scholarly journals Characterizing SPASM/twitch Domain-Containing Radical SAM Enzymes by EPR Spectroscopy

2021 ◽  
Author(s):  
Aidin R. Balo ◽  
Lizhi Tao ◽  
R. David Britt
Keyword(s):  
Epr Spectroscopy ◽  
Continuous Wave ◽  
Spectroscopic Studies ◽  
Paramagnetic Species ◽  
Paramagnetic Resonance ◽  
Iron Sulfur Cluster ◽  
Radical Sam Enzymes ◽  
Iron Sulfur ◽  
Pulse Epr ◽  
Multiple Domains

AbstractOwing to their importance, diversity and abundance of generated paramagnetic species, radical S-adenosylmethionine (rSAM) enzymes have become popular targets for electron paramagnetic resonance (EPR) spectroscopic studies. In contrast to prototypic single-domain and thus single-[4Fe–4S]-containing rSAM enzymes, there is a large subfamily of rSAM enzymes with multiple domains and one or two additional iron–sulfur cluster(s) called the SPASM/twitch domain-containing rSAM enzymes. EPR spectroscopy is a powerful tool that allows for the observation of the iron–sulfur clusters as well as potentially trappable paramagnetic reaction intermediates. Here, we review continuous-wave and pulse EPR spectroscopic studies of SPASM/twitch domain-containing rSAM enzymes. Among these enzymes, we will review in greater depth four well-studied enzymes, BtrN, MoaA, PqqE, and SuiB. Towards establishing a functional consensus of the additional architecture in these enzymes, we describe the commonalities between these enzymes as observed by EPR spectroscopy.

scholarly journals Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly

2021 ◽  
Vol 22 (4) ◽  
pp. 1598
Author(s):  
Amber L. Hendricks ◽  
Christine Wachnowsky ◽  
Brian Fries ◽  
Insiya Fidai ◽  
James A. Cowan
Keyword(s):  
Cluster Assembly ◽  
Paramagnetic Resonance ◽  
Iron Sulfur Cluster ◽  
Sulfur Transfer ◽  
Disease States ◽  
Isolation And Characterization ◽  
Iron Sulfur ◽  
Natural Iron ◽  
Lipoyl Synthase

Lipoyl synthase (LIAS) is an iron–sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe–4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, respectively. Most information concerning LIAS and its mechanism has been determined from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron–sulfur (Fe–S) cluster reconstitution mechanisms. Cluster donation by a number of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe–2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liquid chromatography–mass spectrometry (LC–MS) assay. Electron paramagnetic resonance (EPR) studies of native LIAS and substituted derivatives that lacked the ability to bind one or the other of LIAS’s two [4Fe–4S] clusters revealed a likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe–4S] center. These results detail the trafficking of Fe–S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe–S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe–4S] cluster reconstitution is evident.

scholarly journals Inhomogeneities in PNIPAM Aqueous Solutions: The Inside View by Spin Probe EPR Spectroscopy

Polymers ◽  
2021 ◽  
Vol 13 (21) ◽  
pp. 3829
Author(s):  
Ekaterina M. Zubanova ◽  
Sergei V. Kostjuk ◽  
Peter S. Timashev ◽  
Yury A. Rochev ◽  
Alexander I. Kokorin ◽  
...  
Keyword(s):  
Aqueous Solutions ◽  
Coming Out ◽  
Epr Spectroscopy ◽  
Spin Probe ◽  
Guest Molecule ◽  
Continuous Wave ◽  
Paramagnetic Resonance ◽  
Molten Globules ◽  
The Individual ◽  
Tempo Radical

Coil to globule transition in poly(N-isopropylacrylamide) aqueous solutions was studied using spin probe continuous-wave electronic paramagnetic resonance (CW EPR) spectroscopy with an amphiphilic TEMPO radical as a guest molecule. Using Cu(II) ions as the “quencher” for fast-moving radicals in the liquid phase allowed obtaining the individual spectra of TEMPO radicals in polymer globule and observing inhomogeneities in solutions before globule collapsing. EPR spectra simulations confirm the formation of molten globules at the first step with further collapsing and water molecules coming out of the globule, making it denser.

scholarly journals Functional, structural, and spectroscopic characterization of a glutathione-ligated [2Fe–2S] cluster in poplar glutaredoxin C1

