Characterization of the evolutionarily conserved iron-sulfur cluster of sirohydrochlorin ferrochelatase from Arabidopsis thaliana

Sirohaem is a cofactor of nitrite and sulfite reductases, essential for assimilation of nitrogen and sulfur. Sirohaem is synthesized from the central tetrapyrrole intermediate uroporphyrinogen III by methylation, oxidation and ferrochelation reactions. In Arabidopsis thaliana, the ferrochelation step is catalysed by sirohydrochlorin ferrochelatase (SirB), which, unlike its counterparts in bacteria, contains an [Fe-S] cluster. We determined the cluster to be a [4Fe-4S] type, which quickly oxidizes to a [2Fe-2S] form in the presence of oxygen. We also identified the cluster ligands as four conserved cysteine residues located at the C-terminus. A fifth conserved cysteine residue, Cys(135), is not involved in ligating the cluster directly, but influences the oxygen-sensitivity of the [4Fe-4S] form, and possibly the affinity for the substrate metal. Substitution mutants of the enzyme lacking the Fe-S cluster or Cys(135) retain the same specific activity in vitro and dimeric quaternary structure as the wild-type enzyme. The mutant variants also rescue a defined Escherichia coli sirohaem-deficient mutant. However, the mutant enzymes cannot complement Arabidopsis plants with a null AtSirB mutation, which exhibits post-germination arrest. These observations suggest an important physiological role for the Fe-S cluster in Planta, highlighting the close association of iron, sulfur and tetrapyrrole metabolism.     

Characterization of iron-sulfur cluster assembly protein isca from Acidithiobacillus ferrooxidans

IscA is a key member of the iron-sulfur cluster assembly machinery found in bacteria and eukaryotes, but the mechanism of its function in the biogenesis of iron-sulfur cluster remains elusive. In this paper, we demonstrate that Acidithiobacillus ferrooxidans IscA is a [4Fe-4S] cluster binding protein, and it can bind iron in the presence of DTT with an apparent iron association constant of 4·10²⁰ M^?1. The iron binding in IscA can be promoted by oxygen through oxidizing ferrous iron to ferric iron. Furthermore, we show that the iron bound form of IscA can be converted to iron-sulfur cluster bound form in the presence of IscS and L-cysteine in vitro. Substitution of the invariant cysteine residues Cys35, Cys99, or Cys101 in IscA abolishes the iron binding activity of the protein; the IscA mutants that fail to bind iron are unable to assemble the iron-sulfur clusters. Further studies indicate that the iron-loaded IscA could act as an iron donor for the assembly of iron-sulfur clusters in the scaffold protein IscU in vitro. Taken together, these findings suggest that A. ferrooxidans IscA is not only an iron-sulfur protein, but also an iron binding protein that can act as an iron donor for biogenesis of iron-sulfur clusters.     

Nad+-dependent isocitrate dehydrogenase from Arabidopsis thaliana. Characterization of two closely related subunits

Two cDNA clones which appear to encode different subunits of NAD⁺-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.41) were identified by homology searches from the Arabidopsis EST database. These cDNA clones were obtained and sequenced; both encoded full-length messages and displayed 82.7% nucleotide sequence identity over the coding region. The deduced amino acid sequences revealed preprotein lengths of 367 residues, with an amino acid identity of 86.1%. Genomic Southern blot analysis showed distinct single-copy genes for both IDH subunits. Both IDH subunits were expressed as recombinant proteins in Escherichia coli, and polyclonal antibodies were raised to each subunit. The Arabidopsis cDNA clones were expressed in Saccharomyces cerevisiae mutants which were deficient in either one or both of the yeast NAD⁺-dependent IDH subunits. The Arabidopsis cDNA clones failed to complement the yeast mutations; although both IDH-I and IDH-II were expressed at detectable levels, neither protein was imported into the mitochondria.     

Evolution of chloroplast mononucleotide microsatellites in Arabidopsis thaliana

The level of variation and the mutation rate were investigated in an empirical study of 244 chloroplast microsatellites in 15 accessions of Arabidopsis thaliana. In contrast to SNP variation, microsatellite variation in the chloroplast was found to be common, although less common than microsatellite variation in the nucleus. No microsatellite variation was found in coding regions of the chloroplast. To evaluate different models of microsatellite evolution as possible explanations for the observed pattern of variation, the length distribution of microsatellites in the published DNA sequence of the A. thaliana chloroplast was subsequently used. By combining information from these two analyses we found that the mode of evolution of the chloroplast mononucleotide microsatellites was best described by a linear relation between repeat length and mutation rate, when the repeat lengths exceeded about 7 bp. This model can readily predict the variation observed in non-coding chloroplast DNA. It was found that the number of uninterrupted repeat units had a large impact on the level of chloroplast microsatellite variation. No other factors investigated—such as the position of a locus within the chromosome, or imperfect repeats—appeared to affect the variability of chloroplast microsatellites. By fitting the slippage models to the Genbank sequence of chromosome 1, we show that the difference between microsatellite variation in the nucleus and the chloroplast is largely due to differences in slippage rate. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Characterization of an iron-sulfur cluster assembly protein (ISU1) from Schizosaccharomyces pombe

