共查询到20条相似文献,搜索用时 31 毫秒
1.
Isolation and characterization of an Escherichia coli mutant lacking the cytochrome o terminal oxidase. 总被引:11,自引:9,他引:2
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A respiration-deficient mutant of Escherichia coli has been isolated which is unable to grow aerobically on nonfermentable substrates such as succinate and lactate. Spectroscopic and immunological studies showed that this mutant lacks the cytochrome o terminal oxidase of the high aeration branch of the aerobic electron transport chain. This strain carries a mutation in a gene designated cyo which is cotransducible with the acrA locus. Mutations in cyo were obtained by mutagenizing a strain that was cyd and, thus, was lacking the cytochrome d terminal oxidase. Strain RG99, which carries both the cyd- and cyo- alleles, grows normally under anaerobic conditions in the presence of nitrate. Introduction of the cyd+ allele into the strain restores the respiration function of the strain, indicating that the cytochrome o branch of the respiratory chain is dispensable under normal laboratory growth conditions. 相似文献
2.
Two respiratory mutants of the aerobic bacterium, Vitreoscilla, have been studied: a CO-resistant mutant that can grow in 50% CO-50% oxygen, and a cyanide-resistant mutant that can grow in 1 mM KCN. Wild-type cells are unable to grow under either condition. This report presents evidence that the resistance of the CO mutant is due to an altered membrane-bound cytochrome o [cytochrome o(m)], and that of the cyanide mutant is due to the presence of an increased amount of cytochrome d, which has a lower affinity for cyanide than cytochrome o(m). The evidence was obtained from spectral studies on the three types of intact cells as well as enzymatic and ligand-binding techniques on the cytoplasmic cytochromes o[cytochrome o(s)] and the respiring membrane vesicles isolated from these cells. Carbon monoxide difference spectra of intact cells revealed a 5-nm shift in an absorption maximum of a CO-binding pigment in the CO mutant relative to that of the wild type. The formation of oxygenated cytochrome o(s) and its conversion to the reduced form when the cells became anaerobic due to cellular respiration were inhibited when 1 mM KCN was added to a cell suspension of wild-type cells; the cyanide mutant cells showed resistance to cyanide in this experiment. Cytochrome o(s) purified from all three cell types had identical physical, electron transferring, and ligand binding properties within experimental error. Respiring membrane vesicles isolated from the two mutants showed more resistance to inhibition by cyanide and carbon monoxide than those from the wild type. Carbon monoxide difference spectra of these membrane vesicles revealed that there was a fivefold increase in the amount of cytochrome d in the cyanide mutant relative to the wild type. A CO absorption band of the membrane-bound cytochrome o in the CO mutant membrane vesicles showed a 5-nm shift relative to that of the wild type. 相似文献
3.
The function of the reversible oxygen-binding hemoprotein from Vitreoscilla (VHb), which enhances oxygen-limited cell growth and recombinant protein production when functionally expressed in Escherichia coli, was investigated in wild-type E. coli and in E. coli mutants lacking one of the two terminal oxidases, cytochrome o complex (aerobic terminal oxidase, Cyo) or cytochrome d complex (microaerobic terminal oxidase, Cyd). Deconvolution of VHb, cytochrome o, and cytochrome d bands from in vivo absorption spectra revealed a 5-fold enhancement in cytochrome o content and a 1.5-fold increment in cytochrome d by VHb under microaerobic environments (dissolved oxygen less than 2% air saturation). Based upon oxygen uptake kinetics measurements of these mutants, the apparent oxygen affinity of the Cyo(+), Cyd(-) E. coli was increased in the presence of VHb, but no difference in the apparent K(m) was observed for the Cyo(-), Cyd(+) strain. Results suggest that the expression of VHb in E. coli increases the level and activity of terminal oxidases and thereby improves the efficiency of microaerobic respiration and growth. 相似文献
4.
