si), A ( 1 H), A ( 39 K) and P ( 39 K) suggest it to be an O − centre at the hydroxyl oxygen site and the unpaired electron in the 2 P z orbital. A series of weak satellite peaks accompanying the main absorption line in single-crystal EPR spectra have been shown to arise from four geometrically distinct pairs of neighbouring O − centres ( i.e. , biradicals). The spin Hamiltonian parameters of these O − −O − pairs provide further support for the O − model and its location at the hydroxyl oxygen site. The O − centre in hydroxylapophyllite most likely formed from natural radiation and can be enhanced by gamma-ray irradiation. It is annealed out at 300°C but can be restored readily by gamma-ray irradiation. The presence of these O − −O − pairs in the hydroxylapophyllite is probably attributable to a high abundance of the O − centre in this sample.

Sco is a mononuclear red copper protein involved in the assembly of cytochrome c oxidase. It is spectroscopically similar to red copper nitrosocyanin, but unlike the latter, which has one copper cysteine thiolate, the former has two. In addition to the two cysteine ligands (C45 and C49), the wild-type (WT) protein from Bacillus subtilis (hereafter named BSco) has a histidine (H135) and an unknown endogenous protein oxygen ligand in a distorted tetragonal array. We have compared the properties of the WT protein to variants in which each of the two coordinating Cys residues has been individually mutated to Ala, using UV/visible, Cu and S K-edge X-ray absorption, electron paramagnetic resonance, and resonance Raman spectroscopies. Unlike the Cu(II) form of native Sco, the Cu(II) complexes of the Cys variants are unstable. The copper center of C49A undergoes autoreduction to the Cu(I) form, which is shown by extended X-ray absorption fine structure to be composed of a novel two-coordinate center with one Cys and one His ligand. C45A rearranges to a new stable Cu(II) species coordinated by C49, H135 and a second His ligand recruited from a previously uncoordinated protein side chain. The different chemistry exhibited by the Cys variants can be rationalized by whether a stable Cu(I) species can be formed by autoredox chemistry. For C49A, the remaining Cys and His residues are trans, which facilitates the formation of the highly stable two-coordinate Cu(I) species, while for C45A such a configuration cannot be attained. Resonance Raman spectroscopy of the WT protein indicates a net weak Cu-S bond strength at approximately 2.24 A corresponding to the two thiolate-copper bonds, whereas the single variant C45A shows a moderately strong Cu-S bond at approximately 2.16 A. S K-edge data give a total covalency of 28% for both Cu-S bonds in the WT protein. These data suggest an average covalency per Cu-S bond lower than that observed for nitrosocyanin and close to that expected for type-2 Cu(II)-thiolate systems. The data are discussed relative to the unique Cu-S characteristics of cupredoxins, from which it is concluded that Sco does not contain highly covalent Cu-S bonds of the type expected for long-range electron-transfer reactivity.

We have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis ( K M , V max ), EPR and 1 H, 2 H, 13 C, 31 P, and 57 Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP “parent” molecules, ethylene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on 57 Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organometallic species with HMBPP, a π / σ “metallacycle” or η 2 -alkenyl complex. The complex is poised to interact with H + from E126 (and H124) in reduced wt IspH, resulting in loss of water and formation of an η 1 -allyl complex. After reduction, this forms an η 3 -allyl π -complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne diphosphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by 1 H, 2 H, and 13 C ENDOR, where hyperfine couplings of approximately 6 MHz for 13 C and 10 MHz for 1 H, are observed. Overall, the results are of broad general interest because they provide new insights into IspH catalysis and inhibition, involving organometallic species, and may be applicable to other Fe 4 S 4 -containing proteins, such as IspG.

A natural quartz, annealed first at 800°C and then irradiated with a 3.5 MeV electron beam, has been investigated by single-crystal electron paramagnetic resonance (EPR) spectroscopy at X- and W-band frequencies from 110 K to 298 K. The W-band EPR spectra allow better separation of two previously reported radiation-induced defects (D and E) and improved determinations of their spin Hamiltonian parameters. These defects have similar g tensors with the gmax axes approximately along an O—O pair and gmin axes perpendicular to the short Si—O bonds, but different 27Al hyperfine structures. Centre E is also characterized by a 29Si hyperfine structure (A/geβe = ~0.4 mT). These spin Hamiltonian parameters, together with results from density functional theory (DFT) calculations, suggest centre E to be a new variant of peroxy radicals in quartz, whereas a peroxy radical model for centre D remains tentative. Thermal stabilities and decay kinetics of centres D and E have been investigated by use of isochronal and isothermal annealing experiments on a neutron-irradiated quartz and six smoky quartz crystals in druses from a U deposit.