Genetic studies of bacteria and eukaryotes have led to identification of several gene products that are involved in the biosynthesis of protein-bound iron-sulfur clusters. One of these proteins, ISU, is homologous to the N-terminus of bacterial NifU. The mature forms of His-tagged wild-type and D37A Schizosaccharomyces pombe ISU1 were cloned and overexpressed as inclusion bodies in Escherichia coli. The recombinant D37A protein was purified under denaturing conditions and subsequently reconstituted in vitro. By use of a 5-fold excess of iron and sulfide the reconstituted product was found to be red-brown in color, forming a homodimer of 17 kDa per subunit with approximately two iron atoms per monomer determined by protein and iron quantitation. UV-vis absorption and M?ssbauer spectroscopies (delta = 0.29 +/- 0.05 mm/s; DeltaE(Q) = 0.59 +/- 0.05 mm/s) were used to characterize D37A ISU1 and show the presence of [2Fe-2S](2+) clusters in each subunit. Formation of the holo form of wild-type ISU1 was significantly less efficient using the same reconstitution conditions and is consistent with prior observations that the D37A substitution can stabilize protein-bound clusters. Relative to the human homologue, the yeast ISU is significantly less soluble at ambient temperatures. In both cases the native ISU1 is more sensitive to proton-mediated degradation relative to the D37A derivative. The lability of this family of proteins relative to [2Fe-2S] bearing ferredoxins most likely is of functional relevance for cluster transfer chemistry. M?ssbauer parameters obtained for wild-type ISU1 (delta = 0.31 +/- 0.05 mm/s; DeltaE(Q) = 0.64 +/- 0.05 mm/s) were similar to those obtained for the D37A derivative. Cluster transfer from ISU1 to apo Fd is demonstrated: the first example of transfer from an ISU-type protein. A lower limit for k(2) of 80 M(-1) min(-1) was established for WT cluster transfer and a value of 18 M(-1) min(-1) for the D37A derivative. Finally, we have demonstrated through cross-linking studies that ferredoxin, an electron-transport protein, forms a complex with ISU1 in both apo and holo states. Cross-linking of holo ISU1 with holo Fd is consistent with a role for redox chemistry in cluster assembly and may mimic the intramolecular complex already defined in NifU.     

RAF2 is a RuBisCO assembly factor in Arabidopsis thaliana

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the reaction between gaseous carbon dioxide (CO₂) and ribulose‐1,5‐bisphosphate. Although it is one of the most studied enzymes, the assembly mechanisms of the large hexadecameric RuBisCO is still emerging. In bacteria and in the C4 plant Zea mays, a protein with distant homology to p terin‐4α‐c arbinolamine d ehydratase (PCD) has recently been shown to be involved in RuBisCO assembly. However, studies of the homologous PCD‐like protein (RAF2, RuBisCO assembly factor 2) in the C3 plant Arabidopsis thaliana (A. thaliana) have so far focused on its role in hormone and stress signaling. We investigated whether A. thalianaRAF2 is also involved in RuBisCO assembly. We localized RAF2 to the soluble chloroplast stroma and demonstrated that raf2 A. thaliana mutant plants display a severe pale green phenotype with reduced levels of stromal RuBisCO. We concluded that the RAF2 protein is probably involved in RuBisCO assembly in the C3 plant A. thaliana.     

Characterization of photosystem II assembly complexes containing ONE-HELIX PROTEIN1 in Arabidopsis thaliana

Journal of Plant Research - The assembly process of photosystem II (PSII) requires several auxiliary proteins to form assembly intermediates. In plants, early assembly intermediates comprise D1 and...     

