共查询到20条相似文献,搜索用时 31 毫秒
1.
Ozaki Y Suzuki T Kuruma Y Ueda T Yoshida M 《Biochemical and biophysical research communications》2008,367(3):663-666
In FoF1-ATP synthase, multimeric c-subunits are assembled to a ring (c-ring) in the membranes that rotates as protons flow across Fo. We recently reported that assembly of c-ring of Propionigenium modestum in the membranes of Escherichia coli cells required P. modestum UncI, a product of the conserved uncI gene in the FoF1 operon. However, cooperation with endogenous factors in E. coli remained unclear. Here, P. modestum c-subunit was synthesized in vitro in the presence of liposomes. When c-subunit alone was synthesized, it did not form c-ring. However, when c-subunit and P. modestum UncI were synthesized together, c-ring was formed. Fusion of the two kinds of liposomes, one containing only unassembled c-subunit and the other only UncI, resulted in gradual formation of c-ring. Thus, UncI alone can mediate in vitro post-translational c-ring assembly. 相似文献
2.
Brian D. Cain 《Journal of bioenergetics and biomembranes》2000,32(4):365-371
The a and b subunits constitute the stator elements in the F0 sector of F1F0-ATP synthase.Both subunits have been difficult to study by physical means, so most of the information onstructure and function relationships in the a and b subunits has been obtained using mutagenesisin combination with biochemical methods. These approaches were used to demonstrate thatthe a subunit in association with the ring of c subunits houses the proton channel throughF1F0-ATP synthase. The map of the amino acids contributing to the proton channel is probablycomplete. The two b subunits dimerize, forming an extended flexible unit in the peripheralstalk linking the F1 and F0 sectors. The unique characteristics of specific amino acid substitutionsaffecting the a and b subunits suggested differential effects on rotation during F1F0-ATPaseactivity. 相似文献
3.
For functional characterization, we isolated the F1FO-ATP synthase of the thermophilic cyanobacterium Thermosynechococcus elongatus. Because of the high content of phycobilisomes, a combination of dye-ligand chromatography and anion exchange chromatography was necessary to yield highly pure ATP synthase. All nine single F1FO subunits were identified by mass spectrometry. Western blotting revealed the SDS stable oligomer of subunits c in T. elongatus. In contrast to the mass archived in the database (10,141 Da), MALDI-TOF-MS revealed a mass of the subunit c monomer of only 8238 Da. A notable feature of the ATP synthase was its ability to synthesize ATP in a wide temperature range and its stability against chaotropic reagents. After reconstitution of F1FO into liposomes, ATP synthesis energized by an applied electrochemical proton gradient demonstrated functional integrity. The highest ATP synthesis rate was determined at the natural growth temperature of 55 °C, but even at 95 °C ATP production occurred. In contrast to other prokaryotic and eukaryotic ATP synthases which can be disassembled with Coomassie dye into the membrane integral and the hydrophilic part, the F1FO-ATP synthase possessed a particular stability. Also with the chaotropic reagents sodium bromide and guanidine thiocyanate, significantly harsher conditions were required for disassembly of the thermophilic ATP synthase. 相似文献
4.
Anne M. Gardner Matthew R. Cook Paul R. Gardner 《The Journal of biological chemistry》2010,285(31):23850-23857
Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b5 were found to support a high NOD activity. Cygb-NOD activity shows respective Km values for ascorbate, cytochrome b5, NO, and O2 of 0.25 mm, 0.3 μm, 40 nm, and ∼20 μm and achieves a kcat of 0.5 s−1. Ascorbate and cytochrome b5 reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m−1 s−1 and 3 × 106 m−1 s−1, respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar kcat of 1.2 s−1, a Km(NO) of 40 nm, and a kcat/Km(NO) (k′NOD) value of 3 × 107 m−1 s−1, demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O2] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b5 as reductants. 相似文献
5.
