Biomedical subjects

C A Yu

Publications and source records attributed to C A Yu.

At least 55 records · Page 3Linked to original sources

PubMed1995

Cloning, gene sequencing, and expression of the small molecular mass ubiquinone-binding protein of mitochondrial ubiquinol-cytochrome c reductase.

The cDNA encoding QPc-9.5 kDa (subunit VII) of bovine heart mitochondrial ubiquinol-cytochrome c reductase was cloned and sequenced. This cDNA is 665 base pairs long with an open reading frame of 246 base pairs that encodes an 81-amino acid mature QPc-9.5 kDa. The insert contains 395 base pairs of a 3'-noncoding sequence with a poly(A) tail. The amino acid sequence of QPc-9.5 kDa deduced from this nucleotide sequence is the same as that obtained by protein sequencing except that residue 61 is tryptophan instead of cysteine. The QPc-9.5 kDa was overexpressed in Escherichia coli JM109 cells as a glutathione S-transferase fusion protein (GST-QPc) using the expression vector, pGEX/QPc. The yield of soluble active recombinant GST-QPc fusion protein depends on the induction growth time, temperature, and medium. Maximum yield of recombinant fusion protein was obtained from cells harvested 3 h postinduction of growth at 27 degrees C on LB medium containing betaine and sorbitol. QPc-9.5 kDa was released from the fusion protein by proteolytic cleavage with thrombin. Isolated recombinant QPc-9.5 kDa showed one protein band in SDS-polyacrylamide gel electrophroesis corresponding to subunit VII of mitochondrial ubiquinol-cytochrome c reductase. Although the isolated recombinant QPc-9.5 kDa is soluble in aqueous solution, it is in a highly aggregated form, with an apparent molecular mass of over 1 million. Addition of detergent deaggreates the isolated protein to the monomeric state, suggesting that the recombinant protein exists as a hydrophobic aggregation in aqueous solution. The recombinant QPc-9.5 kDa binds ubiquinone and shows a spectral blue shift. Upon titration of the recombinant protein with ubiquinone, a saturation behavior is observed, suggesting that the binding is specific and that the recombinant protein may be in the functionally active state.

Amino Acid Sequence↗

PubMed1995

Spectroscopic identification of the axial ligands of cytochrome b560 in bovine heart succinate-ubiquinone reductase.

The axial ligands of low potential cytochrome b560 in the five subunit bovine heart succinate-ubiquinone reductase complex and in the isolated quinone binding proteins have been investigated using EPR and near-infrared magnetic circular dichroism spectroscopies. The results are consistent with bis-histidine ligation with near-perpendicular imidazole rings for cytochrome b560 in the four-subunit complex. The pronounced changes in EPR properties that accompany isolation of the cytochrome-b560 containing quinone binding proteins, are attributed to perturbation of the orientation of the imidazole rings of the heme bis-histidine ligands, rather than a change in axial ligation.

Animals↗

PubMed1995

Identification of amino acid residues involved in structural and ubiquinone-binding functions of subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides.

Previous studies established that subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides is involved in structural and ubiquinone-binding functions of the complex. To identify regions or amino acid residues responsible for these functions, deletion, insertion, and substitution mutations at various regions of subunit IV were generated and characterized. Mutational effects on the structural role of subunit IV are indicated by a delay in photosynthetic growth and by a decrease in the cytochrome bc1 complex activity in chromatophores upon detergent treatment. An effect on the ubiquinone-binding function of subunit IV is suggested by an increase in the apparent Km for 2,3-dimethoxy-5-methyl-6-geranyl-1,4-benzoquinol (Q2H2) of the complex. RSIV delta (2-5), in which residues 2-5 are deleted, had photosynthetic growth behavior, tolerance to detergent treatment, and an apparent Km for Q2H2 of its cytochrome bc1 complex similar to those of wild-type or complement cells, indicating that amino acid residues 2-5 are not essential for subunit IV function. RSIV delta (2-11), with residues 2-11 missing, showed a 24-h delay in photosynthetic growth and a 65% inactivation of the cytochrome bc1 complex upon dodecyl maltoside solubilization. However, its apparent Km for Q2H2 was the same as in wild-type cells, indicating that deletion of amino acid residues 6-11 results in loss of the structural but not the ubiquinone-binding function of subunit IV. RSIV delta (113-124), which has 13 amino acid residues deleted from the C terminus, had photosynthetic growth behavior, tolerance to detergent treatment, and ubiquinone-binding kinetics similar to those of wild-type or complement cells, indicating that residues 113-124 are not essential. Point mutants RSIV(W79L) and RSIV(W79F), in which tryptophan 79 was replaced with leucine or phenylalanine, showed a 24-h delay in photosynthetic growth, a decrease of 75% of the cytochrome bc1 complex activity in chromatophores upon detergent solubilization, and a 4-fold increase in the apparent Km for Q2H2, indicating that Trp-79 is essential for the structural and ubiquinone-binding functions of subunit IV.

