In silico pathway reconstruction: Iron-sulfur cluster biogenesis in Saccharomyces cerevisiae

Background  

Current advances in genomics, proteomics and other areas of molecular biology make the identification and reconstruction of novel pathways an emerging area of great interest. One such class of pathways is involved in the biogenesis of Iron-Sulfur Clusters (ISC).     

Iron-sulfur cluster biogenesis in chloroplasts. Involvement of the scaffold protein CpIscA

The chloroplast contains many iron (Fe)-sulfur (S) proteins for the processes of photosynthesis and nitrogen and S assimilation. Although isolated chloroplasts are known to be able to synthesize their own Fe-S clusters, the machinery involved is largely unknown. Recently, a cysteine desulfurase was reported in Arabidopsis (Arabidopsis thaliana; AtCpNifS) that likely provides the S for Fe-S clusters. Here, we describe an additional putative component of the plastid Fe-S cluster assembly machinery in Arabidopsis: CpIscA, which has homology to bacterial IscA and SufA proteins that have a scaffold function during Fe-S cluster formation. CpIscA mRNA was shown to be expressed in all tissues tested, with higher expression level in green, photosynthetic tissues. The plastid localization of CpIscA was confirmed by green fluorescent protein fusions, in vitro import, and immunoblotting experiments. CpIscA was cloned and purified after expression in Escherichia coli. Addition of CpIscA significantly enhanced CpNifS-mediated in vitro reconstitution of the 2Fe-2S cluster in apo-ferredoxin. During incubation with CpNifS in a reconstitution mix, CpIscA was shown to acquire a transient Fe-S cluster. The Fe-S cluster could subsequently be transferred by CpIscA to apo-ferredoxin. We propose that the CpIscA protein serves as a scaffold in chloroplast Fe-S cluster assembly.     

Iron-sulfur cluster sensor-regulators

Regulatory proteins that contain an iron-sulfur cluster cofactor constitute a group that is growing both in number and importance, with a range of functions that include sensing of molecular oxygen, stress response, and iron regulation. In all cases, the cluster plays a central role, as a sensory module, in controlling the activity of the regulator. In some cases, the cluster is required for the protein to attain its regulatory form, while in others the active form requires loss or modification of the cluster. In this way, nature has exploited the inherent reactivity of iron-sulfur clusters. Here, we focus on recent advances that provide new insight into the remarkable chemistries exhibited by these regulators, and how they achieve the required levels of sensitivity and specificity.     

Iron-sulfur cluster proteins: electron transfer and beyond

Iron-sulfur clusters-containing proteins participate in many cellular processes, including crucial biological events like DNA synthesis and processing of dioxygen. In most iron-sulfur proteins, the clusters function as electron-transfer groups in mediating one-electron redox processes and as such they are integral components of respiratory and photosynthetic electron transfer chains and numerous redox enzymes involved in carbon, oxygen, hydrogen, sulfur and nitrogen metabolism. Recently, novel regulatory and enzymatic functions of these proteins have emerged. Iron-sulfur cluster proteins participate in the control of gene expression, oxygen/nitrogen sensing, control of labile iron pool and DNA damage recognition and repair. Their role in cellular response to oxidative stress and as a source of free iron ions is also discussed.     

Iron-sulfur cluster biosynthesis in bacteria: Mechanisms of cluster assembly and transfer

Iron-sulfur [Fe-S] clusters are ubiquitous ancient prosthetic groups that are required to sustain fundamental life processes. Formation of intracellular [Fe-S] clusters does not occur spontaneously but requires a complex biosynthetic machinery. Different types of [Fe-S] cluster assembly systems have been discovered. All of them have in common the requirement of a cysteine desulfurase and the participation of [Fe-S] scaffold proteins. The purpose of this review is to discuss various aspects of the molecular mechanisms of [Fe-S] cluster assembly in living organisms: (i) mechanism of sulfur donor enzymes, namely the cysteine desulfurases; (ii) mechanism by which clusters are preassembled on scaffold proteins and (iii) mechanism of [Fe-S] cluster transfer from scaffold to target proteins.     

Mammalian mitochondrial iron-sulfur cluster biogenesis and transfer and related human diseases

As a cofactor,iron-sulfur (Fe-S) cluster binds to proteins or enzymes that play important roles in vari-ous important biological processes,including DNA synthes...     