SufA from Erwinia chrysanthemi. Characterization of a scaffold protein required for iron-sulfur cluster assembly

SufA is a component of the recently discovered suf operon, which has been shown to play an important function in bacteria during iron-sulfur cluster biosynthesis and resistance to oxidative stress. The SufA protein from Erwinia chrysanthemi, a Gram-negative plant pathogen, has been purified to homogeneity and characterized. It is a homodimer with the ability to assemble rather labile [2Fe-2S] and [4Fe-4S] clusters as shown by M?ssbauer spectroscopy. These clusters can be transferred to apoproteins such as ferredoxin or biotin synthase during a reaction that is not inhibited by bathophenanthroline, an iron chelator. Cluster assembly in these proteins is much more efficient when iron and sulfur are provided by holoSufA than by free iron sulfate and sodium sulfide. We propose the function of SufA is that of a scaffold protein for [Fe-S] cluster assembly and compare it to IscA, a member of the isc operon also involved in cluster biosynthesis in both prokaryotes and eukaryotes. Mechanistic and physiological implications of these results are also discussed.     

The assembly pathway of complex I in Arabidopsis thaliana

All present‐day mitochondria originate from a single endosymbiotic event that gave rise to the last eukaryotic common ancestor more than a billion years ago. However, to date, many aspects of mitochondrial evolution have remained unresolved. Comparative genomics and proteomics have revealed a complex evolutionary origin for many mitochondrial components. To understand the evolution of the respiratory chain, we have examined both the components and the mechanisms of the assembly pathway of complex I. Complex I represents the first enzyme in the respiratory chain, and complex I deficiencies have dramatic consequences in both animals and plants. The complex is located in the mitochondrial inner membrane and possesses two arms: one embedded in the inner membrane and one protruding in the matrix. Here, we describe the assembly pathway of complex I in the model plant Arabidopsis thaliana. Using a proteomics approach called complexome profiling, we have resolved the different steps in the assembly process in plants. We propose a model for the stepwise assembly of complex I, including every subunit. We then compare this pathway with the corresponding pathway in humans and find that complex I assembly in plants follows a different, and likely ancestral, pathway compared with the one in humans. We show that the main evolutionary changes in complex I structure and assembly in humans occurred at the level of the membrane arm, whereas the matrix arm remained rather conserved.     

LPA2 is required for efficient assembly of photosystem II in Arabidopsis thaliana

To elucidate the molecular mechanism of photosystem II (PSII) assembly, we characterized the low psii accumulation2 (lpa2) mutant of Arabidopsis thaliana, which is defective in the accumulation of PSII supercomplexes. The levels and processing patterns of the RNAs encoding the PSII subunits are unaltered in the mutant. In vivo protein-labeling experiments showed that the synthesis of CP43 (for chlorophyll a binding protein) was greatly reduced, but CP47, D1, and D2 were synthesized at normal rates in the lpa2-1 mutant. The newly synthesized CP43 was rapidly degraded in lpa2-1, and the turnover rates of D1 and D2 were higher in lpa2-1 than in wild-type plants. The newly synthesized PSII proteins were assembled into PSII complexes, but the assembly of PSII was less efficient in the mutant than in wild-type plants. LPA2 encodes an intrinsic thylakoid membrane protein, which is not an integral subunit of PSII. Yeast two-hybrid assays indicated that LPA2 interacts with the PSII core protein CP43 but not with the PSII reaction center proteins D1 and D2. Moreover, direct interactions of LPA2 with Albino3 (Alb3), which is involved in thylakoid membrane biogenesis and cell division, were also detected. Thus, the results suggest that LPA2, which appears to form a complex with Alb3, is involved in assisting CP43 assembly within PSII.     

Three different genes encode the iron-sulfur subunit of succinate dehydrogenase in Arabidopsis thaliana

The iron-sulfur protein is an essential component of mitochondrial complex II (succinate dehydrogenase, SDH), which is a functional enzyme of both the citric acid cycle and the respiratory electron transport chain. This protein is encoded by a single-copy nuclear gene in mammals and fungi and by a mitochondrial gene in Rhodophyta and the protist Reclinomonas americana. In Arabidopsis thaliana, the homologous protein is now found to be encoded by three nuclear genes. Two genes (sdh2-1 andsdh2-2) likely arose from a relatively recent duplication event since they have similar structures, encode nearly identical proteins and show similar expression patterns. Both genes are interrupted by a single intron located at a conserved position. Expression was detected in all tissues analysed, with the highest steady-state mRNA levels found in flowers and inflorescences. In contrast, the third gene (sdh2-3) is interrupted by 4 introns, is expressed at a low level, and encodes a SDH2-3 protein which is only 67% similar to SDH2-1 and SDH2-2 and has a different N-terminal presequence. Interestingly, the proteins encoded by these three genes are probably functional because they are highly conserved compared with their homologues in other organisms. These proteins contain the cysteine motifs involved in binding the three iron-sulfur clusters essential for electron transport. Furthermore, the three polypeptides are found to be imported into isolated plant mitochondria.     

