Articoli di riviste: "[4Fe-4S]2+"

1

Azam, Tamanna, Jonathan Przybyla-Toscano, Florence Vignols, Jérémy Couturier, Nicolas Rouhier e Michael K. Johnson. "[4Fe-4S] cluster trafficking mediated by Arabidopsis mitochondrial ISCA and NFU proteins". Journal of Biological Chemistry 295, n. 52 (29 ottobre 2020): 18367–78. http://dx.doi.org/10.1074/jbc.ra120.015726.

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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.

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Duan, Xuewu, Juanjuan Yang, Binbin Ren, Guoqiang Tan e Huangen Ding. "Reactivity of nitric oxide with the [4Fe–4S] cluster of dihydroxyacid dehydratase from Escherichia coli". Biochemical Journal 417, n. 3 (16 gennaio 2009): 783–89. http://dx.doi.org/10.1042/bj20081423.

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Although the NO (nitric oxide)-mediated modification of iron–sulfur proteins has been well-documented in bacteria and mammalian cells, specific reactivity of NO with iron–sulfur proteins still remains elusive. In the present study, we report the first kinetic characterization of the reaction between NO and iron–sulfur clusters in protein using the Escherichia coli IlvD (dihydroxyacid dehydratase) [4Fe–4S] cluster as an example. Combining a sensitive NO electrode with EPR (electron paramagnetic resonance) spectroscopy and an enzyme activity assay, we demonstrate that NO is rapidly consumed by the IlvD [4Fe–4S] cluster with the concomitant formation of the IlvD-bound DNIC (dinitrosyl–iron complex) and inactivation of the enzyme activity under anaerobic conditions. The rate constant for the initial reaction between NO and the IlvD [4Fe–4S] cluster is estimated to be (7.0±2.0)×106 M−2·s−1 at 25 °C, which is approx. 2–3 times faster than that of the NO autoxidation by O2 in aqueous solution. Addition of GSH failed to prevent the NO-mediated modification of the IlvD [4Fe–4S] cluster regardless of the presence of O2 in the medium, further suggesting that NO is more reactive with the IlvD [4Fe–4S] cluster than with GSH or O2. Purified aconitase B [4Fe–4S] cluster from E. coli has an almost identical NO reactivity as the IlvD [4Fe–4S] cluster. However, the reaction between NO and the endonuclease III [4Fe–4S] cluster is relatively slow, apparently because the [4Fe–4S] cluster in endonuclease III is less accessible to solvent than those in IlvD and aconitase B. When E. coli cells containing recombinant IlvD, aconitase B or endonuclease III are exposed to NO using the Silastic tubing NO delivery system under aerobic and anaerobic conditions, the [4Fe–4S] clusters in IlvD and aconitase B, but not in endonuclease III, are efficiently modified forming the protein-bound DNICs, confirming that NO has a higher reactivity with the [4Fe–4S] clusters in IlvD and aconitase B than with O2 or GSH. The results suggest that the iron–sulfur clusters in proteins such as IlvD and aconitase B may constitute the primary targets of the NO cytotoxicity under both aerobic and anaerobic conditions.

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Sutton, Victoria R., Erin L. Mettert, Helmut Beinert e Patricia J. Kiley. "Kinetic Analysis of the Oxidative Conversion of the [4Fe-4S]2+ Cluster of FNR to a [2Fe-2S]2+ Cluster". Journal of Bacteriology 186, n. 23 (1 dicembre 2004): 8018–25. http://dx.doi.org/10.1128/jb.186.23.8018-8025.2004.

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ABSTRACT The ability of FNR to sense and respond to cellular O2 levels depends on its [4Fe-4S]2+ cluster. In the presence of O2, the [4Fe-4S]2+ cluster is converted to a [2Fe-2S]2+ cluster, which inactivates FNR as a transcriptional regulator. In this study, we demonstrate that ∼2 Fe2+ ions are released from the reaction of O2 with the [4Fe-4S]2+ cluster. Fe2+ release was then used as an assay of reaction progress to investigate the rate of [4Fe-4S]2+ to [2Fe-2S]2+ cluster conversion in vitro. We also found that there was no detectable difference in the rate of O2-induced cluster conversion for FNR free in solution compared to its DNA-bound form. In addition, the rate of FNR inactivation was monitored in vivo by measuring the rate at which transcriptional regulation by FNR is lost upon the exposure of cells to O2; a comparison of the in vitro and in vivo rates of conversion suggests that O2-induced cluster conversion is sufficient to explain FNR inactivation in cells. FNR protein levels were also compared for cells grown under aerobic and anaerobic conditions.

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George, S. J., F. A. Armstrong, E. C. Hatchikian e A. J. Thomson. "Electrochemical and spectroscopic characterization of the conversion of the 7Fe into the 8Fe form of ferredoxin III from Desulfovibrio africanus. Identification of a [4Fe–4S] cluster with one non-cysteine ligand". Biochemical Journal 264, n. 1 (15 novembre 1989): 275–84. http://dx.doi.org/10.1042/bj2640275.

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Desulfovibrio africanus ferredoxin III is a protein (Mr 6585) containing one [3Fe-4S]1+,0 and one [4Fe-4S]2+,1+ core cluster when aerobically isolated. The amino acid sequence contains only seven cysteine residues, the minimum required to ligand these two clusters. Cyclic voltammery by means of direct electrochemistry at a pyrolytic-graphite-‘edge’ electrode promoted by neomycin shows that, when reduced, the [3Fe-4S]0 centre reacts rapidly with Fe(II) ion to form a [4Fe-4S]2+ cluster. The latter, which can be reduced at a redox potential similar to that of the other [4Fe-4S] cluster, must include non-thiolate ligation. We propose that the carboxylate side chain of aspartic acid-14 is the most likely candidate, since this amino acid occupies the position of a cysteine residue in the sequence typical of an 8Fe ferredoxin. The magnetic properties at liquid-He temperature of this novel cluster, studied by low-temperature magnetic-c.d. and e.p.r. spectroscopy, are diamagnetic in the oxidized state and S = 3/2 in the one-electron-reduced state. This cluster provides a plausible model for the ligation states of the [4Fe-4S]1+ core in the S = 3/2 cluster of the iron protein of nitrogenase and in Bacillus subtilis glutamine:phosphoribosyl pyrophosphate amidotransferase.

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5

Azam, Tamanna, Jonathan Przybyla-Toscano, Florence Vignols, Jérémy Couturier, Nicolas Rouhier e Michael K. Johnson. "The Arabidopsis Mitochondrial Glutaredoxin GRXS15 Provides [2Fe-2S] Clusters for ISCA-Mediated [4Fe-4S] Cluster Maturation". International Journal of Molecular Sciences 21, n. 23 (3 dicembre 2020): 9237. http://dx.doi.org/10.3390/ijms21239237.

