RESUMO
BACKGROUND: The gut microbiota can impact older adults' health, especially in patients with frailty syndrome. Understanding the association between the gut microbiota and frailty syndrome will help to explain the etiology of age-related diseases. Low-grade systemic inflammation is a factor leading to geriatric disorders, which is known as "inflammaging". Intestinal dysbiosis has a direct relationship with low-grade systemic inflammation because when the natural gut barrier is altered by age or other factors, some microorganisms or their metabolites can cross this barrier and reach the systemic circulation. OBJECTIVES: This review had two general goals: first, to describe the characteristics of the gut microbiota associated with age-related diseases, specifically frailty syndrome. The second aim was to identify potential interventions to improve the composition and function of intestinal microbiota, consequently lessening the burden of patients with frailty syndrome. METHODS: A search of scientific evidence was performed in PubMed, Science Direct, and Redalyc using keywords such as "frailty", "elderly", "nutrient interventions", "probiotics", and "prebiotics". We included studies reporting the effects of nutrient supplementation on frailty syndrome and older adults. These studies were analyzed to identify novel therapeutic alternatives to improve gut microbiota characteristics as well as subclinical signs related to this condition. RESULTS: The gut microbiota participates in many metabolic processes that have an impact on the brain, muscles, and other organs. These processes integrate feedback mechanisms, comprising their respective axis with the intestine and the gut microbiota. Alterations in these associations can lead to frailty. We report a few interventions that demonstrate that prebiotics and probiotics could modulate the gut microbiota in humans. Furthermore, other nutritional interventions could be used in patients with frailty syndrome. CONCLUSION: Probiotics and prebiotics may potentially prevent frailty syndrome or improve the quality of life of patients with this disorder. However, there is not enough information about their appropriate doses and periods of administration. Therefore, further investigations are required to determine these factors and improve their efficacy as therapeutic approaches for frailty syndrome.
Assuntos
Fragilidade , Microbioma Gastrointestinal , Probióticos , Humanos , Idoso , Prebióticos , Qualidade de Vida , Idoso Fragilizado , Probióticos/uso terapêutico , InflamaçãoRESUMO
The ability of the bacterium to use sodium in bioenergetic processes appears to play a key role in both the environmental and pathogenic phases of Vibrio cholerae. Aquatic environments, including fresh, brackish, and coastal waters, are an important factor in the transmission of cholera and an autochthonous source. The organism is considered to be halophilic and has a strict requirement for Na(+) for growth. Furthermore, expression of motility and virulence factors of V. cholerae is intimately linked to sodium bioenergetics and to each other. Several lines of evidence indicated that the activity of the flagellum of V. cholerae might have an impact on virulence gene regulation. As the V. cholerae flagellum is sodium-driven and the Na(+)-NQR enzyme is known to create a sodium motive force across the bacterial membrane, it was recently suggested that the increased toxT expression observed in a nqr-negative strain is mediated by affecting flagella activity. It was suggested that the V. cholerae flagellum might respond to changes in membrane potential and the resulting changes in flagellar rotation might serve as a signal for virulence gene expression. However, we recently demonstrated that although the flagellum of V. cholerae is not required for the effects of ionophores on virulence gene expression, changes in the sodium chemical potential are sensed and thus alternative mechanisms, perhaps involving the TcpP/H proteins, for the detection of these conditions must exist. Analyzing the underlying mechanisms by which bacteria respond to changes in the environment, such as their ability to monitor the level of membrane potential, will probably reveal complex interplays between basic physiological processes and virulence factor expression in a variety of pathogenic species.
