Structural insights into the enzyme catalysis from comparison of three forms of dissimilatory sulphite reductase from Desulfovibrio gigas

The crystal structures of two active forms of dissimilatory sulphite reductase (Dsr) from Desulfovibrio gigas, Dsr‐I and Dsr‐II, are compared at 1.76 and 2.05 Å resolution respectively. The dimeric α₂β₂γ₂ structure of Dsr‐I contains eight [4Fe–4S] clusters, two saddle‐shaped sirohaems and two flat sirohydrochlorins. In Dsr‐II, the [4Fe–4S] cluster associated with the sirohaem in Dsr‐I is replaced by a [3Fe–4S] cluster. Electron paramagnetic resonance (EPR) of the active Dsr‐I and Dsr‐II confirm the co‐factor structures, whereas EPR of a third but inactive form, Dsr‐III, suggests that the sirohaem has been demetallated in addition to its associated [4Fe–4S] cluster replaced by a [3Fe–4S] centre. In Dsr‐I and Dsr‐II, the sirohydrochlorin is located in a putative substrate channel connected to the sirohaem. The γ‐subunit C‐terminus is inserted into a positively charged channel formed between the α‐ and β‐subunits, with its conserved terminal Cysγ104 side‐chain covalently linked to the CHA atom of the sirohaem in Dsr‐I. In Dsr‐II, the thioether bond is broken, and the Cysγ104 side‐chain moves closer to the bound sulphite at the sirohaem pocket. These different forms of Dsr offer structural insights into a mechanism of sulphite reduction that can lead to S₃O₆^2?, S₂O₃^2? and S^2?.     

Novel features of the ISC machinery revealed by characterization of Escherichia coli mutants that survive without iron–sulfur clusters

Biological assembly of iron–sulfur (Fe–S) clusters is mediated by complex systems consisting of multiple proteins. Escherichia coli possesses two distinct systems called the ISC and SUF machineries encoded by iscSUA‐hscBA‐fdx‐iscX and sufABCDSE respectively. Deletion of both pathways results in absence of the biosynthetic apparatus for Fe–S clusters, and consequent lethality, which has hampered detailed genetic studies. Here we report that modification of the isoprenoid biosynthetic pathway can offset the indispensability of the Fe–S cluster biosynthetic systems and show that the resulting Δisc Δsuf double mutants can grow without detectable Fe–S cluster‐containing proteins. We also constructed a series of mutants in which each isc gene was disrupted in the deletion background of sufABCDSE. Phenotypic analysis of the mutants revealed that Fdx, an essential electron‐transfer Fe–S protein in the ISC machinery, is dispensable under anaerobic conditions, which is similar to the situation with IscA. Furthermore, we found that several suppressor mutations in IscU, an Fe–S scaffold protein responsible for the de novo Fe–S cluster assembly, could bypass the essential role of the chaperone system HscA and HscB. These findings pave the way toward a detailed molecular analysis to understand the mechanisms involved in Fe–S cluster biosynthesis.     

ISC-like [2Fe–2S] ferredoxin (FdxB) dimer from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> JCM 20004: structural and electron–nuclear double resonance characterization

The crystal structure of the ISC-like [2Fe–2S] ferredoxin (FdxB), probably involved in the de novo iron-sulfur cluster biosynthesis (ISC) system of Pseudomonas putida JCM 20004, was determined at 1.90-Å resolution and displayed a novel tail-to-tail dimeric form. P. putida FdxB lacks the consensus free cysteine usually present near the cluster of ISC-like ferredoxins, indicating its primarily electron transfer role in the iron-sulfur cluster. Orientation-selective electron–nuclear double resonance spectroscopic analysis of reduced FdxB in conjunction with the crystal structure has identified the innermost Fe2 site with a high positive spin population as the nonreducible iron retaining the Fe³⁺ valence and the outermost Fe1 site as the reduced iron with a low negative spin density. The average g _max direction is skewed, forming an angle of about 27.3° (±4°) with the normal of the [2Fe–2S] plane, whereas the g _int and g _min directions are distributed in the cluster plane, presumably tilted by the same angle with respect to this plane. These results are related to those for other [2Fe–2S] proteins in different electron transport chains (e.g. adrenodoxin) and suggest a significant distortion of the electronic structure of the reduced [2Fe–2S] cluster under the influence of the protein environment around each iron site in general.     