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Iron-sulfur (Fe-S) proteins are crucial for many cellular functions, particularly those involving electron transfer and metabolic reactions. An essential monothiol glutaredoxin GRXS15 plays a key role in the maturation of plant mitochondrial Fe-S proteins. However, its specific molecular function is not clear, and may be different from that of the better characterized yeast and human orthologs, based on known properties. Hence, we report here a detailed characterization of the interactions between Arabidopsis thaliana GRXS15 and ISCA proteins using both in vivo and in vitro approaches. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that GRXS15 interacts with each of the three plant mitochondrial ISCA1a/1b/2 proteins. UV-visible absorption/CD and resonance Raman spectroscopy demonstrated that coexpression of ISCA1a and ISCA2 resulted in samples with one [2Fe-2S]2+ cluster per ISCA1a/2 heterodimer, but cluster reconstitution using as-purified [2Fe-2S]-ISCA1a/2 resulted in a [4Fe-4S]2+ cluster-bound ISCA1a/2 heterodimer. Cluster transfer reactions monitored by UV-visible absorption and CD spectroscopy demonstrated that [2Fe-2S]-GRXS15 mediates [2Fe-2S]2+ cluster assembly on mitochondrial ferredoxin and [4Fe-4S]2+ cluster assembly on the ISCA1a/2 heterodimer in the presence of excess glutathione. This suggests that ISCA1a/2 is an assembler of [4Fe-4S]2+ clusters, via two-electron reductive coupling of two [2Fe-2S]2+ clusters. Overall, the results provide new insights into the roles of GRXS15 and ISCA1a/2 in effecting [2Fe-2S]2+ to [4Fe-4S]2+ cluster conversions for the maturation of client [4Fe-4S] cluster-containing proteins in plants.

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Dridge, Elizabeth J., Carys A. Watts, Brian J. N. Jepson, Kirsty Line, Joanne M. Santini, David J. Richardson e Clive S. Butler. "Investigation of the redox centres of periplasmic selenate reductase from Thauera selenatis by EPR spectroscopy". Biochemical Journal 408, n. 1 (29 ottobre 2007): 19–28. http://dx.doi.org/10.1042/bj20070669.

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Periplasmic SER (selenate reductase) from Thauera selenatis is classified as a member of the Tat (twin-arginine translocase)-translocated (Type II) molybdoenzymes and comprises three subunits each containing redox cofactors. Variable-temperature X-band EPR spectra of the purified SER complex showed features attributable to centres [3Fe–4S]1+, [4Fe–4S]1+, Mo(V) and haem-b. EPR-monitored redox-potentiometric titration of the SerABC complex (SerA–SerB–SerC, a hetero-trimetric complex of αβγ subunits) revealed that the [3Fe–4S] cluster (FS4, iron-sulfur cluster 4) titrated as n=1 Nernstian component with a midpoint redox potential (Em) of +118±10 mV for the [3Fe–4S]1+/0 couple. A [4Fe–4S]1+ cluster EPR signal developed over a range of potentials between 300 and −200 mV and was best fitted to two sequential Nernstian n=1 curves with midpoint redox potentials of +183±10 mV (FS1) and −51±10 mV (FS3) for the two [4Fe–4S]1+/2+ cluster couples. Upon further reduction, the observed signal intensity of the [4Fe–4S]1+ cluster decreases. This change in intensity can again be fitted to an n=1 Nernstian component with a midpoint potential (Em) of about −356 mV (FS2). It is considered likely that, at low redox potential (Em less than −300 mV), the remaining oxidized cluster is reduced (spin S=1/2) and strongly spin-couples to a neighbouring [4Fe–4S]1+ cluster rendering both centres EPR-silent. The involvement of both [3Fe–4S] and [4Fe–4S] clusters in electron transfer to the active site of the periplasmic SER was demonstrated by the re-oxidation of the clusters under anaerobic selenate turnover conditions. Attempts to detect a high-spin [4Fe–4S] cluster (FS0) in SerA at low temperature (5 K) and high power (100 mW) were unsuccessful. The Mo(V) EPR recorded at 60 K, in samples poised at pH 6.0, displays principal g values of g3∼1.999, g2∼1.996 and g1∼1.965 (gav 1.9867). The dominant features at g2 and g3 are not split, but hyperfine splitting is observed in the g1 region of the spectrum and can be best simulated as arising from a single proton with a coupling constant of A1 (1H)=1.014 mT. The presence of the haem-b moiety in SerC was demonstrated by the detection of a signal at g∼3.33 and is consistent with haem co-ordinated by methionine and lysine axial ligands. The combined evidence from EPR analysis and sequence alignments supports the assignment of the periplasmic SER as a member of the Type II molybdoenzymes and provides the first spectro-potentiometric insight into an enzyme that catalyses a key reductive reaction in the biogeochemical selenium cycle.

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BUSCH, Johanneke L. H., Jacques L. BRETON, Barry M. BARTLETT, Fraser A. ARMSTRONG, Richard JAMES e Andrew J. THOMSON. "[3Fe-4S]↔[4Fe-4S] cluster interconversion in Desulfovibrio africanus ferredoxin III: properties of an Asp14→Cys mutant". Biochemical Journal 323, n. 1 (1 aprile 1997): 95–102. http://dx.doi.org/10.1042/bj3230095.

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The 8Fe ferredoxin III from Desulfovibrio africanus is a monomeric protein which contains two [4Fe-4S]2+/1+ clusters, one of which is labile and can readily and reversibly lose one Fe under oxidative conditions to yield a [3Fe-4S]1+/0 cluster. This 4Fe cluster has an S = 3/2 ground spin state instead of S = 1/2 in the reduced +1 state [George, Armstrong, Hatchikian and Thomson (1989) Biochem. J.264, 275-284]. The co-ordination to this cluster is unusual in that an aspartate (Asp14, D14) is found where a cysteine residue normally occurs. Using a mutant protein obtained from the overexpression in Escherichia coli of a synthetic gene in which Asp14, the putative ligand to the removable Fe, has been changed to Cys, we have studied the cluster interconversion properties of the labile cluster. Analysis by EPR and magnetic-circular-dichroism spectroscopies showed that the Asp14 → Cys (D14C) mutant contains two [4Fe-4S]2+/1+ clusters, both with S = 1/2 in the reduced state. Also, unlike in native 8Fe D. africanus ferredoxin III, the 4Fe ↔ 3Fe cluster interconversion reaction was found to be sluggish and did not go to completion. It is inferred that the reversibility of the reaction in the native protein is due to the presence of the aspartate residue at position 14 and that this residue might protect the [3Fe-4S] cluster from further degradation.

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Smith, Eugene T., Dennis W. Bennett e Benjamin A. Feinberg. "Redox properties of 2[4Fe4S] ferredoxins". Analytica Chimica Acta 251, n. 1-2 (ottobre 1991): 27–33. http://dx.doi.org/10.1016/0003-2670(91)87111-j.

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9

Roland, Mélanie, Jonathan Przybyla-Toscano, Florence Vignols, Nathalie Berger, Tamanna Azam, Loick Christ, Véronique Santoni et al. "The plastidial Arabidopsis thaliana NFU1 protein binds and delivers [4Fe-4S] clusters to specific client proteins". Journal of Biological Chemistry 295, n. 6 (6 gennaio 2020): 1727–42. http://dx.doi.org/10.1074/jbc.ra119.011034.