Amino Acid Sequence↗

PubMed1995

An enhanced broad-host-range vector for gram-negative bacteria: avoiding tetracycline phototoxicity during the growth of photosynthetic bacteria.

A mobilizable, broad-host-range (bhr) plasmid was derived from the widely used IncP1 vector pRK415. The new vector, pRKD418, contains an additional resistance gene and an enlarged multiple cloning site (MCS) region. The optimal growth of pRK415-containing bacteria under photosynthetic conditions generally requires the use of optical filters to protect the selective antibiotic tetracycline (Tc) from photooxidation with the resulting production of toxic photoproducts; pRK415 is not stably maintained in the absence of selective pressure. The addition of a trimethoprim-resistant dihydrofolate reductase-encoding gene provided for optimal photosynthetic growth in the presence of a selective antibiotic without any special apparatus. The presence of an antibiotic marker not found in commonly used cloning vectors in many cases facilitates the subcloning of inserts into the bhr plasmid. The new MCS region provides further cloning flexibility with at least sixteen available restriction sites. Easily constructed derivative plasmids, exemplified by pRKD418KmE, provide a convenient screening procedure for the detection of recombinants during subcloning.

Base Sequence↗

PubMed1995

Identification of the ubiquinone-binding domain in QPs1 of succinate-ubiquinone reductase.

An azidoubiquinone derivative, 3-azido-2-methyl-5-methoxy [3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone-protein interaction and to identify ubiquinone-binding proteins in bovine heart mitochondrial succinate-ubiquinone reductase. When the reductase was incubated with [3H]azido-Q and illuminated with long wavelength UV light, the decrease in the enzymatic activity correlated with the amount of azido-Q incorporated into the protein. When the illuminated, [3H]azido-Q-treated reductase was extracted with organic solvent and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radioactivity was found primarily in the QPs1 subunit. The [3H]azido-Q-labeled QPs1 was purified from labeled reductase by a procedure involving ammonium sulfate fractionation, dialysis, organic solvent extraction, lyophilization, preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and cold acetone precipitation. The purified, [3H]azido-Q-labeled QPs1 protein was subjected to reductive carboxymethylation prior to digestion by trypsin. One azido-Q-linked peptide, with a retention time of 66.9 min, was obtained by high performance liquid chromatographic separation. The partial amino-terminal sequence of this peptide is GLTISQL-, indicating that this tryptic peptide comprises amino acid residues 113-140 of the revised amino acid sequence of QPs1. The Q-binding domain, using the proposed structure of QPs1, is probably located in the stretch connecting transmembrane helices 2 and 3 that extrude from the surface of the M side of the inner membrane.

Affinity Labels↗

PubMed1994

Identification of the ubiquinol-binding site in the cytochrome bo3-ubiquinol oxidase of Escherichia coli.

