The electron–electron, or zero-field interaction (ZFI) in the electron paramagnetic resonance (EPR) of high-spin transition ions in metalloproteins and coordination complexes, is commonly described by a simple spin Hamiltonian that is second-order in the spin S: ℋ=𝐷[𝑆2𝑧−𝑆(𝑆+1)/3+𝐸(𝑆2𝑥−𝑆2𝑦). Symmetry considerations, however, allow for fourth-order terms when S ≥ 2. In metalloprotein EPR studies, these terms have rarely been explored. Metal ions can cluster via non-metal bridges, as, for example, in iron-sulfur clusters, in which exchange interaction can result in higher system spin, and this would allow for sixth- and higher-order ZFI terms. For metalloproteins, these have thus far been completely ignored. Single-molecule magnets (SMMs) are multi-metal ion high spin complexes, in which the ZFI usually has a negative sign, thus affording a ground state level pair with maximal spin quantum number mS = ±S, giving rise to unusual magnetic properties at low temperatures. The description of EPR from SMMs is commonly cast in terms of the ‘giant-spin model’, which assumes a magnetically isolated system spin, and in which fourth-order, and recently, even sixth-order ZFI terms have been found to be required. A special version of the giant-spin model, adopted for scaling-up to system spins of order S ≈ 103–104, has been applied to the ubiquitous iron-storage protein ferritin, which has an internal core containing Fe3+ ions whose individual high spins couple in a way to create a superparamagnet at ambient temperature with very high system spin reminiscent to that of ferromagnetic nanoparticles. This scaled giant-spin model is critically evaluated; limitations and future possibilities are explicitly formulated.@en

The development of tungsten biochemistry is sketched from the viewpoint of personal participation. Following its identification as a bio-element, a catalogue of genes, enzymes, and reactions was built up. EPR spectroscopic monitoring of redox states was, and remains, a prominent tool in attempts to understand tungstopterin-based catalysis. A paucity of pre-steady-state data remains a hindrance to overcome to this day. Tungstate transport systems have been characterized and found to be very specific for W over Mo. Additional selectivity is presented by the biosynthetic machinery for tungstopterin enzymes. Metallomics analysis of hyperthermophilic archaeon Pyrococcus furiosus indicates a comprehensive inventory of tungsten proteins.

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Electron paramagnetic resonance spectroscopy is a long-standing method for the exploration of electronic structures of transition ion complexes. The difficulty of its analysis varies considerably, not only with the nature of the spin system, but more so with the relative magnitudes of the magnetic interactions to which the spin is subject, where particularly challenging cases ensue when two interactions are of comparable magnitude. A case in point is the triplet system S = 1 of coordination complexes with two unpaired electrons when the electronic Zeeman interaction and the electronic zero-field interaction are similar in strength. This situation occurs in the X-band spectra of the thermally excited triplet state of dinuclear copper(II) complexes, exemplified by copper acetate monohydrate. In this study, applicability of the recently developed low-frequency broadband EPR spectrometer to S = 1 systems is investigated on the analysis of multi-frequency, 0.5–16 GHz, data from [Cu(CH3COO)2H2O]2. Global fitting affords the spin Hamiltonian parameters gz = 2.365 ± 0.008; gy = 2.055 ± 0.010; gx = 2.077 ± 0.005; Az = 64 gauss; D = 0.335 ± 0.002 cm−1; E = 0.0105 ± 0.0003 cm−1. The latter two define zero-field absorptions at ca. 630, 7730, and 10,360 MHz, which show up in the spectra as one half of a sharpened symmetrical line. Overall, the EPR line shape is Lorentzian, reflecting spin-lattice relaxation, which is a combination of an unusual, essentially temperature-independent, inverted Orbach process via the S = 0 ground state, and a Raman process proportional to T2. Other broadening mechanisms are limited to at best minor contributions from a distribution in E values, and from dipolar interaction with neighboring copper pairs. Monitoring of a first-order double-quantum transition between 8 and 35 GHz shows a previously unnoticed very complex line shape behavior, which should be the subject of future research.@en

A broadband EPR spectrometer is an instrument that can be tuned to many microwave frequencies over several octaves. Its purpose is the collection of multi-frequency data, whose global analysis affords interpretation of complex spectra by means of deconvolution of frequency-dependent and frequency-independent interaction terms. Such spectra are commonly encountered, for example, from transition-metal complexes and metalloproteins. In a series of previous papers, I have described the development of broadband EPR spectrometers around a vector network analyzer. The present study reports on my endeavor to start from an existing X-band spectrometer and to reversibly re-build it into a broadband machine, in a quest to drastically reduce design effort, building costs, and operational complexity, thus bringing broadband EPR within easy reach of a wide range of researchers.

