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1.
The high potential iron–sulfur protein (HiPIP) is involved in the iron respiratory electron transport chain of Acidithiobacillus ferrooxidans but its exact role is unclear. The gene of HiPIP from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, and the protein then purified by one-step affinity chromatography to homogeneity. The molecular mass of the HiPIP monomer
was 7250.43 Da by MALDI-TOF MS, indicating the presence of the [Fe4S4] cluster. The optical and EPR spectra results of the recombinant protein confirmed that the iron–sulfur cluster was correctly
inserted into the active site of the protein. Site-directed mutagenesis results revealed that Cys25, Cys28, Cys37 and Cys50
were involved in ligating to the iron–sulfur cluster. 相似文献
2.
Emile Bol Loes E. Bevers Peter-Leon Hagedoorn Wilfred R. Hagen 《Journal of biological inorganic chemistry》2006,11(8):999-1006
Formaldehyde oxidoreductase (FOR) is one of the tungstopterin iron–sulfur enzymes of the five-membered family of aldehyde oxidoreductases in the hyperthermophilic archaeon Pyrococcus furiosus. In dye-mediated equilibrium redox titrations, the tungsten in active P. furiosus FOR is a two-electron acceptor, W(VI/IV). The intermediate, paramagnetic W(V) state can be trapped only by reduction with substrate, with consecutive one-electron intraprotein electron transfer to the single [4Fe–4S](2+;+) cluster and partial comproportionation of the tungsten over W(IV, V, VI); this is a stable state in the absence of an external electron acceptor. Electron paramagnetic resonance (EPR) spectroscopy reveals a single “low-potential” W(V) spectrum with g
xyz
values 1.847, 1.898, and 1.972, and a [4Fe–4S]+ cubane in a spin mixture of S = 1/2 (10%) and S = 3/2 (90%) of intermediate rhombicity (E/D = 0.21, g
real = 1.91). The development of this intermediate in vitro is slow even at elevated temperature and with a nominal 50:1 excess of substrate over enzyme presumably owing to the very unfavorable hydration equilibrium of the formaldehyde/methylene glycol couple with K
D ≈ 103. Rapid intermediate formation of enzyme at concentrations suitable for EPR spectroscopy (200 μM) is only obtained with extremely high nominal substrate concentration (1 M formaldehyde) and is followed by a slower phase of denaturation. The premise that the free formaldehyde, and not the methylene glycol, is the enzyme’s substrate implies that K
M for formaldehyde is 3 orders of magnitude less that the previously reported value. 相似文献
3.
Christine Wachnowsky Insiya Fidai J. A. Cowan 《Journal of biological inorganic chemistry》2016,21(7):825-836
Human Nfu is an iron–sulfur cluster protein that has recently been implicated in multiple mitochondrial dysfunctional syndrome (MMDS1). The Nfu family of proteins shares a highly homologous domain that contains a conserved active site consisting of a CXXC motif. There is less functional conservation between bacterial and human Nfu proteins, particularly concerning their Iron–sulfur cluster binding and transfer roles. Herein, we characterize the cluster exchange chemistry of human Nfu and its capacity to bind and transfer a [2Fe–2S] cluster. The mechanism of cluster uptake from a physiologically relevant [2Fe–2S](GS)4 cluster complex, and extraction of the Nfu-bound iron–sulfur cluster by glutathione are described. Human holo Nfu shows a dimer-tetramer equilibrium with a protein to cluster ratio of 2:1, reflecting the Nfu-bridging [2Fe–2S] cluster. This cluster can be transferred to apo human ferredoxins at relatively fast rates, demonstrating a direct role for human Nfu in the process of [2Fe–2S] cluster trafficking and delivery. 相似文献
4.
