Overexpression of a monomeric form of the bovine odorant-binding protein protects Escherichia coli from chemical-induced oxidative stress.

Mammalian odorant-binding proteins (OBPs) are soluble lipocalins produced in the nasal mucosa and in other epithelial tissues of several animal species, where they are supposed to serve as scavengers for small structurally unrelated hydrophobic molecules. These would include odorants and toxic aldehydes like 4-hydroxy-2-nonenal (HNE), which are end products of lipid peroxidation; therefore OBP might physiologically contribute to preserve the integrity of epithelial tissues under oxidative stress conditions by removing toxic compounds from the environment and, eventually, driving them to the appropriate degradative pathways. With the aim of developing a biological model based on a living organism for the investigation of the antioxidant properties of OBP, here we asked whether the overexpression of the protein could confer protection from chemical-induced oxidative stress in Escherichia coli. To this aim, bacteria were made to overexpress either GCC-bOBP, a redesigned monomeric mutant of bovine OBP, or its amino-terminal 6-histidine-tagged version 6H-GCC-bOBP. After inducing overexpression for 4 h, bacterial cells were diluted in fresh culture media, and their growth curves were followed in the presence of hydrogen peroxide (H2O2) and tert-Butyl hydroperoxide (tBuOOH), two reactive oxygen species whose toxicity is mainly due to lipid peroxidation, and menadione, a redox-cycling drug producing the superoxide ion. GCC-bOBP and 6H-GCC-bOBP were found to protect bacterial cells from the insulting agents H2O2 and tBuOOH but not from menadione. The obtained data led us to hypothesize that the presence of overexpressed OBP may contribute to protect bacterial cells against oxidative stress probably by sequestering toxic compounds locally produced during the first replication cycles by lipid peroxidation, before bacteria activate their appropriate enzyme-based antioxidative mechanisms.

A case of Kearns-Sayre syndrome with the 4,977-bp common deletion associated with a novel 7,704-bp deletion.

Mitochondria from a patient diagnosed with Kearns-Sayre syndrome (KSS) exhibited severely diminished cytochrome c oxidase activity and at least four mitochondrial DNA (mtDNA) species: 9%-11% of the fulllength mtDNA (16.6 kb), 70%-75% of a 11.7-kb population (harboring the 4,977-bp common deletion), 2%-3% of a 10.5-kb population, and 12%-17% of a 8.9-kb population. The 8.9-kb mtDNA exhibited a secondary deletion that extended 7,704 bp from nucleotide 7,979 in the cox2 gene to nucleotide 15,683 in the cytb gene. To our knowledge, this is the first description of the presence of at least two large-scale deletions of mtDNA in KSS.

Subunit II of cytochrome c oxidase in Chlamydomonad algae is a heterodimer encoded by two independent nuclear genes.

The mitochondrial genomes of Chlamydomonad algae lack the cox2 gene that encodes the essential subunit COX II of cytochrome c oxidase. COX II is normally a single polypeptide encoded by a single mitochondrial gene. In this work we cloned two nuclear genes encoding COX II from both Chlamydomonas reinhardtii and Polytomella sp. The cox2a gene encodes a protein, COX IIA, corresponding to the N-terminal portion of subunit II of cytochrome c oxidase, and the cox2b gene encodes COX IIB, corresponding to the C-terminal region. The cox2a and cox2b genes are located in the nucleus and are independently transcribed into mRNAs that are translated into separate polypeptides. These two proteins assemble with other cytochrome c oxidase subunits in the inner mitochondrial membrane to form the mature multi-subunit complex. We propose that during the evolution of the Chlorophyte algae, the cox2 gene was divided into two mitochondrial genes that were subsequently transferred to the nucleus. This event was evolutionarily distinct from the transfer of an intact cox2 gene to the nucleus in some members the Leguminosae plant family.

Unusual location of a mitochondrial gene. Subunit III of cytochrome C oxidase is encoded in the nucleus of Chlamydomonad algae.