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Distances between Fe ions in multiheme cytochromes are sufficiently short to make the intramolecular dipole-dipole interaction between hemes probable. In the analysis of EPR data from cytochromes, this interaction has thus far been ignored under the assumption that spectra are the simple sum of non-interacting components. Here, we use a recently developed low-frequency broadband EPR spectrometer to establish the extent of dipolar interaction in the example cytochromes, characterize its spectral signatures, and identify present limitations in the analysis. Broadband EPR spectra of Shewanella oneidensis MR-1 small tetraheme cytochrome (STC) have been collected over the frequency range of 0.45 to 13.11 GHz, and they have been compared to similar data from Desulfovibrio vulgaris Hildenborough cytochrome c₃. The two cases are representative examples of two very different heme topologies and corresponding electron-transfer properties in tetraheme proteins. While in cytochrome c₃, the six Fe-Fe distances can be sorted into two well-separated groups, those in STC are diffuse. Since the onset of dipolar interaction between Fe-Fe pairs is already observed in the X-band, the g values are determined in the simulation of the 13.11 GHz spectrum. Low-frequency spectra are analyzed with the inclusion of dipolar interaction based on available structural data on mutual distances and orientations between all hemes. In this procedure, all 24 possible assignments of individual heme spectra to heme topologies are sampled. The 24 configurations can be reduced to a few, but inspection falls short of a unique assignment, due to a remaining lack of understanding of the fine details of these complex spectra. In general, the EPR analysis suggests the four-heme system in c₃ to be more rigid than that in STC, which is proposed to be related to different physiological roles in electron transfer.

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Analysis of citation networks in biomedical research has indicated that belief in a specific scientific claim can gain unfounded authority through citation bias (systematic ignoring of papers that contain content conflicting with a claim), amplification (citation to papers that don't contain primary data), and invention (citing content but claiming it has a different meaning). There is no a priori reason to expect that citation distortion is limited to particular fields of science. This Pespective presents a case study of the literature on maximum iron loading of the ferritin protein to illustrate that the field of metallomics is no exception to the rule that citation distortion is a widespread phenomenon.

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An EPR spectrometer has been developed that can be tuned to many frequencies in the range of ca 0.1–15 GHz. Applicability has been tested on ferrimyoglobin fluoride (MbF) and ferrimyoglobin cyanide (MbCN). MbF has a high-spin (S = 5/2) spectrum with ¹⁹F superhyperfine splitting that is only resolved in X-band along the heme normal. Low-frequency EPR also resolves the splitting in the heme plane. Measurement of linewidth as a function of frequency provides the basis for an analysis of inhomogeneous broadening in terms of g-strain, zero-field distribution, unresolved superhyperfine splittings and dipolar interaction. Rhombicity in the g tensor is found to be absent. MbCN (S = 1/2) has a highly anisotropic low spin (HALS) spectrum for which g_x cannot be determined unequivocally in X-band. Low-frequency EPR allows for measurement of the complete spectrum and determination of the g-tensor. Graphical abstract: [Figure not available: see fulltext.]

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Quantum mechanics postulates that the hydrogen atom has a stable ground state from which it can be promoted to excited states by capture of electromagnetic radiation, with the energy of all possible states given by E_n = −13.598/n² eV, in which n ≥ 1 is a positive integer. It has been previously proposed that the n = 1 state is not the true ground state, and that so-called hydrino states of lower energy can exist, which are characterized by fractional quantum numbers n = 1/p, in which 1 < p ≤ 137 is a limited integer. Electron transition to a hydrino state, H(1/p) is non-radiative and requires a quantized amount of energy, 2mE₁ (m is an integer), to be transferred to a catalyst. Numerous putative hydrino-forming reactions have been previously explored and the products have been characterized by a range of analytical methods. Molecular hydrino has been predicted to be paramagnetic. Here, we give an account of an electron paramagnetic resonance (EPR) study of molecular hydrino H₂(1/4) that was produced as gaseous inclusion in polymeric Ga(O)OH by a plasma reaction of atomic hydrogen with non-hydrogen bonded water as the catalyst. A sharp, complex, multi-line EPR spectrum is found, whose detailed properties prove to be consistent with predictions from hydrino theory. Molecular hydrino was also identified in gas chromatography as a compound faster than molecular hydrogen.

