Isolation and reconstitution of the iron-sulfur protein in ubiquinol-cytochrome c oxidoreductase complex. Phospholipids are essential for the integration of the iron-sulfur protein in the complex

An iron-sulfur protein has been purified from beef heart ubiquinol-cytochrome c oxidoreductase (Complex III) of the mitochondrial respiratory chain by phenyl-Sepharose column chromatography and Sephacryl S-200 gel chromatography. Depletion of most of the endogenous phospholipids in the complex was a prerequisite to the dissociation of the protein from the complex in the former chromatography. The iron-sulfur protein was nearly homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 76 ng atoms of nonheme iron and 66 nmol of acid-labile sulfide/mg of protein. When this preparation was incubated with an iron-sulfur protein-depleted complex in the presence of soybean phospholipids, the enzymic activity was restored up to 90% of that of the parent Complex III, whereas the recovery of the activity was marginal in the absence of the phospholipids. Thus it is clear that the iron-sulfur protein is integrated into the complex with the aid of phospholipids.     

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.     

Immunochemical demonstration of the iron-sulfur protein in Complex III of mitochondrial electron-transfer chain

An iron-sulfur protein of Complex III was purified by phenyl-Sepharose column chromatography and DEAE-Sepharose column chromatography. The purified preparation was homogeneous as demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and a specific antibody directed against this protein was raised in a rabbit. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electrophoretic blotting and immunoperoxidase reaction indicated that Complex III possesses a single polypeptide which reacts with the antibody. It was also found that the iron-sulfur center-containing subunits identified so far in Complex I did not cross-react with the antibody, indicating that they are antigenically unrelated to the iron-sulfur protein of Complex III.     

Integral polypeptide composition of Complex III of the mitochondrial electron-transfer chain

Phospholipids in isolated Complex III of the mitochondrial electron-transfer chain were depleted by hydrophobic chromatography. The complex was further purified by affinity chromatography. The polypeptide composition of the complex was examined using SDS-polyacrylamide gel electrophoresis. Ten polypeptides were demonstrated in the gel pattern of the complex containing more than 10% (w/w) phospholipids; and 9 polypeptides, in the pattern of the complex containing 5% phospholipids. Although the enzymic activity of the complex composed of the 9 polypeptides was about a half of that of the original enzyme, it was fully restored when soybean phospholipid mixture was added. Further depletion of phospholipids to 0.6% makes the iron-sulfur protein dissociable from the complex, resulting in a loss of the enzymic activity (Shimomura, Y. and Ozawa, T. (1982) Biochem. Int. 5, 1-6). These results suggest that Complex III consists of 9 polypeptides, and the smallest polypeptide is a contaminant embedded in phospholipids with respect to the electron-transfer capability of the complex.     

Purification of the iron-sulfur protein, ubiquinone-binding protein, and cytochrome c1 from a single source of mitochondrial complex III

By detergent-exchange chromatography using a phenyl-Sepharose CL-4B column, Complex III of the respiratory chain of beef heart mitochondria was efficiently resolved into five fractions that were rich in the iron-sulfur protein, ubiquinone-binding protein, core proteins, cytochrome c1, and cytochrome b, respectively. Complex III was initially bound to the phenyl-Sepharose column equilibrated with buffer containing 0.25% deoxycholate and 0.2 M NaCl. An iron-sulfur protein fraction was first eluted from the column with buffer containing 1% deoxycholate and no salt after removal of phospholipids from the complex by washing with the buffer for the column equilibration, as reported previously (Y. Shimomura, M. Nishikimi, and T. Ozawa, 1984, J. Biol. Chem. 259, 14059-14063). Subsequently, a fraction containing the ubiquinone-binding protein and another containing two core proteins were eluted with buffers containing 1.5 and 3 M guanidine, respectively. A fraction containing cytochrome c1 was then eluted with buffer containing 1% dodecyl octaethylene glycol monoether. Finally, a cytochrome b-rich fraction was eluted with buffer containing 2% sodium dodecyl sulfate. The fractions of the iron-sulfur protein and ubiquinone-binding protein were further purified by gel chromatography on a Sephacryl S-200 superfine column, and the cytochrome c1 fraction was further purified by ion-exchange chromatography on a DEAE-Sepharose CL-6B column; each of the three purified proteins was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Crystal structure of mitochondrial respiratory membrane protein complex II