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Proteins incorporating iron–sulfur (Fe-S) co-factors are required for a plethora of metabolic processes. Their maturation depends on three Fe-S cluster assembly machineries in plants, located in the cytosol, mitochondria, and chloroplasts. After de novo formation on scaffold proteins, transfer proteins load Fe-S clusters onto client proteins. Among the plastidial representatives of these transfer proteins, NFU2 and NFU3 are required for the maturation of the [4Fe-4S] clusters present in photosystem I subunits, acting upstream of the high-chlorophyll fluorescence 101 (HCF101) protein. NFU2 is also required for the maturation of the [2Fe-2S]-containing dihydroxyacid dehydratase, important for branched-chain amino acid synthesis. Here, we report that recombinant Arabidopsis thaliana NFU1 assembles one [4Fe-4S] cluster per homodimer. Performing co-immunoprecipitation experiments and assessing physical interactions of NFU1 with many [4Fe-4S]-containing plastidial proteins in binary yeast two-hybrid assays, we also gained insights into the specificity of NFU1 for the maturation of chloroplastic Fe-S proteins. Using bimolecular fluorescence complementation and in vitro Fe-S cluster transfer experiments, we confirmed interactions with two proteins involved in isoprenoid and thiamine biosynthesis, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase and 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase, respectively. An additional interaction detected with the scaffold protein SUFD enabled us to build a model in which NFU1 receives its Fe-S cluster from the SUFBC2D scaffold complex and serves in the maturation of specific [4Fe-4S] client proteins. The identification of the NFU1 partner proteins reported here more clearly defines the role of NFU1 in Fe-S client protein maturation in Arabidopsis chloroplasts among other SUF components.

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Stripp, Sven T., Jonathan Oltmanns, Christina S. Müller, David Ehrenberg, Ramona Schlesinger, Joachim Heberle, Lorenz Adrian, Volker Schünemann, Antonio J. Pierik e Basem Soboh. "Electron inventory of the iron-sulfur scaffold complex HypCD essential in [NiFe]-hydrogenase cofactor assembly". Biochemical Journal 478, n. 17 (7 settembre 2021): 3281–95. http://dx.doi.org/10.1042/bcj20210224.

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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.

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Nzuza, Nomfundo, Tiara Padayachee, Wanping Chen, Dominik Gront, David R. Nelson e Khajamohiddin Syed. "Diversification of Ferredoxins across Living Organisms". Current Issues in Molecular Biology 43, n. 3 (30 settembre 2021): 1374–90. http://dx.doi.org/10.3390/cimb43030098.

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Ferredoxins, iron-sulfur (Fe-S) cluster proteins, play a key role in oxidoreduction reactions. To date, evolutionary analysis of these proteins across the domains of life have been confined to observing the abundance of Fe-S cluster types (2Fe-2S, 3Fe-4S, 4Fe-4S, 7Fe-8S (3Fe-4s and 4Fe-4S) and 2[4Fe-4S]) and the diversity of ferredoxins within these cluster types was not studied. To address this research gap, here we propose a subtype classification and nomenclature for ferredoxins based on the characteristic spacing between the cysteine amino acids of the Fe-S binding motif as a subtype signature to assess the diversity of ferredoxins across the living organisms. To test this hypothesis, comparative analysis of ferredoxins between bacterial groups, Alphaproteobacteria and Firmicutes and ferredoxins collected from species of different domains of life that are reported in the literature has been carried out. Ferredoxins were found to be highly diverse within their types. Large numbers of alphaproteobacterial species ferredoxin subtypes were found in Firmicutes species and the same ferredoxin subtypes across the species of Bacteria, Archaea, and Eukarya, suggesting shared common ancestral origin of ferredoxins between Archaea and Bacteria and lateral gene transfer of ferredoxins from prokaryotes (Archaea/Bacteria) to eukaryotes. This study opened new vistas for further analysis of diversity of ferredoxins in living organisms.

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Crack, Jason C., Adrian J. Jervis, Alisa A. Gaskell, Gaye F. White, Jeffrey Green, Andrew J. Thomson e Nick E. Le Brun. "Signal perception by FNR: the role of the iron–sulfur cluster1". Biochemical Society Transactions 36, n. 6 (19 novembre 2008): 1144–48. http://dx.doi.org/10.1042/bst0361144.

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The metabolic flexibility of bacteria is key to their ability to survive and thrive in a wide range of environments. Optimal switching from one metabolic pathway to another is a key requirement for this flexibility. Respiration is a good example: many bacteria utilize O2 as the terminal electron acceptor, but can switch to a range of other acceptors, such as nitrate, when O2 becomes limiting. Sensing environmental levels of O2 is the key step in switching from aerobic to anaerobic respiration. In Escherichia coli, the fumarate and nitrate reduction transcriptional regulator (FNR) controls this switch. Under O2-limiting conditions, FNR binds a [4Fe–4S]2+ cluster, generating a transcriptionally active dimeric form. Exposure to O2 results in conversion of the cluster into a [2Fe–2S]2+ form, leading to dissociation of the protein into inactive monomers. The mechanism of cluster conversion, together with the nature of the reaction products, is of considerable current interest, and a near-complete description of the process has now emerged. The [4Fe–4S]2+ into [2Fe–2S]2+ cluster conversion proceeds via a two-step mechanism. In step 1, a one-electron oxidation of the cluster takes place, resulting in the release of a Fe2+ ion, the formation of an intermediate [3Fe–4S]1+ cluster, together with the generation of a superoxide anion. In step 2, the intermediate [3Fe–4S]1+ cluster rearranges spontaneously to form the [2Fe–2S]2+ cluster, releasing two sulfide ions and an Fe3+ ion in the process. The one-electron activation of the cluster, coupled to catalytic recycling of the superoxide anion back to oxygen via superoxide dismutase and catalase, provides a novel means of amplifying the sensitivity of [4Fe–4S]2+ FNR to its signal molecule.

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Szilagyi, Robert K., Rebecca Hanscam, Eric M. Shepard e Shawn E. McGlynn. "Natural selection based on coordination chemistry: computational assessment of [4Fe–4S]-maquettes with non-coded amino acids". Interface Focus 9, n. 6 (18 ottobre 2019): 20190071. http://dx.doi.org/10.1098/rsfs.2019.0071.