The algae of the family Chlamydomonadaceae lack the gene cox3 that encodes subunit III of cytochrome c oxidase in their mitochondrial genomes. This observation has raised the question of whether this subunit is present in cytochrome c oxidase or whether the corresponding gene is located in the nucleus. Cytochrome c oxidase was isolated from the colorless chlamydomonad Polytomella spp., and the existence of subunit III was established by immunoblotting analysis with an antibody directed against Saccharomyces cerevisiae subunit III. Based partly upon the N-terminal sequence of this subunit, oligodeoxynucleotides were designed and used for polymerase chain reaction amplification, and the resulting product was used to screen a cDNA library of Chlamydomonas reinhardtii. The complete sequences of the cox3 cDNAs from Polytomella spp. and C. reinhardtii are reported. Evidence is provided that the genes for cox3 are encoded by nuclear DNA, and the predicted polypeptides exhibit diminished physical constraints for import as compared with mitochondrial-DNA encoded homologs. This indicates that transfer of this gene to the nucleus occurred before Polytomella diverged from the photosynthetic Chlamydomonas lineage and that this transfer may have occurred in all chlamydomonad algae.

Two unusual amino acid substitutions in cytochrome b of the colorless alga Polytomella spp.: correlation with the atypical spectral properties of the bH heme.

The dithionite-reduced spectra of the purified bc1 complexes from the colorless alga Polytomella spp. and the closely related green alga Chlamydomonas reinhardtii were compared. The spectrum of the bc1 complex from C. reinhardtii showed a profile similar to those of the bc1 complexes from other species. In contrast, the bc1 complex from Polytomella spp. exhibits a double-peak spectrum in the alpha-band region, where the absorption bands of cytochrome c1 and cytochrome b are completely resolved. To further understand the molecular basis of these spectroscopic differences, the mitochondrial gene encoding cytochrome b of Polytomella spp. was cloned, sequenced, and compared with that of C. reinhardtii. The Polytomella spp. cytochrome b gene is 1113 bp long and does not contain introns. The deduced protein sequence exhibits 56% identity and 68% similarity with the cytochrome b of C. reinhardtii, and in a phylogenetic analysis it clearly affiliated with the b-type cytochromes of C. reinhardtii and C. smithii. A comparison of the primary sequences of the Polytomella spp. cytochrome b with other b-type cytochromes, and its analysis based on the structure featuring eight transmembrane stretches, allowed the identification of a tyrosine in position 114, which substitutes for a tryptophan present in all mitochondrial b-type cytochromes sequenced to date. In addition, the primary sequence of the cytochrome b from Polytomella spp. has a serine at position 36, instead of a nonpolar residue (alanine or leucine) found in all other species. In the proposed model for cytochrome b, both residues Tyr114 and Ser36 are in close proximity to the high-potential bH heme. The above data suggest that the polar residues Y114 and S36, each one by itself or in combination, may interact with heme bH of Polytomella spp. and, thus, may be responsible for the unique spectroscopic characteristics of cytochrome b.

An atypical cytochrome b in the colorless alga Polytomella spp.: the high potential bH heme exhibits a double transition in the alpha-peak of its absorption spectrum.

Polytomella spp. is a colorless alga of the family Chlamydomonadaceae that lacks chloroplasts and cell wall. A highly active ubiquinol-cytochrome c oxidoreductase (bc1 complex), sensitive to antimycin and myxothiazol, has been purified and characterized from this alga (Gutiérrez-Cirlos et al., 1994, J. Biol. Chem. 269, 9147-9154). Both in mitochondrial membranes and in the isolated complex, the visible spectrum of cytochrome b from Polytomella spp. exhibits an atypical alpha-band with a maximum at 567 nm. This maximum is shifted 3-4 nm to the red when compared with b-type cytochromes from other organisms. Analysis of the b hemes of the bc1 complex by high performance liquid chromatography revealed no differences in the retention time and in the absorption spectra of the b-type hemes from Polytomella spp. and hemin, indicating that the prosthetic group in this alga is protoheme and thus ruling out the possibility that the red-shift could be due to different chemical substitutions in the porphyrin rings of the bL or bH hemes. The two b hemes were characterized by electrochemical redox titration; at pH 7.8-8.0, the midpoint potential for bL was-143 mV and for bH +25 mV. The spectra of the two b-type hemes were recorded in the presence of different reductants, at selected electrochemical potentials, and in the presence of antimycin A, to distinguish between the contribution of bL and bH to the visible spectrum. Both hemes bL and bH of the algal cytochrome b contribute to the observed bathochromic absorption maximum in the alpha-band of the spectrum. The data also show that the low potential bL heme from Polytomella spp. is spectroscopically similar to that of other organisms, with two transitions in the alpha-peak at 558.7 and 568.4 nm. The high-potential heme bH also exhibits a spectrum with two transitions at 557.2 and 568.9 nm, which surprisingly differs from the spectra of cytochrome bH of mammals, plants, yeasts, and bacteria, which all exhibit a single transition centered around 560 nm.