The cytochrome bo3-ubiquinol oxidase, one of two ubiquinol oxidases in Escherichia coli, is a member of the heme-copper oxidase superfamily. The enzyme contains four protein subunits (I-IV) with apparent molecular masses of 58, 33, 22, and 17 kDa, respectively. Cytochrome bo3 catalyzes the 2-electron oxidation of ubiquinol and the reduction of molecular oxygen to water. Although the primary structures of all four subunits have been determined, the ubiquinol-binding site has not been investigated. The photoreactive radiolabeled azidoubiquinone derivative 3-[3H]azido-2-methyl-5-methoxy-6-geranyl-1,4-benzoquinone (azido-Q), which has been widely used in locating the ubiquinone-binding sites of other enzymes, was used to identify the subunit(s) involved in the binding of quinol to cytochrome bo3. When reduced by dithioerythritol, the azido-Q derivative functioned as a substrate with partial effectiveness, suggesting that azido-Q interacts with a legitimate quinol-binding site. When cytochrome bo3 was incubated with an 8-fold molar excess of azido-Q, illumination by UV light for 10 min resulted in a 50% loss of activity. The uptake of radiolabeled azido-Q by the oxidase complex upon illumination correlated with the photoinactivation. In the presence of the competitive inhibitor 2-heptyl-4-hydroxyquinoline or ubiquinol, the rate of azido-Q uptake and the loss of enzyme activity upon illumination decreased. Analysis of the distribution of radioactivity among the subunits after separation by SDS-polyacrylamide gel electrophoresis showed that subunit II was heavily labeled by azido-Q, but that the other subunits were not. This suggests that the ubiquinol-binding site of the cytochrome bo3 complex is located at least partially on subunit II.

Affinity Labels↗

PubMed1994

Protein ubiquinone interaction. Synthesis and biological properties of 5-alkyl ubiquinone derivatives.

For the investigation of the protein-ubiquinone interaction in the succinate-cytochrome c reductase region of the bovine heart mitochondrial electron transport chain, a series of 5-alkyl-substituted ubiquinone derivatives (5-R-Q0C10) were synthesized and characterized. Syntheses of 5-ethyl-Q0C10, 5-propyl-Q0C10, 5-isopropyl-Q0C10, and 5-butyl-Q0C10, were archived through radical coupling reactions between 2,3-dimethoxy-6-decyl-1,4-benzoquinone (5-H-Q0C10) and the corresponding alkanoyl peroxides. Although the spectral and redox properties of 5-R-Q0C10 are very similar to those of 5-methyl-2,3 dimethoxy-6-decyl-1,4-benzoquinone, the biological electron transfer efficiencies of these derivatives differ significantly. The reducibility of these derivatives by succinate, as measured with succinate-Q reductase and the oxidizability as measured by ubiquinol-cytochrome c reductase, decreased as the size of the substituents increased. 5-Ethyl-Q0C10 has about 50% of the activity of 5-methyl-2,3-dimethoxy-6-decyl-1,4-benzoquinone, whereas molecules with 5-alkyl groups of three or more carbon atoms are virtually inactive as electron acceptors for succinate-Q reductase. Reduced form of the derivative with no substituent at the 5-position, 5-H derivative is more effectively oxidized by ubiquinol-cytochrome c reductase than does the 5-methyl derivative, the native form. The oxidation of 5-H derivative is in a concentration-dependent manner at low concentrations but exhibits a substrate inhibition at higher concentrations. No such substrate inhibition is observed when other 5-substituted Q derivatives are used. 5-H derivative is a better electron acceptor for succinate-Q reductase than any other Q derivatives and does not show substrate inhibition, even at high concentrations. These results indicate that the binding environment of the benzoquinone ring in succinate-Q reductase is more specific than that of ubiquinol-cytochrome c reductase.

Alkylation↗

PubMed1994

Crystallization of mitochondrial cytochrome b-c1 complex from gel with or without reduced pressure.

Cytochrome b-c1 complex (ubiquinol-cytochrome c reductase) of beef heart mitochondria has been crystallized. Crystals grown in capillary tubes diffracted X-rays from a laboratory source to a resolution of 7 A and synchrotron radiation to a resolution of 4.5 A in the presence of mother liquor. However, the movement of crystals in the mother liquor makes data collection very difficult. Removal of the mother liquor from the crystals causes severe loss of diffraction quality. To circumvent these difficulties we have recently developed a method for crystallization of the cytochrome b-c1 complex from a gel. The sizes, shapes and diffraction qualities of crystals grown in gel approach those of crystals obtained from liquid. Preliminary experiments on a Xuong-Hamlin area detector indicate that these crystals have the symmetry of a body centered tetragonal space group with cell constants a = b = 157 A, c = 590 A. Assuming eight cytochrome b-c1 complex dimers per unit cell, the crystals have a solvent content of 70% (v/v). Under reduced pressure the crystallization time is significantly decreased. Although crystals obtained under reduced pressure are generally smaller, the shorter crystallization time provides an opportunity to explore more crystallization conditions.