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Cysteine is the only coded amino acid in biology that contains a thiol functional group. Deprotonated thiolate is essential for anchoring iron–sulfur ([Fe–S]) clusters, as prosthetic groups to the protein matrix. [Fe–S] metalloproteins and metalloenzymes are involved in biological electron transfer, radical chemistry, small molecule activation and signalling. These are key metabolic and regulatory processes that would likely have been present in the earliest organisms. In the context of emergence of life theories, the selection and evolution of the cysteine-specific R–CH 2 –SH side chain is a fascinating question to confront. We undertook a computational [4Fe–4S]-maquette modelling approach to evaluate how side chain length can influence [Fe–S] cluster binding and stability in short 7-mer and long 16-mer peptides, which contained either thioglycine, cysteine or homocysteine. Force field-based molecular dynamics simulations for [4Fe–4S] cluster nest formation were supplemented with density functional theory calculations of a ligand-exchange reaction between a preassembled cluster and the peptide. Secondary structure analysis revealed that peptides with cysteine are found with greater frequency nested to bind preformed [4Fe–4S] clusters. Additionally, the presence of the single methylene group in cysteine ligands mitigates the steric bulk, maintains the H-bonding and dipole network, and provides covalent Fe–S(thiolate) bonds that together create the optimal electronic and geometric structural conditions for [4Fe–4S] cluster binding compared to thioglycine or homocysteine ligands. Our theoretical work forms an experimentally testable hypothesis of the natural selection of cysteine through coordination chemistry.

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Buckel, Wolfgang, Berta M. Martins, Albrecht Messerschmidt e Bernard T. Golding. "Radical-mediated dehydration reactions in anaerobic bacteria". Biological Chemistry 386, n. 10 (1 ottobre 2005): 951–59. http://dx.doi.org/10.1515/bc.2005.111.

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AbstractMost dehydratases catalyse the elimination of water from β-hydroxy ketones, β-hydroxy carboxylic acids or β-hydroxyacyl-CoA. The electron-withdrawing carbonyl functionalities acidify the α-hydrogens to enable their removal by basic amino acid side chains. Anaerobic bacteria, however, ferment amino acids via α- or γ-hydroxyacyl-CoA, dehydrations of which involve the abstraction of a β-hydrogen, which is ostensibly non-acidic (pKca. 40). Evidence is accumulating that β-hydrogens are acidified via transient conversion of the CoA derivatives to enoxy radicals by one-electron transfers, which decrease the pKto 14. The dehydrations of (R)-2-hydroxyacyl-CoA to (E)-2-enoyl-CoA are catalysed by heterodimeric [4Fe-4S]-containing dehydratases, which require reductive activation by an ATP-dependent one-electron transfer mediated by a homodimeric protein with a [4Fe-4S] cluster between the two subunits. The electron is further transferred to the substrate, yielding a ketyl radical anion, which expels the hydroxyl group and forms an enoxy radical. The dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA involves a similar mechanism, in which the ketyl radical anion is generated by one-electron oxidation. The structure of the FAD- and [4Fe-4S]-containing homotetrameric dehydratase is related to that of acyl-CoA dehydrogenases, suggesting a radical-based mechanism for both flavoproteins.

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GREEN, Jeffrey, Brian BENNETT, Peter JORDAN, Edward T. RALPH, Andrew J. THOMSON e John R. GUEST. "Reconstitution of the [4Fe-4S] cluster in FNR and demonstration of the aerobic-anaerobic transcription switch in vitro". Biochemical Journal 316, n. 3 (15 giugno 1996): 887–92. http://dx.doi.org/10.1042/bj3160887.

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The FNR protein of Escherichia coli is a redox-responsive transcription regulator that activates and represses a family of genes required for anaerobic and aerobic metabolism. Reconstitution of wild-type FNR by anaerobic treatment with ferrous ions, cysteine and the NifS protein of Azotobacter vinelandii leads to the incorporation of two [4Fe-4S]2+ clusters per FNR dimer. The UV–visible spectrum of reconstituted FNR has a broad absorbance at 420 nm. The clusters are EPR silent under anaerobic conditions but are degraded to [3Fe-4S]+ by limited oxidation with air, and completely lost on prolonged air exposure. The association of FNR with the iron–sulphur clusters is confirmed by CD spectroscopy. Incorporation of the [4Fe-4S]2+ clusters increases site-specific DNA binding about 7-fold compared with apo-FNR. Anaerobic transcription activation and repression in vitro likewise depends on the presence of the iron–sulphur cluster, and its inactivation under aerobic conditions provides a demonstration in vitro of the FNR-mediated aerobic–anaerobic transcriptional switch.

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Moulis, Jean-Marc, Larry C. Sieker, Keith S. Wilson e Zbigniew Dauter. "Crystal structure of the 2[4Fe-4S] ferredoxin fromChromatium vinosum: Evolutionary and mechanistic inferences for [3/4Fe-4S] ferredoxins". Protein Science 5, n. 9 (settembre 1996): 1765–75. http://dx.doi.org/10.1002/pro.5560050902.

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17

BUSCH, Johanneke L. H., Jacques L. J. BRETON, Barry M. BARTLETT, Richard JAMES, E. Claude HATCHIKIAN e Andrew J. THOMSON. "Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus". Biochemical Journal 314, n. 1 (15 febbraio 1996): 63–71. http://dx.doi.org/10.1042/bj3140063.

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Abstract (sommario):

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of D. africanus ferredoxin III, we have overexpressed it in Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in E. coli as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type D. africanus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]1+/0 and a [4Fe-4S]2+/1+ cluster similar to those found in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin.

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18

Armstrong, F. A., S. J. George, R. Cammack, E. C. Hatchikian e A. J. Thomson. "Electrochemical and spectroscopic characterization of the 7Fe form of ferredoxin III from Desulfovibrio africanus". Biochemical Journal 264, n. 1 (15 novembre 1989): 265–73. http://dx.doi.org/10.1042/bj2640265.

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Abstract (sommario):

Desulfovibrio africanus ferredoxin III is a monomeric protein (Mr 6585) containing seven cysteine residues and 7-8 iron atoms and 6-8 atoms of acid-labile sulphur. It is shown that reversible unmediated electrochemistry of the two iron-sulphur clusters can be obtained by using a pyrolytic-graphite-‘edge’ carbon electrode in the presence of an appropriate aminoglycoside, neomycin or tobramycin, as promoter. Cyclic voltammetry reveals two well-defined reversible waves with E0′ = -140 +/- 10 mV and -410 +/- 5 mV (standard hydrogen electrode) at 2 degrees C. Bulk reduction confirms that each of these corresponds to a one-electron process. Low-temperature e.p.r. and magnetic-c.d. spectroscopy identify the higher-potential redox couple with a cluster of core [3Fe-4S]1+.0 and the lower with a [4Fe-4S]2+.1+ centre. The low-temperature magnetic-c.d. spectra and magnetization properties of the three-iron cluster show that it is essentially identical with that in Desulfovibrio gigas ferredoxin II. We assign cysteine-11, -17 and -51 as ligands of the [3Fe-4S] core and cysteine-21, -41, -44 and -47 to the [4Fe-4S] centre.

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19

BUSCH, Johanneke L. H., Jacques L. BRETON, Sharon L. DAVY, Richard JAMES, Geoffrey R. MOORE, Fraser A. ARMSTRONG e Andrew J. THOMSON. "Ferredoxin III of Desulfovibrio africanus: sequencing of the native gene and characterization of a histidine-tagged form". Biochemical Journal 346, n. 2 (22 febbraio 2000): 375–84. http://dx.doi.org/10.1042/bj3460375.

