scholarly journals Electron Inventory of the Iron-Sulfur Scaffold Complex HypCD Essential in [NiFe]-Hydrogenase Cofactor Assembly

2021 ◽  
Author(s):  
Sven T. Stripp ◽  
Jonathan Oltmanns ◽  
Christina S. Müller ◽  
David Ehrenberg ◽  
Ramona Schlesinger ◽  
...  
Keyword(s):  
Redox Reactions ◽  
Expression And Purification ◽  
Metal Centers ◽  
E Coli ◽  
Iron Sulfur ◽  
Reducing Conditions ◽  
Redox Active ◽  
Purification System ◽  
Efficient Expression

The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN– and CO ligands are transferred and attached to the iron ion. We report an efficient expression and purification system producing the HypCD complex from E. coli with complete metal content. This enabled in-depth spectroscopic characterizations. The results obtained by EPR and Mössbauer spectroscopy demonstrate that the [Fe](CN)2CO cofactor and the [4Fe-4S] cluster of the HypCD complex are redox active. The data indicate a potential-dependent interconversion of the [Fe]2+/3+ and [4Fe-4S]2+/+ couple, respectively. Moreover, ATR FTIR spectroscopy reveals potential-dependent disulfide formation, which hints at an electron confurcation step between the metal centers. MicroScale thermophoresis indicates preferable binding between the HypCD complex and its in vivo interaction partner HypE under reducing conditions. Together, these results provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions required for the assembly of the CN– and CO ligands on the scaffold complex HypCD.

scholarly journals Electron Inventory of the Iron-Sulfur Scaffold Complex HypCD Essential in [NiFe]-Hydrogenase Cofactor Assembly

2021 ◽  
Author(s):  
Sven T. Stripp ◽  
Jonathan Oltmanns ◽  
Christina S. Müller ◽  
David Ehrenberg ◽  
Ramona Schlesinger ◽  
...  
Keyword(s):  
Redox Reactions ◽  
Redox Activity ◽  
Construction Site ◽  
Iron Ion ◽  
Paramagnetic Resonance ◽  
Iron Sulfur ◽  
Reducing Conditions ◽  
Reversible Redox ◽  
Electron Paramagnetic ◽  
Redox Conversion

The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN– and CO ligands are transferred and attached to the iron ion. We developed an efficient protocol for the production and isolation of the functional HypCD complex that facilitated detailed spectroscopic investigations. The results obtained by UV/Vis-, electron paramagnetic Resonance (EPR)-, Resonance Raman-, Fourier-transform infrared (FTIR), and Mössbauer spectroscopy provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions. We demonstrate the redox activity of the HypCD complex reporting the interconversion of the [4Fe-4S]2+/+ couple. Additionally, we observed a reversible redox conversion between the [4Fe-4S]2+ and a [3Fe-4S]+ cluster. MicroScale thermophoresis indicated preferable binding between the HypCD complex and its interaction partner HypEF under reducing conditions. Together, these results suggest a redox cascade involving the [4Fe-4S] cluster and a conserved disulfide bond of HypD that may facilitate the synthesis of the [Fe](CN)2CO cofactor precursor on the HypCD scaffold complex.

scholarly journals On the Conformation of the COOH-terminal Domain of the Large Mechanosensitive Channel MscL

2003 ◽  
Vol 121 (3) ◽  
pp. 227-244 ◽  
Author(s):  
Andriy Anishkin ◽  
Vyacheslav Gendel ◽  
Neda A. Sharifi ◽  
Chien-Sung Chiang ◽  
Lena Shirinian ◽  
...  
Keyword(s):  
Osmotic Shock ◽  
Complete Removal ◽  
Size Exclusion ◽  
Ambient Conditions ◽  
Dynamic Simulations ◽  
E Coli ◽  
Terminal Domain ◽  
Reducing Conditions ◽  
Truncation Mutant

COOH-terminal (S3) domains are conserved within the MscL family of bacterial mechanosensitive channels, but their function remains unclear. The X-ray structure of MscL from Mycobacterium tuberculosis (TbMscL) revealed cytoplasmic domains forming a pentameric bundle (Chang, G., R.H. Spencer, A.T. Lee, M.T. Barclay, and D.C. Rees. 1998. Science. 282:2220–2226). The helices, however, have an unusual orientation in which hydrophobic sidechains face outside while charged residues face inside, possibly due to specific crystallization conditions. Based on the structure of pentameric cartilage protein , we modeled the COOH-terminal region of E. coli MscL to better satisfy the hydrophobicity criteria, with sidechains of conserved aliphatic residues all inside the bundle. Molecular dynamic simulations predicted higher stability for this conformation compared with one modeled after the crystal structure of TbMscL, and suggested distances for disulfide trapping experiments. The single cysteine mutants L121C and I125C formed dimers under ambient conditions and more so in the presence of an oxidant. The double-cysteine mutants, L121C/L122C and L128C/L129C, often cross-link into tetrameric and pentameric structures, consistent with the new model. Patch-clamp examination of these double mutants under moderately oxidizing or reducing conditions indicated that the bundle cross-linking neither prevents the channel from opening nor changes thermodynamic parameters of gating. Destabilization of the bundle by replacing conservative leucines with small polar residues, or complete removal of COOH-terminal domain (Δ110–136 mutation), increased the occupancy of subconducting states but did not change gating parameters substantially. The Δ110–136 truncation mutant was functional in in vivo osmotic shock assays; however, the amount of ATP released into the shock medium was considerably larger than in controls. The data strongly suggest that in contrast to previous gating models (Sukharev, S., M. Betanzos, C.S. Chiang, and H.R. Guy. 2001a. Nature. 409:720–724.), S3 domains are stably associated in both closed and open conformations. The bundle-like assembly of cytoplasmic helices provides stability to the open conformation, and may function as a size-exclusion filter at the cytoplasmic entrance to the MscL pore, preventing loss of essential metabolites.

