Thermophiles ATPases

A crucially important finding is that submitochon-drial particles or vesicles from broken chloroplasts will synthesize ATP from ADP and P , when an artificial pH gradient is imposed.172186 Isolated purified FjF0 ATPase from a thermophilic Bacillus has been coreconstituted into liposomes with the light-driven proton pump bacteiiorhodopsin (Chapter 23). Illumination induced ATP synthesis.187 These observations support Mitchell s proposal that the ATP synthase is both spatially separate from the electron carriers in the membrane and utilizes the protonmotive force to make ATP. Thus, the passage of protons from the outside of the mitochondria back in through the ATP synthase induces the formation of ATP. What is the stoichiometry of this process ... 

The most compelling experiments were performed by Noji et al.202 257-260 They prepared the a3P3y subcomplex of ATPase from a thermophilic bacterium. 

The Mg2+-activated ATPase (or ATP synthase) is made up of two parts. The Fj component is the catalytic, Mg2+-binding, extrinsic membrane protein composed of five different subunits, a, (3, y, S and e. The F0 component is an intrinsic membrane complex that contains three subunits, a, b and c, and mediates proton translocation. The F, protein is bound to the membrane through interaction with F0. The complexity of the F,F0 enzyme has presented many difficulties. Hie greatest advances have been made for the bacterial enzymes, notably for thermophiles, Escherichia coli and Rhodospirillum rubrum, where progress has been made in the purification of subunits and their reconstitution into membranes, and the identification of binding sites for Mg2+ and nucleotides on the Fi subunits.300 FiF0 preparations can be incorporated into liposomes and display H+ translocation, ATP-P, exchange and ATP synthesis.301... 

Va),"3 Bacillus megaterium (Bm),114 Thermophilic bacterium PS3 (PS3),"5 and bovine heart (mitochondria bom)"6 were aligned. Positions with identical amino acid residues in all ATPase are indicated by asterisks. 

A DNA-dependent ATPase activity was found associated with the purified enzyme [77]. In contrast, an ATP-independent relaxation activity detected in partially purified fractions of reverse gyrase [74] was absent from completely purified fractions and probably corresponds to a distinct DNA topoisomerase (see below). A reverse gyrase with similar structure and properties was purified later by Slezarev[78] from another extremely thermophilic archaebacterium, Desulfurococcus amylolyticus[7S],... 

Fig. 5.2. Subunit organization of prokaryotic ATPase. The subunit stoicheiometry is based on that of thermophilic FqF, [51]. /S y 8 a b c = 3 3 l l l l 2 6. The F, portion forms a plug through the phospholipid bilayer. The c subunit assumes a hairpin shape [67].

Y. Shirakihara, A.G.W. Leslie, J.P. Abrahams, J.E. Walker, T. Ueda, Y. Sekimoto, M. Kambara, K. Saika, Y. Kagawa, M. Yoshida, The crystal structure of the nucleotide-free asPg subcomplex of Fi-ATPase from the thermophilic Bacillus PS3 is a symmetric trimer. Structure 5, 825-836 (1997)... 

J.M. Jault, C. Dou, N.B. Grodsky, T. Matsui, M. Yoshida, W.S. Allison, The asPgY subcomplex of the Fi-ATPase from the thermophilic Bacillus PS3 with the P T165S substitution does not entrap inhibitory MgADP in a catalytic site during turnover. J. Biol. Chem. 271, 28818-28824 (1996)... 

C. Don, P.A.G. Fortes, W.S. Alhson, The asQlYSdlWjsY subcomplex of the Fi-ATPase from the Thermophilic Bacillus PS3 fails to dissociate ADP when... 

Y Shirakihara, AGW Leslie, JP Abrahams, JE Walker, T Ueda, Y Sekimoto, M Kambara, K Saika, Y Kagawa and M Yoshida (1997) The crystal structure of the nucleotide-free aaPa subcomplex of F -ATPase from the thermophilic Bacillus PS3 Is a symmetric trimer. Structure 5 825-836... 

Mandal AK, Cheung WD and Arguello JM (2002) Characterization of a thermophilic P-type Ag /Cu -ATPase from the extremophile Archaeo-globusfulgidus. J Biol Chem 277 7201-7208. 

Abbreviations used Fi Fq, H+-translocating ATP synthases from mitochondria, E. coli plasma membranes, plasma membranes of the thermophilic bacterium PS3 and chloroplast membranes CFi, chloroplast coupling factor one ATPase, CFiE. C. 3.6.1.3 SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl... 

Phipps BM, Hoffmann A, Stetter KO, Bainneister W (1991) A novel ATPase complex selectively accumulated upon heat shock is a major cellular component of thermophilic... 

Nechushtai R, Muster P,Binder A, Liveanu V and Nelson N (1983) Photosystem I reaction center from the thermophilic cyanobacterium mastigocladus laminosus, Proc. Natl. Acad. Sci. USA 80, 1179-1183. Nelson N (1983) Structure and synthesis of chloroplast ATPase, Methods Enzymol. 97 510-523. 

ELECTRIC POTENTIAL AND pH GRADIENT FORMATION BY RECONSTITUTED ATPase PROTEO-LIPOSOMES FROM THE THERMOPHILIC CYANOBACTERIUM SYNECHOCOCCUS 6716... 

The proton-translocating ATPase complex has been the object of many studies tc elucidate its energy-transducing function in the direction of both ATP synthesis and hydrolysis. By reconstitution in liposomes, mostly prepared from aso-lectin, a simple and functional model system can be obtained. Recently, a DCCD-sensitive H -ATPase complex has been isolated from the thermophilic cyanobacterium SyneohoooQOUS 6716 (Lubberding et al., submitted) as a Mg " -dependent heat-stable complex, which can only be activated by trypsin. This complex, reconstituted with liposomes from isolated Synechocooous lipids catalyzes DCCD-sensitive exchange and ATP hydrolysis activities and ge-... 

Lubberding HJ, Offerijns F, Vel WAC and De Vries PJR (1981) Characterization of the ATPase of the thermophilic cyanobacterium Synechococcus Z iv idus, In Akoyunoglou G, ed. Photosynthesis II. Electron treuisport and photophosphorylation, pp. 779-788. Balaban International Science Services, Philadelphia. Owens O and Krauss RW (1972) Kinetics of photophosphorylation in c lls of Anacyst is n idulans. Plant Physiol. 49, Suppl. 52. 

Yoshida M, Sone N, Hirata H, Kagawa Y (1977) Reconstitution of ATPase of thermophilic bacterium from purified individual subunits.J.Biol.Chem.252,3480-85... 

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