Iron–sulfur protein maturation in Helicobacter pylori: identifying a Nfu‐type cluster carrier protein and its iron–sulfur protein targets

Helicobacter pylori is anomalous among non nitrogen‐fixing bacteria in containing an incomplete NIF system for Fe–S cluster assembly comprising two essential proteins, NifS (cysteine desulfurase) and NifU (scaffold protein). Although nifU deletion strains cannot be obtained via the conventional gene replacement, a NifU‐depleted strain was constructed and shown to be more sensitive to oxidative stress compared to wild‐type (WT) strains. The hp1492 gene, encoding a putative Nfu‐type Fe–S cluster carrier protein, was disrupted in three different H. pylori strains, indicating that it is not essential. However, Δnfu strains have growth deficiency, are more sensitive to oxidative stress and are unable to colonize mouse stomachs. Moreover, Δnfu strains have lower aconitase activity but higher hydrogenase activity than the WT. Recombinant Nfu was found to bind either one [2Fe–2S] or [4Fe–4S] cluster/dimer, based on analytical, UV–visible absorption/CD and resonance Raman studies. A bacterial two‐hybrid system was used to ascertain interactions between Nfu, NifS, NifU and each of 36 putative Fe–S‐containing target proteins. Nfu, NifS and NifU were found to interact with 15, 6 and 29 putative Fe–S proteins respectively. The results indicate that Nfu, NifS and NifU play a major role in the biosynthesis and/or delivery of Fe–S clusters in H. pylori.     

Evaluation of mitochondrial redox status and energy metabolism of X-irradiated HeLa cells by LC/UV,LC/MS/MS and ESR

To evaluate the metabolic responses in tumour cells exposed to ionizing radiation, oxygen consumption rate (OCR), cellular lipid peroxidation, cellular energy status (intracellular nucleotide pool and ATP production), and mitochondrial reactive oxygen species (ROS), semiquinone (SQ), and iron–sulphur (Fe?S) cluster levels were evaluated in human cervical carcinoma HeLa cells at 12 and 24?h after X-irradiation. LC/MS/MS analysis showed that levels of 8-iso PGF_2α and 5-iPF_2α-VI, lipid peroxidation products of membrane arachidonic acids, were not altered significantly in X-irradiated cells, although mitochondrial ROS levels and OCR significantly increased in the cells at 24?h after irradiation. LC/UV analysis revealed that intracellular AMP, ADP, and ATP levels increased significantly after X-irradiation, but adenylate energy charge (adenylate energy charge (AEC)?=?[ATP?+?0.5?×?ADP]/[ATP?+?ADP?+?AMP]) remained unchanged after X-irradiation. In low-temperature electron spin resonance (ESR) spectra of HeLa cells, the presence of mitochondrial SQ at g?=?2.004 and Fe–S cluster at g?=?1.941 was observed and X-irradiation enhanced the signal intensity of SQ but not of the Fe–S cluster. Furthermore, this radiation-induced increase in SQ signal intensity disappeared on treatment with rotenone, which inhibits electron transfer from Fe–S cluster to SQ in complex I. From these results, it was suggested that an increase in OCR and imbalance in SQ and Fe–S cluster levels, which play a critical role in the mitochondrial electron transport chain (ETC), occur after X-irradiation, resulting in an increase in ATP production and ROS leakage from the activated mitochondrial ETC.     

Genes for iron–sulphur cluster assembly are targets of abiotic stress in rice,Oryza sativa

Iron–sulphur (Fe–S) cluster assembly occurs in chloroplasts, mitochondria and cytosol, involving dozens of genes in higher plants. In this study, we have identified 41 putative Fe–S cluster assembly genes in rice (Oryza sativa) genome, and the expression of all genes was verified. To investigate the role of Fe–S cluster assembly as a metabolic pathway, we applied abiotic stresses to rice seedlings and analysed Fe–S cluster assembly gene expression by qRT‐PCR. Our data showed that genes for Fe–S cluster assembly in chloroplasts of leaves are particularly sensitive to heavy metal treatments, and that Fe–S cluster assembly genes in roots were up‐regulated in response to iron toxicity, oxidative stress and some heavy metal assault. The effect of each stress treatment on the Fe–S cluster assembly machinery demonstrated an unexpected tissue or organelle specificity, suggesting that the physiological relevance of the Fe–S cluster assembly is more complex than thought. Furthermore, our results may reveal potential candidate genes for molecular breeding of rice.     

