ATP hydrolysis by the Escherichia coli F1 ATPase (ECF1) induces a conformational change in the gamma subunit. This change can be monitored by fluorescence changes in N-[4-[7-(diethylamino)-4-methyl]coumarin-3-yl)]maleimide (CM) bound at a cysteine introduced by site-directed mutagenesis into the gamma subunit at position 106 [Turina, P., & Capaldi, R. A. (1994) J. Biol. Chem. 269, 13465-13471]. In studies reported here, the magnitude of the fluorescence change has been determined with the noncleavable nucleotide analogue AMP-PNP and by rapid measurements using the slowly cleavable ATP gamma S. The data indicate that maximal fluorescence change occurs with binding of 1 mol of nucleotide triphosphate per mole of ECF1. During unisite catalysis, ATP binding causes a fluorescence enhancement from CM bound at position 106, which is then followed by fluorescence quenching. The kinetics of these fluorescence changes have been measured using both ATP and ATP gamma S as substrate. With ATP gamma S, these kinetics can be simulated using rate constants similar to those for ATP except for an approximately 30-fold slower rate of the bond cleavage and resynthesis steps, i.e., k+2 and k-2. The observed rates and amplitudes of the fluorescence changes on hydrolysis of ATP and ATP gamma S were analyzed by simulations in which the bond cleavage or the Pi release step was responsible for fluorescence quenching. The results indicate that ATP or ATP gamma S binding causes the fluorescence enhancement of CM bound to the gamma subunit and that this conformational change is reversed upon bond cleavage to yield ADP.Pi or ADP.PiS in catalytic sites.