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Abstract (sommario):

Desulfovibrio africanus ferredoxin III (Da FdIII) contains one [4Fe-4S]2+/1+ cluster and one [3Fe-4S]1+/0 cluster, bound by seven Cys residues, in which the [3Fe-4S] cluster is co-ordinated by the unusual sequence, Cys11-Xaa-Xaa-Asp14-Xaa-Xaa-Cys17-Xaan-Cys51-Glu. The [3Fe-4S] core of this ferredoxin is so far unique in showing rapid bi-directional [3Fe-4S] ↔ [4Fe-4S] cluster interconversion with a wide range of metal ions. In order to obtain protein for mutagenesis studies Da FdIII has been cloned, sequenced, and expressed as a hexa-histidine tagged (ht) polypeptide in Escherichia coli strain BL21(DE3) pLysS. Expression of ht Da FdIII, whether translated from a synthetic gene (pJB10) or from the native nucleotide sequence (pJB11), occurred at similar levels (approx. 6 mg·l-1), but without incorporation of metal clusters. The nucleotide sequence confirms the protein sequence reported previously [Bovier-Lapierre, Bruschi, Bonicel and Hatchikian (1987) Biochim. Biophys. Acta 913, 20-26]. Cluster incorporation was achieved using FeCl3 together with cysteine sulphur transferase, NifS, plus cysteine to generate low levels of sulphide ions. Absorption and EPR spectroscopy show that both [3Fe-4S] and [4Fe-4S] clusters are correctly inserted. Thin-film electrochemistry provides evidence that the [3Fe-4S] cluster undergoes reversible cluster transformation in the presence of Fe(II) and Zn(II) ions with properties identical to the native protein. Nevertheless the protein has lower stability than native Da FdIII during chromatography. The one-dimensional 600 MHz NMR spectrum of the apoprotein indicates an unstructured protein with random coil chemical shifts whereas spectra of the reconstituted ht protein show secondary structural elements and 18 peaks shifted downfield of 9.6 p.p.m. The spectra are unique but have similarities with the shift patterns seen with 7Fe Desulfurolobus ambivalens Fd. The ht does not affect iron-sulphur cluster incorporation, but NMR evidence suggests that excess Fe binds to the tag. This may account for the lower stability of the ht compared with the native protein.

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20

Chen, Minghao, Shin-ichi Asai, Shun Narai, Shusuke Nambu, Naoki Omura, Yuriko Sakaguchi, Tsutomu Suzuki et al. "Biochemical and structural characterization of oxygen-sensitive 2-thiouridine synthesis catalyzed by an iron-sulfur protein TtuA". Proceedings of the National Academy of Sciences 114, n. 19 (24 aprile 2017): 4954–59. http://dx.doi.org/10.1073/pnas.1615585114.

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Abstract (sommario):

Two-thiouridine (s2U) at position 54 of transfer RNA (tRNA) is a posttranscriptional modification that enables thermophilic bacteria to survive in high-temperature environments. s2U is produced by the combined action of two proteins, 2-thiouridine synthetase TtuA and 2-thiouridine synthesis sulfur carrier protein TtuB, which act as a sulfur (S) transfer enzyme and a ubiquitin-like S donor, respectively. Despite the accumulation of biochemical data in vivo, the enzymatic activity by TtuA/TtuB has rarely been observed in vitro, which has hindered examination of the molecular mechanism of S transfer. Here we demonstrate by spectroscopic, biochemical, and crystal structure analyses that TtuA requires oxygen-labile [4Fe-4S]-type iron (Fe)-S clusters for its enzymatic activity, which explains the previously observed inactivation of this enzyme in vitro. The [4Fe-4S] cluster was coordinated by three highly conserved cysteine residues, and one of the Fe atoms was exposed to the active site. Furthermore, the crystal structure of the TtuA-TtuB complex was determined at a resolution of 2.5 Å, which clearly shows the S transfer of TtuB to tRNA using its C-terminal thiocarboxylate group. The active site of TtuA is connected to the outside by two channels, one occupied by TtuB and the other used for tRNA binding. Based on these observations, we propose a molecular mechanism of S transfer by TtuA using the ubiquitin-like S donor and the [4Fe-4S] cluster.

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21

Scott, Anna G., Robert K. Szilagyi, David W. Mulder, Michael W. Ratzloff, Amanda S. Byer, Paul W. King, William E. Broderick, Eric M. Shepard e Joan B. Broderick. "Compositional and structural insights into the nature of the H-cluster precursor on HydF". Dalton Transactions 47, n. 28 (2018): 9521–35. http://dx.doi.org/10.1039/c8dt01654b.

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Abstract (sommario):

Spectroscopic and computational characterization of loaded HydF reveals that the 2Fe subcluster is a coordinatively saturated Fe(i)–Fe(i) species that contains 4 CO and 2 CN ligands, and is anchored to HydF via coordination to a [4Fe–4S] cluster.

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22

Kennedy, Michelle L., e Brian R. Gibney. "Proton Coupling to [4Fe-4S]2+/+and [4Fe-4Se]2+/+Oxidation and Reduction in a Designed Protein". Journal of the American Chemical Society 124, n. 24 (giugno 2002): 6826–27. http://dx.doi.org/10.1021/ja0171613.

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23

Reinhart, F., A. Huber, R. Thiele e G. Unden. "Response of the Oxygen Sensor NreB to Air In Vivo: Fe-S-Containing NreB and Apo-NreB in Aerobically and Anaerobically Growing Staphylococcus carnosus". Journal of Bacteriology 192, n. 1 (23 ottobre 2009): 86–93. http://dx.doi.org/10.1128/jb.01248-09.

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Abstract (sommario):

ABSTRACT The sensor kinase NreB from Staphylococcus carnosus contains an O2-sensitive [4Fe-4S]2+ cluster which is converted by O2 to a [2Fe-2S]2+ cluster, followed by complete degradation and formation of Fe-S-less apo-NreB. NreB·[2Fe-2S]2+ and apoNreB are devoid of kinase activity. NreB contains four Cys residues which ligate the Fe-S clusters. The accessibility of the Cys residues to alkylating agents was tested and used to differentiate Fe-S-containing and Fe-S-less NreB. In a two-step labeling procedure, accessible Cys residues in the native protein were first labeled by iodoacetate. In the second step, Cys residues not labeled in the first step were alkylated with the fluorescent monobromobimane (mBBr) after denaturing of the protein. In purified (aerobic) apoNreB, most (96%) of the Cys residues were alkylated in the first step, but in anaerobic (Fe-S-containing) NreB only a small portion (23%) were alkylated. In anaerobic bacteria, a very small portion of the Cys residues of NreB (9%) were accessible to alkylation in the native state, whereas most (89%) of the Cys residues from aerobic bacteria were accessible. The change in accessibility allowed determination of the half-time (6 min) for the conversion of NreB·[4Fe-4S]2+ to apoNreB after the addition of air in vitro. Overall, in anaerobic bacteria most of the NreB exists as NreB·[4Fe-4S]2+, whereas in aerobic bacteria the (Fe-S-less) apoNreB is predominant and represents the physiological form. The number of accessible Cys residues was also determined by iodoacetate alkylation followed by mass spectrometry of Cys-containing peptides. The pattern of mass increases confirmed the results from the two-step labeling experiments.