Assuntos
Proteínas de Bactérias , Cólera/microbiologia , Sódio/metabolismo , Vibrio cholerae/metabolismo , Sequência de Aminoácidos , Cátions Monovalentes , Membrana Celular/metabolismo , Metabolismo Energético , Humanos , Membranas Intracelulares/metabolismo , Potenciais da Membrana , Modelos Químicos , ATPases Translocadoras de Prótons/metabolismo , Quinona Redutases/química , Quinona Redutases/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/patogenicidade , Virulência/genética , Microbiologia da ÁguaRESUMO
The ubiquinol:cytochrome c2 oxidoreductase (bc-complex) of Rhodobacter sphaeroides has three main subunits, which bear the prosthetic groups, and contribute to three catalytic sites and internal electron transfer pathways which define the modified Q-cycle mechanism. In this paper, we report on progress in modelling the structure of the bc-complex, and experiments using site directed mutagenesis and biophysical assay to probe the structural and function consequences of specific modifications to these subunits.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/química , Rhodobacter sphaeroides/enzimologia , Benzoquinonas/química , Benzoquinonas/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Modelos Moleculares , Mutagênese Sítio-DirigidaRESUMO
We have cloned cDNAs encoding the last iron-sulphur protein of complex I from Neurospora crassa. The cDNA sequence contains an open reading frame that codes for a precursor polypeptide of 226 amino acid residues with a molecular mass of 24972 Da. Our results indicate that the mature protein belongs probably to the peripheral arm of complex I and is rather unstable when not assembled into the enzyme. The protein is highly homologous to the PSST subunit of bovine complex I, the most likely candidate to bind iron-sulphur cluster N-2. All the amino acid residues proposed to bind such a cluster are conserved in the fungal protein.
Assuntos
Proteínas Ferro-Enxofre/genética , NAD(P)H Desidrogenase (Quinona)/genética , Neurospora crassa/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/química , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Rhodobacter sphaeroides expresses a bb3-type quinol oxidase, and two cytochrome c oxidases: cytochrome aa3 and cytochrome cbb3. We report here the characterization of the genes encoding this latter oxidase. The ccoNOQP gene cluster of R. sphaeroides contains four open reading frames with high similarity to all ccoNOQP/fixNOQP gene clusters reported so far. CcoN has the six highly conserved histidines proposed to be involved in binding the low spin heme, and the binuclear center metals. ccoO and ccoP code for membrane bound mono- and diheme cytochromes c. ccoQ codes for a small hydrophobic protein of unknown function. Upstream from the cluster there is a conserved Fnr/FixK-like box which may regulate its expression. Analysis of a R. sphaeroides mutant in which the ccoNOQP gene cluster was inactivated confirms that this cluster encodes the cbb3-type oxidase previously purified. Analysis of proton translocation in several strains shows that cytochrome cbb3 is a proton pump. We also conclude that cytochromes cbb3 and aa3 are the only cytochrome c oxidases in the respiratory chain of R. sphaeroides.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/genética , Oxirredutases/genética , Bombas de Próton/metabolismo , Rhodobacter sphaeroides/genética , Sequência de Aminoácidos , Sequência de Bases , Membrana Celular/enzimologia , Clonagem Molecular , Cobre , Complexo IV da Cadeia de Transporte de Elétrons/química , Genes Bacterianos/genética , Teste de Complementação Genética , Heme , Histidina/química , Dados de Sequência Molecular , Família Multigênica/genética , Mutação , Fases de Leitura Aberta/genética , Oxirredutases/química , Bombas de Próton/química , Bombas de Próton/genética , Mapeamento por Restrição , Rhodobacter sphaeroides/enzimologia , Análise de Sequência de DNARESUMO
The Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is present in the membranes of a number of marine bacteria and pathogenic bacteria. Two of the six subunits of the Na(+)-NQR, NqrB and NqrC, have been previously shown to contain covalently bound flavin adenine mononucleotide (FMN). In the current work, the cloning of nqrC from Vibrio cholerae is reported. The gene has been expressed in V. cholerae and shown to contain one equivalent of covalently bound FMN. In contrast, no covalent flavin was detected when threonine-225 was replaced by leucine. The data show that the FMN attachment does not require assembly of the enzyme and are consistent with the unusual threonine attachment site.