Holger Gohlke Daniel Schlieper Georg Groth 《The Journal of biological chemistry》2012,287(43):36536-36543
The rotation of F1Fo-ATP synthase is powered by the proton motive force across the energy-transducing membrane. The protein complex functions like a turbine; the proton flow drives the rotation of the c-ring of the transmembrane Fo domain, which is coupled to the ATP-producing F1 domain. The hairpin-structured c-protomers transport the protons by reversible protonation/deprotonation of a conserved Asp/Glu at the outer transmembrane helix (TMH). An open question is the proton transfer pathway through the membrane at atomic resolution. The protons are thought to be transferred via two half-channels to and from the conserved cAsp/Glu in the middle of the membrane. By molecular dynamics simulations of c-ring structures in a lipid bilayer, we mapped a water channel as one of the half-channels. We also analyzed the suppressor mutant cP24D/E61G in which the functional carboxylate is shifted to the inner TMH of the c-protomers. Current models concentrating on the “locked” and “open” conformations of the conserved carboxylate side chain are unable to explain the molecular function of this mutant. Our molecular dynamics simulations revealed an extended water channel with additional water molecules bridging the distance of the outer to the inner TMH. We suggest that the geometry of the water channel is an important feature for the molecular function of the membrane part of F1Fo-ATP synthase. The inclination of the proton pathway isolates the two half-channels and may contribute to a favorable clockwise rotation in ATP synthesis mode. 相似文献
6.
In Escherichia coli, the F1FO ATP synthase b subunits house a conserved arginine in the tether domain at position 36 where the subunit emerges from the membrane. Previous
experiments showed that substitution of isoleucine or glutamate result in a loss of enzyme activity. Double mutants have been
constructed in an attempt to achieve an intragenic suppressor of the b
arg36→ile and the b
arg36→glu mutations. The b
arg36→ile mutation could not be suppressed. In contrast, the phenotypic defect resulting from the b
arg36→glu mutation was largely suppressed in the b
arg36→glu,glu39→arg double mutant. E. coli expressing the b
arg36→glu,glu39→arg subunit grew well on succinate-based medium. F1FO ATP synthase complexes were more efficiently assembled and ATP driven proton pumping activity was improved. The evidence
suggests that efficient coupling in F1FO ATP synthase is dependent upon a basic amino acid located at the base of the peripheral stalk. 相似文献
7.
Boris A. Feniouk Alberto Rebecchi Sofie Anefors Armen Y. Mulkidjanian Wolfgang Junge B. Andrea Melandri 《BBA》2007,1767(11):1319-1330
H+-FOF1-ATP synthase couples proton flow through its membrane portion, FO, to the synthesis of ATP in its headpiece, F1. Upon reversal of the reaction the enzyme functions as a proton pumping ATPase. Even in the simplest bacterial enzyme the ATPase activity is regulated by several mechanisms, involving inhibition by MgADP, conformational transitions of the ε subunit, and activation by protonmotive force. Here we report that the Met23Lys mutation in the γ subunit of the Rhodobacter capsulatus ATP synthase significantly impaired the activation of ATP hydrolysis by protonmotive force. The impairment in the mutant was due to faster enzyme deactivation that was particularly evident at low ATP/ADP ratio. We suggest that the electrostatic interaction of the introduced γLys23 with the DELSEED region of subunit β stabilized the ADP-inhibited state of the enzyme by hindering the rotation of subunit γ rotation which is necessary for the activation. 相似文献
8.