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24

Selvaraj, Brinda, Wolfgang Buckel, Bernard T. Golding, G. Matthias Ullmann e Berta M. Martins. "Structure and Function of 4-Hydroxyphenylacetate Decarboxylase and Its Cognate Activating Enzyme". Journal of Molecular Microbiology and Biotechnology 26, n. 1-3 (2016): 76–91. http://dx.doi.org/10.1159/000440882.

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Abstract (sommario):

4-Hydroxyphenylacetate decarboxylase (4Hpad) is the prototype of a new class of Fe-S cluster-dependent glycyl radical enzymes (Fe-S GREs) acting on aromatic compounds. The two-enzyme component system comprises a decarboxylase responsible for substrate conversion and a dedicated activating enzyme (4Hpad-AE). The decarboxylase uses a glycyl/thiyl radical dyad to convert 4-hydroxyphenylacetate into <i>p</i>-cresol (4-methylphenol) by a biologically unprecedented Kolbe-type decarboxylation. In addition to the radical dyad prosthetic group, the decarboxylase unit contains two [4Fe-4S] clusters coordinated by an extra small subunit of unknown function. 4Hpad-AE reductively cleaves S-adenosylmethionine (SAM or AdoMet) at a site-differentiated [4Fe-4S]<sup>2+/+</sup> cluster (RS cluster) generating a transient 5′-deoxyadenosyl radical that produces a stable glycyl radical in the decarboxylase by the abstraction of a hydrogen atom. 4Hpad-AE binds up to two auxiliary [4Fe-4S] clusters coordinated by a ferredoxin-like insert that is C-terminal to the RS cluster-binding motif. The ferredoxin-like domain with its two auxiliary clusters is not vital for SAM-dependent glycyl radical formation in the decarboxylase, but facilitates a longer lifetime for the radical. This review describes the 4Hpad and cognate AE families and focuses on the recent advances and open questions concerning the structure, function and mechanism of this novel Fe-S-dependent class of GREs.

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25

Yan, Aixin, e Patricia J. Kiley. "Dissecting the Role of the N-Terminal Region of the Escherichia coli Global Transcription Factor FNR". Journal of Bacteriology 190, n. 24 (17 ottobre 2008): 8230–33. http://dx.doi.org/10.1128/jb.01242-08.

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Abstract (sommario):

ABSTRACT The role of the N-terminal region of the transcription factor FNR, which immediately precedes the first ligand (Cys20) of the [4Fe-4S] cluster, was investigated. We found that truncation mutants that removed residues 2 to 16 and 2 to 17 had wild-type levels of FNR protein but surprisingly altered O2 regulation.

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26

Suess, Daniel L. M., Ingmar Bürstel, Liliana De La Paz, Jon M. Kuchenreuther, Cindy C. Pham, Stephen P. Cramer, James R. Swartz e R. David Britt. "Cysteine as a ligand platform in the biosynthesis of the FeFe hydrogenase H cluster". Proceedings of the National Academy of Sciences 112, n. 37 (31 agosto 2015): 11455–60. http://dx.doi.org/10.1073/pnas.1508440112.

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Abstract (sommario):

Hydrogenases catalyze the redox interconversion of protons and H2, an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [4Fe–4S]H subcluster coupled to a [2Fe]H subcluster bound by CO, CN–, and azadithiolate ligands. The [2Fe]H subcluster is assembled by the maturases HydE, HydF, and HydG. HydG is a member of the radical S-adenosyl-l-methionine family of enzymes that transforms Fe and l-tyrosine into an [Fe(CO)2(CN)] synthon that is incorporated into the H cluster. Although it is thought that the site of synthon formation in HydG is the “dangler” Fe of a [5Fe] cluster, many mechanistic aspects of this chemistry remain unresolved including the full ligand set of the synthon, how the dangler Fe initially binds to HydG, and how the synthon is released at the end of the reaction. To address these questions, we herein show that l-cysteine (Cys) binds the auxiliary [4Fe–4S] cluster of HydG and further chelates the dangler Fe. We also demonstrate that a [4Fe–4S]aux[CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO)2(CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Thus, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe2+ binding and synthon release.

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27

Smith, Eugene T., Benjamin A. Feinberg, John H. Richards e John M. Tomich. "Physical characterization of a totally synthetic 2[4Fe-4S] clostridial ferredoxin". Journal of the American Chemical Society 113, n. 2 (gennaio 1991): 688–89. http://dx.doi.org/10.1021/ja00002a055.

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28

Martens, C. F., M. C. Feiters, H. L. Blonk, J. G. M. van der Linden e R. J. M. Nolte. "Biomimetic electrochemical behaviour of a semi-encapsulated [4Fe-4S]2+-cluster." Journal of Inorganic Biochemistry 43, n. 2-3 (agosto 1991): 244. http://dx.doi.org/10.1016/0162-0134(91)84234-z.

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29

Bevers, Loes E., Emile Bol, Peter-Leon Hagedoorn e Wilfred R. Hagen. "WOR5, a Novel Tungsten-Containing Aldehyde Oxidoreductase from Pyrococcus furiosus with a Broad Substrate Specificity". Journal of Bacteriology 187, n. 20 (15 ottobre 2005): 7056–61. http://dx.doi.org/10.1128/jb.187.20.7056-7061.2005.

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Abstract (sommario):

ABSTRACT WOR5 is the fifth and last member of the family of tungsten-containing oxidoreductases purified from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimeric protein (subunit, 65 kDa) that contains one [4Fe-4S] cluster and one tungstobispterin cofactor per subunit. It has a broad substrate specificity with a high affinity for several substituted and nonsubstituted aliphatic and aromatic aldehydes with various chain lengths. The highest catalytic efficiency of WOR5 is found for the oxidation of hexanal (V max = 15.6 U/mg, Km = 0.18 mM at 60°C). Hexanal-incubated enzyme exhibits S = 1/2 electron paramagnetic resonance signals from [4Fe-4S]1+ (g values of 2.08, 1.93, and 1.87) and W5+ (g values of 1.977, 1.906, and 1.855). Cyclic voltammetry of ferredoxin and WOR5 on an activated glassy carbon electrode shows a catalytic wave upon addition of hexanal, suggesting that ferredoxin can be a physiological redox partner. The combination of WOR5, formaldehyde oxidoreductase, and aldehyde oxidoreductase forms an efficient catalyst for the oxidation of a broad range of aldehydes in P. furiosus.

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30

Bargagna, Beatrice, Lucia Banci e Francesca Camponeschi. "Understanding the Molecular Basis of the Multiple Mitochondrial Dysfunctions Syndrome 2: The Disease-Causing His96Arg Mutation of BOLA3". International Journal of Molecular Sciences 24, n. 14 (21 luglio 2023): 11734. http://dx.doi.org/10.3390/ijms241411734.