The function of the reversible oxygen-binding hemoprotein from Vitreoscilla (VHb), which enhances oxygen-limited cell growth and recombinant protein production when functionally expressed in Escherichia coli, was investigated in wild-type E. coli and in E. coli mutants lacking one of the two terminal oxidases, cytochrome o complex (aerobic terminal oxidase, Cyo) or cytochrome d complex (microaerobic terminal oxidase, Cyd). Deconvolution of VHb, cytochrome o, and cytochrome d bands from in vivo absorption spectra revealed a 5-fold enhancement in cytochrome o content and a 1.5-fold increment in cytochrome d by VHb under microaerobic environments (dissolved oxygen less than 2% air saturation). Based upon oxygen uptake kinetics measurements of these mutants, the apparent oxygen affinity of the Cyo(+), Cyd(-) E. coli was increased in the presence of VHb, but no difference in the apparent K(m) was observed for the Cyo(-), Cyd(+) strain. Results suggest that the expression of VHb in E. coli increases the level and activity of terminal oxidases and thereby improves the efficiency of microaerobic respiration and growth. (c) 1996 John Wiley & Sons, Inc. 相似文献
5.
The operon (cyo) encoding the Na(+)-pumping respiratory terminal oxidase (cytochrome bo) of the bacterium Vitreoscilla was transformed into Escherichia coli GV100, a deletion mutant of cytochrome bo. This was done for the wild type operon and five mutants in three conserved Cyo subunit I amino acids known to be crucial for H(+) transport in the E. coli enzyme, one near the nuclear center, one in the K-channel, and one in the D-channel. CO-binding, NADH and ubiquinol oxidase, and Na(+)-pumping activities were all substantially inhibited by each mutation. The wild type Vitreoscilla cytochrome bo can pump Na(+) against a concentration gradient, resulting in a transmembrane concentration differential of 2-3 orders of magnitude. It is proposed that Vitreoscilla cytochrome bo pumps four Na(+) through the D-channel to the exterior and transports four H(+) through the K-channel for the reduction of each O(2). 相似文献
6.
The bacterium, Vitreoscilla, produces a delta mu(Na+) across its membrane during respiration. A key enzyme for this function is the cytochrome bo terminal oxidase which, when incorporated into synthetic proteoliposomes, pumps Na(+) across the membrane upon the addition of a substrate. A Vitreoscilla cytochrome bo knock out (cyo(-)) mutant was isolated by transposon mutagenesis using pUT-mini-Tn5Cm. The membranes of this mutant lacked the characteristic 416 nm peak and 432 nm trough in CO difference spectra, which are clearly visible in spectra of the Vitreoscilla wild-type, but peaks at 627, 560, and 530 nm in reduced minus oxidized difference spectra indicate that cytochrome bd is still present. The specific NADH oxidase and ubiquinol-1 oxidase activities of the cyo(-) mutant membranes were less than those of Vitreoscilla wild-type and Escherichia coli membranes, and the stimulation of these activities of the mutant and E. coli membranes by 75 mM NaCl was approximately 50% less than that of Vitreoscilla wild-type membranes. The ubiquinol-1 oxidase activity of the cyo(-) mutant membranes was inhibited by 10 mM KCN to a lesser degree than that of the Vitreoscilla wild-type and E. coli membranes (50, 80, and 85%, respectively). This result is also consistent with the cyo(-) mutant membrane fragments containing only the cytochrome bd terminal oxidase, which is known to be less sensitive to KCN. Although the maximum respiration and growth of the cyo(-) mutant were less than those of the wild-type, this mutant is still capable of growing with cytochrome bd alone. 相似文献
7.
Chung YT Stark BC Webster DA 《Biochemical and biophysical research communications》2006,348(4):1209-1214
The conserved Glu540 in subunit I of Escherichia coli cytochrome bo (a H(+) pump) is replaced by Asp544 in the Vitreoscilla enzyme (a Na(+) pump). Site-directed mutagenesis of the Vitreoscilla cytochrome bo operon changed this Asp to Glu, and both wild type and mutant cyo's were transformed into E. coli strain GV100, which lacks cytochrome bo. Compared to the wild type transformant the Asp544Glu transformant had decreased ability to pump Na(+) as well as decreased stimulation in respiratory activity in the presence of Na(+). Preliminary experiments indicated that this mutant also had increased ability to pump protons, suggesting that this single change may provide cation pumping specificity in this group of enzymes. 相似文献
8.