Enzymes of the Rnf family are believed to be bacterial redox-driven ion pumps, coupling an oxidoreduction process to the translocation of Na+ across the cell membrane. Here we show for the first time that Rnf is a flavoprotein, with FMN covalently bound to threonine-175 in RnfG and a second flavin bound to threonine-187 in RnfD. Rnf subunits D and G are homologous to subunits B and C of Na+-NQR, respectively. Each of these Na+-NQR subunits includes a conserved S(T)GAT motif, with FMN covalently bound to the final threonine. RnfD and RnfG both contain the same motif, suggesting that they bind flavins in a similar way. In order to investigate this, the genes for RnfD and RnfG from Vibrio cholerae were cloned and expressed individually in that organism. In both cases the produced protein fluoresced under UV illumination on an SDS gel, further indicating the presence of flavin. However, analysis of the mutants RnfG-T175L, RnfD-T278L, and RnfD-T187V showed that RnfG-T175 and RnfD-T187 are the likely flavin ligands. This indicates that, in the case of RnfD, the flavin is bound, not to the SGAT sequence but to the final residues of a TMAT sequence, a novel variant of the flavin binding motif. In the case of RnfG, flavin analysis, followed by MALDI-TOF-TOF mass spectrometry, showed that an FMN is covalently attached to threonine-175, the final threonine of the S(T)GAT sequence. Studies by visible, EPR, and ENDOR spectroscopy showed that, upon partial reduction, the isolated RnfG produces a neutral semiquinone intermediate. The semiquinone species disappeared upon full reduction and was not observed in the denatured protein. A topological analysis combining reporter protein fusion and computer predictions indicated that the flavins in RnfG and RnfD are localized in the periplasmic space. In contrast, in NqrC and NqrB the flavins are located in a cytoplasmic loop. This topological analysis suggests that there may be mechanistic differences between the Rnf and Na+-NQR complexes.

The X- and W-band single-crystal electron paramagnetic resonance spectra of an electron-irradiated natural quartz permit quantitative analysis of a 29Si hyperfine structure (A ~12.6 MHz) and an 27Al hyperfine structure (A ≤ 0.8 MHz) for a previously reported hole-like center. The 29Si hyperfine structure arises from interaction with two equivalent Si atoms and is characterized by the direction of the unique A axis close to a Si–O bond direction. The 27Al hyperfine structure, confirmed by pulsed electron nuclear double resonance and electron spin echo envelope modulation spectra, is characterized by the unique A axis approximately along a twofold symmetry axis. These 29Si and 27Al hyperfine data, together with published theoretical results on peroxy radicals in SiO2 as well as our own density functional theory (DFT) calculations on model peroxy centers, suggest this hole-like center to have the unpaired spin on a pair of oxygen atoms linked to two symmetrically equivalent Si atoms and a substitutional Al3+ ion across the c-axis channel, a first peroxy radical in quartz. The nuclear quadrupole matrix P also suggests that the Al3+ ion corresponds closely to the diamagnetic precursor to the [AlO4]0 center.

The cupredoxin fold is an important motif in numerous proteins that are central to several critical cellular processes ranging from aerobic and anaerobic respiration to catalysis and iron homeostasis. Three types of copper sites have been found to date within cupredoxin folds: blue type 1 (T1) copper, red type 2 (T2) copper, and purple Cu A . Although as much as 90% sequence difference has been observed among some members of this superfamily of proteins that span several kingdoms, sequence alignment and phylogenic trees strongly suggest an evolutionary link and common ancestry. However, experimental evidence for such a link has been lacking. We report herein the observation of pH-dependent transformation between blue T1 copper, red T2 copper, and the native purple Cu A centers of nitrous oxide reductase (N 2 OR) from Paracoccus denitrificans . The blue and red copper centers form initially before they are transformed into purple Cu A center. This transformation process is pH-dependent, with lower pH resulting in fewer trapped T1 and T2 coppers and faster transition to purple Cu A . These observations suggest that the purple Cu A site contains the essential elements of T1 and T2 copper centers and that the Cu A center is preferentially formed at low pH. Therefore, this work provides an underlying link between the various cupredoxin copper sites and possible experimental evidence in vitro for the evolutionary relationship between the cupredoxin proteins. The findings also lend physiological relevance to cupredoxin site biosynthesis.

