Nanosecond fluorometric investigation of hydrodynamic properties of adenosine triphosphatase from thermophilic bacterium PS3

The soluble portion (TF1) of proton-translocating ATPase from thermophilic bacterium PS3 was labeled with a fluorescent dye N-(1-pyrene)maleimide. The decay of fluorescence anisotropy of the adduct showed that TF1 in aqueous solution was characterized by a volume of equivalent sphere of 1,120 nm3. This value is 2.4 times the volume calculated from the molecular weight and partial specific volume, indicating a non-spherical shape and/or extensive hydration. A prolate ellipsoid with an axial ratio of 2 to 3 is suggested as a first approximation of the shape of hydrated TF1. The presence or absence of ATP, ADP, or Mg2+ did not alter the volume of the equivalent sphere appreciably; the probable conformational change of TF1 induced by these ligands does not lead to a gross alteration of its hydrodynamic properties.     

Small-angle neutron scattering from the reconstituted TF1 of H(+)-ATPase from thermophilic bacterium PS3 with deuterated subunits

Subunits alpha, beta and gamma of adenosine triphosphatase (H(+)-ATPase) from the thermophilic bacterium PS3 (TF1) have been over-expressed in Escherichia coli. alpha and beta subunits deuterated to the level of 90% were obtained by culturing E. coli in 2H2O medium. Both the subunits and the reconstituted alpha beta gamma complex, TF1, which contain the deuterated components in various combinations, were studied in solution by small-angle neutron scattering. The individual shapes of the subunits and their organization in the alpha beta gamma-TF1 complex were examined using the techniques of selective deuteration and contrast variation. The alpha and beta subunits are well approximated as ellipsoids of revolution having minor semi-axes of 20.4(+/- 0.4) and 20.0(+/- 0.2) A, and major semi-axes of 53.0(+/- 1.4) and 55.8(+/- 0.9) A, respectively. In the TF1 complex, three beta subunits are aligned to form an equilateral triangle, with their major axes tilted by 35 degrees with respect to the 3-fold axis of the complex. The beta-beta distance is about 53 A. Three alpha subunits are similarly arranged, positioned between the beta subunits, and with their direction of tilt opposite to that of the beta subunits. The centers of the alpha and beta subunits lie in the same plane, forming a hexagon. Adjacent subunits overlap in this model, suggesting that they are not simple ellipsoids of revolution.     

Identification of an essential glutamic acid residue in the beta subunit of the adenosine triphosphatase from the thermophilic bacterium PS3

The TF1-ATPase from the thermophilic bacterium, PS3, is inactivated by dicyclohexylcarbodiimide (DCCD). This inactivation is stimulated by ADP and other specific nucleotides and is inhibited by Mg2+. When the inactivation is carried out with [14C]DCCD, about 2 g atoms of 14C are bound/mol of TF1 when the enzyme is nearly completely inactivated. The isolated subunits from TF1 inactivated with [14C]DCCD contain the following amounts of 14C/mol: alpha, 0.12 g atom; beta, 0.47 g atom; gamma, approximately 0.04 g atom; delta, none; and epsilon, 0.05 g atom. Fractionation of tryptic digests have shown that the 14C bound to the alpha subunit is nonspecifically associated with several peptides, and that the 14C bound to the beta subunit is associated with a single tryptic peptide with the amino acid sequence Ala-Gly-Val-Gly-Glu-Arg, where Glu represents the N-gamma-glutamyl derivative of dicyclohexyl[14C]urea.     

Reconstitution of adenosine triphosphatase of thermophilic bacterium from purified individual subunits.

