scholarly journals Iron-Sulfur Flavoprotein (Isf) fromMethanosarcina thermophila Is the Prototype of a Widely Distributed Family

2001 ◽  
Vol 183 (21) ◽  
pp. 6225-6233 ◽  
Author(s):  
Tong Zhao ◽  
Francisco Cruz ◽  
James G. Ferry
Keyword(s):  
Iron Sulfide ◽  
Cell Extract ◽  
Flavin Mononucleotide ◽  
Visible Absorption ◽  
Methanosarcina Thermophila ◽  
Cysteine Motif ◽  
Cell Extracts ◽  
Iron Sulfur ◽  
Uv Visible ◽  
Co Reduction

ABSTRACT A total of 35 homologs of the iron-sulfur flavoprotein (Isf) fromMethanosarcina thermophila were identified in databases. All three domains were represented, and multiple homologs were present in several species. An unusually compact cysteine motif ligating the 4Fe-4S cluster in Isf is conserved in all of the homologs except two, in which either an aspartate or a histidine has replaced the second cysteine in the motif. A phylogenetic analysis of Isf homologs identified four subgroups, two of which were supported by bootstrap data. Three homologs from metabolically and phylogenetically diverse species in the Bacteria and Archaea domains (Af3 from Archaeoglobus fulgidus, Cd1 fromClostridium difficile, and Mj2 from Methanococcus jannaschii) were overproduced in Escherichia coli. Each homolog purified as a homodimer, and the UV-visible absorption spectra were nearly identical to that of Isf. After reconstitution with iron, sulfide, and flavin mononucleotide (FMN) the homologs contained six to eight nonheme iron atoms and 1.6 to 1.7 FMN molecules per dimer, suggesting that two 4Fe-4S or 3Fe-4S clusters and two FMN cofactors were bound to each dimer, which is consistent with Isf data. Homologs Af3 and Mj2 were reduced by CO in reactions catalyzed by cell extract of acetate-grown M. thermophila, but Cd1 was not. Homologs Af3 and Mj2 were reduced by CO in reactions catalyzed by A. fulgidus and M. jannaschii cell extracts. Cell extract of Clostridium thermoaceticum catalyzed CO reduction of Cd1. Our database sequence analyses and biochemical characterizations indicate that Isf is the prototype of a family of iron-sulfur flavoproteins that occur in members of all three domains.

scholarly journals Site-Specific Mutational Analysis of a Novel Cysteine Motif Proposed To Ligate the 4Fe-4S Cluster in the Iron-Sulfur Flavoprotein of the Thermophilic MethanoarchaeonMethanosarcina thermophila

2000 ◽  
Vol 182 (19) ◽  
pp. 5309-5316 ◽  
Author(s):  
Ubolsree Leartsakulpanich ◽  
Mikhail L. Antonkine ◽  
James G. Ferry
Keyword(s):  
Mutational Analysis ◽  
Epr Spectra ◽  
Flavin Mononucleotide ◽  
Wild Type ◽  
Paramagnetic Resonance ◽  
Methanosarcina Thermophila ◽  
Iron Sulfur Clusters ◽  
Cysteine Motif ◽  
Iron Sulfur ◽  
In The Wild

