Redox regulation of the rotation of F(1)-ATP synthase.
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In F(1)-ATPase, the smallest known motor enzyme, unidirectional rotation of the central axis subunit gamma is coupled to ATP hydrolysis. In the present study, we report the redox switching of the rotation of this enzyme. For this purpose, the switch region from the gamma subunit of the redox-sensitive chloroplast F(1)-ATPase was introduced into the bacterial F(1)-ATPase. The ATPase activity of the obtained complex was increased up to 3-fold upon reduction (Bald, D., Noji, H., Stumpp, M. T., Yoshida, M. & Hisabori, T. (2000) J. Biol. Chem. 275, 12757-12762). Here, we successfully observed the modulation of rotation of gamma in this chimeric complex by changes in the redox conditions. In addition we revealed that the suppressed enzymatic activity of the oxidized F(1)-ATPase complex was characterized by more frequent long pauses in the rotation of the gamma subunit. These findings obtained by the single molecule analysis therefore provide new insights into the mechanisms of enzyme regulation. (+info)
Toward an adequate scheme for the ATP synthase catalysis.
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The suggestions from the author's group over the past 25 years for how steps in catalysis by ATP synthase occur are reviewed. Whether rapid ATP hydrolysis requires the binding of ATP to a second site (bi-site activation) or to a second and third site (tri-site activation) is considered. Present evidence is regarded as strongly favoring bi-site activation. Presence of nucleotides at three sites during rapid ATP hydrolysis can be largely accounted for by the retention of ADP formed and/or by the rebinding of ADP formed. Menz, Leslie and Walker ((2001) FEBS Lett., 494, 11-14) recently attained an X-ray structure of a partially closed enzyme form that binds ADP better than ATP. This accomplishment and other considerations form the base for a revised reaction sequence. Three types of catalytic sites are suggested, similar to those proposed before the X-ray data became available. During net ATP synthesis a partially closed site readily binds ADP and Pi but not ATP. At a closed site, tightly bound ADP and Pi are reversibly converted to tightly bound ATP. ATP is released from a partially closed site that can readily bind ATP or ADP. ATP hydrolysis when protonmotive force is low or lacking occurs simply by reversal of all steps with the opposite rotation of the gamma subunit. Each type of site can exist in various conformations or forms as they are interconverted during a 120 degrees rotation. The conformational changes with the ATP synthase, including the vital change when bound ADP and Pi are converted to bound ATP, are correlated with those that occur in enzyme catalysis in general, as illustrated by recent studies of Rose with fumarase. The betaE structure of Walker's group is regarded as an unlikely, or only quite transient, intermediate. Other X-ray structures are regarded as closely resembling but not identical with certain forms participating in catalysis. Correlation of the suggested reaction scheme with other present information is considered. (+info)
Properties of noncatalytic sites of thioredoxin-activated chloroplast coupling factor 1.
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Nucleotide binding properties of two vacant noncatalytic sites of thioredoxin-activated chloroplast coupling factor 1 (CF(1)) were studied. Kinetics of nucleotide binding to noncatalytic sites is described by the first-order equation that allows for two nucleotide binding sites that differ in kinetic features. Dependence of the nucleotide binding rate on nucleotide concentration suggests that tight nucleotide binding is preceded by rapid reversible binding of nucleotides. ADP binding is cooperative. The preincubation of CF(1) with Mg(2+) produces only slight effect on the rate of ADP binding and decreases the ATP binding rate. The ATP and ADP dissociation from noncatalytic sites is described by the first-order equation for similar sites with dissociation rate constants k(-2)(ADP)=1.5 x 10(-1) min(-1) and k(-2)(ATP) congruent with 10(-3) min(-1), respectively. As follows from the study, the noncatalytic sites of CF(1) are not homogeneous. One of them retains the major part of endogenous ADP after CF(1) precipitation with ammonium sulfate. Its other two sites can bind both ADP and ATP but have different kinetic parameters and different affinity for nucleotides. (+info)
In vivo modulation of nonphotochemical exciton quenching (NPQ) by regulation of the chloroplast ATP synthase.
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Nonphotochemical quenching (NPQ) of excitation energy, which protects higher plant photosynthetic machinery from photodamage, is triggered by acidification of the thylakoid lumen as a result of light-induced proton pumping, which also drives the synthesis of ATP. It is clear that the sensitivity of NPQ is modulated in response to changing physiological conditions, but the mechanism for this modulation has remained unclear. Evidence is presented that, in intact tobacco or Arabidopsis leaves, NPQ modulation in response to changing CO(2) levels occurs predominantly by alterations in the conductivity of the CF(O)-CF(1) ATP synthase to protons (g(H)(+)). At a given proton flux, decreasing g(H)(+) will increase transthylakoid proton motive force (pmf), thus lowering lumen pH and contributing to the activation of NPQ. It was found that an approximately 5-fold decrease in g(H)(+) could account for the majority of NPQ modulation as atmospheric CO(2) was decreased from 2,000 ppm to 0 ppm. Data are presented that g(H)(+) is kinetically controlled, rather than imposed thermodynamically by buildup of DeltaG(ATP). Further results suggest that the redox state of the ATP synthase gamma-subunit thiols is not responsible for altering g(H)(+). A working model is proposed wherein g(H)(+) is modulated by stromal metabolite levels, possibly by inorganic phosphate. (+info)
Molecular devices of chloroplast F(1)-ATP synthase for the regulation.
