A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum.

It has been a long-standing hypothesis that the endosymbiotic rhizobia (bacteroids) cope with a concentration of 10 to 20 nM free O2 in legume root nodules by the use of a specialized respiratory electron transport chain terminating with an oxidase that ought to have a high affinity for O2. Previously, we suggested that the microaerobically and anaerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase. Here we report the biochemical characteristics of this terminal oxidase after a 27-fold enrichment from membranes of anaerobically grown B. japonicum wild-type cells. The purified oxidase has TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity. N-terminal amino acid sequencing of its major constituent subunits confirmed that presence of the fixN,fixO, and fixP gene products. FixN is a highly hydrophobic, heme B-binding protein. FixO and FixP are membrane-anchored c-type cytochromes (apparent Mrs of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacrylamide gels. All oxidase properties are diagnostic for it to be a member of the cbb3-type subfamily of heme-copper oxidases. The FixP protein was immunologically detectable in membranes isolated from root nodule bacteroids, and 85% of the total cytochrome c oxidase activity in bacteroid membranes was contributed by the cbb3-type oxidase. The Km values for O2 of the purified enzyme and of membranes from different B. japonicum wild-type and mutant strains were determined by a spectrophotometric method with oxygenated soybean leghemoglobin as the sole O2 delivery system. The derived Km value for O2 of the cbb3-type oxidase in membranes was 7 nM, which is six- to eightfold lower than that determined for the aerobic aa3-type cytochrome c oxidase. We conclude that the cbb3-type oxidase supports microaerobic respiration in endosymbiotic bacteroids.     

The symbiotically essential cbb(3)-type oxidase of Bradyrhizobium japonicum is a proton pump

The male germ line stem cell is the only cell type in the adult that can contribute genes to the next generation and is characterized by postnatal proliferation. It has not been determined whether this cell population can be used to deliberately introduce genetic modification into the germ line to generate transgenic animals or whether human somatic cell gene therapy has the potential to accidentally introduce permanent genetic changes into a patient's germ line. Here we report that several techniques can be used to achieve both in vitro and in vivo gene transfer into mouse male germ line stem cells using a retroviral vector. Expression of a retrovirally delivered reporter lacZ transgene in male germ line stem cells and differentiated germ cells persisted in the testis for more than 6 months. At least one in 300 stem cells could be infected. The experiments demonstrate a system to introduce genes directly into the male germ line and also provide a method to address the potential of human somatic cell gene therapy DNA constructs to enter a patient's germ line.     

Multi-step assembly pathway of the cbb3-type cytochrome c oxidase complex

The cbb3-type cytochrome c oxidases as members of the heme-copper oxidase superfamily are involved in microaerobic respiration in both pathogenic and non-pathogenic proteobacteria. The biogenesis of these multisubunit enzymes, encoded by the ccoNOQP operon, depends on the ccoGHIS gene products, which are proposed to be specifically required for co-factor insertion and maturation of cbb3-type cytochrome c oxidases. Here, the assembly of the cbb3-type cytochrome c oxidase from the facultative photosynthetic model organism Rhodobacter capsulatus was investigated using blue-native polyacrylamide gel electrophoresis. This process involves the formation of a stable but inactive 210 kDa sub-complex consisting of the subunits CcoNOQ and the assembly proteins CcoH and CcoS. By recruiting monomeric CcoP, this sub-complex is converted into an active 230 kDa CcoNOQP complex. Formation of these complexes and the stability of the monomeric CcoP are impaired drastically upon deletion of ccoGHIS. In a ccoI deletion strain, the 230 kDa complex was absent, although monomeric CcoP was still detectable. In contrast, neither of the complexes nor the monomeric CcoP was found in a ccoH deletion strain. In the absence of CcoS, the 230 kDa complex was assembled. However, it exhibited no enzymatic activity, suggesting that CcoS might be involved in a late step of biogenesis. Based on these data, we propose that CcoN, CcoO and CcoQ assemble first into an inactive 210 kDa sub-complex, which is stabilized via its interactions with CcoH and CcoS. Binding of CcoP, and probably subsequent dissociation of CcoH and CcoS, then generates the active 230 kDa complex. The insertion of the heme cofactors into the c-type cytochromes CcoP and CcoO precedes sub-complex formation, while the cofactor insertion into CcoN could occur either before or after the 210 kDa sub-complex formation during the assembly of the cbb3-type cytochrome c oxidase.     