Assuntos
Mononucleotídeo de Flavina/metabolismo , Quinona Redutases/genética , Vibrio cholerae/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Expressão Gênica , Histidina/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Quinona Redutases/biossíntese , Quinona Redutases/metabolismo , Treonina/genética , Vibrio cholerae/metabolismoRESUMO
Cells of the E. coli mutant EP432, which lacks the two Na+/H+ antiporters, NhaA and NhaB, have been reported to have an impaired sodium transport activity (Harel-Bronstein et al. (1995) J. Biol. Chem. 270, 3816-3822). Here we report that active transport of Na+ in EP432 cells can be restored to wild-type levels, either by a high K+ concentration or by an increase in the medium osmolarity. We suggest that this mutant is primarily deficient in osmoregulation rather than in cation transport per se.
Assuntos
Proteínas de Bactérias , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Potássio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Sódio/metabolismo , Divisão Celular , Citoplasma/metabolismo , Escherichia coli/genética , Transporte de Íons , Proteínas de Membrana/genética , Mutação , Trocadores de Sódio-Hidrogênio/genética , Equilíbrio HidroeletrolíticoRESUMO
The energy-converting NADH:ubiquinone oxidoreductase, also known as respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of ions across the membrane. It was assumed that the complex exclusively works as a proton pump. Recently, it has been proposed that complex I from Klebsiella pneumoniae and Escherichia coli work as Na+ pumps. We have used an E. coli complex I preparation to determine the type of ion(s) translocated by means of enzyme activity, generation of a membrane potential and redox-induced Fourier-transform infrared spectroscopy. We did not find any indications for Na+ translocation by the E. coli complex I.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Escherichia coli/enzimologia , Transporte Biológico , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Íons/metabolismo , Cinética , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Two aa3-type cytochromes were purified from membranes of sporulating Bacillus cereus. One of them, an aa3 complex, was found to be composed of two subunits (51 and 31 kDa), two a hemes and three copper atoms, thus being similar to the cytochrome aa3 previously purified from vegetative B. cereus [García-Horsman, J. A., Barquera, B., González-Halphen, D. & Escamilla, J. E. (1991) Mol. Microbiol. 5, 197-205]. The second isoform, a caa3 complex, was expressed in sporulating cells only, and was found to be composed of two subunits (51 and 37 kDa). The 37-kDa subunit (subunit II) is a heme-c-containing polypeptide as shown by its peroxidase activity in SDS/PAGE gels and by its spectral features. Both subunits of the caa3 complex immunologically cross-reacted with antiserum raised against B. cereus cytochrome aa3, suggesting homology between the two enzymes. Also, the heme-c-containing subunit of the caa3 complex was reactive with anti-(bovine cytochrome c) antiserum, but not with anti-(bovine cytochrome c1) antiserum. In addition to one heme c and two hemes a, the caa3 complex contained three copper atoms. Kinetic comparison of aa3 and caa3 complexes revealed that the latter is slightly more active (k = 150 s-1) and has a lower affinity to yeast cytochrome c (Km = 76 microM) and to oxygen (Km = 2 microM) as compared with cytochrome aa3 (100 s-1, 10 microM, and 5 microM, respectively).