Iron–sulfur (Fe/S) cluster containing proteins are widely distributed in nature and are involved in numerous processes including
electron transfer, metabolic reactions, sensing, signaling, and regulation of gene expression. The knowledge about the biogenesis
of Fe/S clusters, and the assembly and maturation of Fe/S cluster containing proteins is still limited, especially in photosynthetic
organisms. In most organisms analyzed so far the biogenesis of Fe/S clusters involves more than one machinery. The additional
compartment in photoautotrophic organisms, the plastids, presents an additional challenge for the regulation of Fe/S cluster
biogenesis. The requirement for Fe/S proteins in multiple chloroplast processes argues that Fe/S cluster assembly is an essential
part of plastid functionality. This review focuses on the interesting and unique aspects of Fe/S cluster biogenesis in photosynthetic
organisms and compares them to what is known in other organisms. 相似文献
5.
《BBA》2013,1827(10):1141-1147
The Escherichia coli respiratory complex II paralogs succinate dehydrogenase (SdhCDAB) and fumarate reductase (FrdABCD) catalyze interconversion of succinate and fumarate coupled to quinone reduction or oxidation, respectively. Based on structural comparison of the two enzymes, equivalent residues at the interface between the highly homologous soluble domains and the divergent membrane anchor domains were targeted for study. This included the residue pair SdhB-R205 and FrdB-S203, as well as the conserved SdhB-K230 and FrdB-K228 pair. The close proximity of these residues to the [3Fe–4S] cluster and the quinone binding pocket provided an excellent opportunity to investigate factors controlling the reduction potential of the [3Fe–4S] cluster, the directionality of electron transfer and catalysis, and the architecture and chemistry of the quinone binding sites. Our results indicate that both SdhB-R205 and SdhB-K230 play important roles in fine tuning the reduction potential of both the [3Fe–4S] cluster and the heme. In FrdABCD, mutation of FrdB-S203 did not alter the reduction potential of the [3Fe–4S] cluster, but removal of the basic residue at FrdB-K228 caused a significant downward shift (> 100 mV) in potential. The latter residue is also indispensable for quinone binding and enzyme activity. The differences observed for the FrdB-K228 and Sdh-K230 variants can be attributed to the different locations of the quinone binding site in the two paralogs. Although this residue is absolutely conserved, they have diverged to achieve different functions in Frd and Sdh. 相似文献
6.
Iron and citrate are essential for the metabolism of most organisms, and regulation of iron and citrate biology at both the
cellular and systemic levels is critical for normal physiology and survival. Mitochondrial and cytosolic aconitases catalyze
the interconversion of citrate and isocitrate, and aconitase activities are affected by iron levels, oxidative stress and
by the status of the Fe–S cluster biogenesis apparatus. Assembly and disassembly of Fe–S clusters is a key process not only
in regulating the enzymatic activity of mitochondrial aconitase in the citric acid cycle, but also in controlling the iron
sensing and RNA binding activities of cytosolic aconitase (also known as iron regulatory protein IRP1). This review discusses
the central role of aconitases in intermediary metabolism and explores how iron homeostasis and Fe–S cluster biogenesis regulate
the Fe–S cluster switch and modulate intracellular citrate flux. 相似文献
7.
This paper presents and discusses evidence suggesting how the diversity of domain folds in existence today might have evolved from peptide ancestors. We apply a structure similarity detection method to detect instances where localized regions of different protein folds contain highly similar sequences and structures. Results of performing an all-on-all comparison of known structures are described and compared with other recently published findings. The numerous instances of local sequence and structure similarities within different protein folds, together with evidence from proteins containing sequence and structure repeats, argues in favor of the evolution of modern single polypeptide domains from ancient short peptide ancestors (antecedent domain segments (ADSs)). In this model, ancient protein structures were formed by self-assembling aggregates of short polypeptides. Subsequently, and perhaps concomitantly with the evolution of higher fidelity DNA replication and repair systems, single polypeptide domains arose from the fusion of ADSs genes. Thus modern protein domains may have a polyphyletic origin. 相似文献
8.