Two conserved non-canonical histidines are essential for activity of the cbb 3-type oxidase in Rhodobacter capsulatus

Cytochrome cbb ₃ oxidase, a member of the heme–copper oxidase superfamily, catalyses the reduction of oxygen to water and generates a proton gradient. Cytochrome c oxidases are characterized by a catalytic subunit (subunit I) containing two hemes and one copper ion ligated by six invariant histidine residues, which are diagnostic of heme–copper oxidases in all type of the heme–copper oxidase superfamily. Alignments of the amino acid sequences of subunit I (FixN or CcoN) of the cbb ₃-type oxidases show that catalytic subunit also contains six non-canonical histidine residues that are conserved in all CcoN subunits of the cbb ₃ oxidase, but not the catalytic subunits of other members of heme–copper oxidases superfamily. The function of these six CcoN-specific conserved histidines of cbb ₃-type oxidase in R. capsulatus is unknown. To analyze the contribution of the two invariant histidines of CcoN, H300 and H394, in activity and assembly of the Rhodobacter capsulatus cbb ₃-type oxidase, they were substituted for valine and alanine, respectively by site-directed mutagenesis. H300V and H394A mutations were analyzed with respect to their activity and assembly. It was found that H394A mutation led to a defect in the assembly of both CcoP and CcoO in the membrane, which results in almost complete loss of activity and that although the H300V mutant is normally assembled in the membrane and retain their stability, its catalytic activity is significantly reduced when compared with wild-type oxidase.     

Operation of the cbb3-type terminal oxidase in Azotobacter vinelandii

A part of the gene encoding cbb ₃-type cytochrome oxidase CcoN subunit was cloned from Azotobacter vinelandii and a mutant strain of this bacterium with disrupted ccoN gene was constructed. In contrast to the wild type strain, this one is unable to oxidize cytochromes c ₄ and c ₅. Thus, the A. vinelandii respiratory chain is shown to contain cbb ₃-type cytochrome c oxidase. It is also shown that the activity of this enzyme is not necessary for diazotrophic growth of A. vinelandii at high oxygen concentrations.     

Heme binding properties of heterologously expressed spinach cytochrome b(6): implications for transmembrane b-type cytochrome formation

In vivo and in vitro requirements for the formation of cytochrome b(6) were examined to analyze the mechanisms of transmembrane b-type cytochrome formation. After heterologous expression of spinach cytochrome b(6), formation of the holo-cytochrome was observed within the E. coli inner membrane. The transmembrane orientation of cytochrome b(6) appeared not to be critical for heme binding and holo-cytochrome formation. Furthermore, in vitro reconstitution of cytochrome b(6) was possible under oxidizing as well as under reducing conditions. Taken together these observations strongly indicate that transmembrane b-type cytochromes can spontaneously assemble in vitro as well as in a membrane.     

A cytochrome c oxidase proton pumping mechanism that excludes the O2 reduction site

A redox-coupled conformational change in Asp51 of subunit I and a hydrogen-bond network connecting Asp51 with the matrix surface have been deduced from X-ray structures of bovine heart cytochrome c oxidase. This has provided evidence that Asp51 may play a role in the proton pumping process. However, the lack of complete conservation of a residue analogous to Asp51, the inclusion of a peptide bond in the hydrogen-bonding network and the lack of apparent involvement of the O2 reduction site have been used as arguments against the involvement of Asp51 in the mechanism of proton pumping. This minireview re-examines these arguments.     

The yeast plasma membrane P4-ATPases are major transporters for lysophospholipids

The transbilayer movement of phospholipids plays an essential role in establishing and maintaining the asymmetric distribution of lipids in biological membranes. The P4-ATPase family has been implicated as the major transporters of the aminoglycerophospholipids in both surface and endomembrane systems. Historically, fluorescent lipid analogs have been used to monitor the lipid transport activity of the P4-ATPases. Recent evidence now demonstrates that lyso-phosphatidylethanolamine (lyso-PtdEtn) and lyso-phosphatidylcholine (lyso-PtdCho) are bona fide biological substrates transported by the yeast plasma membrane ATPases, Dnf1p and Dnf2p, in consort with a second protein Lem3p. Subsequent to transport, the lysophospholipids are acylated by the enzyme Ale1p to produce PtdEtn and PtdCho. The transport of the lysophospholipids occurs at rates sufficient to support all the PtdEtn and PtdCho synthesis required for rapid cell growth. The lysophospholipid transporters also utilize the anti-neoplastic and anti-parasitic ether lipid substrates related to edelfosine. The identification of biological substrates for the plasma membrane ATPases coupled with the power of yeast genetics now provides new tools to dissect the structure and function of the aminoglycerophospholipid transporters.     