The mitochondrial respiratory Complex II or succinate:ubiquinone oxidoreductase (SQR) is an integral membrane protein complex in both the tricarboxylic acid cycle and aerobic respiration. Here we report the first crystal structure of Complex II from porcine heart at 2.4 A resolution and its complex structure with inhibitors 3-nitropropionate and 2-thenoyltrifluoroacetone (TTFA) at 3.5 A resolution. Complex II is comprised of two hydrophilic proteins, flavoprotein (Fp) and iron-sulfur protein (Ip), and two transmembrane proteins (CybL and CybS), as well as prosthetic groups required for electron transfer from succinate to ubiquinone. The structure correlates the protein environments around prosthetic groups with their unique midpoint redox potentials. Two ubiquinone binding sites are discussed and elucidated by TTFA binding. The Complex II structure provides a bona fide model for study of the mitochondrial respiratory system and human mitochondrial diseases related to mutations in this complex.     

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.     

Kinetics of assembly of complex III into the yeast mitochondrial membrane. Evidence for a precursor to the iron-sulfur protein

Complex III immunoprecipitated from yeast cells labeled in vivo with [35S]sulfate or [3H]leucine contained seven subunits with molecular weights ranging from 15,000 to 47,000 when analyzed by electrophoresis on polyacrylamide gels. The subunit composition of the immunoprecipitates was identical with that of the purified complex III isolated from bakers' yeast suggesting that the antiserum recognizes the holoenzyme assembled properly in the membrane (Sidhu, A., and Beattie, D.S. (1982) J. Biol. Chem. 257, 7879-7886). Kinetic studies using double-labeled yeast cells followed by immunoprecipitation of complex III indicated that the subunits of the complex are assembled into the holoenzyme at very different rates. Cytochromes b and c1 and the 15,000-dalton subunit were the first polypeptides to be assembled into the complex with a half-time of labeling of 2.0-2.4 min. Core protein I and the iron-sulfur protein were inserted more slowly into the complex with a half-time of labeling of 4.6 and 5.3 min, respectively. Calculations of precursor pool sizes of the subunits indicated that for both core protein I and the iron-sulfur protein, there are large pools of precursors. The iron-sulfur protein was synthesized in vivo as a larger precursor polypeptide of molecular mass 28,000 Da. The precursor was subsequently cleaved, in a process requiring an energized mitochondrial inner membrane, into an intermediate form 1,500 Da larger than the mature subunit. The conversion of the intermediate to the mature form occurred in the inner mitochondrial membrane.     

Linear and Conformation-Dependent Antigenic Sites on the Nucleoprotein of Rabies Virus

A set of 29 monoclonal antibodies (MAbs) specific for the rabies virus nucleoprotein (N protein) was prepared and used to analyze the topography of antigenic sites. At least four partially overlapping antigenic sites were delineated on the N protein of rabies virus by competitive binding assays. Indirect immunofluorescent antibody tests using MAbs with a series of rabies and rabies-related viruses showed that epitopes shared by various fixed and street strains of rabies virus were mainly localized at antigenic sites II and III, while epitopes representing the genus-specific antigen of Lyssavirus were widely presented at sites I, III and IV. All but one of seven MAbs specific for antigenic sites I, IV and bridge site (I and II) reacted with the antigen that had been denatured by sodium dodecyl sulfate or 2-mercaptoethanol, as well as with the denatured N protein in Western blotting assays. However, none of the MAbs against antigenic sites II and III reacted with the denatured antigen. These data indicate that antigenic sites I and IV, and sites II and III on the N protein of rabies virus are composed of linear and conformation-dependent epitopes, respectively.     