The cysteine desulfurase, IscS, is a highly conserved and essential component of the mitochondrial iron–sulfur cluster (ISC) system that serves as a sulfur donor for Fe–S clusters biogenesis. Fe–S clusters are versatile and labile cofactors of proteins that orchestrate a wide array of essential metabolic processes, such as energy generation and ribosome biogenesis. However, no information regarding the role of IscS or its regulation is available in Leishmania, an evolving pathogen model with rapidly developing drug resistance. In this study, we characterized LdIscS to investigate the ISC system in AmpB-sensitive vs resistant isolates of L. donovani and to understand its regulation. We observed an upregulated Fe–S protein activity in AmpB-resistant isolates but, in contrast to our expectations, LdIscS expression was upregulated in the sensitive strain. However, further investigations showed that LdIscS expression is positively correlated with ROS level and negatively correlated with Fe–S protein activity, independent of strain sensitivity. Thus, our results suggested that LdIscS expression is regulated by ROS level with Fe–S clusters/proteins acting as ROS sensors. Moreover, the direct evidence of a mechanism, in support of our results, is provided by dose-dependent induction of LdIscS-GFP as well as endogenous LdIscS in L. donovani promastigotes by three different ROS inducers: H2O2, menadione, and Amphotericin B. We postulate that LdIscS is upregulated for de novo synthesis or repair of ROS damaged Fe–S clusters. Our results reveal a novel mechanism for regulation of IscS expression that may help parasite survival under oxidative stress conditions encountered during infection of macrophages and suggest a cross talk between two seemingly unrelated metabolic pathways, the ISC system and redox metabolism in L. donovani. 相似文献
9.
Maja Martic Ida Noémi Jakab-Simon Lærke Tvedebrink Haahr Wilfred Raymond Hagen Hans Erik Mølager Christensen 《Journal of biological inorganic chemistry》2013,18(2):261-276
Heterometallic [AgFe3S4] iron–sulfur clusters assembled in wild-type Pyrococcus furiosus ferredoxin and two variants, D14C and D14H, are characterized. The crystal structure of the [AgFe3S4] D14C variant shows that the silver(I) ion is indeed part of the cluster and is coordinated to the thiolate group of residue 14. Cyclic voltammetry shows one redox pair with a reduction potential of +220 mV versus the standard hydrogen electrode which is assigned to the [AgFe3S4]2+/+ couple. The oxidized form of the [AgFe3S4] D14C variant is stable in the presence of dioxygen, whereas the oxidized forms of the [AgFe3S4] wild type and D14H variants convert to the [Fe3S4] ferredoxin form. The monovalent d 10 silver(I) ion stabilizes the [Fe3S4]+/0 cluster fragment, as opposed to divalent d 10 metal ions, resulting in more than 0.4 V difference in reduction potentials between the silver(I) and, e.g., zinc(II) heterometallic [MFe3S4] ferredoxins. The trend in reduction potentials for the variants containing the [AgFe3S4] cluster is wild type ≤ D14C < D14H and shows the same trend as reported for the variants containing the [Fe3S4] cluster, but is different from the D14C < D14H < wild type trend reported for the [Fe4S4] ferredoxin. The similarity in the reduction potential trend for the variants containing the heterometallic [AgFe3S4] cluster and the [Fe3S4] cluster can be rationalized in terms of the electrostatic influence of the residue 14 side chains, rather than the dissociation constant of this residue, as is the case for [Fe4S4] ferredoxins. The trends in reduction potentials are in line with there being no electronic coupling between the silver(I) ion and the Fe3S4 fragment. 相似文献
10.
Abroi A Gough J 《BioEssays : news and reviews in molecular, cellular and developmental biology》2011,33(8):626-635
A crucially important part of the biosphere - the virosphere - is too often overlooked. Inclusion of the virosphere into the global picture of protein structure space reveals that 63 protein domain superfamilies in viruses do not have any structural and evolutionary relatives in modern cellular organisms. More than half of these have functions which are not virus-specific and thus might be a source of new folds and functions for cellular life. The number of viruses on the planet exceeds that of cells by an order of magnitude and viruses evolve up to six orders of magnitude faster. As a result, cellular species are subject to a constitutive 'flow-through' of new viral genetic material. Due to this and the relaxed evolutionary constraints in viruses, the transfer of domains between host-to-virus could be a mechanism for accelerated protein evolution. The virosphere could be an engine for the genesis of protein structures, and may even have been so before the last universal common ancestor of cellular life. 相似文献
11.