Single-crystal electron paramagnetic resonance (EPR) spectra of a natural citrine quartz without any artificial irradiation, measured at W-band frequencies (∼94 GHz) and temperatures of 77, 110 and 298 K, allow better characterization of three previously-reported Centers (#6, #7 and B) and discovery of three new defects (B′, C′ and G′). The W-band EPR spectra reveal that Centers #6 and #7 do not reside on twofold symmetry axes, contrary to results from a previous X-band EPR study. The W-band spectra also show that the previously reported Center B is a mixture of two defects (B and B′) with similar g matrices but different-sized 27Al hyperfine structures. Center C′ has similar principal g values to the previously reported Center C but is distinct from the latter by a larger 27Al hyperfine structure with splittings from 0.10 to 0.22 mT. Also, Center G′ has a similar g matrix to the previously reported Center G but a different 27Al hyperfine structure with splittings from 0.41 to 0.53 mT. These spin-Hamiltonian parameters, together with observed thermal properties and microwave-power dependence, suggest that Centers #6 and #7 probably represent O 2 3− type defects. Centers B and B′ are probably superoxide radicals (O 2 − ) with the unpaired spin localized on the same pair of oxygen atoms around a missing Si atom but linked to a substitutional Al3+ ion each at different neighboring tetrahedral sites. Similarly, Centers G and G′ are most likely superoxide radicals with the unpaired spin localized on another pair of oxygen atoms around a missing Si atom and linked to a substitutional Al3+ ion each at different neighboring tetrahedral sites. Center C′ is probably an ozonide radical associated with a missing Si atom and linked to a substitutional Al3+ ion at the neighboring tetrahedral site. This study exemplifies the value of high-frequency EPR for discrimination of similar defect centers and determination of small local structural distortions that are often difficult to resolve in conventional X- and Q-band EPR studies.

Quartz crystals collected in druses, including one annealed at 400°C, from a fracture in the hanging-wall sandstone at the McArthur River uranium deposit in the Paleoproterozoic Athabasca basin, northern Saskatchewan, have been investigated by single-crystal X-band electron paramagnetic resonance (EPR) spectroscopy at room temperature and 110 K. The single-crystal EPR spectra allow us to distinguish eight paramagnetic defects, including one new center and seven previously reported centers. These centers, most of which were discovered in artificially irradiated crystals and only a few had been suggested to occur in natural quartz by powder EPR, are herewith positively established for the first time as natural radiation-induced defects by single-crystal EPR. The new center is similar in principal g-factor values to the ozonide radical (O 3 − ) in various minerals and synthetic compounds, and is characterized by its g-maximum and g-minimum axes approximately parallel to two O–O edges of the SiO 4 quasi-tetrahedron in the quartz structure. This geometry is also compatible with an ozonide radical formed from a silicon vacancy. The observed linewidths of the new ozonide radical vary from 0.087 to 0.257 mT, which are attributed to unresolved site-splittings or unresolved hyperfine splittings. This new ozonide radical is distinct from a previously reported ozonide radical in citrine quartz, which is characterized by the presence of a small 27 Al hyperfine structure. The new ozonide radical is probably linked to a Si atom in the neighboring tetrahedron and hence represents a general case in quartz, whereas the previously reported ozonide radical linked to a neighboring Al atom is a special variant in Al-bearing quartz. The presence of natural radiation-induced defects in drusy quartz is attributable to the presence of uranium in mineralized assemblages nearby or late hydrothermal fluids.

Single-crystal W-band electron paramagnetic resonance (EPR) spectra of an electron-irradiated quartz, measured at room temperature, 110 and 77 K, disclose three previously reported hole centers (#1, G and an ozonide radical). The W-band EPR spectra of these three centers clearly resolve six magnetically nonequivalent sites each, whereas previous X- and Q-band EPR studies reported Centers #1 and the ozonide radical to consist of only three symmetry-related components and interpreted them to reside on twofold symmetry axes in the quartz structure. The calculated g matrices of Center #1 and the ozonide radical show that deviations from twofold symmetry axes are