Influence of aspirin and iron(III) tetrasulfonated phthalocyanine on bilirubin binding by human serum albumin

The interaction of bilirubin with aspirin-modified human serum albumin (HSA) and the influence of iron tetrasulfonated phthalocyanine on bilirubin binding by the native protein has been studied by difference spectroscopy and circular dichroism measurements. Spectroscopic studies of the systems containing bilirubin and aspirin-modified HSA compared to the analogous systems with the native protein have shown that selective acetylation of albumin at lysine 199 inhibits bilirubin binding by this protein. In both cases, interaction between bilirubin and albumin leads to complex formation at a molar ratio of ligand to protein of 2:1. The studies of the reaction of bilirubin with fragments of albumin produced by reaction with CNBr have demonstrated that one of the strong bilirubin binding sites is located in the M fragment and is close to the high-affinity binding site of aspirin. The other one was found in fragment C. Acetylation of albumin brings about marked conformational change in the protein, which probably accounts for the decrease in its ability to react with anti-HSA antibody. Bilirubin does not change the secondary structure of albumin but, like aspirin, lowers its antigenicity. It has been suggested that the decrease in antigenic properties in this case results from cooperation of the closely neighboring antigenic and bilirubin-binding sites. The studies of the influence of iron(III) tetrasulfonated phthalocyanine on bilirubin binding by HSA suggest that there is no competition between strong sites for iron(III) tetrasulfonated phthalocyanine and bilirubin, but these compounds compete for some of the weaker sites.     

Primary role of mitochondrial Rieske iron-sulfur protein in hypoxic ROS production in pulmonary artery myocytes

This study was designed to determine whether: (1) hypoxia could directly affect ROS production in isolated mitochondria and mitochondrial complex III from pulmonary artery smooth muscle cells (PASMCs) and (2) Rieske iron-sulfur protein in complex III might mediate hypoxic ROS production, leading to hypoxic pulmonary vasoconstriction (HPV). Our data, for the first time, demonstrate that hypoxia significantly enhances ROS production, measured by the standard ROS indicator dichlorodihydrofluorescein/diacetate, in isolated mitochondria from PASMCs. Studies using the newly developed, specific ROS biosensor pHyPer have found that hypoxia increases mitochondrial ROS generation in isolated PASMCs as well. Hypoxic ROS production has also been observed in isolated complex III. Rieske iron-sulfur protein silencing using siRNA abolishes the hypoxic ROS formation in isolated PASM complex III, mitochondria, and cells, whereas Rieske iron-sulfur protein overexpression produces the opposite effect. Rieske iron-sulfur protein silencing inhibits the hypoxic increase in [Ca(2+)](i) in PASMCs and hypoxic vasoconstriction in isolated PAs. These findings together provide novel evidence that mitochondria are the direct hypoxic targets in PASMCs, in which Rieske iron-sulfur protein in complex III may serve as an essential, primary molecule that mediates the hypoxic ROS generation, leading to an increase in intracellular Ca(2+) in PASMCs and HPV.     

Specific roles of protein-phospholipid interactions in the yeast cytochrome bc1 complex structure.

Biochemical data have shown that specific, tightly bound phospholipids are essential for activity of the cytochrome bc1 complex (QCR), an integral membrane protein of the respiratory chain. However, the structure and function of such phospholipids are not yet known. Here we describe five phospholipid molecules and one detergent molecule in the X-ray structure of yeast QCR at 2.3 A resolution. Their individual binding sites suggest specific roles in facilitating structural and functional integrity of the enzyme. Interestingly, a phosphatidylinositol molecule is bound in an unusual interhelical position near the flexible linker region of the Rieske iron-sulfur protein. Two possible proton uptake pathways at the ubiquinone reduction site have been identified: the E/R and the CL/K pathway. Remarkably, cardiolipin is positioned at the entrance to the latter. We propose that cardiolipin ensures structural integrity of the proton-conducting protein environment and takes part directly in proton uptake. Site-directed mutagenesis of ligating residues confirmed the importance of the phosphatidylinositol- and cardiolipin-binding sites.     