Animals↗

PubMed1994

Role of subunit IV in the cytochrome b-c1 complex from Rhodobacter sphaeroides.

Rhodobacter sphaeroides mutants lacking subunit IV (M(r) = 14,384) of the cytochrome b-c1 complex (representative mutant strain, RS delta IV-2) have been constructed by site-specific recombination between the wild-type genomic subunit IV structural gene (fbcQ) and a suicide plasmid containing a defective fbcQ sequence. RS delta IV-2 gives rise to a photosynthetically competent phenotype after a period of adaptation. The chemical compositions, spectral properties, and cytochrome b-c1 complex activities in subunit IV-deficient chromatophores from adapted RS delta IV-2 are similar to those in wild-type chromatophores. However, the apparent Km for Q2H2 for the b-c1 complex in subunit IV-deficient chromatophores from adapted RS delta IV-2 cells is about four times higher than that in chromatophores from wild-type cells. The cytochrome b-c1 complex activity in subunit IV-deficient chromatophores of adapted RS delta IV-2 cells is more labile to detergent treatment than that from wild-type cells. The specific activities of dodecylmaltoside-solubilized fractions of RS delta IV-2, based on cytochrome b, are only one-fourth that of the untreated chromatophores. Introducing a wild-type fbcQ operon on a stable low copy number plasmid, pRK415, into RS delta IV-2 restores photosynthetic growth behavior, the apparent Km value for Q2H2, and tolerance to detergent treatment to that of wild-type cells. Cytochrome b-c1 complex purified from adapted RS delta IV-2 contains only three subunits. It has only 25% of the activity of the four-subunit enzyme. This low activity is accompanied by an increase of the apparent Km for Q2H2 from 3 to 13 microM, suggesting that subunit IV may be involved in quinone binding in addition to its structural role.

Base Sequence↗

PubMed1994

Influence of hydrocarbon tail structure on quinone binding and electron-transfer performance at the QA and QB sites of the photosynthetic reaction center protein.

Binding free energies of 37 functional replacement quinone cofactors with systematically altered hydrocarbon tail structures have been determined for the QA and QB redox catalytic sites of the reaction center protein isolated from Rhodobacter sphaeroides and solubilized in aqueous and in hexane solutions. The first two and part of the third isoprene units of the 10-unit tail of the native ubiquinone-10 cofactor interact with the protein interior at each site. Contributions of the same tail structures to the binding free energies of quinones at the QA and QB sites are comparable, suggesting that the binding domains share common features. Comparison of the affinities of a homologous series of 10 n-alkyl-substituted ubiquinones resolves the binding forces along the length of the tail binding domain and shows that strong steric constraints oppose accommodation of the tail in its extended conformation. Differences in the contributions of identical tail substituents to ubiquinone- and menaquinone-QA site affinities, and tail-induced changes of up to 5-fold in the rates of QA site-mediated electron-transfer reactions, suggest that the tail adjusts the position of the quinone ring. Substitution of ubiquinone with the native 10-unit isoprene tail does not alter the affinity for the sites as determined in hexane solution. However, one- and two-isoprene-substituted quinones bind more tightly than analogs substituted with saturated-alkyl tail substituents. The sites therefore exhibit binding specificity for the native isoprene tail structure. Calculations indicate that the binding specificity arises primarily from a lower integrated torsion potential energy in the bound isoprene tails. The results suggest that the in vivo tail-protein interaction is designed to deter competitive interference of quinone function by amphiphilic species present in the native membrane.

Crystallography, X-Ray↗

PubMed1994

Protein-ubiquinone interaction: synthesis and biological properties of ethoxy ubiquinone derivatives.

