Finding vehicles for biosynthesis of metal clusters with advantageous magnetic and catalytic properties is an important industrial and environmental task. We have found previously that green microalga Chlorella sorokiniana produces a multivalent Mn-O cluster with structure that is similar to photosynthetic oxygen-evolving complex (OEC). Here we reported magnetic and redox properties and the site of accumulation of this cluster, and we proposed the mechanisms of biosynthesis and the protocol for extraction. The cluster was paramagnetic even at room temperature, with an antiferromagnetic transition at ∼13 K. The separation between ground and excited state of ΔE ≈ 15.0 cm⁻¹ matched the separation energy of OEC in S₂ state. Nano X-ray fluorescence microscopy and ³¹P NMR showed that the cluster is accumulated in acidocalcisomes, a lysosome-type organelles rich in polyphosphates. The conditions in these organelles resemble the settings of chemical synthesis of OEC mimics, including mildly acidic pH and the availability of Ca²⁺ ions. Polyphosphates are likely to play a role of stabilizing ligands and modulators of redox properties of Mn²⁺ in the cluster synthesis. The cluster shares redox potentials with OEC and showed catalase-like activity. However, we could not confirm OEC-like performance because the cluster was prone to degradation by oxidizing agents in the presence of organic residue in the extract. The biosynthesis showed an overall yield of ∼25 % and appears to be cost-competitive with chemical synthesis. This study shows that metabolic trades of selected microalgae can be employed in the green synthesis of catalytically functional clusters.

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.

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.

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.

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.]

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.

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.

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.

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.]

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.

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.

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.

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.

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.

The hybrid cluster protein (Hcp) contains a unique 4Fe cluster that is a hybrid of μ-S and μ-O bridges. Escherichia coli Hcp has recently been found to carry NO reductase activity as well as S-nitrosylation activity in NO-based signaling. In other species, the physiological activity has not been established. No reaction mechanism of any Hcp has been proposed. Here, we show that Desulfovibrio vulgaris (Hildenborough) Hcp has nitric oxide reductase activity with benzyl viologen as electron donor. With EPR spectroscopy, we identify three unexpected putative reaction intermediates: both in reduced and oxidized Hcp, dinitrosyl iron complexes are formed. Also, the hybrid cluster in reduced Hcp, but not in oxidized Hcp, binds the product N₂O. Possible implications for a reaction mechanism are discussed.