Iron-sulfur cluster biosynthesis: characterization of Schizosaccharomyces pombe Isa1

Eukaryotic Isa1 is one of several mitochondrial proteins that have been implicated in Fe-S cluster assembly paths in vivo. We report the first biochemical characterization of an eukaryotic member of this family and discuss this in the context of results from in vivo studies and studies of bacterial homologues. Schizosaccharomyces pombe Isa1 is a multimeric protein carrying [2Fe-2S](2+) clusters that have been characterized by M?ssbauer and optical spectroscopic studies. Complex formation with a redox-active ferredoxin has been identified through crosslinking experiments and the coordination chemistry and stability of the native clusters has been investigated through site-directed mutagenesis and spectroscopic analysis. Electronic supplementary material to this paper, containing M?ssbauer and UV-visible spectra for mutant Isa1 proteins, can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0330-2.     

Iron-sulfur cluster biosynthesis. Thermatoga maritima IscU is a structured iron-sulfur cluster assembly protein

Genetic evidence has indicated that Isc proteins play an important role in iron-sulfur cluster biogenesis. In particular, IscU is believed to serve as a scaffold for the assembly of a nascent iron-sulfur cluster that is subsequently delivered to target iron-sulfur apoproteins. We report the characterization of an IscU from Thermatoga maritima, an evolutionarily ancient hyperthermophilic bacterium. The stabilizing influence of a D40A substitution allowed characterization of the holoprotein. M?ssbauer (delta = 0.29 +/- 0.03 mm/s, DeltaE(Q) = 0.58 +/- 0.03 mm/s), UV-visible absorption, and circular dichroism studies of the D40A protein show that T. maritima IscU coordinates a [2Fe-2S]2+ cluster. Thermal denaturation experiments demonstrate that T. maritima IscU is a thermally stable protein with a thermally unstable cluster. This is also the first IscU type domain that is demonstrated to possess a high degree of secondary and tertiary structure. CD spectra indicate 36.7% alpha-helix, 13.1% antiparallel beta-sheet, 11.3% parallel beta-sheet, 20.2% beta-turn, and 19.1% other at 20 degrees C, with negligible spectral change observed at 70 degrees C. Cluster coordination also has no effect on the secondary structure of the protein. The dispersion of signals in 1H-15N heteronuclear single quantum correlation NMR spectra of wild type and D40A IscU supports the presence of significant tertiary structure for the apoprotein, consistent with a scaffolding role, and is in marked contrast to other low molecular weight Fe-S proteins where cofactor coordination is found to be necessary for proper protein folding. Consistent with the observed sequence homology and proposed conservation of function for IscU-type proteins, we demonstrate T. maritima IscU-mediated reconstitution of human apoferredoxin.     

Iron-sulfur cluster in aconitase. Crystallographic evidence for a three-iron center

Native x-ray diffraction data from single crystals of inactive aconitase from pig heart (Mr 80,000) have been collected on oscillation films to 2.7 A. Analysis shows that significant measurements of the anomalous scattering signal from the Fe-S cluster in the enzyme are available in the film data. The 5.0-A resolution anomalous difference Patterson function contains vectors for one Fe-S cluster (one aconitase molecule) per asymmetric unit in space group P2(1)2(1)2 with a = 173.6, b = 72.0, and c = 72.7 A. At 2.7-A resolution, the vector map is best interpreted by three Fe sites separated from each other by less than 3 A. The single-crystal diffraction data thus confirm the presence of a 3Fe center in the inactive form of aconitase. Furthermore, the data provide crystallographic evidence that 3Fe clusters exhibit structural heterogeneity. The Fe-Fe vectors cannot be interpreted in terms of 4-A distances as observed for the [3Fe-3S] cluster in Azotobacter ferrodoxin (Ghosh, D., O'Donnell, S., Furey, W., Robbins, A. H., and Stout, C. D. (1982) J. Mol. Biol. 158, 73-109). The results are therefore in agreement with a [3Fe-4S] cluster having 2.7-A Fe-Fe distances (Beinert, H., Emptage, M. H., Dreyer, J.-L., Scott, R. A., Hahn, J. E., Hodgson, K. O., and Thomson, A. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 393-396). However, the data do not unambiguously discriminate between this model and other 3Fe clusters having short Fe-Fe distances.     

Iron-sulphur cluster biogenesis and mitochondrial iron homeostasis

Iron-sulphur clusters are important cofactors for proteins that are involved in many cellular processes, including electron transport, enzymatic catalysis and regulation. The enzymes that catalyse the formation of iron-sulphur clusters are widely conserved from bacteria to humans. Recent studies in model systems and humans reveal that iron-sulphur proteins have important roles in mitochondrial iron homeostasis and in the pathogenesis of the human disease Friedreich ataxia.     

