A rotor-stator cross-link in the F1-ATPase blocks the rate-limiting step of rotational catalysis

The F0F1-ATP synthase couples the functions of H+ transport and ATP synthesis/hydrolysis through the efficient transmission of energy mediated by rotation of the centrally located gamma, epsilon, and c subunits. To understand the gamma subunit role in the catalytic mechanism, we previously determined the partial rate constants and devised a minimal kinetic model for the rotational hydrolytic mode of the F-1-ATPase enzyme that uniquely fits the pre-steady state and steady state data (Baylis Scanlon, J. A., Al-Shawi, M. K., Le, N. P., and Nakamoto, R. K. (2007) Biochemistry 46, 8785-8797). Here we directly test the model using two single cysteine mutants, beta D380C and beta E381C, which can be used to reversibly inhibit rotation upon formation of a cross-link with the conserved gamma Cys-87. In the pre-steady state, the gamma-beta cross-linked enzyme at high Mg center dot ATP conditions retained the burst of hydrolysis but was not able to release P-i. These data show that the rate-limiting rotation step, k(gamma), occurs after hydrolysis and before P-i release. This analysis provides additional insights into how the enzyme achieves efficient coupling and implicates the beta Glu381 residue for proper formation of the rate-limiting transition state involving gamma subunit rotation.