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Abstract (sommario):

Multiple mitochondrial dysfunctions syndrome type 2 with hyperglycinemia (MMDS2) is a severe disorder of mitochondrial energy metabolism, associated with biallelic mutations in the gene encoding for BOLA3, a protein with a not yet completely understood role in iron-sulfur (Fe-S) cluster biogenesis, but essential for the maturation of mitochondrial [4Fe-4S] proteins. To better understand the role of BOLA3 in MMDS2, we have investigated the impact of the p.His96Arg (c.287A > G) point mutation, which involves a highly conserved residue, previously identified as a [2Fe-2S] cluster ligand in the BOLA3-[2Fe-2S]-GLRX5 heterocomplex, on the structural and functional properties of BOLA3 protein. The His96Arg mutation has been associated with a severe MMDS2 phenotype, characterized by defects in the activity of mitochondrial respiratory complexes and lipoic acid-dependent enzymes. Size exclusion chromatography, NMR, UV-visible, circular dichroism, and EPR spectroscopy characterization have shown that the His96Arg mutation does not impair the interaction of BOLA3 with its protein partner GLRX5, but leads to the formation of an aberrant BOLA3-[2Fe-2S]-GLRX5 heterocomplex, that is not functional anymore in the assembly of a [4Fe-4S] cluster on NFU1. These results allowed us to rationalize the severe phenotype observed in MMDS2 caused by His96Arg mutation.

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31

Mavridis, I. M., P. Giastas, N. Pinotsis, G. Efthymiou, M. Wilmanns, P. Kyritsis e J. M. Moulis. "Structure of the 2[4Fe-4S] ferredoxin fromPseudomonas aeruginosaat 1.32 Å resolution". Acta Crystallographica Section A Foundations of Crystallography 61, a1 (23 agosto 2005): c211—c212. http://dx.doi.org/10.1107/s0108767305090999.

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32

Roy, Anindya, Dayn Joseph Sommer, Robert Arthur Schmitz, Chelsea Lynn Brown, Devens Gust, Andrei Astashkin e Giovanna Ghirlanda. "A De Novo Designed 2[4Fe-4S] Ferredoxin Mimic Mediates Electron Transfer". Journal of the American Chemical Society 136, n. 49 (dicembre 2014): 17343–49. http://dx.doi.org/10.1021/ja510621e.

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33

Elsen, Sylvie, Georgios Efthymiou, Panagiotis Peteinatos, George Diallinas, Panayotis Kyritsis e Jean-Marc Moulis. "A bacteria-specific 2[4Fe-4S] ferredoxin is essential in Pseudomonas aeruginosa". BMC Microbiology 10, n. 1 (2010): 271. http://dx.doi.org/10.1186/1471-2180-10-271.

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34

Bertini, Ivano, Antonio Donaire, Benjamin A. Feinberg, Claudio Luchinat, Mario Picciolp e Huaiping Yuan. "Solution Structure of the Oxidized 2[4Fe-4S] Ferredoxin from Clostridium Pasteurianum". European Journal of Biochemistry 232, n. 1 (agosto 1995): 192–205. http://dx.doi.org/10.1111/j.1432-1033.1995.tb20799.x.

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35

Smith, Laura J., Melanie R. Stapleton, Gavin J. M. Fullstone, Jason C. Crack, Andrew J. Thomson, Nick E. Le Brun, Debbie M. Hunt et al. "Mycobacterium tuberculosis WhiB1 is an essential DNA-binding protein with a nitric oxide-sensitive iron–sulfur cluster". Biochemical Journal 432, n. 3 (25 novembre 2010): 417–27. http://dx.doi.org/10.1042/bj20101440.

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Abstract (sommario):

Mycobacterium tuberculosis is a major pathogen that has the ability to establish, and emerge from, a persistent state. Wbl family proteins are associated with developmental processes in actinomycetes, and M. tuberculosis has seven such proteins. In the present study it is shown that the M. tuberculosis H37Rv whiB1 gene is essential. The WhiB1 protein possesses a [4Fe-4S]2+ cluster that is stable in air but reacts rapidly with eight equivalents of nitric oxide to yield two dinuclear dinitrosyl-iron thiol complexes. The [4Fe-4S] form of WhiB1 did not bind whiB1 promoter DNA, but the reduced and oxidized apo-WhiB1, and nitric oxide-treated holo-WhiB1 did bind to DNA. Mycobacterium smegmatis RNA polymerase induced transcription of whiB1 in vitro; however, in the presence of apo-WhiB1, transcription was severely inhibited, irrespective of the presence or absence of the CRP (cAMP receptor protein) Rv3676, which is known to activate whiB1 expression. Footprinting suggested that autorepression of whiB1 is achieved by apo-WhiB1 binding at a region that overlaps the core promoter elements. A model incorporating regulation of whiB1 expression in response to nitric oxide and cAMP is discussed with implications for sensing two important signals in establishing M. tuberculosis infections.

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36

Gao-Sheridan, H. Samantha, Mary A. Kemper, Reza Khayat, Gareth J. Tilley, Fraser A. Armstrong, Vandana Sridhar, G. Sridhar Prasad, C. David Stout e Barbara K. Burgess. "A T14C Variant ofAzotobacter vinelandiiFerredoxin I Undergoes Facile [3Fe-4S]0to [4Fe-4S]2+Conversionin Vitrobut Notin Vivo". Journal of Biological Chemistry 273, n. 50 (11 dicembre 1998): 33692–701. http://dx.doi.org/10.1074/jbc.273.50.33692.

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37

Cosper, Michele Mader, Guy N. L. Jameson, Roman Davydov, Marly K. Eidsness, Brian M. Hoffman, Boi Hanh Huynh e Michael K. Johnson. "The [4Fe−4S]2+Cluster in Reconstituted Biotin Synthase BindsS-Adenosyl-l-methionine". Journal of the American Chemical Society 124, n. 47 (novembre 2002): 14006–7. http://dx.doi.org/10.1021/ja0283044.

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38

Boll, Matthias, Georg Fuchs, Gareth Tilley, Fraser A. Armstrong e David J. Lowe. "Unusual Spectroscopic and Electrochemical Properties of the 2[4Fe-4S] Ferredoxin ofThauera aromatica†,‡". Biochemistry 39, n. 16 (aprile 2000): 4929–38. http://dx.doi.org/10.1021/bi9927890.

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39

Gao-Sheridan, H. S., e B. K. Burgess. "Purification and characterization of a novel 2 [4Fe4S] ferredoxin from Azotobacter vinelandii". Journal of Inorganic Biochemistry 67, n. 1-4 (luglio 1997): 251. http://dx.doi.org/10.1016/s0162-0134(97)80123-7.

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40

Lotierzo, M., B. Tse Sum Bui, D. Florentin, F. Escalettes e A. Marquet. "Biotin synthase mechanism: an overview". Biochemical Society Transactions 33, n. 4 (1 agosto 2005): 820–23. http://dx.doi.org/10.1042/bst0330820.

