The characteristics of the (alpha V371C)3(beta R337C)3 gamma double mutant subcomplex of the TF1-ATPase indicate that the catalytic site at the alpha TP-beta TP interface with bound MgADP in crystal structures of MF1 represents a catalytic site containing inhibitory MgADP.

In the MF 1 crystal structure with the MgADP-fluoroaluminate complex bound to two catalytic sites [Menz, R. I., Walker, J. E., and Leslie, A. G. W. (2001) Cell 106, 331-341], the guanidinium of βR 3 3 7 is within 2.9 A of the α-oxygen of αS 3 7 0 and 3.7 A of a methyl group of αV 3 7 1 at the α E -β H C interface. To examine the functional role of this contact, the (αV 3 7 1 C) 3 (βR 3 3 7 C) 3 γ subcomplex of the TF 1 -ATPase was prepared and characterized. Steady state ATPase activity of the reduced double-mutant is 30% of that of the wild type. Inactivation of the double mutant containing empty catalytic sites or MgADP bound to one catalytic site with CuCl 2 cross-linked two α-β pairs, whereas a single α-β pair cross-linked when at least two catalytic sites contained MgADP. The reduced double mutant hydrolyzed substoichiometric ATP 100-fold more rapidly than the enzyme containing two cross-linked α-β pairs. Addition of AlCl 3 and NaF to the reduced double mutant after incubation with stoichiometric MgADP or 200 μM MgADP irreversibly inactivated the steady state ATPase activity with rate constants of 1.5 x 10 - 2 and 4.1 x 10 - 2 min - 1 , respectively. In contrast, addition of AlCl 3 and NaF to the cross-linked enzyme after incubation with stoichiometric or 200 μM MgADP irreversibly inactivated ATPase activity with a common rate constant of ∼10 - 4 min - 1 . Correlation of these results with crystal structures of MF 1 suggests that the catalytic site at the α T P -β T P interface is loaded first upon addition of nucleotides to nucleotide-depleted F 1 -ATPases and that the catalytic site at the α T P -β T P interface with bound MgADP in crystal structures represents a catalytic site containing inhibitory MgADP.