Assuntos
Bacillus cereus/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Isoenzimas/isolamento & purificação , Bacillus cereus/fisiologia , Western Blotting , Membrana Celular/enzimologia , Cromatografia , Cromatografia DEAE-Celulose , Cromatografia em Gel , Cromatografia por Troca Iônica , Durapatita , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hidroxiapatitas , Isoenzimas/metabolismo , Cinética , Peso Molecular , Espectrofotometria , Esporos Bacterianos/enzimologiaRESUMO
Aspartate-75 (D75) was recently suggested to participate in a ubiquinone-binding site in subunit I of cytochrome bo(3) from Escherichia coli on the basis of a structural model [Abramson, J., Riistama, S., Larsson, G., Jasaitis, A., Svensson-Ek, M., Laakkonen, L., Puustinen, A., Iwata, S., and Wikström, M. (2000) Nat. Struct. Biol. 7 (10), 910-917]. We studied the protonation state of D75 for the reduced and oxidized forms of the enzyme, using a combined site-directed mutagenesis, electrochemical, and FTIR spectroscopic approach. The D75H mutant is catalytically inactive, whereas the more conservative D75E substitution has quinol oxidase activity equal to that of the wild-type enzyme. Electrochemically induced FTIR difference spectra of the inactive D75H mutant enzyme show a clear decrease in the spectroscopic region characteristic of protonated aspartates and glutamates. Strong variations in the amide I region of the FTIR difference spectrum, however, reflect a more general perturbation due to this mutation of both the protein and the bound quinone. Electrochemically induced FTIR difference spectra on the highly conservative D75E mutant enzyme show a shift from 1734 to 1750 cm(-1) in direct comparison to wild type. After H/D exchange, the mode at 1750 cm(-1) shifts to 1735 cm(-1). These modes, concomitant with the reduced state of the enzyme, can be assigned to the nu(C=O) vibrational mode of protonated D75 and E75, respectively. In the spectroscopic region where signals for deprotonated acidic groups are expected, band shifts for the nu(COO(-))(s/as) modes from 1563 to 1554-1539 cm(-1) and from 1315 to 1336 cm(-1), respectively, are found for the oxidized enzyme. These signals indicate that D75 (or E75 in the mutant) is deprotonated in the oxidized form of cytochrome bo(3) and is protonated upon full reduction of the enzyme. It is suggested that upon reduction of the bound ubiquinone at the high affinity site, D75 takes up a proton, possibly sharing it with ubiquinol.
Assuntos
Ácido Aspártico/metabolismo , Citocromos/metabolismo , Escherichia coli/enzimologia , Prótons , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , Amidas , Ácido Aspártico/genética , Sítios de Ligação/genética , Grupo dos Citocromos b , Citocromos/genética , Eletroquímica , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Histidina/genética , Mutagênese Sítio-Dirigida , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
Two genes in the Escherichia coli genome, b4065 (yjcE) and b1191 (ycgO), are similar to genes encoding eukaryotic Na+/H+ exchangers. Mutants were constructed in which yjcE (GRN11), ycgO (GRF55) or both (GRD22) were inactivated. There was no change in respiration-driven Na+ efflux in any of the mutants when grown in media containing 50-500 mM Na+. The only striking finding was that growth of GRF55 was impaired at low osmolarity. In complex low-salt medium, GRF55 grew at a wild-type rate for three to four generations but then stopped; the growth was partially recovered after a pause, the length of which was dependent on salt concentration. Measurement of cytoplasmic alkali cations showed that an abrupt loss of about one-half of the intracellular K+ preceded the pause. When grown in low-salt medium with only 20 mM added Na+, GRF55 also lost the ability to maintain a sodium concentration gradient. However, this phenomenon appears to be a secondary effect of the ycgO deletion. The double mutant GRD22 has the same properties as GRF55; no additional effect was found. The data indicate that neither ycgO nor yjeE participates in respiration-driven Na+ extrusion. Instead, ycgO is required for growth at low osmolarity. Hence it is concluded that ycgO participates in cell volume regulation, and accordingly it is suggested that ycgO be renamed cvrA.
Assuntos
Proteínas de Escherichia coli/fisiologia , Escherichia coli/metabolismo , Trocadores de Sódio-Hidrogênio/fisiologia , Sódio/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Proteínas de Transporte de Cátions , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Genes Bacterianos , Concentração de Íons de Hidrogênio , Potenciais da Membrana , Dados de Sequência Molecular , Mutação , Concentração Osmolar , Oxigênio/metabolismo , Fenótipo , Plasmídeos/genética , Potássio/metabolismo , Homologia de Sequência de Aminoácidos , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
The human mitochondrial ND1/3460 mutation changes Ala52 to Thr in the ND1 subunit of Complex I, and causes Leber's hereditary optic neuropathy (LHON) [Huoponen et al. (1991) Am. J. Hum. Genet. 48, 1147]. We have used a bacterial counterpart of Complex I, NDH-1 from Paracoccus denitrificans, for studying the effect of mutations in the ND1 subunit on the enzymatic activity. The LHON mutation as well as several other mutations in strictly conserved amino acids in its vicinity were introduced into the NQO8 subunit of NDH-1, a bacterial homologue of ND1. The enzymatic activity of the mutants in the presence of hexammineruthenium (rotenone-insensitive) and ubiquinone-1 (rotenone-sensitive) were assayed. In addition, the kinetics of the interaction of selected mutant enzymes with ubiquinone-1, ubiquinone-2, and decylubiquinone was studied. The results suggest that the mutated residues play an important role in ubiquinone reduction by Complex I.