ABSTRACT Isf (iron-sulfur flavoprotein) from Methanosarcina thermophila has been produced in Escherichia coli as a dimer containing two 4Fe-4S clusters and two FMN (flavin mononucleotide) cofactors. The deduced sequence of Isf contains six cysteines (Cys 16, Cys 47, Cys 50, Cys 53, Cys 59, and Cys 180), four of which (Cys 47, Cys 50, Cys 53, and Cys 59) comprise a motif with high identity to a motif (CX2CX2CX4–7C) present in all homologous Isf sequences available in the databases. The spacing of the motif is highly compact and atypical of motifs coordinating known 4Fe-4S clusters; therefore, all six cysteines in Isf from M. thermophila were altered to either alanine or serine to obtain corroborating biochemical evidence that the motif coordinates the 4Fe-4S cluster and to further characterize properties of the cluster dependent on ligation. All except the C16S variant were produced in inclusion bodies and were void of iron-sulfur clusters and FMN. Reconstitution of the iron-sulfur cluster and FMN was attempted for each variant. The UV-visible spectra of all reconstituted variants indicated the presence of iron-sulfur clusters and FMN. The reduced C16A/S variants showed the same electron paramagnetic resonance (EPR) spectra as wild-type Isf, whereas the reduced C180A/S variants showed EPR spectra identical to those of one of the two 4Fe-4S species present in the wild-type Isf spectrum. Conversely, EPR spectra of the oxidized C50A and C59A variants showed g values characteristic of a 3Fe-4S cluster. The spectra of the C47A and C53A variants indicated a 4Fe-4S cluster with g values and linewidths different from those for the wild type. The combined results of this study support a role for the novel CX2CX2CX4–7C motif in ligating the 4Fe-4S clusters in Isf and Isf homologues.

scholarly journals Structures of the Iron-Sulfur Flavoproteins from Methanosarcina thermophila and Archaeoglobus fulgidus

2005 ◽  
Vol 187 (11) ◽  
pp. 3848-3854 ◽  
Author(s):  
Susana L. A. Andrade ◽  
Francisco Cruz ◽  
Catherine L. Drennan ◽  
Vijay Ramakrishnan ◽  
Douglas C. Rees ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Three Dimensional ◽  
Archaeoglobus Fulgidus ◽  
Flavin Mononucleotide ◽  
Methanosarcina Thermophila ◽  
Sulfate Reducing ◽  
Iron Sulfur ◽  
Redox Active ◽  
Sequence Homologies ◽  
Insight Into

ABSTRACT Iron-sulfur flavoproteins (ISF) constitute a widespread family of redox-active proteins in anaerobic prokaryotes. Based on sequence homologies, their overall structure is expected to be similar to that of flavodoxins, but in addition to a flavin mononucleotide cofactor they also contain a cubane-type [4Fe:4S] cluster. In order to gain further insight into the function and properties of ISF, the three-dimensional structures of two ISF homologs, one from the thermophilic methanogen Methanosarcina thermophila and one from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus, were determined. The structures indicate that ISF assembles to form a tetramer and that electron transfer between the two types of redox cofactors requires oligomerization to juxtapose the flavin mononucleotide and [4Fe:4S] cluster bound to different subunits. This is only possible between different monomers upon oligomerization. Fundamental differences in the surface properties of the two ISF homologs underscore the diversity encountered within this protein family.

scholarly journals [4Fe-4S] cluster trafficking mediated by Arabidopsis mitochondrial ISCA and NFU proteins

2020 ◽  
Vol 295 (52) ◽  
pp. 18367-18378 ◽  
Author(s):  
Tamanna Azam ◽  
Jonathan Przybyla-Toscano ◽  
Florence Vignols ◽  
Jérémy Couturier ◽  
Nicolas Rouhier ◽  
...  
Keyword(s):  
Bimolecular Fluorescence Complementation ◽  
Back Reaction ◽  
Cluster Assembly ◽  
Carrier Proteins ◽  
Respiratory Chain Complexes ◽  
Visible Absorption ◽  
Iron Sulfur ◽  
Cluster Transfer ◽  
Activity Measurements ◽  
Uv Visible