2007 ◽  
Vol 104 (18) ◽  
pp. 7379-7384 ◽  
Author(s):  
Nicolas Rouhier ◽  
Hideaki Unno ◽  
Sibali Bandyopadhyay ◽  
Lluis Masip ◽  
Sung-Kun Kim ◽  
...  
Keyword(s):  
Intracellular Localization ◽  
Spectroscopic Characterization ◽  
Spectroscopic Studies ◽  
Iron Sulfur Cluster ◽  
X Ray Crystallography ◽  
Iron Sulfur ◽  
Catalytic Cysteine ◽  
A Subunit ◽  
Stress Sensing

When expressed in Escherichia coli, cytosolic poplar glutaredoxin C1 (CGYC active site) exists as a dimeric iron–sulfur-containing holoprotein or as a monomeric apoprotein in solution. Analytical and spectroscopic studies of wild-type protein and site-directed variants and structural characterization of the holoprotein by using x-ray crystallography indicate that the holoprotein contains a subunit-bridging [2Fe–2S] cluster that is ligated by the catalytic cysteines of two glutaredoxins and the cysteines of two glutathiones. Mutagenesis data on a variety of poplar glutaredoxins suggest that the incorporation of an iron–sulfur cluster could be a general feature of plant glutaredoxins possessing a glycine adjacent to the catalytic cysteine. In light of these results, the possible involvement of plant glutaredoxins in oxidative stress sensing or iron–sulfur biosynthesis is discussed with respect to their intracellular localization.

scholarly journals Biochemical and Spectroscopic Studies of Epoxyqueuosine Reductase: A Novel Iron–Sulfur Cluster- and Cobalamin-Containing Protein Involved in the Biosynthesis of Queuosine

Biochemistry ◽  
2015 ◽  
Vol 54 (31) ◽  
pp. 4927-4935 ◽  
Author(s):  
Zachary D. Miles ◽  
William K. Myers ◽  
William M. Kincannon ◽  
R. David Britt ◽  
Vahe Bandarian
Keyword(s):  
Spectroscopic Studies ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur

Human Ferredoxin:  Overproduction inEscherichia coli, Reconstitutionin Vitro, and Spectroscopic Studies of Iron−Sulfur Cluster Ligand Cysteine-to-Serine Mutants†

Biochemistry ◽  
1996 ◽  
Vol 35 (29) ◽  
pp. 9488-9495 ◽  
Author(s):  
Bin Xia ◽  
Hong Cheng ◽  
Vahe Bandarian ◽  
George H. Reed ◽  
John L. Markley
Keyword(s):  
Spectroscopic Studies ◽  
Inescherichia Coli ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur

scholarly journals An EPR Investigation of Defect Structure and Electron Transfer Mechanism in Mixed-Conductive LiBO2-V2O5 Glasses

2021 ◽  
Author(s):  
Jacob N. Spencer ◽  
Andrea Folli ◽  
Hong Ren ◽  
Damien M. Murphy
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Electron Transfer ◽  
Epr Spectroscopy ◽  
Defect Structure ◽  
Continuous Wave ◽  
Transfer Mechanism ◽  
Paramagnetic Resonance ◽  
Electron Transfer Mechanism ◽  
Electron Paramagnetic

Continuous Wave (CW) Electron Paramagnetic Resonance (EPR) spectroscopy was used to study the defect structure and electron transfer mechanism in a series of LiBO2-V2O5 mixed conductive glasses of varying V2O5...

scholarly journals Molecular-Level Release of Coumarin-3-Carboxylic Acid and Warfarin-Derivatives from BSA-Based Hydrogels

Pharmaceutics ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1661
Author(s):  
Niuosha Sanaeifar ◽  
Karsten Mäder ◽  
Dariush Hinderberger
Keyword(s):  
Carboxylic Acid ◽  
Epr Spectroscopy ◽  
Continuous Wave ◽  
Molecular Level ◽  
Controlled Drug Delivery ◽  
Gel Formation ◽  
Release Behavior ◽  
Paramagnetic Resonance ◽  
Electron Paramagnetic ◽  
Bsa Interaction

This investigation aimed at developing BSA hydrogels as a controlled release system to study the release behavior of spin-labeled coumarin-3-carboxylic acid (SL-CCS) and warfarin (SL-WFR). The release profiles of these spin-labeled (SL-) pharmaceuticals from BSA hydrogels prepared with different procedures are compared in detail. The mechanical properties of the gels during formation and release were studied via rheology, while a nanoscopic view on the release behavior was achieved by analyzing SL-drugs–BSA interaction using continuous wave electron paramagnetic resonance (CW EPR) spectroscopy. The influence of type of drug, drug concentration, duration of gel formation, and gelation methods on release behavior were characterized by CW EPR spectroscopy, EPR imaging (EPRI), and dynamic light scattering (DLS), which provide information on the interaction of BSA with SL-drugs, the percentage of drug inside the hydrogel and the nature and size of the released structures, respectively. We found that the release rate of SL-CCS and SL-WFR from BSA hydrogels is tunable through drug ratios, hydrogel incubation time and gelation procedures. All of the results indicate that BSA hydrogels can be potentially exploited in controlled drug delivery applications.