Large-scale top-down proteomics of the Arabidopsis thaliana leaf and chloroplast proteomes

We present a large-scale top-down proteomics (TDP) study of plant leaf and chloroplast proteins, achieving the identification of over 4700 unique proteoforms. Using capillary zone electrophoresis coupled with tandem mass spectrometry analysis of offline size-exclusion chromatography fractions, we identify 3198 proteoforms for total leaf and 1836 proteoforms for chloroplast, with 1024 and 363 proteoforms having post-translational modifications, respectively. The electrophoretic mobility prediction of capillary zone electrophoresis allowed us to validate post-translational modifications that impact the charge state such as acetylation and phosphorylation. Identified modifications included Trp (di)oxidation events on six chloroplast proteins that may represent novel targets of singlet oxygen sensing. Furthermore, our TDP data provides direct experimental evidence of the N- and C-terminal residues of numerous mature proteoforms from chloroplast, mitochondria, endoplasmic reticulum, and other sub-cellular localizations. With this information, we suggest true transit peptide cleavage sites and correct sub-cellular localization signal predictions. This large-scale analysis illustrates the power of top-down proteoform identification of post-translational modifications and intact sequences that can benefit our understanding of both the structure and function of hundreds of plant proteins.     

Network of protein-protein interactions among iron-sulfur cluster assembly proteins in Escherichia coli

The assembly of iron-sulfur (Fe-S) clusters is mediated by complex machinery which, in Escherichia coli, is encoded by the iscRSUA-hscBA-fdx-ORF3 gene cluster. Here, we demonstrate the network of protein-protein interactions among the components involved in the machinery. We have constructed (His)(6)-tagged versions of the components and identified their interacting partners that were co-purified from E. coli extracts with a Ni-affinity column. Direct associations of the defined pair of proteins were further examined in yeast cells using the two-hybrid system. In accord with the previous in vitro binding and kinetic experiments, interactions were observed for the combinations of IscS and IscU, IscU and HscB, IscU and HscA, and HscB and HscA. In addition, we have identified previously unreported interactions between IscS and Fdx, IscS and ORF3, IscA and HscA, and HscA and Fdx. We also found, by site-directed mutational analysis combined with the two-hybrid system, that two cysteine residues in IscU are essential for binding with HscB but not with IscS. Despite the complex network of interactions in various combinations of components, heteromultimeric complexes were not observed in our experiments except for the putative oligomeric form of IscU-IscS-ORF3. Thus, the sequential association and dissociation among the IscS, IscU, IscA, HscB, HscA, Fdx, and ORF3 proteins may be a critical process in the assembly of Fe-S clusters.     

Characterization of Adaptation in Phototropism of Arabidopsis thaliana

Phototropic curvature has been measured for etiolated Arabidopsis thaliana seedlings with and without a preirradiation. A bilateral preirradiation with 450-nm light at a fluence greater than about 0.1 micromole per square meter causes a rapid desensitization to a subsequent 450-nanometer unilateral irradiation at 0.5 micromole per square meter. Following a refractory period, the capacity to respond phototropically recovers to the predesensitization level, and the response is then enhanced. The length of the refractory period is between 10 and 20 minutes. Both the time needed for recovery and the extent of enhancement increase with increasing fluence of the bilateral preirradiation. Based on the relative spectral sensitivities of desensitization and enhancement, these responses can be separated. Desensitization is induced by blue light but not by red light. Enhancement, however, is induced by both blue and red light. Thus, enhancement can be induced without desensitization but not vice versa. Both desensitization and enhancement affect only the magnitude of the response and do not affect the fluence threshold.     

拟南芥ABA不敏感型突变体及其ABA结合特性

拟南芥的脱落醚（ABA）不敏感型突变体abi2,在对ABA的敏感性、气孔开度及种子休眠方面,与野生型有明显差异。通过3H－ABA与野生型对的亚细胞组分的结合分析,表明38000×g组分特异结合活性最高,结合最适温度为20℃,最适保温时间：20℃时为70min;0℃时为90min。由饱和曲线的Scatchard分析表明：abi2存在一种ABA结合位点,野生型有两种ABA结合位点。对3H－ABA结合的38000×g组分的SDS－PAGE电泳分析表明：野生型有3个结合活性峰,而abi2只有1个结合活性峰。