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Abstract (sommario):

Biotin synthase, a member of the ‘radical SAM’ (S-adenosylmethionine) family, converts DTB (dethiobiotin) into biotin. The active form of the Escherichia coli enzyme contains two (Fe-S) centres, a (4Fe-4S) and a (2Fe-2S). The (4Fe-4S)2+/+ mediates the electron transfer required for the reductive cleavage of SAM into methionine and a DOA• (deoxyadenosyl radical). Two DOA•, i.e. two SAM molecules, are consumed to activate the positions 6 and 9 of DTB. A direct transfer of isotope from the labelled substrate into DOAH (deoxyadenosine) has been observed with 2H, although not quantitatively, but not with tritium. The source of the sulphur introduced to form biotin is still under debate. We have shown that the (2Fe-2S)2+ cluster can be reconstituted in the apoenzyme with S2− and Fe2+. When S2− was replaced by [34S2−], [35S2−] or Se2−, biotin containing mostly the sulphur isotopes or selenium was obtained. This leads us to favour the hypothesis that the (2Fe-2S) centre is the sulphur donor, which may explain the absence of turnover of the enzyme. DTBSH (9-mercaptodethiobiotin), which already contains the sulphur atom of biotin, was shown to be an alternative substrate of biotin synthase both in vivo and with a crude extract. When this compound was tested with a well-defined in vitro system, the same turnover of one and similar reaction rates were observed for DTB and DTBSH. We postulate that the same intermediate is formed from both substrates.

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41

Mulholland, Stephen E., Brian R. Gibney, Francesc Rabanal e P. Leslie Dutton. "Determination of Nonligand Amino Acids Critical to [4Fe-4S]2+/+Assembly in Ferredoxin Maquettes†". Biochemistry 38, n. 32 (agosto 1999): 10442–48. http://dx.doi.org/10.1021/bi9908742.

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42

Hinckley, Glen T., e Perry A. Frey. "Cofactor Dependence of Reduction Potentials for [4Fe-4S]2+/1+in Lysine 2,3-Aminomutase†". Biochemistry 45, n. 10 (marzo 2006): 3219–25. http://dx.doi.org/10.1021/bi0519497.

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43

Hinckley, Glen T., e Perry A. Frey. "Cofactor Dependence of Reduction Potentials for [4Fe-4S]2+/1+in Lysine 2,3-Aminomutase". Biochemistry 45, n. 22 (giugno 2006): 6996. http://dx.doi.org/10.1021/bi0680126.

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44

Stack, T. D. P., e R. H. Holm. "Subsite-specific functionalization of the [4Fe-4S]2+ analog of iron-sulfur protein clusters". Journal of the American Chemical Society 109, n. 8 (aprile 1987): 2546–47. http://dx.doi.org/10.1021/ja00242a067.

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45

Cammack, R., D. S. Patil e J. H. Weiner. "Evidence that centre 2 in Escherichia coli fumarate reductase is a [4Fe-4S]cluster". Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 870, n. 3 (aprile 1986): 545–51. http://dx.doi.org/10.1016/0167-4838(86)90264-5.

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46

Tan, Ming-Liang, Yan Luo e Toshiko Ichiye. "Tuning the Intramolecular Electron Transfer in 2[4Fe-4S] Ferredoxin: A Molecular Dynamics Study". Biophysical Journal 100, n. 3 (febbraio 2011): 132a. http://dx.doi.org/10.1016/j.bpj.2010.12.924.

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47

Nasta, Veronica, Dafne Suraci, Spyridon Gourdoupis, Simone Ciofi‐Baffoni e Lucia Banci. "A pathway for assembling [4Fe‐4S] 2+ clusters in mitochondrial iron–sulfur protein biogenesis". FEBS Journal 287, n. 11 (3 dicembre 2019): 2312–27. http://dx.doi.org/10.1111/febs.15140.

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48

Chen, Kaisheng, Gareth J. Tilley, Vandana Sridhar, G. Sridhar Prasad, C. David Stout, Fraser A. Armstrong e Barbara K. Burgess. "Alteration of the Reduction Potential of the [4Fe-4S]2+/+Cluster ofAzotobacter vinelandiiFerredoxin I". Journal of Biological Chemistry 274, n. 51 (17 dicembre 1999): 36479–87. http://dx.doi.org/10.1074/jbc.274.51.36479.

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49

Moulis, Jean-Marc. "Molecular cloning and expression of the gene encoding Chromatium vinosum 2[4Fe-4S] ferredoxin". Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression 1308, n. 1 (luglio 1996): 12–14. http://dx.doi.org/10.1016/0167-4781(96)00082-6.

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50

CHEN, Dawei, Frank J. RUZICKA e Perry A. FREY. "A novel lysine 2,3-aminomutase encoded by the yodO gene of Bacillus subtilis: characterization and the observation of organic radical intermediates". Biochemical Journal 348, n. 3 (7 giugno 2000): 539–49. http://dx.doi.org/10.1042/bj3480539.

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Abstract (sommario):

The yodO gene product of Bacillus subtilis has been cloned and overexpressed in Escherichia coli and purified. The nucleotide sequence encodes a protein of 471 amino acids with a calculated molecular mass of 54071 Da. The translated amino acid sequence is more than 60% identical to that of the lysine 2,3-aminomutase from Clostridium subterminale SB4. Analytical HPLC gel-permeation chromatography leads to an estimate of an overall molecular mass of 224000±21000 Da, which corresponds to a tetrameric protein. The purified protein contains iron, sulphide and pyridoxal 5ʹ-phosphate (PLP) and displays an optical absorption band extending to 700 nm, suggesting the presence of an iron-sulphide cluster. After reductive incubation with L-cysteine anaerobically, the protein catalyses the transformation of L-lysine into β-lysine in the presence of S-adenosylmethionine (AdoMet) and sodium dithionite. The Km value for L-lysine is estimated to be 8.0±2.2 mM. The iron-sulphur centre is stable in air, allowing aerobic purification. EPR spectroscopy at 10 K of the purified enzyme revealed an EPR signal similar to that of the [4Fe-4S]3+ cluster observed in the clostridial lysine 2,3-aminomutase. Incubation with cysteine under anaerobic conditions converts the iron-sulphur centre into the EPR-silent [4Fe-4S]2+. Unlike the clostridial enzyme, the fully reduced [4Fe-4S]+ could not be characterized by further reduction with dithionite in the presence of AdoMet, although both dithionite and AdoMet were required to activate the enzyme. Upon addition of L-lysine, dithionite and AdoMet to the reduced enzyme and freezing the solution to 77 K, the EPR spectrum revealed the presence of an organic free-radical signal (g = 2.0023), which displayed multiple hyperfine transitions very similar to the spectrum of the β-lysine-related radical in the mechanism of the clostridial lysine 2,3-aminomutase. Experiments with isotopically substituted L-lysine and lysine analogues verified the association of spin density with the carbon skeleton of lysine. The data indicate that the protein encoded by the yodO gene of B. subtilis is a novel lysine 2,3-aminomutase. The E. coli homologue of clostridial lysine 2,3-aminomutase was also expressed in E. coli and purified. This protein contained iron and sulphide but not PLP, it did not display lysine 2,3-aminomutase activity, and addition of PLP did not induce 2,3-aminomutase activity.

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