ADP-fluoroaluminate complexes are formed cooperatively at two catalytic sites of wild-type and mutant alpha3beta3gamma subcomplexes of the F1-ATPase from the thermophilic Bacillus PS3.
Addition of Al3+ and F- to the alpha3beta3gamma subcomplex of the TF1-ATPase containing MgADP in one catalytic site causes slow, complete inactivation as the ADP-fluoroaluminate complex is formed. This conflicts with the "bisite" stochastic model suggested earlier (Issartel, J. P., Dupuis, A., Lunardi, J. & Vignais, P. V. (1991) Biochemistry 30, 4726-4733] on the finding that complete inactivation of the bovine mitochondrial F1-ATPase by Al3+, F-, Mg2+, and excess ADP occurs as ADP-fluoroaluminate complexes form in two catalytic sites. When Al3+ and F- were added to alpha3beta3gamma containing MgADP in two catalytic sites, inactivation accelerated 8-fold, indicating catalytic to catalytic site cooperativity. When added to alpha3beta3gamma containing MgADP bound to one or two catalytic sites prior to addition of Al3+ and F-, phosphate inhibits formation of the ADP-fluoroaluminate complex. When introduced after adding 200 microM ADP plus Mg2+ to alpha3beta3gamma, but before adding Al3+ and F-, phosphate accelerated formation of the ADP-fluoroaluminate complex 3-fold. Sulfite accelerated formation of the ADP-fluoroaluminate complex 9-fold when 200 microM ADP plus Mg2+ was added to alpha3beta3gamma before adding Al3+ and F-. The accelerations induced by phosphate or sulfite in the presence of excess ADP and Mg2+ suggest noncatalytic to catalytic site cooperativity. When Al3+ and F- were added to the (alphaD261N)3beta3gamma subcomplex containing MgADP in a single catalytic site, the ADP-fluoroaluminate complex formed at least 10-fold more slowly than observed with wild-type under the same conditions. Therefore, the catalytic site containing MgADP recognizes the alphaD261N substitution when noncatalytic sites are empty. Cross-linking alpha to gamma or beta to gamma by oxidizing the (alphaA396C)3beta3(gammaA22C) and alpha3(betaD390C)3(gammaS90C) subcomplexes, respectively, abolishes cooperative formation of ADP-fluoroaluminate complexes in two catalytic sites. ADP-fluoroaluminate complex formation is restricted to a single catalytic site in the oxidized double mutants. The alpha3beta3delta subcomplex does not form an inhibitory ADP-fluoroaluminate complex under any of the conditions examined for the alpha3beta3gamma subcomplexes.
Dou
C
C
Department of Chemistry & Biochemistry, School of Medicine, University of California at San Diego, La Jolla 92093, USA.
Grodsky
N B
NB
Matsui
T
T
Yoshida
M
M
Allison
W S
WS
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000535:Aluminum / Q000378:metabolism*; D001407:Bacillus; D001665:Binding Sites; D005461:Fluorine / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008956:Models, Chemical; D010710:Phosphates / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013447:Sulfites / Q000378:metabolism
D046911:Macromolecular Substances; D010710:Phosphates; D013447:Sulfites; C053629:fluoroaluminum; D005461:Fluorine; D000244:Adenosine Diphosphate; D000535:Aluminum; D006180:Proton-Translocating ATPases