Overexpression and reconstitution of a Rieske iron-sulfur protein from the cyanobacterium Synechocystis PCC 6803

Chloroplast cyt b6f complexes as well as mitochondrial and bacterial cyt bc1 complexes contain a high potential Rieske iron-sulfur protein which is essential for their function. To characterise the isolated Rieske protein from the mesophilic cyanobacterium Synechocystis PCC6803 we cloned the encoding gene into an expression vector and overexpressed the protein in E. coli. In cells overexpressing the protein no typical Rieske type EPR signal was detected neither in membranes nor in inclusion bodies where the majority of the protein was deposited. The inclusion bodies were isolated from the E. coli cells and denaturated with 8 M urea. With a single anion exchange chromatographic step a pure protein could be obtained which was used for further experiments. The NifS like protein IscS was recently reported to mediate the incorporation of iron-sulfur clusters into ferredoxin in vitro. We used the recombinant IscS protein for the incorporation of the cluster into the folded Rieske apoprotein. Spectroscopic characterisation of the resultant protein by CD and EPR spectroscopy showed the presence of a typical Rieske iron-sulfur centre.     

Density functional calculation of pK a values and redox potentials in the bovine Rieske iron-sulfur protein

The redox potential of the Rieske iron-sulfur protein depends on pH. It has been proposed that the histidines coordinating one of the irons are responsible for this pH dependence, but an experimental proof for this proposal is still lacking. In this work, we present a density functional/continuum electrostatics calculation of the p K(a) values of the histidines in the Rieske iron-sulfur center. The calculated apparent p K(a) values are 6.9 and 8.8 in the oxidized state, which are in good agreement with the corresponding experimental values of 7.5 and 9.2 and the measured pH dependence of the redox potential. Neither of these two p K(a) values can, however, be assigned to only one of the histidines. We find that both histidines titrate over a wide pH range in the oxidized state. Reduction of the iron-sulfur center shifts the p K(a) values to 11.3 and 12.8, thus above 10.0 as found experimentally. The results provide a complete picture of the coupling of proton and electron binding, showing strongly cooperative binding of protons at electrode potentials near the redox midpoint potential of the cluster. The potential biological function of the low p K(a) value of the histidines and the shift upon reduction are briefly discussed.     

The iron-sulfur cluster of the Rieske iron-sulfur protein functions as a proton-exiting gate in the cytochrome bc(1) complex

The destruction of the Rieske iron-sulfur cluster ([2Fe-2S]) in the bc(1) complex by hematoporphyrin-promoted photoinactivation resulted in the complex becoming proton-permeable. To study further the role of this [2Fe-2S] cluster in proton translocation of the bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with mutations at the histidine ligands of the [2Fe-2S] cluster were generated and characterized. These mutants lacked the [2Fe-2S] cluster and possessed no bc(1) activity. When the mutant complex was co-inlaid in phospholipid vesicles with intact bovine mitochondrial bc(1) complex or cytochrome c oxidase, the proton ejection, normally observed in intact reductase or oxidase vesicles during the oxidation of their corresponding substrates, disappeared. This indicated the creation of a proton-leaking channel in the mutant complex, whose [2Fe-2S] cluster was lacking. Insertion of the bc(1) complex lacking the head domain of the Rieske iron-sulfur protein, removed by thermolysin digestion, into PL vesicles together with mitochondrial bc(1) complex also rendered the vesicles proton-permeable. Addition of the excess purified head domain of the Rieske iron-sulfur protein partially restored the proton-pumping activity. These results indicated that elimination of the [2Fe-2S] cluster in mutant bc(1) complexes opened up an otherwise closed proton channel within the bc(1) complex. It was speculated that in the normal catalytic cycle of the bc(1) complex, the [2Fe-2S] cluster may function as a proton-exiting gate.     