1. Five subunits (alpha, beta, gamma, delta, and epsilon) of an ATPase from a thermophilic bacterium PS3 were purified in the presence of 8 M urea by ion exchange chromatography. Then the ATPase activity was reconstituted by mixing the subunit solutions and incubating them at 20-45 degrees, at pH 6.3 to 7.0. 2. Mixtures containing beta + gamma or alpha + beta + delta regained ATP-hydrolyzing activity, but mixtures of alpha + beta and beta + delta did not. Combinations not including beta were all inactive. 3. The ATPase activity reconstituted from alpha + beta + delta was thermolabile and insensitive to NaN3, whereas the activities obtained from mixtures containing beta and gamma were thermostable and sensitive to NaN3, like the native ATPase. 4. The assemblies containing both beta and gamma subunits had the same mobility as the native ATPase molecule on gel electrophoresis, those without the gamma subunit moved more rapidly toward the anode. 5. Subunits epsilon and delta did not inhibit the ATPase activity of either the assembly (alpha + beta + gamma) or the native ATPase.     

Purification and properties of a dicyclohexylcarbodiimide-sensitive adenosine triphosphatase from a thermophilic bacterium.

1. A stable ATPase complex with sensitivity to dicyclohexylcarbodiimide (TFo-F1) was purified from the membranes of the thermophilic aerobic bacterium PS3, by ion exchange chromatography in the presence of Triton X-100. 2. The ATPase of TFo-F1 was maximal at 70 degrees at pH 8.6 and was stable after monomerization in 4 M urea and 0.5% Triton X-100 at 25 degrees. The activity was dependent on Mg2+, Co2+, or Mn2+, and it became insensitive to dicyclohexylcarbodiimide when Ca2+ or Cd2+ was added instead. 3. TFo-F1 required P-lipids of this bacterium contained branched fatty acyl groups but no unsaturated groups and were stable against oxidation and heat. 4. Studies by electron microscopy, gel electrophoresis, and use of anti-ATPase antibody and [3H]acetyl-ATPase indicated that the TFo-F1 complex was composed of an ATPase moiety (TF1, five different subunits) and a hydrophobic moiety (TFo, three different subunits. TFo conferred TF1 with sensitivity to dicyclohexylcarbodiimide. 5. Vesicles catalyzing 32Pi-ATP exchange and ATP-driven enhancement of fluorescence of anilinonaphthalene sulfonate were reconstituted by dialyzing pure TFo-F1 and P-lipids together, and were active even at 50-75 degrees. The vesicles reconstituted from TFo-F1 and bacterial P-lipids were more stable than those reconstituted from TFo-F1 and soybean P-lipids.     

Small-angle X-ray scattering studies of Mg.AT(D)P-induced hexamer to dimer dissociation in the reconstituted alpha 3 beta 3 complex of ATP synthase from thermophilic bacterium PS3.

The alpha 3 beta 3 complex of ATP synthase obtained from a thermophilic bacterium PS3 was isolated and found to show the ATPase activity (Kagawa, Y., Ohta, S., and Otawara-Hamamoto, Y. (1989) FEBS Lett. 249, 67-69). The structure and the nucleotide binding effects of the alpha 3 beta 3 complex were investigated by means of small-angle x-ray scattering and high performance liquid chromatography. The scattering profile from the alpha 3 beta 3 complex was explained with a model in which the complex is made of an ellipsoid of revolution with the axes of 121.8, 121.8, and 72.0 A having an elliptical hollow cavity with the axes of 35.4, 35.4, and 72.0 A. By the addition of Mg.AT(D)P, significant changes in the scattering profile were observed, in which the radius of gyration decreased from 44 to 35 A. This change was found by gel filtration to be caused by the dissociation reaction from the alpha 3 beta 3 hexamer to the alpha beta dimer. The dissociation of the alpha 3 beta 3 complex was not induced by unhydrolyzable ATP analogue, nor by Pi, Mg2+, and Pi + Mg2+. The structure of the dimer was well explained by the triaxial ellipsoidal model with the axes of 105.2, 39.4, and 108.2 A. The dissociation into the dimer is considered to be related to the ATPase activity because the AT(D)P-induced dissociation is observed only in the presence of Mg2+ ions.     