For investigation of the protein-ubiquinone interaction in the succinate-cytochrome c reductase region of the bovine heart mitochondrial electron-transport chain, ethoxy-substituted ubiquinone derivatives, 2-ethoxy-3-methoxy- or 3-ethoxy-2-methoxy-5-methyl-6-decyl-1,4-benzoquinone (EtOQ0C10) and 2,3-diethoxy-5-methyl-6-decyl-1,4-benzoquinone [(EtO)2Q0C10], were synthesized and characterized. These compounds were synthesized from 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (Q0C10) by reaction with sodium ethoxide/ethanol in hexane under anaerobic conditions. The products, EtOQ0C10 and (ETO)2Q0C10, were separated by thin-layer chromatography using hexane/ether (3.5:1) as the developing solvent. The Rf values for diethoxy and monoethoxy derivatives are 0.7 and 0.6, respectively. The spectral and redox properties of EtOQ0C10 and (ETO)2Q0C10 are very similar to those of Q0C10. The reducibility of these derivatives by succinate was measured with succinate-Q reductase (SQR), and their oxidizability was measured by ubiquinol-cytochrome c reductase (QCR). Ethoxy ubiquinone derivatives exhibit concentration-dependent inhibition of SQR activity, with (ETO)2Q0C10 being the more potent inhibitor. These derivatives do not inhibit QCR and are reduced by succinate-cytochrome c reductase in an antimycin-insensitive manner. When used as substrate for QCR, EtOQ0C10H2 has about 55%, and (ETO)2Q0C10H2 about 15%, of the activity of Q0C10H2, but with lower apparent Km values. The low efficiency of these compounds as electron donors is apparently not due to their weak binding to QCR. These results indicate that the binding environment of the benzoquinone ring in succinate-Q reductase is very specific and differs from that in ubiquinol-cytochrome c reductase.

Animals↗

PubMed1994

Ubiquinone binding domains in bovine heart mitochondrial cytochrome b.

Cytochrome b was identified as one of the ubiquinone-binding proteins in bovine heart mitochondrial ubiquinol-cytochrome c reductase by photoaffinity labeling using 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]-octyl)-1,4-benzoquinone ([3H]azido-Q). The [3H]azido-Q-labeled cytochrome b protein was purified to homogeneity from the azido-Q-labeled ubiquinol-cytochrome c reductase by a procedure involving Triton X-100 and urea treatment, calcium phosphate column chromatography, acetone precipitation, decanoyl-N-methylglucamide-cholate extraction, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified cytochrome b protein containing 0.5 mol of azido-Q/mol of protein was subjected to reductive carboxymethylation and succinylation prior to digestion by chymotrypsin. Two azido-Q-linked peptides with retention times of 47.1 and 49.0 min were obtained by high performance liquid chromatographic separation. Partial amino-terminal amino acid sequences of these two peptides were determined to be GATVI- and ALVADL-, indicating that these two chymotryptic peptides are from amino residues 142-155 and 326-336. Monospecific polyclonal antibodies against two synthetic ubiquinone-binding peptides, NH2-G-A-T-V-I-T-N-L-L-S-COOH (P-47) and NH2-W-A-L-V-A-D-L-L-T-L-T-W-I-COOH (P-49), were generated in rabbits and purified. Western blotting and enzyme-linked immunosorbent assays showed that the purified antibodies against P-47 reacted with cytochrome b-containing reductases and purified cytochrome b protein. Antibodies against P-47 inhibited activities of succinate-cytochrome c and ubiquinol-cytochrome c reductases only when they were incubated with phospholipid-depleted reductases prior to the replenishment with phospholipid. No inhibition was observed with incubation with phospholipid-containing reductases, indicating that this peptide involved in ubiquinone binding is buried in a phospholipid environment.

Amino Acid Sequence↗

PubMed1994

Effects of novel antiarrhythmic agents, BRB-I-28 and its derivatives, on the heart mitochondrial respiratory chain and sarcoplasmic reticulum Ca(2+)-ATPase.