The hallmark of a Type-I photosynthetic reaction center (RC) is the presence of three [4Fe–4S]2+/1+ clusters, named FX, FA, and FB that act as terminal electron acceptors. Their function is to increase the distance, and hence the lifetime, of the initial charge-separated state so that diffusion-mediated processes, such as the reduction of ferredoxin, can occur. Type-I homodimeric RCs, such as those found in heliobacteria, green-sulfur bacteria, and Candidatus Chloracidobacterium thermophilum, are less well understood than Photosystem I, the prototypical Type-I heterodimeric RC found in cyanobacteria and plants. Here, we review recent progress that has been made in elucidating the spectroscopic and biochemical properties of the bound Fe/S clusters and their cognate proteins in homodimeric Type-I RCs. In Heliobacterium modesticaldum, the identification and characterization of two loosely bound polypeptides, PshBI and PshBII that harbor the FA and FB clusters threatens to break the long-accepted assumption that Type-I RCs harbor one tightly bound FA/FB-containing protein. Additionally, the detection of the FX cluster in S = 1/2 and S = 3/2 ground spin states has resolved the long-standing issue of its missing EPR spectrum. In Chlorobaculum tepidum, the focus is on the biochemical properties of the unusual extrinsic Fe/S protein, PscB, which is readily dissociable from the RC core. The C-terminal domain of PscB is constructed as a bacterial ferredoxin, harboring the FA and FB clusters, but the N-terminal domain contains a number of PxxP motifs and is rich in Lys, Pro, and Ala residues, features characteristic of proteins that interact with SH3 domains. Little is known about Candidatus Chloracidobacterium thermophilum except that the photosynthetic RC is predicted to be a Type-I homodimer with an FX-binding site. These findings are placed in a context that promises to unify the acceptor side of homodimeric Type-I RCs in prokaryotic phototrophs. 相似文献
12.
John A. Kyndt John C. Fitch Robert E. Berry Matt C. Stewart Kevin Whitley Terry E. Meyer F. Ann Walker Michael A. Cusanovich 《BBA》2012,1817(5):811-818
A triad of tyrosine residues (Y152–154) in the cytochrome c1 subunit (C1) of the Rhodobacter capsulatus cytochrome bc1 complex (BC1) is ideally positioned to interact with cytochrome c2 (C2). Mutational analysis of these three tyrosines showed that, of the three, Y154 is the most important, since its mutation to alanine resulted in significantly reduced levels, destabilization, and inactivation of BC1. A second-site revertant of this mutant that regained photosynthetic capacity was found to have acquired two further mutations—A181T and A200V. The Y152Q mutation did not change the spectral or electrochemical properties of C1, and showed wild-type enzymatic C2 reduction rates, indicating that this mutation did not introduce major structural changes in C1 nor affect overall activity. Mutations Y153Q and Y153A, on the other hand, clearly affect the redox properties of C1 (e.g. by lowering the midpoint potential as much as 117 mV in Y153Q) and the activity by 90% and 50%, respectively. A more conservative Y153F mutant on the other hand, behaves similarly to wild-type. This underscores the importance of an aromatic residue at position Y153, presumably to maintain close packing with P184, which modeling indicates is likely to stabilize the sixth heme ligand conformation. 相似文献
13.
Iron–sulfur (Fe–S) clusters are the oldest and most versatile inorganic cofactors that are required to sustain fundamental life processes. Bacteria have three systems of [Fe–S] cluster biogenesis, designated ISC, NIF, and SUF. In contrast, the Thermus thermophiles HB8 has only one system, formed mostly by SUF homologs that contain six proteins: SufA, SufB, SufC, SufD, SufS and SufE. The kinetics of SufC ATPase was studied using a linked enzyme assay method. In the presence of SufB, SufD or SufBD complexes, the activity of SufC was enhanced. The cysteine desulfurase activity of SufS was also stimulated by the presence of the SufBCD complex. The results obtained through enzymology revealed that aconitase activity was activated by [Fe–S] clusters reconstituted on the SufBCD complex. Consolidated results from spectral and enzymatic analysis suggest that the SufBCD complex is a novel type of Fe–S scaffold system that can assemble Fe/S clusters de novo. 相似文献
14.