Sequence and over-expression of subunits of adenosine triphosphate synthase in thermophilic bacterium PS3

The primary structures of all the subunits of thermophilic ATP synthase were determined, and its alpha, beta and gamma subunits could be over-expressed in Escherichia coli, because these subunits were stable and reconstitutable. DNA of 7500 base pairs in length was found to contain a cluster of nine genes for subunits of ATP synthase. The order of their reading frames (size in base pairs) was: I(381): a(630): c(216): b(489): delta(537): alpha(1507): gamma(858): beta(1419): epsilon(396), I being a gene for a small hydrophobic, basic protein expressed in vitro. All the termini of TF0F1 subunits were confirmed by peptide sequencing. Large quantities of the overexpressed thermophilic alpha, beta and gamma subunits were prepared from the extract of E. coli, by a few purification steps.     

Pulsed cytochrome c oxidase from the thermophilic bacterium PS3.

A caa3-type terminal cytochrome c oxidase (EC 1.9.3.1) from the thermophilic bacterium PS3 containing three subunits showed conversion from resting into pulsed form. Upon pulsing (reduction and re-oxidation), the cytochrome c oxidase activity increased over 10-fold. This enhanced activity of the pulsed enzyme gradually decayed. Addition of phospholipids, necessary for the enzyme activity, did not affect this decay process. Small changes in the absorption spectrum were observed for the resting-into-pulsed transition and for H2O2 ligation to the pulsed enzyme. The e.p.r. spectrum of the resting enzyme was very similar to that of mitochondrial enzyme, but the transient g = 5, 1.78 and 1.69 set of e.p.r. signals, associated with the pulsed bovine heart oxidase, were not observed in the case of pulsed bacterium-PS3 enzyme.     

Functional arginine residues and carboxyl groups in the adenosine triphosphatase of the thermophilic bacterium PS-3

Treatment of purified ATPase of the thermophilic bacterium PS-3 with the arginine reagent phenylglyoxal or with Woodward's reagent K, gave complete inactivation of the enzyme. The inactivation rates followed apparent first-order kinetics. The apparent order of reaction with respect to inhibitor concentrations gave values near to 1 with both reagents, suggesting that inactivation was a consequence of modifying one arginine or carboxyl group per active site. ADP and ATP strongly protected the thermophilic ATPase against both reagents. GDP and IDP protected less, whilst CTP did not protect. Experiments in which the incorporation of [14C]phenylglyoxal into the enzyme was measured show that extrapolation of incorporation to 100% inactivation of the enzyme gives 8-9 mol [14C]phenylglyoxal per mol ATPase, whilst ADP or ATP prevent modification of about one arginine per mol.     

Purified proton conductor in proton translocating adenosine triphosphatase of a thermophilic bacterium.

1. The membrane-integrated portion (TF0) of the proton translocating ATPase complex (TF0-F1) of the thermophilic bacterium PS3 was highly purified. Its proton-conducting activity was investigated in vesicles reconstituted from TF0 and phospholipids (TF0 vesicles). 2. The rate of proton conduction through TF0 was proportional to the membrane potential imposed (6H+ uptake/s/TF0 molecule with 103 mV at pH 8.0). The pH profile of the rate revealed that a proton, not a hydroxy ion, was the true substrate conducted and that there was a monoprotic proton binding site in TF0 (pKa = 6.8). The temperature coefficient of proton conductance of TF0 showed a considerable variation depending on the phospholipids of the vesicles with respective transition temperatures. 3. Passive proton conduction through TF0 was inhibited stoichiometrically by addition of either the soluble ATPase portion (TF1) of TF0-F1, or an energy transfer inhibitor dicyclohexylcarbodiimide or an antibody against TF0. 4. The proton conductance of TF0 was concluded to represent its intrinsic activity in the original TF0-F1 complex.     

Cytochrome oxidase from thermophilic bacterium PS3 contains a fourth protein subunit

Monoclonal antibodies prepared against subunits II and IV of beef heart cytochrome oxidase were found to cross-react with thermophilic bacterial PS3 oxidase. Each individual antibody affects the enzymatic activity. "Western" blot analyses showed that subunit II antibodies of beef heart recognized subunit II of PS3 and subunit IV antibody likewise recognized a fourth protein subunit on slab gels. This fourth subunit previously thought to be a contaminant or a degradation product has a molecular weight of about 10,500 on SDS-gels, and appears to exist in stoichiometric amount. We have extracted this subunit from slab gels and compared its amino acid composition with that of subunit III.     