Compartmentalisation of [FeFe]‐hydrogenase maturation in Chlamydomonas reinhardtii

Molecular hydrogen (H₂) can be produced in green microalgae by [FeFe]‐hydrogenases as a direct product of photosynthesis. The Chlamydomonas reinhardtii hydrogenase HYDA1 contains a catalytic site comprising a classic [4Fe4S] cluster linked to a unique 2Fe sub‐cluster. From in vitro studies it appears that the [4Fe4S] cluster is incorporated first by the housekeeping FeS cluster assembly machinery, followed by the 2Fe sub‐cluster, whose biosynthesis requires the specific maturases HYDEF and HYDG. To investigate the maturation process in vivo, we expressed HYDA1 from the C. reinhardtii chloroplast and nuclear genomes (with and without a chloroplast transit peptide) in a hydrogenase‐deficient mutant strain, and examined the cellular enzymatic hydrogenase activity, as well as in vivo H₂ production. The transformants expressing HYDA1 from the chloroplast genome displayed levels of H₂ production comparable to the wild type, as did the transformants expressing full‐length HYDA1 from the nuclear genome. In contrast, cells equipped with cytoplasm‐targeted HYDA1 produced inactive enzyme, which could only be activated in vitro after reconstitution of the [4Fe4S] cluster. This indicates that the HYDA1 FeS cluster can only be built by the chloroplastic FeS cluster assembly machinery. Further, the expression of a bacterial hydrogenase gene, CPI, from the C. reinhardtii chloroplast genome resulted in H₂‐producing strains, demonstrating that a hydrogenase with a very different structure can fulfil the role of HYDA1 in vivo and that overexpression of foreign hydrogenases in C. reinhardtii is possible. All chloroplast transformants were stable and no toxic effects were seen from HYDA1 or CPI expression.     

Physiological and transcriptional responses in the iron–sulphur cluster assembly pathway under abiotic stress in peach (Prunus persica L.) seedlings

As one of the most indispensable element in mineral nutrition of plants, iron (Fe) is closely related to fruits quality and yield. However, molecular mechanisms towards Fe metabolism in fruit trees is largely unclear. In higher plants, iron–sulphur (Fe–S) cluster assembly occurs in chloroplasts, mitochondria and cytosol involving dozens of genes. In this study, we identified 44 putative Fe–S cluster assembly genes in peach (Prunus persica cv. ‘Xiahui6’), and analyzed Fe–S cluster assembly gene expression profiles in response to abiotic stresses. Peach seedlings were more sensitive to iron deficiency, drought and salinity stress, evidenced in reduced photosynthetic performance and altered activity of nitrite reductase, succinate dehydrogenase and aconitase. In addition, Fe–S cluster assembly genes are differentially regulated by abiotic stresses. Iron depletion and drought stress are likely to affect Fe–S cluster assembly genes in leaves. Excess iron toxicity mainly induces Fe–S cluster assembly gene expression in roots, whereas salinity stress massively inhibits Fe–S cluster assembly gene expression in roots. Interestingly, we found that un-functional scaffolds are more prone to disappear during the long-term evolution in perennial woody plants. Our findings directly provide molecular basis for Fe metabolism in peach, and favorably reveal potential candidate genes for further functional determination.     

Bacillithiol has a role in Fe–S cluster biogenesis in Staphylococcus aureus

Staphylococcus aureus does not produce the low‐molecular‐weight (LMW) thiol glutathione, but it does produce the LMW thiol bacillithiol (BSH). To better understand the roles that BSH plays in staphylococcal metabolism, we constructed and examined strains lacking BSH. Phenotypic analysis found that the BSH‐deficient strains cultured either aerobically or anaerobically had growth defects that were alleviated by the addition of exogenous iron (Fe) or the amino acids leucine and isoleucine. The activities of the iron–sulfur (Fe–S) cluster‐dependent enzymes LeuCD and IlvD, which are required for the biosynthesis of leucine and isoleucine, were decreased in strains lacking BSH. The BSH‐deficient cells also had decreased aconitase and glutamate synthase activities, suggesting a general defect in Fe–S cluster biogenesis. The phenotypes of the BSH‐deficient strains were exacerbated in strains lacking the Fe–S cluster carrier Nfu and partially suppressed by multicopy expression of either sufA or nfu, suggesting functional overlap between BSH and Fe–S carrier proteins. Biochemical analysis found that SufA bound and transferred Fe–S clusters to apo‐aconitase, verifying that it serves as an Fe–S cluster carrier. The results presented are consistent with the hypothesis that BSH has roles in Fe homeostasis and the carriage of Fe–S clusters to apo‐proteins in S. aureus.     

