Mutations in the Nucleotide Binding Domain of the α Subunits of the F1-ATPase from Thermophilic Bacillus PS3 That Affect Cross-Talk between Nucleotide Binding Sites

Inactivation of MF1 (bovine mitochondrial F1-ATPase) with 5‘-p-fluorosulfonylbenzoylethenoadenosine is caused by labeling αY244 [Verburg, J. G., and Allison, W. S. (1990) J. Biol. Chem. 265, 8065−8074]. In the crystal structure [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621−628], αY244 is hydrogen bonded to αR304 which is also hydrogen bonded to αY300. The catalytic properties of mutant α3β3γ subcomplexes of the TF1-ATPase from the thermophilic Bacillus PS3 containing the αF244C, αR304C, and αY300C substitutions have been examined. Each has unique features for hydrolyzing ATP and forming inhibitory ADP−fluoroaluminate complexes in catalytic sites. Unlike wild-type, the (αR304C)3β3γ and (αY300C)3β3γ subcomplexes entrap inhibitory MgADP in a catalytic site during turnover which fails to dissociate when ATP binds to noncatalytic sites. Although the hydrolytic properties of the (αF244C)3β3γ subcomplex and wild-type are similar, the mutant forms ADP−fluoroaluminate complexes 7 times faster than wild-type when Al3+ and F- are added to it in the presence of excess ADP and Mg2+. It also resists inhibition by high Mg2+ concentrations in the assay medium. At least one noncatalytic site of the (αF244C)3β3γ subcomplex has increased affinity for ADP, indicating that the enhanced rate of formation of the ADP−fluoroaluminate complex reflects augmented cooperativity between noncatalytic and catalytic sites. The rate of formation of the ADP−fluoroaluminate complex in (αY300C)3β3γ increases only 40% when MgADP in bound to two catalytic sites rather than one, compared to a 9-fold increase exhibited by wild type. When Al3+ and F- are added to the (αY300C)3β3γ subcomplex after incubation with excess ADP and Mg2+, ADP−fluoroaluminate complexes are formed in three catalytic sites rather than two observed with the other subcomplexes. Reconciliation of the catalytic properties of the mutant subcomplexes in terms of the crystal structure suggests that αF244, αR304, and αY300 of TF1 are part of a pathway that propagates conformational signals from one catalytic site to another.