Reconstitution of vesicles capable of energy transformation from phospholipids and adenosine triphosphatase of a thermophilic bacterium.

1. A stable ATPase [EC 3.6.1.3] complex (TF0-F1) from the thermophilic bacterium PS3 was reconstituted into vesicles capable of energy transformation,measured as ATP-dependent enhancement of fluorescence of 8-anilinonoaphthalene-1-sulfonate. 2. The factors necessary for obtaining highly active vesicles were investigated. Cholate and deoxycholate were both required for solubilization of TF0-F1 and P-lipids, and removal of the detergents by dialysis resulted in vesicle formation. Medium of around pH 8 and low ionic strength containing 2.5 mM MgSO4 was found suitable for dialysis. The optimal temperature for reconstitution was 30 degrees with soybean P-lipids and 45 degree with PS3 P-lipids. The optimal ratio of protein to lipid was about 1/50. 3. The vesicles obtained under these conditions were mainly 100-200 nm in diameter, covered with 9.5 nm spheres, and had a bouyant density of 1.06 in sucrose andan internal volume of about 0.5 mul per mg of P-lipids.     

Resonance Raman study of the aa3-type cytochrome oxidase of thermophilic bacterium PS3

Resonance Raman spectra of the aa3-type cytochrome oxidase of thermophilic bacterium PS3, which has a simpler subunit composition than the mitochondrial enzymes but very similar enzymatic properties, are investigated under various conditions and compared with those of mitochondrial enzymes. The intensities of the two marker lines of reduced cytochrome a3 at 1667 and 213 cm-1 had different dependences on the incubation temperatures and pH. With regard to the incubation temperature dependence, the intensity of the 1667-cm-1 line, the peripheral CH = O stretching mode of the a3 heme, behaved in nearly the same way as that of the oxidase activity whereas the intensity of the 213-cm-1 line, the Fe-histidine stretching mode of the a3 heme, exhibited a similar dependence to that of the proton pumping activity. The 213-cm-1 line disappeared upon binding of carbon monoxide, upon raising the pH above 9.2, or after incubating above 55 degrees C. The Raman line at 1611 cm-1, which was recently suggested to probe the proton pump activity [Babcock, G.T., & Callahan, P.M. (1983) Biochemistry 22, 2314-2319], remained unaltered after incubation at 60 degrees C for 20 min despite a reduction of proton pumping activity to one-third. This argues against the proposed mechanism. The frequencies of the Raman lines were the same for the intact membrane and the isolated enzyme in the reduced state. The Raman spectra of cytochrome oxidase isolated from bacterium, yeast, and bovine heart were different in the lower frequency region below 600 cm-1 but closely alike in the higher frequency region above 1200 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of hydrogen-deuterium exchange in ATPase from a thermophilic bacterium PS3.

The kinetics of the hydrogen-deuterium exchange reaction in a stable ATPase (TF₁) from a thermophilic bacterium PS3 was followed by infrared absorption measurements. The rates of the hydrogen-deuterium exchange reactions decreased in following order; free form, TF₁·ADP, TF₁·ATP and TF₁·AMP-P(NH)P. TF₁ does not dissociate into subunits even in the absence of nucleotides, thus differences in exchange likely reflect differences in conformations of subunits. These results indicate that the structure is most restricted when ATP or AMP-P(NH)P is bound to the enzyme.     

Small-angle X-ray scattering studies of enzymes

Enzyme function requires conformational changes to achieve substrate binding, domain rearrangements, and interactions with partner proteins, but these movements are difficult to observe. Small-angle X-ray scattering (SAXS) is a versatile structural technique that can probe such conformational changes under solution conditions that are physiologically relevant. Although it is generally considered a low-resolution structural technique, when used to study conformational changes as a function of time, ligand binding, or protein interactions, SAXS can provide rich insight into enzyme behavior, including subtle domain movements. In this perspective, we highlight recent uses of SAXS to probe structural enzyme changes upon ligand and partner-protein binding and discuss tools for signal deconvolution of complex protein solutions.