A novel mitochondrial DNA point mutation associated with mitochondrial encephalocardiomyopathy.

A novel mitochondrial DNA (mtDNA) mutation at position nt 4320 in the tRNA(Ile) gene was associated with severe encephalopathy in a 7-month-old infant, who died of intractable hypertrophic cardiomyopathy. The mutation was present in heteroplasmic fashion (88%) in muscle and fulfills accepted criteria for pathogenicity. This is the fourth pathogenic mutation identified in this gene, which appears to be a "hotspot" for deleterious mutations affecting the heart. This report adds to the evidence of genetic heterogeneity in hypertrophic cardiomyopathies.

Characterization of a 5025 base pair mitochondrial DNA deletion in Kearns-Sayre syndrome.

We characterized a mitochondrial DNA deletion in a patient with Kearns-Sayre syndrome. Southern blot hybridization showed that 86 to 93% of the mitochondrial genome harbored a 5.0 kb deletion. The percentage of affected genomes is higher than in previously described cases. Direct sequencing of the breakpoint region revealed that the deletion extended 5025 bp from nt 10,050 in the tRNA Gly gene to nt 15,076 in the cytochrome b gene, thus 30% of the total mitochondrial genome was lost by this deletion. A pair of extremely short mirror sequences flanking the mitochondrial DNA breakpoints were identified. These flanking sequences differ from previously published consensus 'hot-spots', known to give rise to deletions in human mitochondrial DNA.

A highly active ubiquinol-cytochrome c reductase (bc1 complex) from the colorless alga Polytomella spp., a close relative of Chlamydomonas. Characterization of the heme binding site of cytochrome c1.

The alga Polytomella spp. offers extraordinary advantages in the preparation of mitochondria since it lacks chloroplasts and a cell wall. In this work the mitochondrial bc1 complex from Polytomella spp. was solubilized and purified by ion exchange chromatography. The complex was found to be composed of 10 polypeptides and exhibited high rates of ubiquinol-cytochrome c oxidoreductase activity (> 300 s-1) sensitive to antimycin and myxothiazol. The molecular mass of the bc1 complex from Polytomella spp. was assayed by gel filtration and estimated to be of 256,300 Da. Therefore, this complex exhibits the unique property of behaving as a monomer. Amino-terminal sequencing of cytochrome c1 identified 7 residues, from which a deoxyoligonucleotide was designed. A second deoxyoligonucleotide was constructed based on a highly conserved region of the c1 type cytochromes. With these probes, a fragment of the cytochrome c1 gene was amplified by polymerase chain reaction and sequenced. The deduced sequence of the apoprotein exhibited a consensus binding site CXXCH. The data suggest that the cytochrome c1 from Polytomella spp. differs from other protoctists like Crithidia and Euglena, i.e. it exhibits a heme binding domain structurally related to the bovine, yeast, and Neurospora c1 type cytochromes.

Subunit structures of purified beef mitochondrial cytochrome bc1 complex from liver and heart.

The existence of tissue-specific isozymes of cytochrome c oxidase has been widely documented. We have now studied if there are differences between subunits of mitochondrial bc1 complexes isolated from liver and heart. For this purpose, we have developed a method for the purification of an active ubiquinol-cytochrome c oxidoreductase from adult bovine liver that includes solubilization of submitochondrial particles with deoxycholate, ammonium acetate fractionation, resolubilization with dodecyl maltoside, and ion exchange chromatography. The electrophoretic pattern of the liver preparation showed the presence of 11 subunits, with apparent molecular weights identical to the ones reported for the heart complex. Western blot analysis and isoelectric focusing followed by two-dimensional gels of bc1 complexes from liver and heart were compared, and no qualitative differences were observed. In addition, the high-molecular-weight subunits of the purified complexes from both tissues, subunits I, II, V, and VI, were isolated by PAGE in the presence of Coomasie Blue and subjected to limited proteolysis and to chemical digestion with cyanogen bromide and BNPS-skatol, and the peptide patterns were compared. Finally, two of the small-molecular-weight subunits from the liver complex were isolated (subunits VII and X), partially analyzed by amino terminal sequencing, and found to be identical with the reported sequence of their heart counterparts. The data suggest that, in contrast to the case of cytochrome c oxidase, bc1 complexes from liver and heart do not exhibit tissue-specific differences.

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.