Giraud MF Paumard P Sanchez C Brèthes D Velours J Dautant A 《Journal of structural biology》2012,177(2):490-497
The F1FO-ATP synthase is a rotary molecular nanomotor. F1 is a chemical motor driven by ATP hydrolysis while FO is an electrical motor driven by the proton flow. The two stepping motors are mechanically coupled through a common rotary shaft. Up to now, the three available crystal structures of the F1c10 sub-complex of the yeast F1FO-ATP synthase were isomorphous and then named yF1c10(I). In this crystal form, significant interactions of the c10-ring with the F1-head of neighboring molecules affected the overall conformation of the F1-c-ring complex. The symmetry axis of the F1-head and the inertia axis of the c-ring were tilted near the interface between the F1-central stalk and the c-ring rotor, resulting in an unbalanced machine. We have solved a new crystal form of the F1c10 complex, named yF1c10(II), inhibited by adenylyl-imidodiphosphate (AMP-PNP) and dicyclohexylcarbodiimide (DCCD), at 6.5 Å resolution in which the crystal packing has a weaker influence over the conformation of the F1-c-ring complex. yF1c10(II) provides a model of a more efficient generator. yF1c10(II) and bovine bF1c8 structures share a common rotor architecture with the inertia center of the F1-stator close to the rotor axis. 相似文献
9.
The insertion of inner membrane proteins in Escherichia coli occurs almost exclusively via the SecYEG pathway, while some membrane proteins require the membrane protein insertase YidC. In vitro analysis demonstrates that subunit a of the F1F0 ATP synthase (F0a) is strictly dependent on Ffh, SecYEG and YidC for its membrane insertion but independent of the proton motive force. The insertion of the first transmembrane segment of F0a also depends on Ffh and SecYEG but not on YidC, whereas the insertion is strongly dependent on the proton motive force, unlike the full-length F0a protein. These data demonstrate an extensive role of YidC in the assembly of the F0 sector of the F1F0 ATP synthase. 相似文献
10.
Alain Dautant Jean Velours Marie-France Giraud 《The Journal of biological chemistry》2010,285(38):29502-29510
The F1c10 subcomplex of the yeast F1F0-ATP synthase includes the membrane rotor part c10-ring linked to a catalytic head, (αβ)3, by a central stalk, γδϵ. The Saccharomyces cerevisiae yF1c10·ADP subcomplex was crystallized in the presence of Mg·ADP, dicyclohexylcarbodiimide (DCCD), and azide. The structure was solved by molecular replacement using a high resolution model of the yeast F1 and a bacterial c-ring model with 10 copies of the c-subunit. The structure refined to 3.43-Å resolution displays new features compared with the original yF1c10 and with the yF1 inhibited by adenylyl imidodiphosphate (AMP-PNP) (yF1(I–III)). An ADP molecule was bound in both βDP and βTP catalytic sites. The αDP-βDP pair is slightly open and resembles the novel conformation identified in yF1, whereas the αTP-βTP pair is very closed and resembles more a DP pair. yF1c10·ADP provides a model of a new Mg·ADP-inhibited state of the yeast F1. As for the original yF1 and yF1c10 structures, the foot of the central stalk is rotated by ∼40 ° with respect to bovine structures. The assembly of the F1 central stalk with the F0 c-ring rotor is mainly provided by electrostatic interactions. On the rotor ring, the essential cGlu59 carboxylate group is surrounded by hydrophobic residues and is not involved in hydrogen bonding. 相似文献
11.
A synthetic study in the Cd-Se-O-Cl system led to formation of the new oxochloride compound Cd4(SeO3)2OCl2 via solid state reactions. The compound crystallizes in the orthorhombic space group Fmmm with cell parameters a = 7.3610(3) Å, b = 15.4936(2) Å, c = 17.5603(3) Å, Z = 8, S = 0.969, F(000) = 2800, R = 0.0185, Rw = 0.0384. Single crystal X-ray data were collected at 293 K. The crystal structure can be considered as layered and the building units are distorted [Cd(1)O6] octahedra, distorted [Cd(2)O8] cubes, irregular [Cd(3)O4Cl2] polyhedra and SeO3E trigonal pyramids. There are two crystallographically unique Cl atoms that both are half occupied. Thermogravimetric studies show that the compound starts to decompose at 500°C. The crystal structure of the new compound is closely related to the previously described compound Cd4(SeO3)2Cl4(H2O). 相似文献
12.