The iron-sulfur protein is necessary for the complete assembly of the low-molecular-weight subunits into the cytochrome b-c1 complex of yeast mitochondria

A strain of yeast lacking the gene for the Rieske iron-sulfur protein (RIP) of the cytochrome b-c1 complex was used to study the assembly of this complex in the mitochondrial membrane. This strain lacks the mRNA for the iron-sulfur protein as evidenced by both Northern hybridization using a probe containing the coding region of the gene plus in vitro translation of total RNA followed by immunoprecipitation with a specific antibody against the iron-sulfur protein. In addition, isolated mitochondria from this strain lacked cytochrome c reductase activity with either succinate or the decyl analog of ubiquinol as substrate. Immunoblotting studies with antiserum against the cytochrome b-c1 complex revealed that mitochondria from the iron-sulfur protein-deficient strain have levels of core protein I, core protein II, and cytochrome c1 equal to those of wild-type mitochondria; however, a decrease in cytochrome b was evident from both immunoblotting and spectral analysis. Moreover, it is evident from the immunoprecipitates of radiolabeled mitochondria that the amounts of the low-molecular-weight subunits (17, 14, and 11 kDa) are decreased 53, 65, and 50%, respectively, in mitochondria lacking the iron-sulfur protein. These results suggest that the iron-sulfur protein is required for the complete assembly of the low-molecular-weight subunits into the cytochrome b-c1 complex.     

Identification of a g equals 1.90 high-potential iron-sulfur protein in chloroplasts.

A new bound iron-sulfur protein has been identified in spinach chloroplasts. In the reduced form, this protein has an electron paramagnetic resonance spectrum at 20°K with g-values of 2.02 and 1.90. The midpoint oxidation-reduction potential (E_m) of the protein, which is pH-independent, is +290 mV. These properties are similar to those of the “Rieske” g = 1.90 iron-sulfur protein of mitochondrial Complex III.     

A new neutralizing antibody against botulinum neurotoxin B recognizes the protein receptor binding sites for synaptotagmins II

Botulinum neurotoxins (BoNts) pose a biological hazard to humans and a serious potential bioweapon threat. Given the safety concern regarding the currently used equine antitoxin therapy for botulism, it is imperative to develop agents that are effective binding inhibitors. The aim of this study was to identify a novel neutralizing antibody against botulinum neurotoxin B (BoNtb) that recognizes the protein receptor binding sites for synaptotagmins II. This antibody showed significant dose-dependent protection against lethal toxin challenge in vivo at an intraperitoneal (i.p.) dose 10 times the half lethal dose (LD50). We proved that the efficacy of SC12 was based on its counteraction on the recognition and binding of BoNtb to target cells, resulting from the combination of antibody with the high affinity (KD: 1.34 nM) to protein receptor binding sites of BoNt by targeting a 25-mer dominant antigenic site on Hcc region (residues 1253–1277). The structure of the site targeted by this antibody overlaps the pocket-like protein receptor binding sites located at the distal tip of toxin molecule. Information gained from this study will facilitate the development of potent inhibitors that prevent the binding of BoNts with its receptors.     

The role of phospholipids in the binding of antimycin to yeast Complex III

Treatment of yeast Complex III with hexane or repeated fractionation with ammonium sulfate-cholate abolishes the ability of antimycin to bind to the complex. Antimycin binding is partially restored by addition of phospholipids to the depleted complex; this action of phospholipids can be enhanced by including Q6 in the reconstitution mixture.     

Intermediate length Rieske iron-sulfur protein is present and functionally active in the cytochrome bc1 complex of Saccharomyces cerevisiae

To investigate the relationship between post-translational processing of the Rieske iron-sulfur protein of Saccharomyces cerevisiae and its assembly into the mitochondrial cytochrome bc1 complex we used iron-sulfur proteins in which the presequences had been changed by site-directed mutagenesis of the cloned iron-sulfur protein gene, so that the recognition sites for the matrix processing peptidase or the mitochondrial intermediate peptidase (MIP) had been destroyed. When yeast strain JPJ1, in which the gene for the iron-sulfur protein is deleted, was transformed with these constructs on a single copy expression vector, mitochondrial membranes and bc1 complexes isolated from these strains accumulated intermediate length iron-sulfur proteins in vivo. The cytochrome bc1 complex activities of these membranes and bc1 complexes indicate that intermediate iron-sulfur protein (i-ISP) has full activity when compared with that of mature sized iron-sulfur protein (m-ISP). Therefore the iron-sulfur cluster must have been inserted before processing of i-ISP to m-ISP by MIP. When iron-sulfur protein is imported into mitochondria in vitro, i-ISP interacts with components of the bc1 complex before it is processed to m-ISP. These results establish that the iron-sulfur cluster is inserted into the apoprotein before MIP cleaves off the second part of the presequence and that this second processing step takes place after i-ISP has been assembled into the bc1 complex.     