scholarly journals Rapid-scan electron paramagnetic resonance using an EPR-on-a-Chip sensor

2021 ◽  
Vol 2 (2) ◽  
pp. 673-687
Author(s):  
Silvio Künstner ◽  
Anh Chu ◽  
Klaus-Peter Dinse ◽  
Alexander Schnegg ◽  
Joseph E. McPeak ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Continuous Wave ◽  
Great Promise ◽  
Paramagnetic Species ◽  
Voltage Controlled Oscillator ◽  
Paramagnetic Resonance ◽  
Microwave Source ◽  
Rapid Scan ◽  
Planar Coil ◽  
Electron Paramagnetic

Abstract. Electron paramagnetic resonance (EPR) spectroscopy is the method of choice to investigate and quantify paramagnetic species in many scientific fields, including materials science and the life sciences. Common EPR spectrometers use electromagnets and microwave (MW) resonators, limiting their application to dedicated lab environments. Here, novel aspects of voltage-controlled oscillator (VCO)-based EPR-on-a-Chip (EPRoC) detectors are discussed, which have recently gained interest in the EPR community. More specifically, it is demonstrated that with a VCO-based EPRoC detector, the amplitude-sensitive mode of detection can be used to perform very fast rapid-scan EPR experiments with a comparatively simple experimental setup to improve sensitivity compared to the continuous-wave regime. In place of a MW resonator, VCO-based EPRoC detectors use an array of injection-locked VCOs, each incorporating a miniaturized planar coil as a combined microwave source and detector. A striking advantage of the VCO-based approach is the possibility of replacing the conventionally used magnetic field sweeps with frequency sweeps with very high agility and near-constant sensitivity. Here, proof-of-concept rapid-scan EPR (RS-EPRoC) experiments are performed by sweeping the frequency of the EPRoC VCO array with up to 400 THz s−1, corresponding to a field sweep rate of 14 kT s−1. The resulting time-domain RS-EPRoC signals of a micrometer-scale BDPA sample can be transformed into the corresponding absorption EPR signals with high precision. Considering currently available technology, the frequency sweep range may be extended to 320 MHz, indicating that RS-EPRoC shows great promise for future sensitivity enhancements in the rapid-scan regime.

scholarly journals Peek Inside the Water Mixtures of Ionic Liquids at Molecular Level: Microscopic Properties Probed by EPR Spectroscopy

2021 ◽  
Vol 22 (21) ◽  
pp. 11900
Author(s):  
Mikhail Yu. Ivanov ◽  
Yuliya F. Polienko ◽  
Igor A. Kirilyuk ◽  
Sergey A. Prikhod’ko ◽  
Nicolay Yu. Adonin ◽  
...  
Keyword(s):  
Ionic Liquids ◽  
Epr Spectroscopy ◽  
Continuous Wave ◽  
Molecular Level ◽  
Glassy State ◽  
Residual Water ◽  
Pulse Electron Paramagnetic Resonance ◽  
Spin Probes ◽  
Paramagnetic Resonance ◽  
Electron Paramagnetic

Many ionic liquids (ILs) can be mixed with water, forming either true solutions or emulsions. This favors their applications in many respects, but at the same time might strongly alter their physicochemical properties. A number of methods exist for studying the macroscopic properties of such mixtures, whereas understanding their characteristics at micro/nanoscale is rather challenging. In this work we investigate microscopic properties, such as viscosity and local structuring, in binary water mixtures of IL [Bmim]BF4 in liquid and glassy states. For this sake, we use continuous wave and pulse electron paramagnetic resonance (EPR) spectroscopy with dedicated spin probes, located preferably in IL-rich domains or distributed in IL- and water-rich domains. We demonstrate that the glassy-state nanostructuring of IL-rich domains is very similar to that in neat ILs. At the same time, in liquid state the residual water makes local viscosity in IL-rich domains noticeably different compared to neat ILs, even though the overwhelming amount of water is contained in water-rich domains. These results have to be taken into account in various applications of IL-water mixtures, especially in those cases demanding the combinations of optimum micro- and macroscopic characteristics.

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