Eran Levin Brit Plotnik Eran Amichai Luzie J. Braulke Shmulik Landau Yoram Yom-Tov Noga Kronfeld-Schor 《Proceedings. Biological sciences / The Royal Society》2015,282(1804)
We report that two species of mouse-tailed bats (Rhinopoma microphyllum and R. cystops) hibernate for five months during winter in geothermally heated caves with stable high temperature (20°C). While hibernating, these bats do not feed or drink, even on warm nights when other bat species are active. We used thermo-sensitive transmitters to measure the bats’ skin temperature in the natural hibernacula and open flow respirometry to measure torpid metabolic rate at different ambient temperatures (Ta, 16–35°C) and evaporative water loss (EWL) in the laboratory. Bats average skin temperature at the natural hibernacula was 21.7 ± 0.8°C, and no arousals were recorded. Both species reached the lowest metabolic rates around natural hibernacula temperatures (20°C, average of 0.14 ± 0.01 and 0.16 ± 0.04 ml O2 g−1 h−1 for R. microphyllum and R. cystops, respectively) and aroused from torpor when Ta fell below 16°C. During torpor the bats performed long apnoeas (14 ± 1.6 and 16 ± 1.5 min, respectively) and had a very low EWL. We hypothesize that the particular diet of these bats is an adaptation to hibernation at high temperatures and that caves featuring high temperature and humidity during winter enable these species to survive this season on the northern edge of their world distribution. 相似文献
13.
Robert H. Fillingame 《Journal of bioenergetics and biomembranes》1992,24(5):485-491
The F0 sector of the ATP synthase complex facilitates proton translocation through the membrane, and via interaction with the F1 sector, couples proton transport to ATP synthesis. The molecular mechanism of function is being probed by a combination of mutant analysis and structural biochemistry, and recent progress on theEscherichia coli F0 sector is reviewed here. TheE. coli F0 is composed of three types of subunits (a, b, andc) and current information on their folding and organization in F0 is reviewed. The structure of purified subunitc in chloroform-methanol-H2O resembles that in native F0, and progress in determining the structure by NMR methods is reviewed. Genetic experiments suggest that the two helices of subunitc must interact as a functional unit around an essential carboxyl group as protons are transported. In addition, a unique class of suppressor mutations identify a transmembrane helix of subunita that is proposed to interact with the bihelical unit of subunitc during proton transport. The role of multiple units of subunitc in coupling proton translocation to ATP synthesis is considered. The special roles of Asp61 of subunitc and Arg210 of subunita in proton translocation are also discussed. 相似文献
14.
Low-temperature leaf photosynthesis of a Miscanthus germplasm collection correlates positively to shoot growth rate and specific leaf area 总被引:1,自引:0,他引:1
Xiurong Jiao Kirsten K?rup Mathias Neumann Andersen Karen Koefoed Petersen Thomas Prade Stanis?aw Je?owski Szymon Ornatowski Barbara Górynowicz Idan Spitz Poul Erik L?rke Uffe J?rgensen 《Annals of botany》2016,117(7):1229-1239
Background and Aims The C4 perennial grass miscanthus has been found to be less sensitive to cold than most other C4 species, but still emerges later in spring than C3 species. Genotypic differences in miscanthus were investigated to identify genotypes with a high cold tolerance at low temperatures and quick recovery upon rising temperatures to enable them to exploit the early growing season in maritime cold climates. Suitable methods for field screening of cold tolerance in miscanthus were also identified.Methods Fourteen genotypes of M. sacchariflorus, M. sinensis, M. tinctorius and M. × giganteus were selected and grown under warm (24 °C) and cold (14 °C) conditions in a controlled environment. Dark-adapted chlorophyll fluorescence, specific leaf area (SLA) and net photosynthetic rate at a photosynthetically active radiation (PAR) of 1000 μmol m–2 s–1 (A1000) were measured. Photosynthetic light and CO2 response curves were obtained from 11 of the genotypes, and shoot growth rate was measured under field conditions.Key Results A positive linear relationship was found between SLA and light-saturated photosynthesis (Asat) across genotypes, and also between shoot growth rate under cool field conditions and A1000 at 14 °C in a climate chamber. When lowering the temperature from 24 to 14 °C, one M. sacchariflorus exhibited significantly higher Asat and maximum photosynthetic rate in the CO2 response curve (Vmax) than other genotypes at 14 °C, except M. × giganteus ‘Hornum’. Several genotypes returned to their pre-chilling A1000 values when the temperature was increased to 24 °C after 24 d growth at 14 °C.Conclusions One M. sacchariflorus genotype had similar or higher photosynthetic capacity than M. × giganteus, and may be used for cultivation together with M. × giganteus or for breeding new interspecies hybrids with improved traits for temperate climates. Two easily measured variables, SLA and shoot growth rate, may be useful for genotype screening of productivity and cold tolerance. 相似文献
15.