Precise determination of protein antigenic structures has unravelled the molecular immune recognition of proteins and provided a prototype for synthetic mimicking of other protein binding sites

Summary Intensive research in the author's laboratory had culminated in the determination and synthesis of all the antigenic sites of myoglobin in 1975 and of lysozyme in 1978. Very recently most of the antigenic sites of serum albumin were also localized and synthesized. These investigations provided the first unique insight into the molecular features responsible for the immune recognition of protein antigens and of the factors which determine and regulate the antigenicity of the sites. But moreover, these studies have charted a multi-approach chemical strategy for investigation and synthetic duplication of protein binding sites. Furthermore, the concept of surface-simulation synthesis, which we introduced and developed during our determination of the antigenic structure of lysozyme, has provided a remarkable dimension of unlimited versatility for the synthetic mimicking of any type of protein binding sites. In this concept, the spatially adjacent residues of a protein binding site are linked directly via peptide bonds with appropriate spacers into a single peptide which does not exist in the protein but mimicks a surface region of it. This has proved to be a powerful concept in protein molecular recognition and has opened up many untapped avenues in investigation, duplication and perhaps manipulation of a variety of protein activities. In fact, binding sites representing other protein activities (including antibody combining sites) have or are now being mimicked synthetically in our laboratory by the concept of surface-simulation synthesis.     

Differential labeling of the subunits of respiratory Complex III with [3H]succinic anhydride, [14C]succinic anhydride,andp-diazobenzene-[35S]sulfonate

Exposure of antimycin-treated Complex III (ubiquinol-cytochromec reductase) purified from bovine heart mitochondria to [³H]succinic anhydride plus [³⁵S]p-diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [³H]succinic anhydride. In contrast, relative labeling by [³⁵S]DABS was similar to [³H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex III depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [³H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by¹⁴C- and³H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7, 8, and 9. Two additional polypeptides of molecular masses 23 and 12 kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of¹⁴C/³H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in¹⁴C/³H labeling ratios of core proteins I and II, cytochromec ₁, and a polypeptide of molecular mass 13 kDa identified as an antimycin-binding protein.     

Assembly of the iron-sulfur protein into the cytochrome b-c1, complex of yeast mitochondria.

The assembly of the iron-sulfur protein into the cytochrome bc1 complex after import and processing of the precursor form into mitochondria in vitro was investigated by immunoprecipitation of the radiolabeled iron-sulfur protein from detergent-solubilized mitochondria with specific antisera. After import in vitro, the labeled mature form of the iron-sulfur protein was immunoprecipitated by antisera against both the iron-sulfur protein and the entire bc1 complex from mitochondria solubilized with either Triton X-100 or dodecyl maltoside. After sodium dodecyl sulfate solubilization of mitochondria, however, the antiserum against the iron-sulfur protein, but not that against the bc1 complex, immunoprecipitated the radiolabeled iron-sulfur protein. These results suggest that in mitochondria the mature form of the iron-sulfur protein is assembled with other subunits of the bc1 complex that are recognized by the antiserum against the bc1 complex. By contrast, the intermediate and precursor forms of the iron-sulfur protein that accumulated in the matrix when proteolytic processing was blocked with EDTA and o-phenanthroline were not efficiently assembled into the bc1 complex. The import and processing of the iron-sulfur protein also occurred in mitochondria lacking either cytochrome b (W-267) or the iron-sulfur protein (JPJ1). The mature form of the iron-sulfur protein was immunoprecipitated by antisera against the bc1 complex or core protein I after import in vitro into these mitochondria, suggesting that the mature form is associated with other subunits of the bc1 complex in these strains.