A high-affinity iron transport system of Rhizobium meliloti may be required for efficient nitrogen fixation in planta

We have analyzed the ability of single site insertion mutants of Rhizobium meliloti 1021 defective in various components of a high-affinity iron transport system to produce nodules, fix nitogen and promote plant growth. Our results indicate that a high-affinity iron transport system may significantly increase the ability of the differentiated form of the bacterium to fix nitrogen and induce an increase in plant growth.Abbreviations EDDA ethylenediamine-N,N-bis(2-hydroxyphenylacetic acid) - CAS chrome azurol S     

The assignment of the 655 nm spectral band of cytochrome oxidase

The spectral characteristics of the ‘655 nm’ band of cytochrome oxidase were found to be affected by ligands of the binuclear centre, including formate and chloride, and by the resting/pulsed transition. The band titrated with near n=1 characteristics at a midpoint of about 400 mV, in contrast to haem a₃, which exhibits strong redox interaction and a titration range at significantly lower potential. Thus, although the total reduced-oxidised difference spectrum of haem a₃, shows a trough at about 655 nm, this characteristic is absent in the low potential region. The 655 nm feature may arise from a charge transfer band of ferric high-spin haem a₃, which is modulated by the redox state of Cu_B, as suggested by Beinert et al. [(1976) Biochim. Biophys. Acta 423, 339–355].     

The effect of pH on the oxygen kinetics of cytochrome c oxidase proteoliposomes

The effect of pH on the oxygen kinetics of cytochrome c oxidase incorporated into phospholipid vesicles is studied. The pH profiles of the oxygen kinetics of energized and deenergized oxidase vesicles are similar. An effect of pH on the slope of the reciprocal plot of rate against oxygen concentration is observed, and this may indicate that protons are involved in the rate limiting step of the reaction between oxygen and reduced oxidase. In contrast to the pH dependence of the oxygen kinetics, the binding of CO to the oxidase is not pH dependent.     

The use of specific lysine modifications to locate the reaction site of cytochrome c with sulfite oxidase

The reduction of cytochrome c by beef liver sulfite oxidase was found to be strongly inhibited by high ionic strength, indicating the importance of electrostatic interactions to the reaction. The reaction rates of sulfite oxidase with singly trifluoroacetylated or trifluoromethylphenylcarbamylated cytochrome c derivatives were studied to determine the role of individual lysines in the reaction. The reaction rate was decreased by modification of the lysines immediately surrounding the heme crevice, the decreases following the order: Lys 13 > Lys 25 Lys 79 ≈ Lys 87 > Lys 8 ≈ Lys 27 ≈ Lys 72. Modification of lysines 22, 55, 88, 99, and 100 had no effect on the reaction rate. These results indicate that the interaction site on cytochrome c for sulfite oxidase is at the heme crevice region, and overlaps considerable with that for cytochrome oxidase.     

The distance between cytochromes a and a3 in the azide compound of bovine-heart cytochrome oxidase

The electron-spin relaxation rates of the two species of cytochrome a³⁺₃-azide found in the azide compound of bovine-heart cytochrome oxidase were measured by progressive microwave saturation at T = 10 K. It has been shown previously that Cyt a⁺³₃-azide gives rise to two distinct EPR resonances, depending upon the oxidation state of Cyt a. When Cyt a is ferrous, Cyt a³⁺₃-azide has g = 2.88, 2.19 and 1.64; upon oxidation of Cyt a, the a³⁺₃-azide g-values become g = 2.77, 2.18, and 1.74 (Goodman, G. (1984) J. Biol. Chem. 259, 15094–15099). The relaxation effect of Cyt a on Cyt a₃ could be measured as the difference in microwave field saturation parameter H_1/2 between the g = 2.77 and g = 2.88 species. For each signal the spin-lattice relaxation time T₁ was determined from H_1/2 using the transverse relaxation time T₂. The value of T₂ at 10 K was extrapolated from a plot of line-width vs. temperature at higher temperature. The dipolar contribution to T₁ was related to the Cyt a-Cyt a₃ spin-spin distance utilizing available information on the relative orientation of Cyt a₃-azide and Cyt a (Erecinska, M., Wilson, D.F. and Blasie, J.K. (1979) Biochim. Biophys. Acta 545, 352–364). By taking into account the relaxation parameters for both g_x and g_z components of the Cyt a₃-azide g-tensor, the angle between the g_z components of the Cyt a and Cyt a₃g-tensors was determined to be between 0 and 18°, and the Cyt a-Cyt a₃ spin-spin distance was found to be 19 ± 8 Å.     