Assuntos
Sequência Conservada/genética , DNA Mitocondrial/genética , Mutagênese Sítio-Dirigida , NAD(P)H Desidrogenase (Quinona)/genética , Paracoccus denitrificans/genética , Alanina/genética , Sequência de Aminoácidos , Animais , Ácido Aspártico/genética , DNA Mitocondrial/metabolismo , Complexo I de Transporte de Elétrons , Glutamina/genética , Humanos , Lisina/genética , Dados de Sequência Molecular , NAD(P)H Desidrogenase (Quinona)/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Atrofias Ópticas Hereditárias/enzimologia , Atrofias Ópticas Hereditárias/genética , Paracoccus denitrificans/enzimologia , Quinona Redutases/genética , Quinona Redutases/metabolismoRESUMO
The respiratory systems of the Bacillus cereus mother cell, forespore, and dormant and germinated spore were studied. The results indicated that the electron transfer capacity during sporulation, dormancy, and germination is related to the menaquinone levels in the membrane. During the maturation stages of sporulation (stages III to VI), forespore NADH oxidase activity underwent inactivation concomitant with a sevenfold decrease in the content of menaquinone and without major changes in the content of cytochromes and segment transfer activities. During the same period, NADH oxidase and menaquinone levels in the mother cell compartment steadily decreased to about 50% at the end of stage VI. Dormant spore membranes contained high levels of NADH dehydrogenase and cytochromes, but in the presence of NADH, they exhibited very low levels of O2 uptake and cytochrome reduction. Addition of menadione to dormant spore membranes restored NADH-dependent respiration and cytochrome reduction. During early germination, NADH-dependent respiration and cytochrome reduction were restored simultaneously with a fourfold increase in the menaquinone content; during germination, no significant changes in cytochrome levels or segment electron transfer activities of the respiratory system took place.
Assuntos
Bacillus cereus/fisiologia , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Vitamina K/farmacologia , Bacillus cereus/efeitos dos fármacos , Bacillus cereus/metabolismo , Membrana Celular/enzimologia , Citocromos/metabolismo , Ativação Enzimática , Cinética , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/metabolismo , Esporos Bacterianos/fisiologiaRESUMO
Site-directed mutations of conserved residues in the domain binding the 2Fe-2S cluster of the Rieske subunit of the ubiquinol:cytochrome c2 oxidoreductase (bc1 complex) of Rhodobacter sphaeroides have been constructed. The substitution of aspartate for glycine at position 133 in the Rb. sphaeroides sequence (mutant FG133D), which mimicked a mutation previously isolated and characterized in yeast by Gatti et al. [Gatti, D.L., Meinhardt, S.W., Ohnishi, T., & Tzagoloff, A. (1989) J. Mol. Biol. 205, 421-435], allowed more detailed studies of thermodynamic behavior and the kinetics of the ubiquinol:cytochrome c2 oxidoreductase on flash activation of the photosynthetic chain. The impaired catalysis in this mutant complex is localized to the quinol oxidizing site. The apparent second-order rate constant for reduction of cytochrome bH via the quinol oxidizing site is about 20-fold lower than that of the wild-type and correlates with its apparent activation barrier being increased relative to that of the wild-type. Substitutions for the cysteines and a histidine which are conserved in the putative 2Fe-2S binding domain of the Rieske subunit selectively knock out the 2Fe-2S cluster and quinol oxidizing activity, while leaving the cytochromes and other catalytic sites essentially intact. Reversion properties of these strains are consistent with the mutated residues being essential.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/química , Hidroquinonas/metabolismo , Proteínas Ferro-Enxofre/química , Mutagênese Sítio-Dirigida , Rhodobacter sphaeroides/química , Sequência de Aminoácidos , Sítios de Ligação , Citocromos c1/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Cinética , Luz , Dados de Sequência Molecular , Oxirredução , Rhodobacter sphaeroides/genética , Espectrofotometria , TermodinâmicaRESUMO