G Wu H D Williams M Zamanian F Gibson R K Poole 《Journal of general microbiology》1992,138(10):2101-2112
We report the isolation and characterization of a mutant of Escherichia coli unable to grow aerobically on non-fermentable substrates, except for very slow growth on glycerol. The mutant contains cytochrome oxidases o and d, and grows anaerobically with alternative electron acceptors. Oxygen consumption rates of cell-free extracts were low relative to activities in an isogenic control strain, but were restored in vitro by adding ubiquinone-1 to cell-free extracts. Transformation with a cloned 2.8 kb ClaI-EcoRV fragment of chromosomal DNA restored the ability of this mutant (AN2571) to grow on succinate and also restored cellular quinone levels in this strain. The plasmid also complemented a previously isolated ubiG mutant (AN151) for aerobic growth on succinate. The nucleotide sequence revealed a 0.7 kb portion of gyrA. Unidirectional nested deletions from this fragment and complementation analysis identified an open reading frame encoding a protein with a predicted molecular mass of 26.5 kDa. This gene (ubiG) encodes the enzyme 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone methyltransferase, which catalyses the terminal step in the biosynthesis of ubiquinone. The open reading frame is preceded by a putative Shine-Dalgarno sequence and followed by three palindromic unit sequences. Comparison of the inferred amino acid sequence of UbiG with the sequence of other S-adenosylmethionine (AdoMet)-dependent methyltransferases reveals a highly conserved AdoMet-binding region. The cloned 2.8 kb fragment also contains a sequence encoding the C-terminus of a protein with 42-44% identity to fungal acetyl-CoA synthetases. 相似文献
9.
Purification of a cytochrome bd terminal oxidase encoded by the Escherichia coli app locus from a delta cyo delta cyd strain complemented by genes from Bacillus firmus OF4. 总被引:4,自引:3,他引:1
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Escherichia coli GK100, with deletions in the operons encoding its two terminal oxidases, cytochrome bo and ctyochrome bd, was complemented for growth on succinate by a recombinant plasmid (pMS100) containing a 3.4-kb region of DNA from alkaliphilic Bacillus firmus OF4. The complementing DNA was predicted to encode five proteins, but neither sequence analysis nor complementation experiments with subclones provided insight into the basis for the complementation. Cytochrome difference spectra of everted membrane vesicles from the transformed strain had characteristics of a cytochrome bd spectrum but with features different from those observed for alkaliphile membranes. To determine the bacterial source and identity of the structural genes for the cytochrome bd in the transformed mutant, the complex was extracted and partially purified. On sodium dodecyl sulfate-polyacrylamide gels, two polypeptides were resolved from the preparation, 43 (subunit I) and 27 (subunit II) kDa. An internal peptide from subunit I was sequenced, and it yielded the same primary sequence as is found in positions 496 to 510 of E. coli appC. Consistent with the microsequencing results pMS100 failed to complement a triple mutant of E. coli carrying a deletion in appB as well as in the cyo and cyd loci. The deduced sequence of AppBC had been predicted to be very similar to the sequence of CydAB (J. Dassa et al., Mol. Gen. Genet. 229:341-352, 1991) but this is the first demonstration that the former is indeed a cytochrome bd terminal oxidase. The enzyme catalyzed oxygen uptake coupled to quinol or N,N,N',N'-tetramethyl-p-phenylenediamine oxidation, and the activity was sensitive to cyanide. No cross-reactivity to subunit-specific polyclonal antibodies directed against the two individual subunits of cyd-encoded cytochrome bd was detected. Since this is the second cytochrome bd discovered in E. coli, it is proposed that the two complexes be designated cytochrome bd-I (cydAB-encoded enzyme) and cytochrome bd-II (appBC-encoded enzyme). In addition, cbdAB is suggested as a more appropriate gene designation for cytochrome bd than either appBC or cyxAB. 相似文献
10.
谷氨酸棒状杆菌SA001是缺失了乳酸脱氢酶基因 (ldhA) 的菌株。为了增加厌氧条件下经异柠檬酸到丁二酸的代谢通量,以提高丁二酸的产量。将来自大肠杆菌Escherichia coli K12的异柠檬酸裂解酶基因导入谷氨酸棒状杆菌SA001 (SA001/pXMJ19-aceA) 中。该菌经0.8 mmol/L的IPTG有氧诱导12 h后,转入厌氧发酵16 h,丁二酸的产量为10.38 g/L,丁二酸的生产强度为0.83 g/(L·h)。与出发菌株比较,异柠檬酸裂解酶的酶活提高了5.8倍,丁二酸的产量提高了48%。结果表明过量表达异柠檬酸裂解酶可以增加由乙醛酸途径流向丁二酸的代谢流。 相似文献
11.