Iron–sulfur cluster exchange reactions mediated by the human Nfu protein

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.     

An EPR/HYSCORE, Mössbauer, and resonance Raman study of the hydrogenase maturation enzyme HydF: a model for N-coordination to [4Fe–4S] clusters

The biosynthesis of the organometallic H cluster of [Fe–Fe] hydrogenase requires three accessory proteins, two of which (HydE and HydG) belong to the radical S-adenosylmethionine enzyme superfamily. The third, HydF, is an Fe–S protein with GTPase activity. The [4Fe–4S] cluster of HydF is bound to the polypeptide chain through only the three, conserved, cysteine residues present in the binding sequence motif CysXHisX_(46-53)HisCysXXCys. However, the involvement of the two highly conserved histidines as a fourth ligand for the cluster coordination is controversial. In this study, we set out to characterize further the [4Fe–4S] cluster of HydF using Mössbauer, EPR, hyperfine sublevel correlation (HYSCORE), and resonance Raman spectroscopy in order to investigate the influence of nitrogen ligands on the spectroscopic properties of [4Fe–4S]^2+/+ clusters. Our results show that Mössbauer, resonance Raman, and EPR spectroscopy are not able to readily discriminate between the imidazole-coordinated [4Fe–4S] cluster and the non-imidazole-bound [4Fe–4S] cluster with an exchangeable fourth ligand that is present in wild-type HydF. HYSCORE spectroscopy, on the other hand, detects the presence of an imidazole/histidine ligand on the cluster on the basis of the appearance of a specific spectral pattern in the strongly coupled region, with a coupling constant of approximately 6 MHz. We also discovered that a His-tagged version of HydF, with a hexahistidine tag at the N-terminus, has a [4Fe–4S] cluster coordinated by one histidine from the tag. This observation strongly indicates that care has to be taken in the analysis of data obtained on tagged forms of metalloproteins.     

Nitrite,sodium nitroprusside,potassium ferricyanide and hydrogen peroxide release dormancy of <Emphasis Type="Italic">Amaranthus retroflexus</Emphasis> seeds in a nitric oxide-dependent manner

Nitric oxide (NO) and reactive oxygen species (ROS) are important regulators involving various processes of plant growth and development. Amaranthus retroflexus L. seeds possess a relative dormancy property that means freshly collected seeds can only germinate over a limited, high temperature range. Here, we show that the relative dormancy of A. retroflexus seeds could be significantly released following treatments with exogenous NO/cyanide (CN) donors such as nitrite, gases evolved from acidified nitrite, sodium nitroprusside (SNP), potassium ferricyanide (Fe(III)CN) and gases evolved from SNP or Fe(III)CN solutions, as well as exogenously supplied ROS, hydrogen peroxide (H₂O₂). However, the effectiveness varied among these chemicals. Gases evolved from acidified nitrite displayed maximum effect while H₂O₂ had minimum effect. We also show that the effects of these compounds could be significantly inhibited by NO specific scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), indicating that NO signaling pathway might play a central role in the dormancy release and germination of A. retroflexus seeds, while both ROS and CN might act through NO-dependent signaling cascades.     

Characterization of a modified nitrogenase Fe protein from Klebsiella pneumoniae in which the 4Fe4S cluster has been replaced by a 4Fe4Se cluster

The Azotobacter vinelandii nifS gene product has been used with selenocysteine to reconstitute Klebsiella pneumoniae nitrogenase Fe protein. Chemical analysis and extended X-ray absorption fine structure (EXAFS) spectroscopy show that the 4Fe4S cluster present in the native protein is replaced by a 4Fe4Se cluster. As well, EXAFS spectroscopy shows that the bond lengths to the cysteine thiolate ligands shrink by 0.05 Å (from 2.28 to 2.23 Å) upon reduction, whereas the Fe–Fe distance is essentially unchanged. Thus, the core of the 4Fe4Se cluster remains essentially static on reduction, whilst the external cysteine thiolate ligands are pulled in towards the cluster. Compared with native (S)–Fe protein, the (Se)–Fe protein has a 20-fold increased rate of MgATP-induced Fe chelation, a sixfold decreased specific activity for acetylene reduction, a fivefold decreased rate of MgATP-dependent electron transfer from (Se)–Fe protein to MoFe protein, and a fourfold increase in the ATP to 2e ^? ratio. The high ATP to 2e ^? ratio and decreased specific activity are consistent with a lower rate of dissociation of oxidized (Se)–Fe protein from reduced MoFe protein. Thus, the relatively small adjustments in the Fe protein structure necessary to accommodate the 4Fe4Se cluster are transmitted both to adjacent residues that dock at the surface of the MoFe protein and to the ATP hydrolysis sites located approximately 19 Å away.     