The cytochrome b subunit of the bc1 complex contains two heme components, cytochrome bL and cytochrome bH, and is the locus of both a quinol oxidizing site (Qo or Qz) and a quinone reducing site (Qi or Qc). The quinone reductase site has been previously characterized as the site of interaction for a set of inhibitors including antimycin A, diuron, funiculosin, and HQNO. In this paper, four highly conserved residues in the cytochrome b subunit of Rhodobacter sphaeroides (A52, H217, K251, and D252) were targeted for site-directed mutagenesis. These residues were chosen as being likely to be at or near the quinone reductase site, on the basis of known locations of missense mutations in the homologous yeast subunit that confer resistance to Qc-directed inhibitors. The site-directed mutants all exhibit a normal rate of reduction of cytochrome bH, suggesting a fully functional quinol oxidizing site. However, each of the mutants is impaired, to varying degrees, in the rate of reoxidation of cytochrome bH. Two mutants (H217A and D252A) are unable to grow photosynthetically, indicating a severe defect in the bc1 complex. In both cases, the cause of the defect is the lack of reoxidation of cytochrome bH by ubiquinone. This is the first report of mutations that selectively impair the rate of electron transfer from cytochrome bH to the Qc-site. This set of mutations will be useful not only for modeling the structure of the quinone reducing site but also in elucidating the catalytic mechanism of this portion of the Q-cycle.
Assuntos
Grupo dos Citocromos b/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Estrutura Secundária de Proteína , Rhodobacter sphaeroides/enzimologia , Sequência de Aminoácidos , Antimicina A/análogos & derivados , Antimicina A/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular/métodos , Códon/genética , Grupo dos Citocromos b/química , Grupo dos Citocromos b/genética , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/genética , Escherichia coli/genética , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NAD(P)H Desidrogenase (Quinona)/química , Oligodesoxirribonucleotídeos , Plasmídeos , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rhodobacter sphaeroides/genéticaRESUMO
The cytochrome b subunit of the ubiquinol:cytochrome c oxidoreductase (the bc1 complex) contains two heme prosthetic groups, cytochrome bL and cytochrome bH. In addition, this subunit also provides major elements of the quinol oxidation site (Qo) and a separate quinone reductase site (Qi), which are thought to be located on opposite sides of the membrane. Site-directed mutagenesis has been used to explore the role(s) of specific amino acid residues in this subunit from the photosynthetic bacterium Rhodobacter sphaeroides. Previous work identified five residues, Gly48 (Gly33), Ala52 (Gly37), His217 (His202), Lys251 (Lys228), and Asp252 (Asp229), as being either at or near the quinone reductase site (the residue numbers in parentheses designate the equivalent positions in the yeast mitochondrial enzyme). These residues are predicted to be near the cytoplasmic boundaries of transmembrane helices: helix A (G48, A52), helix D (H217), or helix E (K251, D252). In the current work, the importance of two additional highly conserved residues, which are also predicted to be near the cytoplasmic boundaries of transmembrane helices, is explored by site-directed mutagenesis. R114 (helix B) has been substituted with K, Q, and A, and W129 (helix C) has been changed to A and F. The results suggest that a positively charged residue at position 114 is important. The R114K mutation causes only subtle effects, which appear to be localized to cytochrome bH and the quinone reductase site. In contrast, R114Q is not assembled, and R114A, although partially assembled, is nonfunctional and appears to have a very low amount of cytochrome b associated with the complex. Both mutants at position 129 (W129A and W129F) are able to support the photosynthetic growth of the organism, but show abnormal characteristics. The defects associated with the W129A mutation appear to be primarily associated with the quinone reductase site and cytochrome bH, whereas the W129F mutation appears to result in more global defects that also perturb the cytochrome bL locus. The results are consistent with the placement of residues R114 and W129 near the cytoplasmic side of the membrane, but suggest that these residues are important for the assembly and overall stability of the complex.