The effects of BRB-I-28 and its derivatives (GLG-V-13, SAZ-VII-22 and SAZ-VII-23), a novel group of antiarrhythmic agents, were investigated on the rat heart mitochondrial respiratory chain. The results indicate that BRB-I-28 and its derivatives have concentration-dependent inhibitory effects on NADH oxidase and NADH-CoQ reductase (complex I), but they have no significant effects on succinate oxidase, succinate dehydrogenase (complex II), CoQ-cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), and NADH-K3Fe(CN)6 reductase. The site of inhibition of BRB-I-28 and its derivatives on the respiratory chain was localized between flavoprotein n (FPn) and CoQ, which is similar to the effect of rotenone and several other antiarrhythmic drugs such as amiodarone, propranolol, etc. BRB-I-28 and its derivatives also have significant inhibitory effects on mitochondrial ATPase activity as reported for other antiarrhythmic drugs such as amiodarone, propranolol, quinidine, and lidocaine. However, BRB-I-28 and its derivatives have no direct effects on sarcoplasmic reticulum Ca(2+)-ATPase activity. The inhibitory effects of BRB-I-28 and its derivatives on mitochondrial oxidative phosphorylation may result in the depletion of ATP. This effect, in combination with their effects on Na+,K(+)-ATPase, could possibly produce an increase in Ca2+ concentration in cytosol. This may be another mechanism by which these DHBCN derivatives produce an increase in systemic arterial blood pressure and contractile force of isolated cardiac muscle. On the other hand, inhibition on mitochondrial respiration may account for some of the potential toxic effects of these diheterabicyclo[3.3.1]nonane derivatives.

Adenosine Triphosphatases↗

PubMed1993

Intracomplex electron transfer between ruthenium-cytochrome c derivatives and cytochrome c1.

The reactions of a beef heart cytochrome c1 preparation containing the hinge protein with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)(bisbipyridine)ruthenium(II) (Ru(II)) were studied by flash photolysis. All of the ruthenium-cytochrome c derivatives formed complexes with cytochrome c1 in low ionic strength buffer (5 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a 0.4-microseconds laser flash resulted in rapid electron transfer to the ferric heme group in cytochrome c, followed by electron transfer from the ferrous heme group of cytochrome c to the ferric heme group of cytochrome c1. The kinetic difference spectra displayed maxima at 546 nm and minima at 554 nm characteristic of electron transfer between the two cytochromes. The rate constants were independent of concentration at low ionic strength, indicating intracomplex electron transfer. The rate constants were 4,800, 6,800, 22,000, and 22,000 s-1 for cytochrome c derivatives modified at lysines 13, 27, 25, and 72, respectively. The observed rate constants were independent of ionic strength up to about 50 nM and then decreased progressively with further increases in ionic strength indicating dissociation of the complex. Second-order kinetics were observed at 310 mM ionic strength, with rate constants of 1.0 x 10(6), 1.6 x 10(7), 1.2 x 10(8), and 3.0 x 10(7) M-1 s-1 for the derivatives modified at lysines 13, 27, 25, and 72, respectively. The ionic strength dependence of the second-order rate constants is comparable to that involving native horse cytochrome c and is consistent with electron transfer reactions between oppositely charged proteins.

Animals↗

PubMed1993

Structural characterization of isolated mitochondrial cytochrome c1.

Resonance Raman spectroscopy (RRS) has been employed to characterize cytochromes c1 isolated from bc1 complexes of beef heart mitochondria and Rhodopseudomonas sphaeroides. The data obtained in this study extend the physical characterization of cytochromes c1 and focus on the effects of the local protein environment on the heme active site. While the general characteristics of the cytochromes c1 are similar to those of smaller soluble cytochromes c, the behavior of several core-size and ligation-sensitive heme modes reveal that significant systematic differences exist between those species. These, most likely, result from changes in the heme axial-ligand interactions.

Animals↗

PubMed1993

The mitochondrial targeting presequence of the Rieske iron-sulfur protein is processed in a single step after insertion into the cytochrome bc1 complex in mammals and retained as a subunit in the complex.

The amino acid sequence of subunit 9 of the bovine heart cytochrome bc1 complex is identical to the 78-amino acid presequence that is removed post-translationally from the Rieske iron-sulfur protein as it is imported and targeted to the mitochondrial cytochrome bc1 complex. Iron-sulfur protein precursor, generated by in vitro transcription and translation, is processed to mature size in a single step when incubated with rat liver mitochondria, and generates a peptide that comigrates on SDS-polyacrylamide gel electrophoresis with subunit 9. These results suggest that the Rieske protein is processed in a single proteolytic step after it is inserted into the cytochrome bc1 complex in mammals, and that the processed presequence remains as a subunit of the complex. This is apparently the first instance in which a cleaved targeting presequence has been shown to be retained in the cell, possibly exhibiting a second function in addition to its function in protein trafficking.

