ATP hydrolysis-driven structural changes in the gamma-subunit of Escherichia coli ATPase monitored by fluorescence from probes bound at introduced cysteine residues.

Abstract

Four mutants of the Escherichia coli F1ATPase, gamma S8-C, gamma T106-C, gamma S179-C, and gamma V286-C, which have a cysteine introduced at different sites in the gamma-subunit by site-directed mutagenesis, were reacted with the fluorescent reagent N-(4-7-(diethylamino)4-methylcoumarin-3-yl)-maleimide (CM) under conditions that selectively label the introduced Cys residue. With each mutant the effect of nucleotide binding on the fluorescence of the probe has been monitored. The results obtained with the mutants gamma S8-C and gamma T106-C are similar. In both cases, there was a spectral shift and change in fluorescence intensity on adding AMP.PNP or ATP to enzyme emptied of nucleotide from catalytic sites, while no change in the fluorescence spectrum was observed upon adding ADP. The fluorescence spectral changes obtained with ATP were transient and involved an initial rapid fluorescence enhancement followed by a subsequent fluorescence quenching. The kinetics of these ATP-induced fluorescence changes and the kinetics of ATP hydrolysis as monitored by the rates of ATP binding and of Pi formation were the same under conditions of unisite catalysis, indicating that the conformational changes in the gamma-subunit being measured by the fluorescent probe are driven by ATP hydrolysis in catalytic sites. No nucleotide-dependent fluorescence changes were observed with CM bound at a Cys at position 179. Nucleotide-dependent changes in fluorescence were seen with CM bound at position 286, but these appear to reflect structural changes due to binding of ADP or ATP in noncatalytic sites. The fluorescence changes observed in mutants gamma S8-C and gamma T106-C were not seen in subunit epsilon-free E. coli F1ATPase, although such enzyme preparations are highly active ATPases. We conclude that the structural changes monitored by the fluorescent probe are a part of the conformational coupling, whereby catalytic site events are linked to proton channeling.