Assuntos
Arginina/química , Grupo dos Citocromos b/química , Complexo III da Cadeia de Transporte de Elétrons/química , Rhodobacter sphaeroides/enzimologia , Triptofano/química , Sequência de Aminoácidos , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Arginina/genética , Sequência de Bases , Grupo dos Citocromos b/genética , Primers do DNA/química , Eletroquímica , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Estabilidade Enzimática/genética , Cinética , Luz , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Plasmídeos , Conformação Proteica , Estrutura Secundária de Proteína , Mapeamento por Restrição , Espectrofotometria Ultravioleta , Titulometria , Triptofano/genéticaRESUMO
Cytochrome c-oxidase type aa3 (EC 1.9.3.1) was purified to homogeneity from vegetative Bacillus cereus by ion-exchange and hydroxylapatite chromatography in the presence of Triton X-100. Gel filtration analysis suggested a dimeric structure apparently 172 kDa in size; however, only a monomer of 81 kDa was detected when analysed by non-denaturing gel electrophoresis. Denaturing gel electrophoresis analysis of the protein showed the presence of two subunits (51 and 30 kDa). Atomic absorption and visible spectroscopy showed typical aa3 redox centres with haem a iron and copper in a ratio of 22 nmol and 35 ng-atom per mg protein, respectively. No haem c was found associated with the purified enzyme in the conditions reported here. Oxidase activity was fully reconstituted by phospholipids in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine or reduced yeast cytochrome c (but not horse cytochrome c) as electron donors. This activity was abolished by cyanide and carbon monoxide.
Assuntos
Bacillus cereus/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Bacillus/enzimologia , Cardiolipinas/farmacologia , Cromatografia por Troca Iônica , Grupo dos Citocromos c/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Glucosídeos/farmacologia , Cinética , Peso Molecular , Fosfatidilcolinas , Fosfolipídeos/farmacologia , Análise EspectralRESUMO
The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi was purified and studied by EPR and visible spectroscopy. Two EPR signals in the NADH-reduced enzyme were detected: one, a radical signal, and the other a line around g = 1.94, which is typical for a [2Fe-2S] cluster. An E(m) of -267 mV was found for the Fe-S cluster (n = 1), independent of sodium concentration. The spin concentration of the radical in the enzyme was approximately the same under a variety of redox conditions. The time course of Na+-NQR reduction by NADH indicated the presence of at least two different flavin species. Reduction of the first species (most likely, a FAD near the NADH dehydrogenase site) was very rapid in both the presence and absence of sodium. Reduction of the second flavin species (presumably, covalently bound FMN) was slower and strongly dependent on sodium concentration, with an apparent activation constant for Na+ of approximately 3.4 mM. This is very similar to the Km for Na+ in the steady-state quinone reductase reaction catalyzed by this enzyme. These data led us to conclude that the sodium-dependent step within the Na+-NQR is located between the noncovalently bound FAD and the covalently bound FMN.