The Na+-pumping NADH-ubiquinone oxidoreductase has six polypeptide subunits (NqrA–F) and a number of redox cofactors, including a noncovalently bound FAD and a 2Fe-2S center in subunit F, covalently bound FMNs in subunits B and C, and a noncovalently bound riboflavin in an undisclosed location. The FMN cofactors in subunits B and C are bound to threonine residues by phosphoester linkages. A neutral flavin-semiquinone radical is observed in the oxidized enzyme, whereas an anionic flavin-semiquinone has been reported in the reduced enzyme. For this work, we have altered the binding ligands of the FMNs in subunits B and C by replacing the threonine ligands with other amino acids, and we studied the resulting mutants by EPR and electron nuclear double resonance spectroscopy. We conclude that the sodium-translocating NADH:quinone oxidoreductase forms three spectroscopically distinct flavin radicals as follows: 1) a neutral radical in the oxidized enzyme, which is observed in all of the mutants and most likely arises from the riboflavin; 2) an anionic radical observed in the fully reduced enzyme, which is present in wild type, and the NqrC-T225Y mutant but not the NqrB-T236Y mutant; 3) a second anionic radical, seen primarily under weakly reducing conditions, which is present in wild type, and the NqrB-T236Y mutant but not the NqrC-T225Y mutant. Thus, we can tentatively assign the first anionic radical to the FMN in subunit B and the second to the FMN in subunit C. The second anionic radical has not been reported previously. In electron nuclear double resonance spectra, it exhibits a larger line width and larger 8α-methyl proton splittings, compared with the first anionic radical.

X-band electron paramagnetic resonance (EPR) spectra of gamma-irradiated crystals of carbonate-bearing fluorapatite from the Levant mine, Cornwall, England, revealed the presence of two previously characterized centers (i.e., an O - defect and an O-□ F defect, where □ F represents a vacancy at the F site), a CO - 2 radical, and a new oxygen-associated hole-like center in the anion column. The O-□ F center in carbonate-bearing fluorapatite is stable at room temperature, whereas in carbonate-free fluorapatite the stability of this radical is shifted to lower temperatures (

Gamma-irradiated chlorapatite, synthesized from a CaCl2 fl ux, has been investigated by powder and single-crystal electron paramagnetic resonance (EPR) spectroscopy at X- and W-band frequencies, including in situ high-T X-band EPR. The powder EPR spectra, particularly high-T X-band spectra and high-resolution W-band spectra, reveal a new hole-like center, H(III), in addition to two previously reported hole-like centers, H(I) and H(II). Center H(III) is characterized by an electron spin ½ and hyperfi ne interaction with one 35 Cl nucleus, suggesting a structural model consisting of a hole trapped by a substitutional oxygen ion adjacent to a Cl – ion vacancy in the anion column. This discovery of center H(III) also lends support to the structural model already proposed by other authors for center H(II). Single-crystal X-band EPR spectra also disclose a new electronic center, E(I). The structure model for center E(I) includes an electron trapped at an isolated Cl – ion vacancy in the anion column, corresponding to center F E(II) in fl uorapatite and similar to the well-known F center in alkali halides.

The effects of chloride on the redox properties of an engineered binuclear heme-copper center in myoglobin (Cu(B)Mb) were studied by UV-vis spectroelectrochemistry and EPR spectroscopy. A low-spin heme Fe(III)-Cu(I) intermediate was observed during the redox titration of Cu(B)Mb only in the presence of both Cu(II) and chloride. Upon the first electron transfer to the Cu(B) center, one of the His ligands of Cu(B) center dissociates and coordinates to the heme iron, forming a six-coordinate low-spin ferric heme center and a reduced Cu(B) center. The second electron transfer reduces the ferric heme and causes the release of the coordinated His ligand. Thus, the fully reduced state of the heme-copper center contains a five-coordinate ferrous heme and a reduced Cu(B) center, ready for O(2) binding and reduction to water to occur. In the absence of a chloride ion, formation of the low-spin heme species was not observed. These redox reactions are completely reversible. These results indicate that binding of chloride to the Cu(B) center can induce redox-dependent structural changes, and the bound chloride and hydroxide in the heme-copper center may play different roles in the redox-linked enzymatic reactions of heme-copper oxidases, probably because of their different binding affinity to the copper center and the relatively high concentration of chloride under physiological conditions.