Delphine G. Bernard Daili J. A. Netz Thibaut J. Lagny Antonio J. Pierik Janneke Balk 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2013,368(1622)
The assembly of iron–sulfur (Fe–S) clusters requires dedicated protein factors inside the living cell. Striking similarities between prokaryotic and eukaryotic assembly proteins suggest that plant cells inherited two different pathways through endosymbiosis: the ISC pathway in mitochondria and the SUF pathway in plastids. Fe–S proteins are also found in the cytosol and nucleus, but little is known about how they are assembled in plant cells. Here, we show that neither plastid assembly proteins nor the cytosolic cysteine desulfurase ABA3 are required for the activity of cytosolic aconitase, which depends on a [4Fe–4S] cluster. In contrast, cytosolic aconitase activity depended on the mitochondrial cysteine desulfurase NFS1 and the mitochondrial transporter ATM3. In addition, we were able to complement a yeast mutant in the cytosolic Fe–S cluster assembly pathway, dre2, with the Arabidopsis homologue AtDRE2, but only when expressed together with the diflavin reductase AtTAH18. Spectroscopic characterization showed that purified AtDRE2 could bind up to two Fe–S clusters. Purified AtTAH18 bound one flavin per molecule and was able to accept electrons from NAD(P)H. These results suggest that the proteins involved in cytosolic Fe–S cluster assembly are highly conserved, and that dependence on the mitochondria arose before the second endosymbiosis event leading to plastids. 相似文献
15.
Meike Stelter Ana M. P. Melo Gudmundur O. Hreggvidsson Sigridur Hjorleifsdottir Lígia M. Saraiva Miguel Teixeira Margarida Archer 《Journal of biological inorganic chemistry》2010,15(3):303-313
The aerobic respiratory chain of the thermohalophilic bacterium Rhodothermus marinus, a nonphotosynthetic organism from the Bacteroidetes/Chlorobi group, contains a high-potential iron–sulfur protein (HiPIP) that transfers electrons from a bc 1 analog complex to a caa 3 oxygen reductase. Here, we describe the crystal structure of the reduced form of R. marinus HiPIP, solved by the single-wavelength anomalous diffraction method, based on the anomalous scattering of the iron atoms from the [4Fe–4S]3+/2+ cluster and refined to 1.0 Å resolution. This is the first structure of a HiPIP isolated from a nonphotosynthetic bacterium involved in an aerobic respiratory chain. The structure shows a similar environment around the cluster as the other HiPIPs from phototrophic bacteria, but reveals several features distinct from those of the other HiPIPs of phototrophic bacteria, such as a different fold of the N-terminal region of the polypeptide due to a disulfide bridge and a ten-residue-long insertion. 相似文献
16.
《Inorganica chimica acta》1988,154(1):59-62
Magnetic Circular dichroism (MCD) spectra were obtained for bis(o-xylyl-dithiolato)ferrate(III) ([Fe(S2-o-xylyl)2]−) and bis[o-xylyl-dithiolato-μ2-sulfidoferrate(III)] ([Fe2S*2(S2-o-xylyl)2]2−) ions. The MCD magnitude of the dimeric [Fe2S*2(S2-o-xylyl)2]2− ion was found to be only one half of that for the monomeric [Fe(S2-o-xylyl)2]− ion. The difference in MCD magnitudes was attributed to the change in the thermal populations of ground state sublevels derived from the magnetic exchange interaction. 相似文献
17.