Assuntos
Quinona Redutases/metabolismo , Sódio/metabolismo , Vibrio/enzimologia , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Cinética , Oxirredução , Bombas de Próton/metabolismo , Quinona Redutases/isolamento & purificação , EspectrofotometriaRESUMO
Quinol oxidation by the bc(1) complex of Rhodobacter sphaeroides occurs from an enzyme-substrate complex formed between quinol bound at the Q(o) site and the iron-sulfur protein (ISP) docked at an interface on cytochrome b. From the structure of the stigmatellin-containing mitochondrial complex, we suggest that hydrogen bonds to the two quinol hydroxyl groups, from Glu-272 of cytochrome b and His-161 of the ISP, help to stabilize the enzyme-substrate complex and aid proton release. Reduction of the oxidized ISP involves H transfer from quinol. Release of the proton occurs when the acceptor chain reoxidizes the reduced ISP, after domain movement to an interface on cytochrome c(1). Effects of mutations to the ISP that change the redox potential and/or the pK on the oxidized form support this mechanism. Structures for the complex in the presence of inhibitors show two different orientations of Glu-272. In stigmatellin-containing crystals, the side chain points into the site, to hydrogen bond with a ring hydroxyl, while His-161 hydrogen bonds to the carbonyl group. In the native structure, or crystals containing myxothiazol or beta-methoxyacrylate-type inhibitors, the Glu-272 side chain is rotated to point out of the site, to the surface of an external aqueous channel. Effects of mutation at this residue suggest that this group is involved in ligation of stigmatellin and quinol, but not quinone, and that the carboxylate function is essential for rapid turnover. H(+) transfer from semiquinone to the carboxylate side chain and rotation to the position found in the myxothiazol structure provide a pathway for release of the second proton.
Assuntos
Grupo dos Citocromos b/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Hidroquinonas/metabolismo , Rhodobacter sphaeroides/enzimologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Galinhas , Grupo dos Citocromos b/química , Citocromos c1/química , Citocromos c1/metabolismo , Estabilidade Enzimática , Ligação de Hidrogênio , Cinética , Mitocôndrias Cardíacas/enzimologia , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Polienos/química , Polienos/metabolismo , Conformação ProteicaRESUMO
Structures of mitochondrial ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) from several animal sources have provided a basis for understanding the functional mechanism at the molecular level. Using structures of the chicken complex with and without inhibitors, we analyze the effects of mutation on quinol oxidation at the Q(o) site of the complex. We suggest a mechanism for the reaction that incorporates two features revealed by the structures, a movement of the iron sulfur protein between two separate reaction domains on cytochrome c(1) and cytochrome b and a bifurcated volume for the Q(o) site. The volume identified by inhibitor binding as the Q(o) site has two domains in which inhibitors of different classes bind differentially; a domain proximal to heme b(L), where myxothiazole and beta-methoxyacrylate- (MOA-) type inhibitors bind (class II), and a distal domain close to the iron sulfur protein docking interface, where stigmatellin and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiaole (UHDBT) bind (class I). Displacement of one class of inhibitor by another is accounted for by the overlap of their volumes, since the exit tunnel to the lipid phase forces the hydrophobic "tails" to occupy common space. We conclude that the site can contain only one "tailed" occupant, either an inhibitor or a quinol or one of their reaction products. The differential sensitivity of strains with mutations in the different domains is explained by the proximity of the affected residues to the binding domains of the inhibitors. New insights into mechanism are provided by analysis of mutations that affect changes in the electron paramagnetic resonance (EPR) spectrum of the iron sulfur protein, associated with its interactions with the Q(o)-site occupant. The structures show that all interactions with the iron sulfur protein must occur at the distal position. These include interactions between quinone, or class I inhibitors, and the reduced iron sulfur protein and formation of a reaction complex between quinol and oxidized iron sulfur protein. The step with high activation energy is after formation of the reaction complex, likely in formation of the semiquinone and subsequent dissociation of the complex into products. We suggest that further progress of the reaction requires a movement of semiquinone to the proximal position, thus mapping the bifurcated reaction to the bifurcated volume. We suggest that such a movement, together with a change in conformation of the site, would remove any semiquinone formed from further interaction with the oxidized [2Fe-2S] center and also from reaction with O(2) to form superoxide anion. We also identify two separate reaction paths for exit of the two protons released in quinol oxidation.