scholarly journals In vivo evidence for the iron-binding activity of an iron–sulfur cluster assembly protein IscA in Escherichia coli

2010 ◽  
Vol 432 (3) ◽  
pp. 429-436 ◽  
Author(s):  
Wu Wang ◽  
Hao Huang ◽  
Guoqiang Tan ◽  
Fan Si ◽  
Min Liu ◽  
...  
Keyword(s):  
Growth Conditions ◽  
Binding Activity ◽  
Anaerobic Growth ◽  
Iron Binding ◽  
Cluster Assembly ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur

IscA is a key member of the iron–sulfur cluster assembly machinery in prokaryotic and eukaryotic organisms; however, the physiological function of IscA still remains elusive. In the present paper we report the in vivo evidence demonstrating the iron-binding activity of IscA in Escherichia coli cells. Supplement of exogenous iron (1 μM) in M9 minimal medium is sufficient to maximize the iron binding in IscA expressed in E. coli cells under aerobic growth conditions. In contrast, IscU, an iron–sulfur cluster assembly scaffold protein, or CyaY, a bacterial frataxin homologue, fails to bind any iron in E. coli cells under the same experimental conditions. Interestingly, the strong iron-binding activity of IscA is greatly diminished in E. coli cells under anaerobic growth conditions. Additional studies reveal that oxygen in medium promotes the iron binding in IscA, and that the iron binding in IscA in turn prevents formation of biologically inaccessible ferric hydroxide under aerobic conditions. Consistent with the differential iron-binding activity of IscA under aerobic and anaerobic conditions, we find that IscA and its paralogue SufA are essential for the iron–sulfur cluster assembly in E. coli cells under aerobic growth conditions, but not under anaerobic growth conditions. The results provide in vivo evidence that IscA may act as an iron chaperone for the biogenesis of iron–sulfur clusters in E. coli cells under aerobic conditions.

Efficient Expression and Purification of Bombina maxima Trefoil Factors 2 Variant in Escherichia coli as Fusion Protein

2014 ◽  
Vol 926-930 ◽  
pp. 1187-1190
Author(s):  
Tong Yi Sun
Keyword(s):  
Escherichia Coli ◽  
Wound Healing ◽  
Fusion Protein ◽  
Insoluble Protein ◽  
Expression And Purification ◽  
Trefoil Factors ◽  
E Coli ◽  
Healing Agent ◽  
Efficient Expression ◽  
Bombina Maxima

Bm-TFF2 was isolated from Bombina maxima. The B.maxima Trefoil factors 2 Variant (vBm-TFF2) could be developed as a novel administered wound healing agent. The challenge is its preparation from refolding insoluble protein expressed in Escherichia coli. A recombinant vBm-TFF2 was overexpressed in E. coli cells as a fusion protein with bacterial N-utilizing substance A (NusA). The fusion protein NusA-Bm-TFF2 can promote the wound healing.

scholarly journals Laboratory study of spectral induced polarization responses of magnetite — Fe2+ redox reactions in porous media

Geophysics ◽  
2014 ◽  
Vol 79 (1) ◽  
pp. D21-D30 ◽  
Author(s):  
Christopher G. Hubbard ◽  
L. Jared West ◽  
Juan Diego Rodriguez-Blanco ◽  
Samuel Shaw
Keyword(s):  
Redox Reactions ◽  
Induced Polarization ◽  
Phase Response ◽  
Field Surveys ◽  
Spectral Induced Polarization ◽  
Reducing Conditions ◽  
Redox Active ◽  
Large Phase ◽  
Ph Range