The oxidation of ubiquinol by the isolated Rieske iron-sulfur protein in solution

The pre-steady-state redox reactions of the Rieske iron-sulfur protein isolated from beef heart mitochondria have been characterized. The rates of oxidation by c-type cytochromes is much faster than the rate of reduction by ubiquinols. This enables the monitoring of the oxidation of ubiquinols by the Rieske protein through the steady-state electron transfer to cytochrome c in solution. The pH and ionic strength dependence of this reaction indicate that the ubiquinol anion is the direct reductant of the oxidized cluster of the iron-sulfur protein. The second electron from ubiquinol is diverted to oxygen by the isolated Rieske protein, and forms oxygen radicals that contribute to the steady-state reduction of cytochrome c. Under anaerobic conditions, however, the reduction of cytochrome c catalyzed by the protein becomes mechanicistically identical to the chemical reduction by ubiquinols. The present kinetic work outlines that: (i) the electron transfer between the ubiquinol anion and the Rieske cluster has a comparable rate when the protein is isolated or inserted into the parent cytochrome c reductase enzyme; (ii) the Rieske protein may be a relevant generator of oxygen radicals during mitochondrial respiration.     

Dephosphorylation of the Rieske iron-sulfur protein after induction of the mitochondrial permeability transition

In the mitochondrial permeability transition (MPT), MPT pores open to cause the mitochondrial inner membrane to become non-selectively permeable to molecules of mass up to 1500 Da. In this study, we used proteomics to investigate protein changes after MPT induction. Isolated rat liver mitochondria were incubated with various MPT inducers, including CaCl2, tert-butylhydroperoxide, and phenylarsine oxide, in the presence and absence of the MPT inhibitor, cyclosporin A. MPT induction was confirmed by an absorbance swelling assay. Mitochondrial membrane proteins prepared from control and treated mitochondria were separated by two-dimensional (2D) gel electrophoresis and stained with SyproRuby or Coomassie blue. Proteins of interest were further identified by mass spectrometry. 2D gel electrophoresis by isoelectric focusing and SDS-PAGE consistently showed a protein spot that shifted to a more basic isoelectric point after the MPT. This shift was prevented by CsA but did not occur after protonophoric uncoupling. Mass spectrometry identified this protein as the Rieske iron-sulfur protein (RISP) of ubiquinol-cytochrome c reductase (Complex III). Phosphatase treatment of sonicated mitochondria caused the same shift in RISP as occurred in MPT inducer-treated mitochondria. 2D gel electrophoresis by blue-native-PAGE and SDS-PAGE showed that RISP existed as an apparent monomer in mitochondrial membranes in addition to forming a complex with ubiquinol-cytochrome c reductase. These findings suggest that RISP may be part of MPT pores and that dephosphorylation of RISP may play a role in regulation of the MPT.     

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.     

The chloroplast Rieske iron-sulfur protein. At the crossroad of electron transport and signal transduction

We have addressed the functional and structural roles of three domains of the chloroplast Rieske iron-sulfur protein; that is, the flexible hinge that connects the transmembrane helix to the soluble cluster-bearing domain, the N-terminal stromal protruding domain, and the transmembrane helix. To this aim mutants were generated in the green alga Chlamydomonas reinhardtii. Their capacities to assemble the cytochrome b6f complex, perform plastoquinol oxidation, and signal redox-induced activation of the light-harvesting complex II kinase during state transition were tested in vivo. Deletion of one residue and extensions of up to five residues in the flexible hinge had no significant effect on complex accumulation or electron transfer efficiency. Deletion of three residues (Delta3G) dramatically decreased reaction rates by a factor of approximately 10. These data indicate that the chloroplast iron-sulfur protein-linking domain is much more flexible than that of its counterpart in mitochondria. Despite greatly slowed catalysis in the Delta3G mutant, there was no apparent delay in light-harvesting complex II kinase activation or state transitions. This indicates that conformational changes occurring in the Rieske protein did not represent a limiting step for kinase activation within the time scale tested. No phenotype could be associated with mutations in the N-terminal stromal-exposed domain. In contrast, the N17V mutation in the Rieske protein transmembrane helix resulted in a large decrease in the cytochrome f synthesis rate. This reveals that the Rieske protein transmembrane helix plays an active role in assembly-mediated control of cytochrome f synthesis. We propose a structural model to interpret this phenomenon based on the C. reinhardtii cytochrome b6f structure.     

Novel isolation and reconstitution of the iron-sulfur protein of mitochondrial Complex III

An iron-sulfur protein was isolated from mitochondrial Complex III as a single band on SDS-polyacrylamide gel electrophoresis by hydrophobic interaction chromatography, in which the complex was dissociated into the iron-sulfur protein and the rest of the complex with a loss of enzymic activity. The critical condition for the dissociation was depletion of boundary phospholipids to 0.6% (w/w). After gel filtration of the isolated iron-sulfur protein, 76 ng atom of non-heme iron and 66 nmol of acid-labile sulfide were found per mg of the protein. By incubating the protein with the iron-sulfur protein depleted-complex in the presence of a soybean phospholipid mixture, the enzymic activity was fully recovered. The electrophoretic pattern of the reconstituted complex showed a polypeptide composition similar to that of the original Complex III. These results indicate that the iron-sulfur protein is attached in the complex with the aid of boundary phospholipids.     