Ramandeep Hwang KW Raje M Kim KJ Stark BC Dikshit KL Webster DA 《The Journal of biological chemistry》2001,276(27):24781-24789
The obligate aerobic bacterium, Vitreoscilla, synthesizes elevated quantities of a homodimeric hemoglobin (VHb) under hypoxic growth conditions. Expression of VHb in heterologous hosts often enhances growth and product formation. A role in facilitating oxygen transfer to the respiratory membranes is one explanation of its cellular function. Immunogold labeling of VHb in both Vitreoscilla and recombinant Escherichia coli bearing the VHb gene clearly indicated that VHb has a cytoplasmic (not periplasmic) localization and is concentrated near the periphery of the cytosolic face of the cell membrane. OmpA signal-peptide VHb fusions were transported into the periplasm in E. coli, but this did not confer any additional growth advantage. The interaction of VHb with respiratory membranes was also studied. The K(d) values for the binding of VHb to Vitreoscilla and E. coli cell membranes were approximately 5-6 microm, a 4-8-fold higher affinity than those of horse myoglobin and hemoglobin for these same membranes. VHb stimulated the ubiquinol-1 oxidase activity of inverted Vitreoscilla membranes by 68%. The inclusion of Vitreoscilla cytochrome bo in proteoliposomes led to 2.4- and 6-fold increases in VHb binding affinity and binding site number, respectively, relative to control liposomes, suggesting a direct interaction between VHb and cytochrome bo. 相似文献
12.
The purification and characterization of the cytochrome d terminal oxidase complex of the Escherichia coli aerobic respiratory chain 总被引:22,自引:0,他引:22
The aerobic respiratory chain of Escherichia coli is branched. In aerobically grown cells harvested in midexponential phase, a respiratory chain containing only b-type cytochromes is predominant. This chain contains a terminal oxidase which is a b-type cytochrome, referred to as cytochrome o. However, when the bacteria are grown under conditions of oxygen limitation, additional components of the respiratory chain are induced, as evidenced by the appearance of new spectroscopic species. These include a new b-type cytochrome, cytochrome b558, as well as cytochrome a1 and cytochrome d. In this paper, a purification protocol and the initial characterization of the terminal oxidase complex containing cytochrome d are reported. Solubilization of the membrane is effected by Zwittergent 3-12, and purification is accomplished by chromatography with DEAE-Sepharose CL-6B and hydroxyapatite. The complex contains cytochrome b558, a1, and d. Analysis by sodium dodecyl sulfate-polyacrylamide gels indicates that the complex contains only two types of polypeptides with the molecular weights estimated to be 57,000 and 43,000. The purified complex has oxidase activity in the presence of detergents, utilizing substrates including ubinquinol-1, N,N,N',N'-tetramethyl-p-phenylenediamine, and 2,3,5,6-tetramethyl-p-phenylenediamine. The cytochrome d complex contains protoheme IX and iron, but does not contain nonheme iron or copper. Approximately half of the cytochromes which are thought to participate in E. coli aerobic respiration are accounted for by this single complex. These results suggest that the E. coli aerobic respiratory chain is organized around a relatively small number of cytochrome-containing complexes. 相似文献
13.
Respiratory Mechanisms in the Flexibacteriaceae: Terminal Oxidase Systems of Saprospira grandis and Vitreoscilla Species 总被引:4,自引:2,他引:2
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Particles from both Saprospira grandis and Vitreoscilla species, obtained by high-pressure extrusion and sonic treatment, respectively, actively catalyze the oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate with O(2). These activities are inhibited by cyanide but not by antimycin; Saprospira is also amytal- and rotenone-insensitive. Vitreoscilla preparations were unable to oxidize mammalian ferrocytochrome c and reduced tetramethyl-p-phenylenediamine, whereas the Saprospira preparations did so actively. Low-temperature (77 K) difference spectroscopy of Vitreoscilla cells and particles indicates the presence of three maxima in the cytochrome alpha-region at 554, 558, and 562 nm. All three cytochromes are active in NADH and succinate oxidation, but none is ascorbate reducible. Cytochrome o is the only CO-binding pigment present and is probably the terminal oxidase; it has properties similar to the cytochrome o isolated in solubilized form from this organism. Saprospira cells and membranes exhibit four cytochrome absorption bands whose maxima are at 550, 554, 558, and 603 nm at 77 K. The latter component has not been noted previously. NADH and succinate reduce all four cytochromes, but ascorbate reduces only the 550- and 603-nm pigments. CO spectra indicate the presence of cytochrome a,a(3) which is probably the oxidase. A second CO-binding pigment is present which is not a peroxidase but may be a cytochrome. 相似文献
14.