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A hybrid cluster (HC) is a 4Fe cluster with both sulfur and oxygen bridges. Hybrid cluster proteins (Hcps) contain two 4Fe clusters, a one electron transferring iron-sulfur cluster and a hybrid cluster. The structural gene, hcp, is diffusely found in bacteria, archaea, and monocellular eukarya, and the HC binding motif involving amino acids H, E, C, C, C, C, E, (K) appears to be fully conserved. HC is the active site of the enzyme Hcp. Of several reported Hcp enzymatic activities the conversion of nitric oxide into nitrous oxide, NO reductase, has been established as physiologically relevant. Other activities, notably signal transduction by NO transfer to other proteins, are controversial. The HC undergoes a complex structural change associated with single-electron iron based redox chemistry as well as electron-pair redox chemistry of a persulfidocysteine sulfur atom. A mechanistic scheme is proposed for the HC encompassing its structural, magnetic, and enzymatic properties.

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A virus hijacks host cellular machineries and metabolites in order to reproduce. In response, the innate immune system activates different processes to fight back. Although many aspects of these processes have been well investigated, the key roles played by iron–sulfur [FeS] clusters, which are among the oldest classes of bio-inorganic cofactors, have barely been considered. Here we discuss how several [FeS] cluster-containing proteins activate, support and modulate the innate immune response to restrict viral infections, and how some of these proteins simultaneously support the replication of viruses. We also propose models of function of some proteins in the innate immune response and argue that [FeS] clusters in many of these proteins act as biological ‘fuses’ to control the response. We hope this overview helps to inspire future research in the emerging field of bio-inorganic virology/immunology and that such studies may reveal new molecular insight into the links between viral infections and diseases like cancer and neurodegeneration. [Figure not available: see fulltext.]

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A previously developed spectrometer for broadband electron paramagnetic resonance (EPR) spectroscopy of dilute randomly oriented systems has been considerably modified to extend the frequency reach down to the hundred MHz range and to boost concentration sensitivity by 1 to 2 orders of magnitude. The instrument is now suitable for the study of biological systems in particular metalloproteins. As a proof of concept, examples from the class of low-spin ferric hemoproteins are studied in terms of frequency-dependent changes in their EPR spectra. Mono-heme cytochrome c EPR is determined by g-strain over a wide frequency range, whereas a combination of unresolved ligand hyperfine interaction and concentration-dependent intermolecular dipolar interaction becomes dominant at very low frequencies. In the four heme containing cytochrome c3, g-strain combines with intramolecular dipolar interaction over the full-studied frequency range of 0.23-12.0 GHz. It is concluded that the point-dipole approach is inappropriate to describe magnetic interactions between low-spin ferric heme systems and that a body of literature on redox interactions in multi-heme proteins will be affected by this conclusion.

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The heme enzyme chlorite dismutase (Cld) catalyzes O-O bond formation as part of the conversion of the toxic chlorite (ClO2-) to chloride (Cl-) and molecular oxygen (O2). Enzymatic O-O bond formation is rare in nature, and therefore, the reaction mechanism of Cld is of great interest. Microsecond timescale pre-steady-state kinetic experiments employing Cld from Azospira oryzae (AoCld), the natural substrate chlorite, and the model substrate peracetic acid (PAA) reveal the formation of distinct intermediates. AoCld forms a complex with PAA rapidly, which is cleaved heterolytically to yield Compound I, which is sequentially converted to Compound II. In the presence of chlorite, AoCld forms an initial intermediate with spectroscopic characteristics of a 6-coordinate high-spin ferric substrate adduct, which subsequently transforms at kobs = 2-5 × 104 s-1 to an intermediate 5-coordinated high-spin ferric species. Microsecond-Timescale freeze-hyperquench experiments uncovered the presence of a transient low-spin ferric species and a triplet species attributed to two weakly coupled amino acid cation radicals. The intermediates of the chlorite reaction were not observed with the model substrate PAA. These findings demonstrate the nature of physiologically relevant catalytic intermediates and show that the commonly used model substrate may not behave as expected, which demands a revision of the currently proposed mechanism of Clds. The transient triplet-state biradical species that we designate as Compound T is, to the best of our knowledge, unique in heme enzymology. The results highlight electron paramagnetic resonance spectroscopic evidence for transient intermediate formation during the reaction of AoCld with its natural substrate chlorite. In the proposed mechanism, the heme iron remains ferric throughout the catalytic cycle, which may minimize the heme moiety's reorganization and thereby maximize the enzyme's catalytic efficiency.

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Biocatalytic copper centers are generally involved in the activation and reduction of dioxygen, with only few exceptions known. Here we report the discovery and characterization of a previously undescribed copper center that forms the active site of a copper-containing enzyme thiocyanate dehydrogenase (suggested EC 1.8.2.7) that was purified from the haloalkaliphilic sulfur-oxidizing bacterium of the genus Thioalkalivibrio ubiquitous in saline alkaline soda lakes. The copper cluster is formed by three copper ions located at the corners of a near-isosceles triangle and facilitates a direct thiocyanate conversion into cyanate, elemental sulfur, and two reducing equivalents without involvement of molecular oxygen. A molecular mechanism of catalysis is suggested based on high-resolution three-dimensional structures, electron paramagnetic resonance (EPR) spectroscopy, quantum mechanics/molecular mechanics (QM/MM) simulations, kinetic studies, and the results of site-directed mutagenesis.