Spectral induced polarization (SIP) phase anomalies in field surveys at contaminated sites have previously been shown to correlate with the occurrence of chemically reducing conditions and/or semiconductive minerals, but the reasons for this are not fully understood. We report a systematic laboratory investigation of the role of the semiconductive mineral magnetite and its interaction with redox-active versus redox-inactive ions in producing such phase anomalies. The SIP responses of quartz sand with 5% magnetite in solutions containing redox-inactive [Formula: see text] and [Formula: see text] versus redox-active [Formula: see text] were measured across the pH ranges corresponding to adsorption of these metals to magnetite. With redox inactive ions [Formula: see text] and [Formula: see text], SIP phase response showed no changes across the pH range 4–10, corresponding to their adsorption, showing [Formula: see text] anomalies peaking at [Formula: see text]–74 Hz. These large phase anomalies are probably caused by polarization of the magnetite-solution interfaces. With the redox-active ion [Formula: see text], frequency of peak phase response decreased progressively from [Formula: see text] to [Formula: see text] as effluent pH increased from four to seven, corresponding to progressive adsorption of [Formula: see text] to the magnetite surface. The latter frequency (3 Hz) corresponds approximately with those of phase anomalies detected in field surveys reported elsewhere. We conclude that pH sensitivity arises from redox reactions between [Formula: see text] and magnetite surfaces, with transfer of electrical charge through the bulk mineral, as reported in other laboratory investigations. Our results confirm that SIP measurements are sensitive to redox reactions involving charge transfers between adsorbed ions and semiconductive minerals. Phase anomalies seen in field surveys of groundwater contamination and biostimulation may therefore be indicative of iron-reducing conditions, when semiconductive iron minerals such as magnetite are present.

scholarly journals Electron Inventory of the Iron-Sulfur Scaffold Complex HypCD Essential in [NiFe]-Hydrogenase Cofactor Assembly

2021 ◽  
Author(s):  
Sven T. Stripp ◽  
Jonathan Oltmanns ◽  
Christina S. Müller ◽  
David Ehrenberg ◽  
Ramona Schlesinger ◽  
...  
Keyword(s):  
Redox Reactions ◽  
Redox Activity ◽  
Construction Site ◽  
Iron Ion ◽  
Paramagnetic Resonance ◽  
Iron Sulfur ◽  
Reducing Conditions ◽  
Reversible Redox ◽  
Electron Paramagnetic ◽  
Redox Conversion

The [4Fe-4S] cluster containing scaffold complex HypCD is the central construction site for the assembly of the [Fe](CN)2CO cofactor precursor of [NiFe]-hydrogenase. While the importance of the HypCD complex is well established, not much is known about the mechanism by which the CN– and CO ligands are transferred and attached to the iron ion. We developed an efficient protocol for the production and isolation of the functional HypCD complex that facilitated detailed spectroscopic investigations. The results obtained by UV/Vis-, electron paramagnetic Resonance (EPR)-, Resonance Raman-, Fourier-transform infrared (FTIR), and Mössbauer spectroscopy provide comprehensive evidence for an electron inventory fit to drive multi-electron redox reactions. We demonstrate the redox activity of the HypCD complex reporting the interconversion of the [4Fe-4S]2+/+ couple. Additionally, we observed a reversible redox conversion between the [4Fe-4S]2+ and a [3Fe-4S]+ cluster. MicroScale thermophoresis indicated preferable binding between the HypCD complex and its interaction partner HypEF under reducing conditions. Together, these results suggest a redox cascade involving the [4Fe-4S] cluster and a conserved disulfide bond of HypD that may facilitate the synthesis of the [Fe](CN)2CO cofactor precursor on the HypCD scaffold complex.

Efficient Expression and Purification of Cryptochrome1 from Columbia livia in E. coli

2018 ◽  
Vol 25 (11) ◽  
pp. 986-995
Author(s):  
Xiaoxia Yuan ◽  
Wenhao Wang ◽  
Wenjian Wu ◽  
Jing Wang
Keyword(s):  
Expression And Purification ◽  
E Coli ◽  
Efficient Expression ◽  
Columbia Livia

Design and Experiment of Sinonovacula constricta Purification Bacteria System

2013 ◽  
Vol 690-693 ◽  
pp. 3401-3405
Author(s):  
Wei Fang Zhong ◽  
Yue Hong Wang ◽  
Hong Ying Liu
Keyword(s):  
Chlorine Dioxide ◽  
Product Yield ◽  
Rate Of Change ◽  
Sinonovacula Constricta ◽  
E Coli ◽  
Purification System ◽  
Test Platform ◽  
Pollution Emissions ◽  
Chlorine Dioxide Disinfection

A test platform of purification experiment based on with UV and chlorine dioxide disinfection was established. so as to purify the E. coli in vivo shellfish. Based on achieve UV and chlorine dioxide disinfection purposes in closed recirculation-seawater purification system scheme. Sinonovacula constricta were used as experiment materials for circulating seawater purification sterilization experiments online. The results indicated that the purification system to achieve the purpose of purifying shellfish microorganisms also has the advantage of saving energy and reducing pollution emissions. Based on the rate of change for the evaluation of e.coli, Sinonovacula constricta purified in 8-12 hours. E.coli reached to DB35/575-2004 purification of marine shellfish standard requirements ( 300MPN/100g), product yield is 95~98%.

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