The stationary-phase-exit defect of cydC (surB) mutants is due to the lack of a functional terminal cytochrome oxidase.
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The surB gene was identified as a gene product required for Escherichia coli cells to exit stationary phase at 37 degrees C under aerobic conditions. surB was shown to be the same as cydC, whose product is required for the proper assembly and activity of cytochrome d oxidase. Cytochrome d oxidase, encoded by the cydAB operon, is one of two alternate terminal cytochrome oxidases that function during aerobic electron transport in E. coli. Mutations inactivating the cydAB operon also cause a temperature-sensitive defect in exiting stationary phase, but the phenotype is not as severe as it is for surB mutants. In this study, we examined the phenotypes of surB1 delta(cydAB) double mutants and the ability of overexpression of cytochrome o oxidase to suppress the temperature-sensitive stationary-phase-exit defect of surB1 and delta(cydAB) mutants and analyzed spontaneous suppressors of surB1. Our results indicate that the severe temperature-sensitive defect in exiting stationary phase of surB1 mutants is due both to the absence of terminal cytochrome oxidase activity and to the presence of a defective cytochrome d oxidase. Membrane vesicles prepared from wild-type, surB1, and delta(cydAB) strains produced superoxide radicals at the same rate in vitro. Therefore, the aerobic growth defects of the surB1 and delta(cydAB) strains are not due to enhanced superoxide production resulting from the block in aerobic electron transport. 相似文献
15.
Cloning and expression of the Thiobacillus ferrooxidans glutamine synthetase gene in Escherichia coli. 总被引:8,自引:4,他引:4
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The glutamine synthetase (GS) gene glnA of Thiobacillus ferrooxidans was cloned on recombinant plasmid pMEB100 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as the sole source of nitrogen. High levels of GS-specific activity were obtained in the E. coli glnA deletion mutants containing the T. ferrooxidans GS gene. The cloned T. ferrooxidans DNA fragment containing the glnA gene activated histidase activity in an E. coli glnA glnL glnG deletion mutant containing the Klebsiella aerogenes hut operon. Plasmid pMEB100 also enabled the E. coli glnA glnL glnG deletion mutant to utilize arginine or low levels of glutamine as the sole source of nitrogen. There was no detectable DNA homology between the T. ferrooxidans glnA gene and the E. coli glnA gene. 相似文献
16.
透明颤菌血红蛋白的表达对酵母中麦角固醇合成的影响 总被引:1,自引:0,他引:1
构建了含透明颤菌(Vistreoscilla)血红蛋白基因vgb和酵母遗传霉素(G418)抗性基因的重组质粒pVgbkanMX4,转化至酿酒酵母Saccharomyces cerevisiae 1190中,经过分析,基因vgb在酵母细胞中得到表达。对重组菌和野生菌进行了摇瓶培养及5 L发酵罐培养的研究。在摇瓶实验中,重组菌的麦角固醇产量比野生菌有显著提高,在野生菌中的含量为0.573%、而在重组菌中的产量为1.07%。 经过30 h发酵罐培养的实验,野生菌中麦角固醇含量为0.9%,重组菌中其含量为1.38%,验证了摇瓶实验的结果。结果证明vgb基因有利于酵母中麦角固醇的合成。 相似文献
17.
The lpd gene product functions as the L protein in the Escherichia coli glycine cleavage enzyme system. 总被引:5,自引:5,他引:0
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The lpd-encoded lipoamide dehydrogenase, common to the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes, also functions as the lipoamide dehydrogenase (L protein) in the Escherichia coli glycine cleavage (GCV) enzyme complex. Inducible GCV enzyme activity was not detected in an lpd deletion mutant; lpd+ transductants had normal levels of inducible GCV enzyme activity. A serA lpd double mutant was unable to utilize glycine as a serine source and lacked detectable GCV enzyme activity, the phenotype of a serA gcv mutant. Transformation of the double mutant with a plasmid encoding a functional lpd gene restored the ability of the mutant to use glycine as a serine source and restored inducible GCV enzyme activity to normal levels. The presence of acetate and succinate in the growth medium of a strain wild type for lpd and gcv resulted in a 50% reduction in inducible GCV enzyme activity. Enzyme levels were restored to normal under these growth conditions when the strain was transformed with a plasmid encoding a functional lpd gene. 相似文献
18.