Isolation and characterization of the nuclear gene encoding the Rieske iron-sulfur protein (RIP1) from Saccharomyces cerevisiae

The nuclear gene encoding the Rieske iron-sulfur protein of the cytochrome bc1 complex of the mitochondrial respiratory chain has been isolated and characterized from Saccharomyces cerevisiae. We used a segment of the iron-sulfur protein gene from Neurospora crassa (Harnisch, U., Weiss, H., and Sebald, W. (1985) Eur. J. Biochem. 149, 95-99) to detect the yeast gene by Southern analysis. Five different but overlapping clones were then isolated by probing a yeast genomic library carried on YEp 13 by colony lift hybridization. Several approaches confirmed that the isolated DNA contained the gene for the Rieske iron-sulfur protein. The yeast gene, which contains no introns, can be expressed in Escherichia coli. A 900-base pair HindIII-EcoRI fragment was subcloned into pUC19 and directed the synthesis of immunodetectable protein. The gene was also identified by disruption of its chromosomal copy by homologous integration. A 400 base pair PstI-EcoRI fragment cloned adjacent to a HIS3 marker in pUC18 was used as an integrating vector. HIS+ transformants were obtained which were unable to grow on the nonfermentable carbon source glycerol. Southern analysis of the respiration deficient (gly-) strains confirmed that the chromosomal copy of the gene was disrupted, and immunoblots of extracts of the transformants indicated a lack of iron-sulfur protein. A respiration-deficient integrant was transformed to GLY+ by a 2-kilobase pair HindIII-BglII fragment, including a complete copy of the gene, carried on a multicopy episomal vector. Immunoblots with monoclonal antibodies to the iron-sulfur protein indicated overproduction of the protein in the complemented strain and revealed expression of approximately equal amounts of mature iron-sulfur protein and of a protein approximately 3 kDa larger than the mature protein in the complemented strain. A 1.2-kilobase pair segment of DNA from the clone which complemented the disrupted strains was sequenced and found to contain an open reading frame of 645 nucleotides, capable of encoding a 21,946-dalton protein. The gene is flanked by consensus signal sequences for initiation and termination which are common in yeast and is preceded by a possible upstream activating sequence. Amino acid sequence analysis of the amino-terminal end of the mature iron-sulfur protein agreed exactly with that predicted by the nucleotide sequence starting at Lys-31.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cloning and sequencing of a cDNA encoding the Rieske iron-sulfur protein of bovine heart mitochondrial ubiquinol-cytochrome c reductase

Two cDNA clones encoding bovine heart mitochondrial Rieske iron-sulfur protein were obtained by immunological screening of a bovine heart cDNA expression library in lambda gt11 with antiserum directed against Rieske iron-sulfur protein isolated from bovine heart mitochondrial ubiquinol-cytochrome c reductase. The cDNA inserts were 1005 and 1100 base pairs with an open reading frame of 807 base pairs which encoded a 196-amino acid mature Rieske iron-sulfur protein and a 73-amino acid presequence. The amino acid sequence of Rieske iron-sulfur protein deduced from nucleotide sequencing is the same as that obtained from protein sequencing except at residues #73 and #191 which are Ser and Asp instead of Ala and Gly, respectively.     

On the interaction of mitochondrial complex III with the Rieske iron-sulfur protein (subunit V).

Limited proteolysis of solubilized beef heart mitochondrial complex III with trypsin yields a product previously identified as fragment V" (González-Halphen, D., Lindorfer, M. A., and Capaldi, R. A. (1988) Biochemistry 27, 7021-7031). In this work, fragment V" was generated by trypsin treatment of both the intact complex III and the purified Rieske iron-sulfur protein. Thus, in its bound or isolated form, the same sites of subunit V are sensitive to protease action. Fragment V" was a soluble protein that retained its iron-sulfur moiety. It was purified by exclusion from a hydrophobic phenyl-Sepharose CL-4B column followed by gel filtration. In contrast to the pure, intact subunit V, fragment V" did not reconstitute oxidoreductase activity when combined with complex III devoid of subunit V. However, a 20-amino acid synthetic peptide carrying the sequence between amino acids Lys33 and Lys52 of the Rieske iron-sulfur protein competed with intact subunit V in reconstitution assays. The results obtained suggest that the iron-sulfur protein binds to complex III by hydrophobic protein-protein interactions, and that a nontransmembrane 18-amino acid amphipathic stretch accounts for the association of this subunit to the rest of the complex.     