Yasser Mohseni Behbahani Paul Saighi Flavia Corsi Elodie Laine Alessandra Carbone 《Proteomics》2023,23(17):2200159
Physical interactions between proteins are central to all biological processes. Yet, the current knowledge of who interacts with whom in the cell and in what manner relies on partial, noisy, and highly heterogeneous data. Thus, there is a need for methods comprehensively describing and organizing such data. LEVELNET is a versatile and interactive tool for visualizing, exploring, and comparing protein–protein interaction (PPI) networks inferred from different types of evidence. LEVELNET helps to break down the complexity of PPI networks by representing them as multi-layered graphs and by facilitating the direct comparison of their subnetworks toward biological interpretation. It focuses primarily on the protein chains whose 3D structures are available in the Protein Data Bank. We showcase some potential applications, such as investigating the structural evidence supporting PPIs associated to specific biological processes, assessing the co-localization of interaction partners, comparing the PPI networks obtained through computational experiments versus homology transfer, and creating PPI benchmarks with desired properties. 相似文献
18.
Counts of acidophilic iron‐oxidizing bacteria, ratios of S2O3=—S/SO4=—S and Fe+3/Fe+2, and S2O3=—S oxidation potentials were examined over a two‐year period in coal refuse (acid gob) treated with limestone and/or sewage sludge. A non‐amended treatment was used as a control. No significant difference in population counts of acidophilic iron‐oxidizing bacteria were observed between treatments in either year of the study. S2O3=—S/SO4=—S and Fe+3/Fe+2 ratios indicated active sulfur and iron oxidation suggesting that limestone and/or sewage sludge may be ineffective in suppressing pyrite oxidation. Under optimal conditions, S2O3=—S oxidation potentials (in vitro) showed a logarithmic increase in SO4=—S formation for all four treatments over time. The final pH of the treatments following twenty days of perfusion ranged from 3.06 to 3.59. 相似文献
19.
An original experimental method of direct molecular fishing has been developed for identification of potential partners of protein–protein and protein–peptide interactions. It is based on combination of surface plasmon resonance technology (SPR), size exclusion and affinity chromatography and mass spectrometric identification of proteins (LC-MS/MS). Previously, we demonstrated applicability of this method for protein interactomics using experimental model system, as well as in the pilot study in the frame of the Human Proteome Project (HPP). In the present paper, this method was successfully applied to identify possible molecular partners of 7 target proteins encoded by genes of 18 chromosome (also in the frame of the HPP). Fishing on the affinity sorbents with immobilized target proteins as ligands was carried out using total lysate of human liver tissue as well as pooled sets of fractions (individual for each bait-protein) obtained by means of a combination of size exclusion chromatography and SPR analysis for the presence of potential prey-proteins in each fraction. As a result we obtained lists of possible molecular partners of all 7 proteins and performed a comparative evaluation of direct fishing specificity for these target proteins. Direct molecular fishing was also successfully used for search of potential protein partners interacting with different isoforms of amyloid-beta peptide, playing a key role in the development of Alzheimer’s disease. The synthetic peptides that are analogues of the metal-binding domain isoforms of beta-amyloid were used as molecular baits and the fishing was performed in various fractions of immortalized human neural cells. As a result, 13 potential partner proteins were identified in the cytosol fraction of the cells by fishing on amyloid-beta peptide (1-16). 相似文献
20.
A key element in eukaryotic immune defenses against invading microbes is the production of reactive oxygen and nitrogen species.
One of the main targets of these species are the iron–sulfur clusters, which are essential prosthetic groups that confer to
proteins the ability to perform crucial roles in biological processes. Microbes have developed sophisticated systems to eliminate
nitrosative and oxidative species and promote the repair of the damages inflicted. The Ric (Repair of Iron Centers) proteins
constitute a novel family of microbial di-iron proteins with a widespread distribution among microbes, including Gram-positive
and Gram-negative bacteria, protozoa and fungi. The Ric proteins are encoded by genes that are up-regulated by nitric oxide
and hydrogen peroxide. Recent studies have shown that the active di-iron center is involved in the restoration of Fe–S clusters
damaged by exposure to nitric oxide and hydrogen peroxide. 相似文献