透明颤菌血红蛋白基因在阿维链霉菌中的表达 总被引:7,自引:0,他引:7
将含有自身启动子的透明颤菌血红蛋白基因( vhb) 克隆至大肠杆菌—链霉菌穿梭质粒载体pIJ653 中构建成表达载体p WY101 和p WY102 ,用它们转化阿维菌素(avermectins) 产生菌———阿维链霉菌( Streptomyces avermitilis) ,经Western blotting 分析并未检测到vhb 基因表达,但用穿梭载体pHZ1252( 其中的vhb 基因位于受硫链丝菌素诱导的链霉菌强启动子PtipA之下) 转化阿维链霉菌并经硫链丝菌素诱导,则在该菌中表达出了有活性的VHb 蛋白。pHZ1252 在阿维链霉菌中发生了重组缺失,但缺失的pHZ1252 上仍含有完整的vhb 基因及诱导型强启动子,且可在阿维链霉菌中稳定遗传,却不能再转化大肠杆菌。 相似文献
19.
Vitreoscilla hemoglobin is a good oxygen trapping agent and its presence in genetically engineered Escherichia coli helps this bacterium to grow better. Here, the potential use of this hemoglobin, for improving the growth and the oxygen transfer properties of Pseudomonas aeruginosa as well as Escherichia coli, was investigated. To stably maintain it in both bacteria, a broad-host range cosmid vector (pHG1), containing the entire coding sequence for Vitreoscilla hemoglobin gene and its native promoter on a 2.3 kb fragment, was constructed. Though at different levels, both bacteria produced hemoglobin and while the oxygen uptake rates of vgb-bearing strains were 2-3-fold greater than that of non-vgb-bearing strains in both bacteria, the growth advantage afforded by the presence of Vitreoscilla hemoglobin was somewhat varied. As an alternative to the traditional method of the improvement of oxygen transfer properties of the environment in which cells are grown, the genetic manipulation applied here improved the oxygen utilization properties of cells themselves. 相似文献
20.
The respiratory chain of Rhodopseudomonas capsulata, strain St. Louis and of two respiration deficient mutants (M6 and M7) has been investigated by examining the redox and spectral characteristics of the cytochromes and their response to substrates and to specific respiratory inhibitors. Since the specific lesions of M6 and M7 have been localized on two different branches of the multiple oxidase system of the wild type strain, the capability for aerobic growth of these mutants can be considered as a proof of the physiological significance of both branched systems "in vivo". Using M6 and M7 mutants the response of the branched chain to respiratory inhibitors could be established. Cytochrome oxidase activity, a specific function of an high potential cytochrome b (E'0 = +413 mV) is sensitive to low concentrations of KCN (5-10(-5) M); CO is a specific inhibitor of an alternative oxidase, which is also inhibited by high concentrations of KCN (10(-3) M). Antimycin A inhibits preferentially the branch of the chain affected by low concentrations of cyanide. Redox titrations and spectral data indicate the presence in the membrane of three cytochromes of b type (E'0 = +413, +260, +47 vM) and two cytochromes of c type (E'0 = +342, +94 mV). A clear indication of the involvement in respiration of cytochrome b413, cytochrome c342 and cytochrome b47 has been obtained. Only 50% of the dithionite reducible cytochrome b can be reduced by respiratory substrates also in the presence of high concentrations of KCN or in anaerobiosis. The presence and function of quinones in the respiratory electron transport system has been clearly demonstrated. Quinones, which are reducible by NADH and succinate to about the same extent can be reoxidized through both branches of the respiratory chain, as shown by the response of their redox state to KCN. The possible site of the branching of the electron transport chain has been investigated comparing the per cent level of reduction of quinones and of cytochromes b and c as a function of KCN concentrations in membranes from wild type and M6 mutants cells. The site of the branching has been localized at the level of quinones-cytochrome b47. A tentative scheme of the respiratory chains operating in Rhodopseudomonas capsulata, St. Louis and in the two respiration deficient mutants, M6 and M7 is presented. 相似文献