Colchicine-binding protein from phloem and xylem of a higher plant

Summary Colchicine has been demonstrated by ion-exchange and by gelfiltration assay to bind to a protein fraction derived from the higher plant Heracleum mantegazzianum. Colchicine-binding protein from a plant source was much more unstable than tubulin from animal preparations. The tissues of Heracleum vary in their content of colchicine-binding activity. No activity was obtained from non-vascular tissue. Phloem has at most, twice as much activity as xylem. The significance of these results is discussed in relation to a proposed degree of homology between P protein of phloem and microtubule protein.     

Failure to insert the iron-sulfur cluster into the Rieske iron-sulfur protein impairs both center N and center P of the cytochrome bc1 complex

Mutation of a serine that forms a hydrogen bond to the iron-sulfur cluster of the Rieske iron-sulfur protein to a cysteine results in a respiratory-deficient yeast strain due to formation of iron-sulfur protein lacking the iron-sulfur cluster. The Rieske apoprotein lacking the iron-sulfur cluster is inserted into both monomers of the dimeric cytochrome bc(1) complex and processed to mature size, but the protein lacking iron-sulfur cluster is more susceptible to proteolysis. In addition, the protein environment of center P in one half of the dimer is affected by failure to insert the iron-sulfur cluster as indicated by the fact that only one molecule of myxothiazol can be bound to the cytochrome bc(1) dimer. Although the bc(1) complex lacking the Rieske iron-sulfur cluster cannot oxidize ubiquinol through center P, rates of reduction of cytochrome b by menaquinol through center N are normal. However, less cytochrome b is reduced through center N, and only one molecule of antimycin can be bound at center N in the bc(1) dimer lacking iron-sulfur cluster. These results indicate that failure to insert the [2Fe-2S] cluster impairs assembly of the Rieske protein into the bc(1) complex and that this interferes with proper assembly of both center P and center N in one half of the dimeric enzyme.     

Cloning and sequence analysis of a cDNA encoding the Rieske iron-sulfur protein of rat mitochondrial cytochrome bc1 complex

We have isolated a cDNA clone for the Rieske iron-sulfur protein of rat cytochrome bc1 complex, by screening a rat liver cDNA expression library using antiserum directed against the corresponding protein of bovine. The amino acid sequence deduced from the nucleotide sequence of the cDNA indicated that the mature polypeptide of the rat protein consists of 196 amino acid residues with a molecular weight of 21,465, and that it is formed as a precursor with an amino-terminal extension. Northern blot analysis indicated that rat liver possibly contains different sizes of mRNAs for the Rieske iron-sulfur protein, and Southern blot analysis demonstrated that rats and mice possess a single gene for this protein.     

Cells lacking Rieske iron-sulfur protein have a reactive oxygen species-associated decrease in respiratory complexes I and IV

Mitochondrial respiratory complexes of the electron transport chain (CI, CIII, and CIV) can be assembled into larger structures forming supercomplexes. We analyzed the assembly/stability of respiratory complexes in mouse lung fibroblasts lacking the Rieske iron-sulfur protein (RISP knockout [KO]cells), one of the catalytic subunits of CIII. In the absence of RISP, most of the remaining CIII subunits were able to assemble into a large precomplex that lacked enzymatic activity. CI, CIV, and supercomplexes were decreased in the RISP-deficient cells. Reintroduction of RISP into KO cells restored CIII activity and increased the levels of active CI, CIV, and supercomplexes. We found that hypoxia (1% O(2)) resulted in increased levels of CI, CIV, and supercomplex assembly in RISP KO cells. In addition, treatment of control cells with different oxidative phosphorylation (OXPHOS) inhibitors showed that compounds known to generate reactive oxygen species (ROS) (e.g., antimycin A and oligomycin) had a negative impact on CI and supercomplex levels. Accordingly, a superoxide dismutase (SOD) mimetic compound and SOD2 overexpression provided a partial increase in supercomplex levels in the RISP KO cells. Our data suggest that the stability of CI, CIV, and supercomplexes is regulated by ROS in the context of defective oxidative phosphorylation.     