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In chloroplasts, synthesis of ATP is energetically coupled with the utilization of a proton gradient formed by photosynthetic electron transport. The involved enzyme, the chloroplast ATP synthase, can potentially hydrolyze ATP when the magnitude of the transmembrane electrochemical potential difference of protons (Delta(micro)H(+)) is small, e.g. at low light intensity or in the dark. To prevent this wasteful consumption of ATP, the activity of chloroplast ATP synthase is regulated as the occasion may demand. As regulation systems Delta(micro)H(+) activation, thiol modulation, tight binding of ADP and the role of the intrinsic inhibitory subunit epsilon is well documented. In this article, we discuss recent progress in understanding of the regulation system of the chloroplast ATP synthase at the molecular level. (+info)
Respiratory chain supercomplexes of mitochondria and bacteria.
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Respiratory chain complexes are fragments of larger structural and functional units, the respiratory chain supercomplexes or "respirasomes", which exist in bacterial and mitochondrial membranes. Supercomplexes of mitochondria and bacteria contain complexes III, IV, and complex I, with the notable exception of Saccharomyces cerevisiae, which does not possess complex I. These supercomplexes often are stable to sonication but sensitive to most detergents except digitonin. In S. cerevisiae, a major component linking complexes III and IV together is cardiolipin.In Paracoccus denitrificans, complex I itself is rather detergent-sensitive and thus could not be obtained in detergent-solubilized form so far. However, it can be isolated as part of a supercomplex. Stabilization of complex I by binding to complex III was also found in human mitochondria. Further functional roles of the organization in a supercomplex are catalytic enhancement by reducing diffusion distances of substrates or, depending on the organism, channelling of the substrates quinone and cytochrome c. This makes redox reactions less dependent of midpoint potentials of substrates, and permits electron flow at low degree of substrate reduction.A dimeric state of ATP synthase seems to be specific for mitochondria. Exclusively, monomeric ATP synthase was found in Acetobacterium woodii, in P. denitrificans, and in spinach chloroplasts. (+info)
Maize ABI4 binds coupling element1 in abscisic acid and sugar response genes.
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Significant progress has been made in elucidating the mechanism of abscisic acid (ABA)-regulated gene expression, including the characterization of an ABA-responsive element (ABRE), which is regulated by basic domain/Leu zipper transcription factors. In addition to the ABRE, a coupling element (CE1) has been demonstrated to be involved in ABA-induced expression. However, a trans factor that interacts with CE1 has yet to be characterized. We report the isolation of a seed-specific maize ABI4 homolog and demonstrate, using a PCR-based in vitro selection procedure, that the maize ABI4 protein binds to the CE-1 like sequence CACCG. Using electrophoretic mobility shift assays, we demonstrate that recombinant ZmABI4 protein binds to the CE1 element in a number of ABA-related genes. ZmABI4 also binds to the promoter of the sugar-responsive ADH1 gene, demonstrating the ability of this protein to regulate both ABA- and sugar-regulated pathways. ZmABI4 complements Arabidopsis ABI4 function, because abi4 mutant plants transformed with the ZmABI4 gene have an ABA- and sugar-sensitive phenotype. Identification of the maize ABI4 ortholog and the demonstration of its binding to a known ABA response element provide a link between ABA-mediated kernel development and the regulation of ABA response genes. (+info)
The Chlamydomonas reinhardtii organellar genomes respond transcriptionally and post-transcriptionally to abiotic stimuli.
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The Chlamydomonas reinhardtii plastid and mitochondrial transcriptomes were surveyed for changes in RNA profiles resulting from growth in 12 culture conditions representing 8 abiotic stimuli. Organellar RNA abundance exhibited marked changes during nutrient stress and exposure to UV light, as revealed by both RNA gel blot and DNA microarray analyses. Of particular note were large increases in tufA and clpP transcript abundance during nutrient limitation. Phosphate and sulfur limitation resulted in the most global, yet opposite, effects on organellar RNA abundance, changes that were dissected further using run-on transcription assays. Removal of sulfate from the culture medium, which is known to reduce photosynthesis, resulted in 2-fold to 10-fold decreases in transcription rates, which were reflected in lower RNA abundance. The decrease in transcriptional activity was completely reversible and recovered to twice the control level after sulfate replenishment. Conversely, phosphate limitation resulted in a twofold to threefold increase in RNA abundance that was found to be a post-transcriptional effect, because it could be accounted for by increased RNA stability. This finding is consistent with the known metabolic slowdown under phosphate stress. Additionally, inhibitor studies suggested that unlike those in higher plants, Chlamydomonas chloroplasts lack a nucleus-encoded plastid RNA polymerase. The apparently single type of polymerase could contribute to the rapid and genome-wide transcriptional responses observed within the chloroplast. (+info)