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The luminescent solar concentrator has the potential of widespread use as a generator of electricity from sunlight well-suited for use in the urban environment owing to its adaptability in shape and coloration. The device performance is heavily dependent on the ability to transport light long distances to the edges. A common organic luminophore used in the device is based on a perylene core. In this work, we describe an overlooked effect of UV illumination on the appearance and efficiency of these devices. An additional absorption peak appears upon polymerization under intense UV in nitrogen atmosphere which significantly reduces the edge emissions from the device. The additional absorbance peak disappears after exposure to air, indicating the presence of a radical anion being formed during the UV light exposure. This suggests newly-produced LSC devices should be allowed to stand a period of time under ambient conditions before their performance characteristics are determined, which could have implications in potential future commercialization of the technology.

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Biliverdin (BV), a product of heme catabolism, is known to interact with transition metals, but the details of such interactions under physiological conditions are scarce. Herein, we examined coordinate/redox interactions of BV with Cu ²⁺ in phosphate buffer at pH 7.4, using spectrophotometry, HESI-MS, Raman spectroscopy, ¹ H NMR, EPR, fluorimetry, and electrochemical methods. BV formed a stable coordination complex with copper in 1:1 stoichiometry. The structure of BV was more planar and energetically stable in the complex. The complex showed strong paramagnetic effects that were attributed to an unpaired delocalized e ^- . The delocalized electron may come from BV or Cu ²⁺ , so the complex is formally composed either of BV radical cation and Cu ¹⁺ or of BV radical anion and Cu ³⁺ . The complex underwent oxidation only in the presence of both O ₂ and an excess of Cu ²⁺ , or a strong oxidizing agent, and it was resistant to reducing agents. The biological effects of the stable BV metallocomplex containing a delocalized unpaired electron should be further examined, and may provide an answer to the long-standing question of high energy investment in the catabolism of BV, which represents a relatively harmless molecule per se.

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Escherichia coli ZraP (zinc resistance associated protein) is the major Zn containing soluble protein under Zn stress conditions. ZraP is the accessory protein of a bacterial two-component, Zn²⁺ sensitive signal transduction system ZraSR. ZraP has also been reported to act as a Zn²⁺ dependent molecular chaperone. An explanation why ZraP is the major Zn protein under the stress condition of Zn²⁺ overload (0.2 mM) has remained elusive. We have recombinantly produced E. coli ZraP and measured Zn²⁺ and Cu²⁺ affinity in-vitro using Isothermal Titration Calorimetry. ZraP has a significantly higher affinity for Cu²⁺ than for Zn²⁺. Mutation of the conserved Cys¹⁰² to Ala or Ser resulted in a change of the oligomeric state of the protein. Mutation of the conserved His¹⁰⁷ to Ala did not affect the zinc binding affinity or the oligomeric state of the protein. Deletion of the ZraP coding gene from the E. coli genome resulted in a phenotype with tolerance to very high zinc concentrations (up to 2.5 mM) that were lethal to wild type E. coli. These results exclude a direct role for ZraP in Zn²⁺ tolerance in E. coli.

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Analysis of the electron paramagnetic resonance (EPR) of transition ion complexes requires data taken at different microwave frequencies because the spin Hamiltonian contains operators linear in the frequency as well as operators independent of the frequency. In practice, data collection is hampered by the fact that conventional EPR spectrometers have always been designed to operate at a single frequency. Here, a broadband instrument is described and tested that operates from 0.5 to 12 GHz and whose sensitivity approaches that of single-frequency spectrometers. Multifrequency EPR from triclinic substitutional (0.5%) Cu(II) in ZnSO₄ is globally analyzed to illustrate a novel approach to reliable determination of the molecular electronic structure of transition ion complexes from field-frequency 2D data sets.

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The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.

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Over half a century ago the hypothesis was put forth that redox-active metal ions and multidentate protein ligands may combine to form a local state of entasis: an irregular symmetry intermediate between those dictated by coordination chemistry for the two redox states involved. Such an energetically poised domain would be at the basis of high activity (notably electron-transfer rates) in biological systems. Today the concept of the entatic state has become textbook material. Based on EPR spectroscopic data it is proposed here that poised, entatic states may only be of marginal existence; rather the occurrence of relatively wide distributions of coordination geometries (or: ecstatic states) afford a stochastic tuning of structure towards low-energy unimolecular transition states.@en