斜纹夜蛾铁硫亚基蛋白的表达及功能鉴定

铁硫亚基蛋白(rieske iron-sulfur protein, RISP) 是线粒体复合物Ⅲ的关键蛋白亚基之一, 在呼吸链电子传递过程中起到重要作用。本研究通过RT-PCR克隆得到斜纹夜蛾Spodoptera litura RISP基因SlitRISP的ORF, 构建了pET32a-SlitRISP原核表达载体, SDS-PAGE和Western blot检测结果显示, SlitRISP原核表达蛋白以包涵体的形式存在于菌体沉淀中, 且RISP抗体可成功用于该蛋白的免疫印迹检测。为了进一步鉴定SlitRISP在斜纹夜蛾离体细胞系SL-1中的功能, 通过向细胞内转染siRNA, 利用RNAi技术沉默SL-1中的SlitRISP。qRT-PCR结果表明, 分别经50 nmol/L和100 nmol/L siRNA处理48 h后, SL-1中SlitRISP的表达均几乎完全被抑制; Western blot结果显示, SL-1中SlitRISP含量显著低于CK。当SL-1 SlitRISP被成功沉默后, 通过检测SL-1线粒体膜电位、 细胞ATP含量和细胞增殖抑制率鉴定RISP在线粒体电子传递过程中的重要作用。流式细胞仪测定结果表明, 经50 nmol/L和100 nmol/L siRNA处理24 h后, SL-1线粒体膜电位相对于CK分别降低23.52%和11.32%, 而处理48 h后, SL-1线粒体膜电位则分别升高5.58%和27.66%; siRNA处理24 h和48 h后, SL-1 ATP含量相对于CK分别降低82.71%和84.50%, 最终导致SL-1细胞增殖抑制率分别为53.64%和67.94%。这些结果表明SlitRISP在SL-1中参与线粒体膜电位的形成和细胞ATP的合成。介于RISP在线粒体电子传递链中的重要作用, 其可能成为新型杀虫作用靶标, 这可为研制新型呼吸抑制剂提供参考。     

The structure of the soluble domain of an archaeal Rieske iron-sulfur protein at 1.1 A resolution

The first crystal structure of an archaeal Rieske iron-sulfur protein, the soluble domain of Rieske iron-sulfur protein II (soxF) from the hyperthermo-acidophile Sulfolobus acidocaldarius, has been solved by multiple wavelength anomalous dispersion (MAD) and has been refined to 1.1 A resolution. SoxF is a subunit of the terminal oxidase supercomplex SoxM in the plasma membrane of S. acidocaldarius that combines features of a cytochrome bc(1) complex and a cytochrome c oxidase. The [2Fe-2S] cluster of soxF is most likely the primary electron acceptor during the oxidation of caldariella quinone by the cytochrome a(587)/Rieske subcomplex. The geometry of the [2Fe-2S] cluster and the structure of the cluster-binding site are almost identical in soxF and the Rieske proteins from eucaryal cytochrome bc(1) and b(6)f complexes, suggesting a strict conservation of the catalytic mechanism. The main domain of soxF and part of the cluster-binding domain, though structurally related, show a significantly divergent structure with respect to topology, non-covalent interactions and surface charges. The divergent structure of soxF reflects a different topology of the soxM complex compared to eucaryal bc complexes and the adaptation of the protein to the extreme ambient conditions on the outer membrane surface of a hyperthermo-acidophilic organism.     

The primary structure of human Rieske iron-sulfur protein of mitochondrial cytochrome bc1 complex deduced from cDNA analysis

We isolated a cDNA encoding human Rieske Fe-S protein of mitochondrial cytochrome bc1 complex from a fibroblast cDNA library by colony hybridization. The cDNA contains the nucleotide sequence encoding all of the amino acids (274 residues) comprising the putative precursor to the protein. Based on the known amino acid sequence of bovine Rieske Fe-S protein, the N-terminal extension sequence is presumed to be composed of 78 amino acids with a molecular weight of 8053. The mature protein consists of the same number of amino acid residues as that of its rat and bovine counterparts, having a homology of about 92% with the latter.