Sari Palmroth Gabriel G. Katul Chris A. Maier Eric Ward Stefano Manzoni Giulia Vico 《Annals of botany》2013,111(3):467-477
Background and Aims
Water and nitrogen (N) are two limiting resources for biomass production of terrestrial vegetation. Water losses in transpiration (E) can be decreased by reducing leaf stomatal conductance (gs) at the expense of lowering CO2 uptake (A), resulting in increased water-use efficiency. However, with more N available, higher allocation of N to photosynthetic proteins improves A so that N-use efficiency is reduced when gs declines. Hence, a trade-off is expected between these two resource-use efficiencies. In this study it is hypothesized that when foliar concentration (N) varies on time scales much longer than gs, an explicit complementary relationship between the marginal water- and N-use efficiency emerges. Furthermore, a shift in this relationship is anticipated with increasing atmospheric CO2 concentration (ca).Methods
Optimization theory is employed to quantify interactions between resource-use efficiencies under elevated ca and soil N amendments. The analyses are based on marginal water- and N-use efficiencies, λ = (∂A/∂gs)/(∂E/∂gs) and η = ∂A/∂N, respectively. The relationship between the two efficiencies and related variation in intercellular CO2 concentration (ci) were examined using A/ci curves and foliar N measured on Pinus taeda needles collected at various canopy locations at the Duke Forest Free Air CO2 Enrichment experiment (North Carolina, USA).Key Results
Optimality theory allowed the definition of a novel, explicit relationship between two intrinsic leaf-scale properties where η is complementary to the square-root of λ. The data support the model predictions that elevated ca increased η and λ, and at given ca and needle age-class, the two quantities varied among needles in an approximately complementary manner.Conclusions
The derived analytical expressions can be employed in scaling-up carbon, water and N fluxes from leaf to ecosystem, but also to derive transpiration estimates from those of η, and assist in predicting how increasing ca influences ecosystem water use. 相似文献16.
Tristan Nicke Tobias Schnitzer Karin Münch Julia Adamczack Kristin Haufschildt Sabine Buchmeier Martin Kucklick Undine Felgentr?ger Lothar J?nsch Katharina Riedel Gunhild Layer 《Bioscience reports》2013,33(3)
The periplasmic cytochrome cd1 nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors haem c and haem d1. Whereas the haem c is incorporated into NirS by the cytochrome c maturation system I, nothing is known about the insertion of the haem d1 into NirS. Here, we show by co-immunoprecipitation that NirS interacts with the potential haem d1 insertion protein NirN in vivo. This NirS–NirN interaction is dependent on the presence of the putative haem d1 biosynthesis enzyme NirF. Further, we show by affinity co-purification that NirS also directly interacts with NirF. Additionally, NirF is shown to be a membrane anchored lipoprotein in P. aeruginosa. Finally, the analysis by UV–visible absorption spectroscopy of the periplasmic protein fractions prepared from the P. aeruginosa WT (wild-type) and a P. aeruginosa ΔnirN mutant shows that the cofactor content of NirS is altered in the absence of NirN. Based on our results, we propose a potential model for the maturation of NirS in which the three proteins NirS, NirN and NirF form a transient, membrane-associated complex in order to achieve the last step of haem d1 biosynthesis and insertion of the cofactor into NirS. 相似文献
17.