Amino Acid Sequence↗

PubMed1993

Mitochondrial ubiquinol-cytochrome c reductase complex: crystallization and protein: ubiquinone interaction.

The ubiquinol-cytochrome c reductase complex was crystallized in a thin plate form, which diffracts X-rays to 7 A resolution in the presence of mother liquor. This crystalline complex contains ten protein subunits and 140 nmol phospholipid per milligram protein. Over 90% of the phospholipid and ubiquinone in the reductase can be removed by repeated ammonium sulfate precipitation in the presence of 0.5% sodium cholate. The delipidated complex has no enzymatic activity and shows significant changes in the circular dichroism spectrum in the near UV region and in the EPR characteristics of both cytochromes b. Enzyme activity and spectral characteristics can be restored by replenishing the phospholipid and ubiquinone. The structural requirements of ubiquinone for electron transport were studied by measuring the ability of a variety of synthetic ubiquinone derivatives to restore the enzymatic activity and native spectroscopic signatures to the delipidated complex. Q-binding proteins and binding domains were identified using photoaffinity labeled Q-derivatives and HPLC separation of photolabeled peptides. Interaction between ubiquinol-cytochrome c reductase and succinate-Q reductase was established by differential scanning calorimetry and saturation transfer EPR using spin-labeled ubiquinol-cytochrome c reductase. Involvement of iron-sulfur protein in proton translocation by ubiquinol-cytochrome c reductase was investigated by hematorporphyrin-promoted photoinactivation of the complex. The cDNAs encoding the Rieske iron-sulfur protein and a small molecular mass Q-binding protein (QPc-9.5 kDa) were isolated and their nucleotide sequences determined. These will be useful in future structural and mechanistic studies of ubiquinol-cytochrome c reductase via in vitro reconstitution between an over-expressed, mutated subunit and a specific subunit-depleted reductase.

Amino Acid Sequence↗

PubMed1992

Cytochrome b560 (QPs1) of mitochondrial succinate-ubiquinone reductase. Immunochemistry, cloning, and nucleotide sequencing.

Mitochondrial succinate-ubiquinone reductase is composed of two parts, a water-soluble succinate dehydrogenase and a two-polypeptide membrane-anchoring protein fraction (QPs). The larger polypeptide of QPs is believed to be associated with cytochrome b560 (QPs1). The structure of QPs1 was studied by immunochemistry and molecular cloning and sequencing. Antibodies against QPs1 were raised in rabbits, purified, and characterized by enzyme-linked immunosorbent assay and Western blotting. The purified antibodies inhibited 75% of the reconstitutive activity of QPs and reacted with both submitochondrial particles (SMP) and mitoplasts. The binding of these antibodies to SMP was greatly increased when succinate dehydrogenase was removed from SMP by alkaline treatment, indicating that QPs1 is a transmembranous protein and that some of its specific epitopes are covered by succinate dehydrogenase. Anti-QPs1 antibodies were used to screen one cDNA clone encoding QPs1 from a bovine heart cDNA lambda gt11 expression library. The cDNA insert is 946 base pairs with an open reading frame of 396 base pairs that encodes for 132 amino acid residues. The molecular weight of QPs1, calculated from the deduced amino acid sequence, is 14,320. Although the apparent molecular weight of QPs1, estimated by high resolution SDS-polyacrylamide gel electrophoresis, is approximately 11,000, the existence of a presequence was ruled out by mass spectrometric analysis of protein fragments. QPs1 is a very hydrophobic protein. Three probable membrane-spanning segments were revealed by a hydropathy plot of the sequence. QPs1 has a higher sequence similarity to the sdhC peptide of Escherichia coli than to the sdhC peptide (cytochrome b558) of Bacillus subtilis. Like the bacterial proteins, QPs1 has 2 conserved histidines at positions 34 and 90. The conserved nature and similar location of these 2 histidines, on the matrix-side surface of the membrane, suggest that they are involved in heme ligation of cytochrome b560.

Amino Acid Sequence↗