The cydD gene product, component of a heterodimeric ABC transporter, is required for assembly of periplasmic cytochrome c and of cytochrome bd in Escherichia coli

Abstract The cydD gene of Escherichia coli encodes a protein which, together with the CydC protein, probably constitutes a heterodimeric, ABC-family membrane transporter, necessary for biosynthesis of the cytochrome bd quinol oxidase. Here, we demonstrate that a cydD mutant also fails to synthesise periplasmic c -type cytochrome(s), suggesting that the transporter exports haem or some other component involved in assembly of cytochromes that are found in, or exposed to, the periplasm. The CydDC system appears to be the first example of a transporter required for periplasmic cytochrome assembly processes requiring more than one type of haem. A mutant defective in trxB (adjacent to the cydDC operon, and encoding thioredoxin reductase) was unaffected in cytochrome c or bd assembly.     

Cloning, sequencing and mutational analysis of the cytochrome c552 gene (cycB) from Bradyrhizobium japonicum strain 110

We report the cloning and nucleotide sequence analysis of the cytochrome c552 gene (cycB) of Bradyrhizobium japonicum strain 110. The gene was identified with help of an oligonucleotide that was designed on the basis of the amino acid sequence determined for purified cytochrome c552 of B. japonicum strain CC705. The cycB gene product has an N-terminal 23-amino acid signal peptide that is missing in the mature cytochrome c552 protein. A B. japonicum cycB insertion mutant was constructed which had no observable phenotypic defects in denitrification and symbiotic nitrogen fixation. Thus, the function of c552 remains unknown.     

导入dctABD和parCBA／DE基因提高大豆慢生根瘤菌固氮皎效率和稳定？…

以ｐＬＡＦＲ３为载体构建重组质粒ｐＨＮ２０７,携带有来自苜蓿根瘤菌（Ｓｉｎｏｒｈｉｚｏｂｉｕｍ ｍｅｌｉｌｏｔｉ）的四碳二羧酸转移酶基因ｄｃｔＡＢＤ、来自ｐＴＲ１０２的ｐａｒＣＢＦＡ／ＤＥ基因和标记发光酶基因ｌｕｘＡＢ。利用２亲本杂交法,将重组质粒ｐＨＮ２０７导入大豆慢生根瘤菌（Ｂｒａｄｙｒｈｉｚｏｂｉｕｍ ｊａｐｏｎｉｃｕｍ）ＴＡ１１和ＣＢ１８０９,分别考察了转移接合子中外源重组质粒在人工培养条     

Decreased cytochrome oxidase activity in hepatic mitochondria after chronic ethanol consumption and the possible role of decreased cytochrome aa3 content and changes in phospholipids

In ethanol-fed baboons, hepatic mitochondrial cytochrome oxidase activity and cytochrome aa₃ content were significantly decreased by 58.3 and 50.5%, respectively, compared to their pair-fed controls. However, there was no significant correlation between the two, suggesting that other factors in addition to cytochrome aa₃ may be responsible for the depression in cytochrome oxidase activity. The total phospholipid content of the mitochondrial membranes was significantly decreased (0.24 ± 0.03 μmol of phospholipid phosphorus/mg of protein vs. 0.32 ± 0.04 in controls). This change was accounted for, in part, by the significant decrease in the levels of phosphatidylcholine and cardiolipin. In addition, the fatty acid pattern of the phospholipids was changed. There was a marked increase in the relative amounts of oleic and linoleic acid and a decrease in arachidonic acid. These changes were associated with an increase in the activity of phospholipase A₂. The reactivation rate of phospholipid-depleted cytochrome oxidase by endogenous phospholipids from ethanol-fed baboons was significantly lower than that by phospholipid from pair-fed controls, when measured at an optimal phospholipid to protein ratio. Thus, it appears that alterations in the phospholipid composition of the mitochondrial membranes are responsible, at least in part, for the depression of cytochrome oxidase activity produced by chronic ethanol consumption.