Iron-sulfur cluster assembly: NifU-directed activation of the nitrogenase Fe protein

The NifU protein is a homodimer that is proposed to provide a molecular scaffold for the assembly of [Fe-S] clusters uniquely destined for the maturation of the nitrogenase catalytic components. There are three domains contained within NifU, with the N-terminal domain exhibiting a high degree of primary sequence similarity to a related family of [Fe-S] cluster biosynthetic scaffolds designated IscU. The C-terminal domain of NifU exhibits sequence similarity to a second family of proposed [Fe-S] cluster biosynthetic scaffolds designated Nfu. Genetic experiments described here involving amino acid substitutions within the N-terminal and C-terminal domains of NifU indicate that both domains can separately participate in nitrogenase-specific [Fe-S] cluster formation, although the N-terminal domain appears to have the dominant function. These in vivo experiments were supported by in vitro [Fe-S] cluster assembly and transfer experiments involving the activation of an apo-form of the nitrogenase Fe protein.     

CpNifS-dependent iron-sulfur cluster biogenesis in chloroplasts

Iron-sulfur (Fe-S) clusters are important prosthetic groups in all organisms. The biosynthesis of Fe-S clusters has been studied extensively in bacteria and yeast. By contrast, much remains to be discovered about Fe-S cluster biogenesis in higher plants. Plant plastids are known to make their own Fe-S clusters. Plastid Fe-S proteins are involved in essential metabolic pathways, such as photosynthesis, nitrogen and sulfur assimilation, protein import, and chlorophyll transformation. This review aims to summarize the roles of Fe-S proteins in essential metabolic pathways and to give an overview of the latest findings on plastidic Fe-S assembly. The plastidic Fe-S biosynthetic machinery contains many homologues of bacterial mobilization of sulfur (SUF) proteins, but there are additional components and properties that may be plant-specific. These additional features could make the plastidic machinery more suitable for assembling Fe-S clusters in the presence of oxygen, and may enable it to be regulated in response to oxidative stress, iron status and light.     

Iron-sulfur cluster dynamics in biotin synthase: A new [2Fe-2S] cluster

Biotin synthase (BioB) catalyses the final step in the biosynthesis of biotin. Aerobically purified biotin synthase contains one [2Fe-2S]²⁺ cluster per monomer. However, active BioB contains in addition a [4Fe-4S]²⁺ cluster which can be formed either by reconstitution with iron and sulfide, or on reduction with sodium dithionite. Here, we use EPR spectroscopy to show that mutations in the conserved YNHNLD sequence of Escherichia coli BioB affect the formation and stability of the [4Fe-4S]¹⁺ cluster on reduction with dithionite and report the observation of a new [2Fe-2S]¹⁺ cluster. These results serve to illustrate the dynamic nature of iron-sulfur clusters in biotin synthase and the role played by the protein in cluster interconversion.     

Iron-sulfur cluster biosynthesis. Molecular chaperone DnaK promotes IscU-bound [2Fe-2S] cluster stability and inhibits cluster transfer activity

IscU functions as a scaffold for Fe-S cluster assembly and transfer, and is known to be a substrate protein for molecular chaperones. Kinetic studies of Fe-S cluster transfer from holo IscU to apo Fd in the presence of chaperone DnaK demonstrate an inhibitory effect on the rate of Fe-S cluster transfer from IscU. Binding of DnaK reduces the rate of formation of the IscU-Fd complex (greater than 8-fold), but has little influence on the intrinsic rate of iron-sulfur cluster transfer to apo Fd. Apparently the molecular chaperone DnaK does not facilitate the process of Fe-S cluster transfer from IscU. Rather, DnaK has a modest influence on the stability of the IscU-bound Fe-S cluster that may reflect a more important role in promoting cluster assembly. In accord with prior observations the cochaperone DnaJ stimulates the ATPase activity of DnaK, but has a minimal influence on IscU cluster transfer activity, either alone or in concert with DnaK.     