Numerous iron-sulfur (Fe-S) proteins with diverse functions are present in the matrix and respiratory chain complexes of mitochondria. Although [4Fe-4S] clusters are the most common type of Fe-S cluster in mitochondria, the molecular mechanism of [4Fe-4S] cluster assembly and insertion into target proteins by the mitochondrial iron-sulfur cluster (ISC) maturation system is not well-understood. Here we report a detailed characterization of two late-acting Fe-S cluster-carrier proteins from Arabidopsis thaliana, NFU4 and NFU5. Yeast two-hybrid and bimolecular fluorescence complementation studies demonstrated interaction of both the NFU4 and NFU5 proteins with the ISCA class of Fe-S carrier proteins. Recombinant NFU4 and NFU5 were purified as apo-proteins after expression in Escherichia coli. In vitro Fe-S cluster reconstitution led to the insertion of one [4Fe-4S]2+ cluster per homodimer as determined by UV-visible absorption/CD, resonance Raman and EPR spectroscopy, and analytical studies. Cluster transfer reactions, monitored by UV-visible absorption and CD spectroscopy, showed that a [4Fe-4S]2+ cluster-bound ISCA1a/2 heterodimer is effective in transferring [4Fe-4S]2+ clusters to both NFU4 and NFU5 with negligible back reaction. In addition, [4Fe-4S]2+ cluster-bound ISCA1a/2, NFU4, and NFU5 were all found to be effective [4Fe-4S]2+ cluster donors for maturation of the mitochondrial apo-aconitase 2 as assessed by enzyme activity measurements. The results demonstrate rapid, unidirectional, and quantitative [4Fe-4S]2+ cluster transfer from ISCA1a/2 to NFU4 or NFU5 that further delineates their respective positions in the plant ISC machinery and their contributions to the maturation of client [4Fe-4S] cluster-containing proteins.

scholarly journals Novel Functions of an Iron-Sulfur Flavoprotein from Trichomonas vaginalis Hydrogenosomes

2014 ◽  
Vol 58 (6) ◽  
pp. 3224-3232 ◽  
Author(s):  
Tamara Smutná ◽  
Katerina Pilarová ◽  
Ján Tarábek ◽  
Jan Tachezy ◽  
Ivan Hrdý
Keyword(s):  
Oxidative Stress ◽  
Trichomonas Vaginalis ◽  
Defense Mechanism ◽  
Electron Donors ◽  
Flavin Mononucleotide ◽  
Methanosarcina Thermophila ◽  
Content Type ◽  
Sexually Transmitted ◽  
Iron Sulfur ◽  
Reducing Activity

ABSTRACTIron-sulfur flavoproteins (Isf) are flavin mononucleotide (FMN)- and FeS cluster-containing proteins commonly encountered in anaerobic prokaryotes. However, with the exception of Isf fromMethanosarcina thermophila, which participates in oxidative stress management by removing oxygen and hydrogen peroxide, none of these proteins has been characterized in terms of function.Trichomonas vaginalis, a sexually transmitted eukaryotic parasite of humans, was found to express several iron-sulfur flavoprotein (TvIsf) homologs in its hydrogenosomes. We show here that in addition to having oxygen-reducing activity, the recombinant TvIsf also functions as a detoxifying reductase of metronidazole and chloramphenicol, both of which are antibiotics effective against a variety of anaerobic microbes. TvIsf can utilize both NADH and reduced ferredoxin as electron donors. Given the prevalence of Isf in anaerobic prokaryotes, we propose that these proteins are central to a novel defense mechanism against xenobiotics.

Remarkable kinetic stability of α-thiocarbamoyl substituted 4-methoxybenzylcations

1998 ◽  
Vol 76 (12) ◽  
pp. 1910-1915 ◽  
Author(s):  
Robert A McClelland ◽  
Victoria E Licence ◽  
John P Richard ◽  
Kathleen B Williams ◽  
Shrong-Shi Lin
Keyword(s):  
Key Words ◽  
Strong Interaction ◽  
Rate Constants ◽  
Flash Photolysis ◽  
Visible Absorption ◽  
Methyl Group ◽  
Thioamide Group ◽  
Azide Ion ◽  
Uv Visible ◽  
Leaving Groups