Many marine and pathogenic bacteria have a unique sodium-translocating NADH:ubiquinone oxidoreductase (Na(+)-NQR), which generates an electrochemical Na(+) gradient during aerobic respiration. Na(+)-NQR consists of six subunits (NqrA-F) and contains five known redox cofactors: two covalently bound FMNs, one noncovalently bound FAD, one riboflavin, and one 2Fe-2S center. A stable neutral flavin-semiquinone radical is observed in the air-oxidized enzyme, while the NADH- or dithionite-reduced enzyme exhibits a stable anionic flavin-semiquinone radical. The NqrF subunit has been implicated in binding of both the 2Fe-2S cluster and the FAD. Four conserved cysteines (C70, C76, C79, and C111) in NqrF match the canonical 2Fe-2S motif, and three conserved residues (R210, Y212, S246) have been predicted to be part of a flavin binding domain. In this work, these two motifs have been altered by site-directed mutagenesis of individual residues and are confirmed to be essential for binding, respectively, the 2Fe-2S cluster and FAD. EPR spectra of the FAD-deficient mutants in the oxidized and reduced forms exhibit neutral and anionic flavo-semiquinone radical signals, respectively, demonstrating that the FAD in NqrF is not the source of either radical signal. In both the FAD and 2Fe-2S center mutants the line widths of the neutral and anionic flavo-semiquinone EPR signals are unchanged from the wild-type enzyme, indicating that neither of these centers is nearby or coupled to the radicals. Measurements of steady-state turnover using NADH, Q-1, and the artificial electron acceptor ferricyanide strongly support an electron transport pathway model in which the noncovalently bound FAD in the NqrF subunit is the initial electron acceptor and electrons then flow to the 2Fe-2S center.

Electron paramagnetic resonance spectra of manganese-containing plasticine putty have been obtained both at 9.4 and 94.2 GHz (room temperature). It has been possible to analyze both, quantitatively by computer-based simulation and exact diagonalization of a general spin-Hamiltonian, by use of an appropriate powder technique. A fine fit was obtained for both frequencies with an isotropicg-2.00117, uniaxial55Mn hyperfine valuesA ∥/g c β c = −93.5 G (where ‖ implies direction (ℤ) andA ⊥/g c β c = −94.3 G, a nuclear quadrupole effectP z(55Mn)=0, and a uniaxial electronic quadrupole matrix (D/g c β c = 3D Z/2g c β c = −82.8 G). The electronic octupole energy was taken into account via the single parameterB 4 0 /g c β c = −0.024 G. The plasticine has been demonstrated to afford a fine intensity and magnetic-field standard, say, for use with free radicals such as spin-trap adducts. Examples provided are 2,2-diphenyl-l-picrylhydrazyl, and hydroxyl radical generated by the Fenton reaction and spin-trapped with 5,5-dimethyl-1-pyroline-N-oxide.

Room-temperature, single-crystal W-band (~94 GHz) electron paramagnetic resonance (EPR) spectra of a flux-grown fluorapatite (AP30-0) containing 0.8(1) ppm Gd and 0.9 ppm Mn disclose the presence of two even-isotope Gd3+ centers (electron spin number S = 7/2 and nuclear spin number I = 0) and a 55Mn2+ center (S = I = 5/2). The relative abundance of the two Gd3+ centers (corresponding to recently established centers “a” and “b” containing Gd3+ ions at the Ca2 and Ca1 sites, respectively) in AP30-0 has been estimated to be 0.20, indicating that “a” in this sample arises from the presence of ~0.2 ppm Gd at the Ca2 site. In addition, high-resolution W-band spectra of this sample at ~120 and ~77 K disclose the 155Gd and 157Gd spectra of “a,” in which these isotopes (I = 3/2) are only ~0.02 ppm in abundance. To the best of our knowledge, this is the first-ever demonstration of structural characterization of sub-ppm-level trace elements in minerals and their synthetic analogs. Moreover, the fact that the Gd3+ and 55Mn2+ centers in AP30-0 are detected despite the multiplicity of lines arising from their complex fine structures, hyperfine structures, and magnetic-site splittings, suggests that the W-band EPR technique is potentially capable of characterizing trace elements with a single unpaired electron (S = 1/2) and zero nuclear spin (I = 0) at even lower concentrations. The spin-Hamiltonian parameters of the 55Mn2+ center, including matrices g , D , A , and P , and high-spin term of type S4, have been determined by optimization using the single-crystal W-band EPR spectra of a Gd-doped fluorapatite containing 3.0(4) ppm Mn. The principal-axis directions of D and the pseudo-symmetry axes calculated from the S4 parameters confirm that this center corresponds to occupancy of 55Mn2+ ions at the Ca1 site. Also, the optimized parameters suggest that the Mn2+-substituted Ca1 site in the flux-grown fluorapatite has rhombic (i.e., triclinic) local symmetry [e.g., D/geβe = 436.2(6) G, E/geβe = 1.1(1) G], slightly different from the trigonal symmetry of the ideal Ca1 site.