Both human ferredoxins 1 and 2 and ferredoxin reductase are important for iron-sulfur cluster biogenesis

Ferredoxins are iron-sulfur proteins that have been studied for decades because of their role in facilitating the monooxygenase reactions catalyzed by p450 enzymes. More recently, studies in bacteria and yeast have demonstrated important roles for ferredoxin and ferredoxin reductase in iron-sulfur cluster assembly. The human genome contains two homologous ferredoxins, ferredoxin 1 (FDX1) and ferredoxin 2 (FDX2--formerly known as ferredoxin 1L). More recently, the roles of these two human ferredoxins in iron-sulfur cluster assembly were assessed, and it was concluded that FDX1 was important solely for its interaction with p450 enzymes to synthesize mitochondrial steroid precursors, whereas FDX2 was used for synthesis of iron-sulfur clusters, but not steroidogenesis. To further assess the role of the FDX-FDXR system in mammalian iron-sulfur cluster biogenesis, we performed siRNA studies on FDX1 and FDX2, on several human cell lines, using oligonucleotides identical to those previously used, along with new oligonucleotides that specifically targeted each gene. We concluded that both FDX1 and FDX2 were important in iron-sulfur cluster biogenesis. Loss of FDX1 activity disrupted activity of iron-sulfur cluster enzymes and cellular iron homeostasis, causing mitochondrial iron overload and cytosolic iron depletion. Moreover, knockdown of the sole human ferredoxin reductase, FDXR, diminished iron-sulfur cluster assembly and caused mitochondrial iron overload in conjunction with cytosolic depletion. Our studies suggest that interference with any of the three related genes, FDX1, FDX2 or FDXR, disrupts iron-sulfur cluster assembly and maintenance of normal cytosolic and mitochondrial iron homeostasis.     

过氧化物酶体的生物发生与疾病

过氧化物酶体的膜蛋白和酶分子由核基因编码,在游离的核糖体上合成之后,由定位信号引导靶向运输并组装到过氧化物酶体的。本文就过氧化物酶体膜蛋白信号ｍＰＴＳ、酶分子信号ＰＴＳ１Ｔ ＰＴＳ２、酶分子运进过氧化物酶体的模型以及由于过氧化物酶体生物发生的障碍而引起的疾病加以讨论。     

Iron-sulfur cluster reconstitution of spinach chloroplast Rieske protein requires a partially prefolded apoprotein

The Rieske 2Fe-2S protein is a central component of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for expression in Escherichia coli of full-length and truncated Spinacia oleracea Rieske (PetC) proteins fused to the MalE, maltose binding protein. The expressed Rieske fusion proteins were found predominantly in soluble form in the E. coli cytoplasm. These proteins could be readily purified for further experimentation. In vitro reconstitution of the characteristic, "Rieske-type" 2Fe-2S cluster into these fused proteins was accomplished by a chemical method employing reduced iron and sulfide. Cluster incorporation was monitored by electron paramagnetic resonance and optical circular dichroism (CD) spectroscopy. CD spectral analysis in the ultraviolet region suggests that the spinach Rieske apoprotein must be in a partially folded conformation to incorporate an appropriate iron-sulfur cluster. These data further suggest that upon cluster integration, further folding occurs, allowing the Rieske protein to attain a final, native structure. The data presented here are the first to demonstrate successful chemical reconstitution of the 2Fe-2S cluster into a Rieske apoprotein from higher plant chloroplasts.     

Iron-sulfur cluster biosynthesis: biochemical characterization of the conformational dynamics of Thermotoga maritima IscU and the relevance for cellular cluster assembly

Important for the understanding of the functional properties of the iron-sulfur scaffold IscU is knowledge of the structure and dynamics of this protein class. Structural characterization of Thermotoga maritima IscU by CD (Mansy, S. S., Wu, G., Surerus, K. K., and Cowan, J. A. (2002) J. Biol. Chem. 277, 21397-21404) and high resolution NMR (Bertini, I., Cowan, J. A., Del Bianco, C., Luchinat, C., and Mansy, S. S. (2003) J. Mol. Biol. 331, 907-924) yielded data indicating a high degree of secondary structure. However, the latter also revealed IscU to exist in a dynamic equilibrium between two or more distinct conformations, possibly existing in a molten globule state. Herein, we further characterize the molten globule characteristics of T. maritima IscU by near-ultraviolet circular dichroism, 1-anilino-8-naphthalenesulfonic acid binding, free energy of unfolding, hydrodynamic radius measurements, and limited tryptic digestion. The data suggest unusual dynamic behavior that is not fully consistent with typical protein states such as fully folded, fully unfolded, or molten globule. For instance, the existence of a stable tertiary fold is supported by near-UV CD spectra and hydrodynamic radius measurements, whereas other data are less clearly interpretable and may be viewed as consistent with either a molten globule or fully folded state. However, all of the data are consistent with our previous hypothesis of a protein sampling multiple discrete tertiary conformations in which these structural transitions occur on a "slow" time scale. To describe such proteins, we introduce the term multiple discrete conformers.