The kinetics of the cytochrome (cyt) components of the bc1 complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The “traditional” set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt ctot (cyt c1 + cyt c2), cyt bL, cyt bH, and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c1 and c2 is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c1, c2, bL, and bH) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc1 complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc1 complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range. 相似文献
18.
Stahlberg H Müller DJ Suda K Fotiadis D Engel A Meier T Matthey U Dimroth P 《EMBO reports》2001,2(3):229-233
Synthesis of adenosine triphosphate (ATP) by the F1F0 ATP synthase involves a membrane-embedded rotary engine, the F0 domain, which drives the extra-membranous catalytic F1 domain. The F0 domain consists of subunits a1b2 and a cylindrical rotor assembled from 9–14 α-helical hairpin-shaped c-subunits. According to structural analyses, rotors contain 10 c-subunits in yeast and 14 in chloroplast ATP synthases. We determined the rotor stoichiometry of Ilyobacter tartaricus ATP synthase by atomic force microscopy and cryo-electron microscopy, and show the cylindrical sodium-driven rotor to comprise 11 c-subunits. 相似文献
19.
Yuan-Gang Qu Fei Zhou Linda Yu Chang-An Yu 《The Journal of biological chemistry》2013,288(2):1047-1054
Proton transfer involving internal water molecules that provide hydrogen bonds and facilitate proton diffusion has been identified in some membrane proteins. Arg-94 in cytochrome b of the Rhodobacter sphaeroides bc1 complex is fully conserved and is hydrogen-bonded to the heme propionate and a chain of water molecules. To further elucidate the role of Arg-94, we generated the mutations R94A, R94D, and R94N. The wild-type and mutant bc1 complexes were purified and then characterized. The results show that substitution of Arg-94 decreased electron transfer activity and proton pumping capability and increased O2˙̄ production, suggesting the importance of Arg-94 in the catalytic mechanism of the bc1 complex in R. sphaeroides. This also suggests that the transport of H+, O2, and O2˙̄ in the bc1 complex may occur by the same pathway. 相似文献
20.
Min Sang Fei Ma Jie Xie Xiao-Bo Chen Ke-Bin Wang Xiao-Chun Qin Wen-Da Wang Jing-Quan Zhao Liang-Bi Li Jian-Ping Zhang Ting-Yun Kuang 《Biophysical chemistry》2010
Electron paramagnetic resonance (EPR) spectroscopy was used to detect the light-induced formation of singlet oxygen (1O2*) in the intact and the Rieske-depleted cytochrome b6f complexes (Cyt b6f) from Bryopsis corticulans, as well as in the isolated Rieske Fe–S protein. It is shown that, under white-light illumination and aerobic conditions, chlorophyll a (Chl a) bound in the intact Cyt b6f can be bleached by light-induced 1O2*, and that the 1O2* production can be promoted by D2O or scavenged by extraneous antioxidants such as l-histidine, ascorbate, β-carotene and glutathione. Under similar experimental conditions, 1O2* was also detected in the Rieske-depleted Cyt b6f complex, but not in the isolated Rieske Fe–S protein. The results prove that Chl a cofactor, rather than Rieske Fe–S protein, is the specific site of 1O2* formation, a conclusion which draws further support from the generation of 1O2* with selective excitation of Chl a using monocolor red light. 相似文献