Structure and function of the hybrid cluster protein

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.BT/Biocatalysi

Hypothesis: entatic versus ecstatic states in metalloproteins

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.BT/Biocatalysi

Very Low-Frequency Broadband Electron Paramagnetic Resonance Spectroscopy of Metalloproteins

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. BT/Biocatalysi

Conversion of a Single-Frequency X-Band EPR Spectrometer into a Broadband Multi-Frequency 0.1–18 GHz Instrument for Analysis of Complex Molecular Spin Hamiltonians

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.BT/Biocatalysi

Low-frequency EPR of ferrimyoglobin fluoride and ferrimyoglobin cyanide: a case study on the applicability of broadband analysis to high-spin hemoproteins and to HALS hemoproteins

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 gx 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.]BT/Biocatalysi

Broadband EPR Spectroscopy of the Triplet State: Multi-Frequency Analysis of Copper Acetate Monohydrate

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.BT/Biocatalysi

EPR spectroscopy of complex biological iron–sulfur systems

From the very first discovery of biological iron–sulfur clusters with EPR, the spectroscopy has been used to study not only purified proteins but also complex systems such as respiratory complexes, membrane particles and, later, whole cells. In recent times, the emphasis of iron–sulfur biochemistry has moved from characterization of individual proteins to the systems biology of iron–sulfur biosynthesis, regulation, degradation, and implications for human health. Although this move would suggest a blossoming of System-EPR as a specific, non-invasive monitor of Fe/S (dys)homeostasis in whole cells, a review of the literature reveals limited success possibly due to technical difficulties in adherence to EPR spectroscopic and biochemical standards. In an attempt to boost application of System-EPR the required boundary conditions and their practical applications are explicitly and comprehensively formulated.BT/Biocatalysi

Broadband Tunable Electron Paramagnetic Resonance Spectroscopy of Dilute Metal Complexes

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.BT/Biocatalysi

Maximum iron loading of ferritin: half a century of sustained citation distortion

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.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.BT/Biocatalysi