HdrC2 from Acidithiobacillus ferrooxidans Owns Two Iron–Sulfur Binding Motifs But Binds Only One Variable Cluster Between [4Fe–4S] and [3Fe–4S]

The heterodisulfide reductase complex HdrABC from Acidithiobacillus ferrooxidans was suggested to own novel features that act in reverse to convert the sulfane sulfur of GS _n H species (n > 1) into sulfite in sulfur oxidation. The HdrC subunit is potentially encoded by two different highly upregulated genes sharing only 29 % identity in A. ferrooxidans grown in sulfur-containing medium, which were named as HdrC1 and HdrC2, respectively and had been confirmed to contain iron–sulfur cluster by expression and characterization, especially the HdrC1 which had been showed to bind only one [4Fe–4S] cluster by mutations. However, the mutations of the HdrC2 remain to be done and the detailed binding information of it is still unclear. Here, we report the expression, mutations, and molecular modeling of the HdrC2 from A. ferrooxidans. This HdrC2 had two identical motifs (Cx₂Cx₂Cx₃C) containing total of eight cysteine residues potentially for iron–sulfur cluster binding. This purified HdrC2 was exhibited to contain one variable cluster converted between [4Fe–4S] and [3Fe–4S] according to different conditions by the UV-scanning and EPR spectra. The site-directed mutagenesis results of these eight residues further confirmed that the HdrC2 in reduction with Fe²⁺ condition loaded only one [4Fe–4S]⁺ with spin S = 1/2 ligated by the residues of Cys73, Cys109, Cys112, and Cys115; the HdrC2 in natural aeration condition lost the Fe atom ligated by the residue of Cys73 and loaded only one [3Fe–4S]⁰ with spin S = 0; the HdrC2 in oxidation condition loaded only one [3Fe–4S]⁺ with spin S = 1/2. Molecular modeling results were also in line with the experiment results.     

The active site of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans. I. Light sensitivity and magnetic hyperfine interactions as observed by electron paramagnetic resonance

The hydrogen-activating cluster (H cluster) in [FeFe]-hydrogenases consists of two moieties. The [2Fe]_H subcluster is a (L)(CO)(CN)Fe(μ-RS₂)(μ-CO)Fe(CysS)(CO)(CN) centre. The Cys-bound Fe is called Fe1, the other iron Fe2. The Cys-thiol forms a bridge to a [4Fe–4S] cluster, the [4Fe–4S]_H subcluster. We report that electron paramagnetic resonance (EPR) spectra of the ⁵⁷Fe-enriched enzyme from Desulfovibrio desulfuricans in the H_ox–CO state are consistent with a magnetic hyperfine interaction of the unpaired spin with all six Fe atoms of the H cluster. In contrast to the inactive aerobic enzyme, the active enzyme is easily destroyed by light. The [2Fe]_H subcluster in some enzyme molecules loses CO by photolysis, whereupon other molecules firmly bind the released CO to form the H_ox–CO state giving rise to the so-called axial 2.06 EPR signal. Though not destroyed by light, the H_ox–CO state is affected by it. As demonstrated in the accompanying paper [49] two of the intrinsic COs, both bound to Fe2, can be exchanged by extrinsic ¹³CO during illumination at 2 °C. We found that only one of the three ¹³COs, the one at the extrinsic position, gives an EPR-detectable isotropic superhyperfine interaction of 0.6 mT. At 30 K both the inhibiting extrinsic CO bound to Fe2 and one more CO can be photolysed. EPR spectra of the photolysed products are consistent with a 3d ⁷ system of Fe with the formal oxidation state +1. The damaged enzyme shows a light-sensitive g=5 signal which is ascribed to an S=3/2 form of the [2Fe]_H subcluster. The light sensitivity of the enzyme explains the occurrence of the g=5 signal and the axial 2.06 signal in published EPR spectra of nearly all preparations studied thus far.