4-Methoxybenzyl cations bearing α-(N,N-dimethylcarbamoyl) and α-(N,N-dimethylthiocarbamoyl) substituents have been generated photochemically upon irradiation of precursors with pentafluorobenzoate or 4-methoxybenzoate leaving groups. The ions have been observed with flash photolysis in 40:60 acetonitrile:water and in 50:50 methanol:water, and rate constants were measured for their decay in solvent alone and for their capture by azide ion. The cations so studied and their lifetimes in 40% acetonitrile are 6, ArC+H-CONMe2, 0.6 μs; 2, ArC+H-CSNMe2, 7 ms; and 4, ArC+(CH3)-CSMe2, 6 ms, where Ar = 4-MeOC6H4. The cation 4 reacts with solvent by elimination of a proton from the α-methyl group, and the rate constant for solvent addition must be less than 1 s-1. The CSNMe2 substituted cations are 105-107-fold longer lived than analogs where the thioamide group has been replaced with an α-methyl. The UV-visible absorption spectra of these two cations also show significant differences from those of typical 4-methoxybenzyl cations. Thus, both the lifetimes and spectra point to a strong interaction of the benzylic centre with the thioamide group. Key words: flash photolysis, thiocarbamoyl stabilized carbocation, photosolvolysis.

scholarly journals Water-soluble β-aminobisulfonate building blocks for pH and Cu2+ indicators

RSC Advances ◽  
2016 ◽  
Vol 6 (88) ◽  
pp. 84712-84721 ◽  
Author(s):  
Maria A. Cardona ◽  
Marina Kveder ◽  
Ulrich Baisch ◽  
Michael R. Probert ◽  
David C. Magri
Keyword(s):  
Nmr Spectroscopy ◽  
Building Blocks ◽  
Water Soluble ◽  
Visible Absorption ◽  
H Nmr ◽  
Uv Visible

Two phenyl β-aminobisulfonate ligands characterised by UV-visible absorption, EPR and 1H NMR spectroscopy exhibit evidence for binding with Cu2+ in water and methanol.

Study on the Inclusion Behavior of Cucurbit [n] uril with Phenylalanine

2011 ◽  
Vol 197-198 ◽  
pp. 1153-1156
Author(s):  
Ning Chen ◽  
Ya Bin Li
Keyword(s):  
1H Nmr ◽  
Interaction Mechanism ◽  
Acetate Buffer Solution ◽  
Molar Ratio ◽  
High Concentration ◽  
Buffer Solution ◽  
Visible Absorption ◽  
Nmr Spectrum ◽  
Application Range ◽  
Uv Visible

The characteristics of host-guest complexes between cucurbit[n]uril (CB [n]) and phenylalanine were investigated by UV-visible absorption spectroscopy in acetate buffer solution at room temperature. It was found that the UV-visible absorption increased steadily with constantly dropping the high concentration of cucurbit[6]uril (CB [6]) and cucurbit[8]uril (CB [8]) in the phenylalanine solution which indicates that there are some interaction betweenCB [n] and phenylalanine.Then CB [6] and phenylalanine at molar ratio of 1:1 to weigh while CB [8] and phenylalanine at molar ratio of 1:2, respectively, are both demonstrated by 1H NMR spectra. 1H NMR spectrum of complexes was obtained, indicating an enthalpic driving force for host-guest complexes. The possible interaction mechanism and inclusion mode were also discussed. This work may extend the application range of CB [n] in supramolecular and pharmaceutical analysis.

UV-Visible Absorption Studies of Gossypol-Metal Cation Complexes in Acetonitrile Solution

1991 ◽  
Vol 24 (10) ◽  
pp. 1265-1273 ◽  
Author(s):  
Bronislaw Marciniak ◽  
Halina Kozubek ◽  
Bogumil Brzezinski
Keyword(s):  
Metal Cation ◽  
Acetonitrile Solution ◽  
Visible Absorption ◽  
Absorption Studies ◽  
Uv Visible ◽  
Cation Complexes
Sign in / Sign up

Export Citation Format

Share Document